R/Cas_Type.R
In mrb20045/CaSilico: In silico crRNA design for different CRISPR-Cas subtypes
Defines functions
VDotplot
mafftR
CaSilico
Documented in
CaSilico
mafftR
VDotplot
#' In silico crRNA design for CRISPR-Cas12 (A, B and F1 subtypes) and CRISPR-Cas13 (A, B and D subtypes).
#'
#'
#' @param ResultsFolder Name of the folder to save the final results; Default: ReaultsFolder="CaSilico_output".
#' @param TargetFasta Full path to the target sequence(s) to design crRNA for; Example: TargetFasta="/usr/mrb/casilico/genome.fasta"
#' @param TargetAccession Genome/gene/transcript accession numbers(s)(RefSeq or ENSEMBL ID)for the target sequence(s) to design crRNA for; Example: TargetAccession=c("U15717", "U15718").
#' @param TargetCoordinate Genomic coordinate(s)for the target sequence(s) to design crRNA for. CaSilico automatically retrieves the sequences based on the coordinates and their species; Example: TargetCoordinate=list(chromosome=c("1","5"), start=c("2000","12000"), end=c("2150","15500"), strand=c("-","-"), species = c("bos_taurus","bos_taurus")).
#' @param CrisprTypes A vector of CRSPR subtypes to design crRNAs based on their characteristics. One or more subtypes can be selected; Example: CrisprTypes=c("casVI_A","casVI_B","casVI_D","casV_A","casV_B","casV_F1").
#' @param ConservationMethod Desired method (1 or 2) to detect the conserved regions when more than one target sequence is submitted. See detailes for more information; Default: 1.
#' @param ConservationThreshold Desired threshold (between 0 and 1) to detect the conserved regions when more than one target sequence is submitted; Default: 0.95.
#' @param OffTarget If TRUE, CaSilico search for off-targets in the sequences specified by the Organism or LocalOff argument. This analysis may take a long time based on the number of subtypes in CrisprTypes argument and number of the selected sequences to be searched (Organism or LocalFasta arguments); Default: FALSE.
#' @param OffAsk If TRUE, in online mode before starting the off target analysis for each subtype, CaSilico ask from the user if he/she wants to continue or not; Default: FALSE.
#' @param Organism A vector of desired organisms for online off target analysis. CaSilico automatically search the identified crRNAs on the sequences(s)(NR or Refseq database, based on the CRISPR-Cas subtype) of the specified organismes; Example: Organism=c("Homo sapiens","Bos taurus",...).
#' @param LocalOff If TRUE, CaSilico search for off targets in local mode and Organism argument dose not works. LocalFile have to be set. Default: FALSE.
#' @param LocalFasta Full path to the fasta file sequence(s) to be used for local off target analysis. One or more fasta file can be addressed; Example: LocalFasta=c("/yourpath1/filename1.fasta","/yourpath2/filename2.fasta").
#' @param LocalName A vector including desired names related to the LocalFasta argument to determine the name of the results. If it is'nt specified by the user, Casilico considers sequence1, sequence2 and etc based on the number of fasta files in LocalFasta argument; Example: LocalName=c("Name1", "Name2").
#' @param Threads Number of threads to use, default value is 4.; Example: Threads=8..
#' @return Casilico presents the results in HTML and PDF files that can be found in ReaultsFolder.
#' @section References:
#' ...
#' @section Further details:
#' Casilico accepts three kinds of input formats as target sequence to be scanned for all potential target sites: 1) direct sequence(s) (TargetFasta argument); 2) Accession nember (TargetAccession argument) and 3) genomic coordinate (TargetCoordinate argument). One of these arguments have to be determined by the user as TargetFasta is recommended. Also, there are two different approaches to identify conserved regions (using ConservationMethod argument): 1) Most or all positions in the fragments (with length of w) have to be conserved and ≤2 positions can be polymorphic (based on the defined conservation score), as these positions can be occurred in the specific locations of the window, 2) Most or all positions in the fragments (with length of w) have to be conserved and ≤2 positions can be polymorphic (based on the defined conservation score) in everywhere of the fragment (with length of w).
#' @export
#' @import seqinr DT RRNA ape htmltools httr tibble dplyr readr
#' @examples
#
#'
#'############# (1) #############
#'# Run with TargetFasta and without off-target analysis
#'
#'# Full path of target sequences
#'data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")
#'
#'# Run CaSilico for subtype VI_A with ConservationThreshold = 0.98 and ConservationMethod = 1
#'CaSilico(ResultsFolder="Example",
#' TargetFasta=data,
#' TargetAccession=NULL,
#' CrisprTypes=c("casVI_A"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = F,
#' OffAsk = F)
#'##############################
#'#
#'#
#'#
#'############# (2) #############
#'
#'# Run with TargetFasta and offline off-target analysis
#'
#'# Full path of target sequences
#'data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")
#'
#'# Full path to fasta file sequences for local off target analysis
#'genome_dir <- paste0(system.file(package = "CaSilico"),"/data/genomes_off_target")
#'Local_Fasta <- file.path(genome_dir,list.files(genome_dir))
#'
#'
#'# Names related to the LocalFasta argument to determine the name of the results. (optional)
#'Local_Name <- c("Genome1","Genome2","Genome3")
#'
#'# Run CaSilico for subtypes V_A and V_B with ConservationThreshold = 0.98 and ConservationMethod = 1
#'CaSilico(ResultsFolder="Example1",
#' TargetFasta=data,
#' TargetAccession=NULL,
#' CrisprTypes=c("casV_A","casV_B"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = T,
#' OffAsk = F,
#' Organism=NULL,
#' LocalOff=T,
#' LocalFasta=Local_Fasta,
#' LocalName=Local_Name)
#'##############################
#'#
#'#
#'#
#'############# (3) #############
#'# Run with TargetFasta and online off-target analysis
#'
#'# Full path of target sequences
#'data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")
#'
#'# Run CaSilico for subtype VI_A with ConservationThreshold = 0.98 and ConservationMethod = 1
#'# and online off-target analysis for Homo sapiens
#'CaSilico(ResultsFolder="Example2",
#' TargetFasta=data ,
#' TargetAccession=NULL,
#' CrisprTypes=c("casVI_A"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = T,
#' OffAsk = F,
#' Organism="Homo sapiens",
#' LocalOff=F)
#'##############################
#'#
#'#
#'#
#'############# (4) #############
#'# Run with accession numbers
#'
#'# Run CaSilico for subtypes VI_A and VI_B with ConservationThreshold = 0.98 and ConservationMethod = 1
#'# The accession numbers used are: "U15717" and "U15718"
#' CaSilico(ResultsFolder="Example3",
#' TargetAccession=c("U15717", "U15718"),
#' CrisprTypes=c("casVI_A","casVI_B"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = F,
#' OffAsk = F)
#'##############################
#'#
#'#
#'#
#'############# (5) #############
#' # Run with sequence coordinate
#'
#' # Run CaSilico for subtypes VI_A and VI_B with ConservationThreshold = 0.98 and ConservationMethod = 1
#' # The desired coordinates are:
#' # (1) chromosome = 1, start = 2000, end = 2150, strand = "-", species = "bos_taurus"
#' # (2) chromosome = 1, start = 2000, end = 2150, strand = "-", species = "bos_taurus"
#' CaSilico(ResultsFolder="Example4",
#' TargetCoordinate=list(chromosome=c("1","1"),
#' start=c("2000","2000"),
#' end=c("2150","2150"),
#' strand=c("-","-"),
#' species = c("bos_taurus","bos_taurus")),
#' CrisprTypes=c("casVI_B","casVI_A"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = F,
#' OffAsk = F)
#'##############################
#'
CaSilico=function(ResultsFolder="CaSilico_output",
TargetFasta=NULL,
TargetAccession=NULL,
TargetCoordinate=list(chromosome=NA, start=NA, end=NA, strand=NA, species = NA ),
CrisprTypes,
ConservationMethod = 1,
ConservationThreshold=0.95,
OffTarget = F,
OffAsk=F,
Organism=NULL,
LocalOff=F,
LocalFasta=c("Path1","Path2"),
LocalName=c("Organism1","Organism2"),
Threads=4){
library("seqinr")
library("DT")
library("RRNA")
library("ape")
library("htmltools")
library("tibble")
library("dplyr")
library("readr")
defaultW <- getOption("warn")
options(warn = -1)
host_system=Sys.info()[['sysname']]
if (host_system %in% c("Windows","Linux")==F) {
stop("Currently CaSilico only supports Windows and Linux systems")
}
first_main_dir=getwd()
package_location<-system.file(package = "CaSilico")
if (host_system=="Windows") {
MAFFT_location=paste0(package_location,"/","dependency_win/mafft-win")
RNAfold_path = paste0(package_location,"/","dependency_win/Blastn/RNAfold.exe")
Blast_location=paste0(package_location,"/","dependency_win/Blastn/blastn.exe")
makeblastdb_location=paste0(package_location,"/","dependency_win/Blastn/makeblastdb.exe")
RNAplot_location=paste0(package_location,"/","dependency_win/Blastn/RNAplot.exe")
}
############## change mod sotware ###########
if (host_system=="Linux") {
MAFFT_location=paste0(package_location,"/dependency_linux/mafft-linux64")
RNAfold_path = paste0(package_location,"/dependency_linux/Blastn/RNAfold")
Blast_location=paste0(package_location,"/dependency_linux/Blastn/blastn")
makeblastdb_location=paste0(package_location,"/dependency_linux/Blastn/makeblastdb")
RNAplot_location=paste0(package_location,"/dependency_linux/Blastn/RNAfold")
path_software=c(RNAfold_path,
Blast_location,
makeblastdb_location,
RNAplot_location,
paste0(MAFFT_location,"/mafft.bat",collapse = ""))
for (path in path_software) {
chmod_command=paste0("chmod 777 ","'",path,"'")
lapply(chmod_command, system)
}
lapply(paste0("chmod -R 777 ",MAFFT_location), system)
}
#############################################
is_empty=function(X){
length(X)==0
}
# input seq is path
if (is_empty(TargetFasta)==FALSE) {
fasta_file=read.fasta(TargetFasta)
if (exists("fasta_file")==F | length(fasta_file)==0){
stop("There is a problem in your file or path of file. please check it.")
}
TargetAccession=NULL
TargetCoordinate=NULL
}
# input seq is accession number
if (length(TargetAccession)>=1) {
input_genes_fasta=read.GenBank(TargetAccession)
if (is_empty(input_genes_fasta)==T) {
stop("The access number entered is incorrect. please check it")
}
write.dna(input_genes_fasta,
file ="Input_Genes.fasta",
format = "fasta",
append = FALSE,
nbcol = 6,
colsep = "",
colw = 10)
fasta_file=read.fasta("Input_Genes.fasta")
file.remove("Input_Genes.fasta")
TargetFasta=NULL
TargetCoordinate=NULL
}
# input seq is coordinate
if (all(is.na(TargetCoordinate))==F) {
library("httr")
getseq=function(chromosome, start, end, strand, species = "Homo_sapiens"){
url = paste0("https://useast.ensembl.org/", species, "/Export/Output/Location?db=core;flank3_display=0;flank5_display=0;output=fasta;r=",
chromosome, ":", start, "-", end, ";strand=", strand,
";utr5=yes;cdna=yes;intron=yes;utr3=yes;peptide=yes;coding=yes;genomic=unmasked;exon=yes;_format=Text")
r=GET(url)
w=content(r, "text")
s=strsplit(w,split = "\r\n")[[1]]
paste0(s[-c(1,length(s))],collapse = "")
}
seqs_of_cordinate=c()
for (no_cor in 1:length(TargetCoordinate$chromosome)) {
seqs_of_cordinate=c(seqs_of_cordinate,
paste0(TargetCoordinate$chromosome[no_cor],"@",
TargetCoordinate$start[no_cor],"@",
TargetCoordinate$end[no_cor],"@",
TargetCoordinate$strand[no_cor],"@",
TargetCoordinate$species[no_cor],"@"))
}
input_coordinate_seq=c()
for (cordinate_seq in 1:length(seqs_of_cordinate)) {
temp_seq=seqs_of_cordinate[cordinate_seq]
temp_seq=strsplit(temp_seq,split = "@")[[1]]
temp_sequence="ddd"
iter=0
while(all(iter<=150 , temp_sequence=="ddd")){
try({
temp_sequence=getseq(temp_seq[1],temp_seq[2],temp_seq[3],temp_seq[4],temp_seq[5])
},silent = T)
iter=iter+1
}
input_coordinate_seq=c(input_coordinate_seq,temp_sequence)
}
if (any(input_coordinate_seq=="ddd")) {
stop("CaSilico is unable to download your coordinate sequences due to a problem on the Ensembl site.
Please try again later.")
}
write_line_in=c()
for (i in 1:length(input_coordinate_seq)) {
write_line_in=c(write_line_in,paste0(">",i),input_coordinate_seq[i])
}
file_Conaction<-file("Input_coordinate.fasta")
writeLines(write_line_in, file_Conaction)
close(file_Conaction)
fasta_file=read.fasta("Input_coordinate.fasta")
file.remove("Input_coordinate.fasta")
TargetFasta=NULL
TargetCoordinate=NULL
}
final_file_list=list()
# calculated total nucleotide for mafft limitation (mafft limitation > 1500 sequences * 30kb)
total_Nucleotide=0
for (h in 1:length(fasta_file)) {
total_Nucleotide=total_Nucleotide+length(fasta_file[[h]])
empty_pos=which(fasta_file[[h]]==" ")
if (length(empty_pos)>0) {
final_file_list[[h]]=fasta_file[[h]][-empty_pos]
}
else {final_file_list[[h]]=fasta_file[[h]]}
}
if ( total_Nucleotide > 40000000 ) {
print("Restrictions on Multiple Alignment")
}
if (total_Nucleotide <= 40000000) {
if (length(final_file_list)==1) {
write.fasta(list(final_file_list[[1]],final_file_list[[1]]), c(names(fasta_file),names(fasta_file)), "final_fasta_file.fasta")
}else{
write.fasta(final_file_list, names(fasta_file), "final_fasta_file.fasta")
}
setwd(MAFFT_location)
if (file.exists("final_fasta_file.fasta")) {
file.remove("final_fasta_file.fasta")
}
if (file.exists("final_fasta_file.aln")) {
file.remove("final_fasta_file.aln")
}
setwd(first_main_dir)
file.copy("final_fasta_file.fasta",MAFFT_location)
file.remove("final_fasta_file.fasta")
}
# Run mafft
setwd(MAFFT_location)
if (host_system=="Linux") {
MAFFT_list<- paste0("./mafft.bat --auto "," --thread ",Threads, " --inputorder final_fasta_file.fasta > final_fasta_file.aln")
}
if (host_system=="Windows") {
MAFFT_list<- paste0("mafft --auto "," --thread ",Threads, " --inputorder final_fasta_file.fasta > final_fasta_file.aln")
}
lapply(MAFFT_list, system) ###run MAFFT for all input .txt files
FMDValn <- read.fasta(file = "final_fasta_file.aln", as.string = T)
file.copy("final_fasta_file.aln",first_main_dir)
file.remove("final_fasta_file.fasta")
file.remove("final_fasta_file.aln")
setwd(first_main_dir)
topoTypes_sequences <- FMDValn
topoTypes_names <- names(FMDValn)
################# calculate frequency matrix for each base in sequences #########
score_matrix=matrix(0,5,length(strsplit(topoTypes_sequences[[1]],split = "")[[1]]))
rownames(score_matrix)=c("g","t","c","a","-")
for (i in 1:length(topoTypes_sequences)) {
seq=strsplit(topoTypes_sequences[[i]],split = "")[[1]]
for (j in 1:length(seq)) {
# one Nucleotide symbols
if(seq[j]=="g" | seq[j]=="G"){
score_matrix[1,j]= score_matrix[1,j]+1}
else if(seq[j]=="t" | seq[j]=="T" | seq[j]=="U" | seq[j]=="u"){
score_matrix[2,j]= score_matrix[2,j]+1}
else if(seq[j]=="c" | seq[j]=="C"){
score_matrix[3,j]= score_matrix[3,j]+1}
else if(seq[j]=="a" |seq[j]=="A"){
score_matrix[4,j]= score_matrix[4,j]+1}
else if(seq[j]=="-"){
score_matrix[5,j]= score_matrix[5,j]+1}
# four Nucleotide symbols
else if(seq[j]=="n" | seq[j]=="N" ){
score_matrix[1:4,j]= score_matrix[1:4,j]+0.25}
# two Nucleotide symbols
else if(seq[j]=="r" | seq[j]=="R" ){
score_matrix[c(1,4),j]= score_matrix[c(1,4),j]+0.5}
else if(seq[j]=="y" | seq[j]=="Y" ){
score_matrix[c(2,3),j]= score_matrix[c(2,3),j]+0.5}
else if(seq[j]=="k" | seq[j]=="K" ){
score_matrix[1:2,j]= score_matrix[1:2,j]+0.5}
else if(seq[j]=="m" | seq[j]=="M" ){
score_matrix[3:4,j]= score_matrix[3:4,j]+0.5}
else if(seq[j]=="s" | seq[j]=="S" ){
score_matrix[c(1,3),j]= score_matrix[c(1,3),j]+0.5}
else if(seq[j]=="w" | seq[j]=="W" ){
score_matrix[c(2,4),j]= score_matrix[c(2,4),j]+0.5}
# three Nucleotide symbols
else if(seq[j]=="b" | seq[j]=="B" ){
score_matrix[1:2,j]= score_matrix[1:2,j]+(0.33)
score_matrix[3,j]= score_matrix[3,j]+(0.34)}
else if(seq[j]=="d" | seq[j]=="D" ){
score_matrix[1:2,j]= score_matrix[1:2,j]+(0.33)
score_matrix[4,j]= score_matrix[4,j]+(0.34)}
else if(seq[j]=="h" | seq[j]=="H" ){
score_matrix[2:3,j]= score_matrix[2:3,j]+(0.33)
score_matrix[4,j]= score_matrix[4,j]+(0.34)}
else if(seq[j]=="v" | seq[j]=="V" ){
score_matrix[3:4,j]= score_matrix[3:4,j]+(0.33)
score_matrix[1,j]= score_matrix[1,j]+(0.34)}
}
}
#################################################################################
################### score matrix normalization ####################
normal_score_matrix=score_matrix/length(topoTypes_sequences)
normal_score_matrix1=normal_score_matrix
row_name_matrix=c("g","t","c","a","Del")
rownames(normal_score_matrix1)=row_name_matrix
####################################################################
######################### creATE msa sequence #####################
extract_higher_frequency=function(x){
for (g in 1:nrow(normal_score_matrix)) {
if(normal_score_matrix[g,x]==max(normal_score_matrix[,x]))
break
}
normal_score_matrix[g,x]
}
final_score=c()
for (k in 1:ncol(normal_score_matrix)) {
final_score=c(final_score,extract_higher_frequency(k))
}
final_str=toString(names(final_score),)
scores=as.data.frame(final_score)[[1]]
str_frags=strsplit(final_str,split = ", ")[[1]]
final_str_frags=c()
final_score1=c()
pso_del=c()
for (i in 1:length(str_frags)) {
if (str_frags[i]!="-") {
final_str_frags=c(final_str_frags,str_frags[i])
final_score1=c(final_score1,scores[i])
}
else if (str_frags[i]=="-") {
pso_del=c(pso_del,i)
}
}
if (length(pso_del)==0) {
original_pos_in_matrix=1:length(str_frags)
}
if (length(pso_del)!=0) {
original_pos_in_matrix=setdiff(1:length(str_frags),pso_del)
}
###############################################################
########################### Functions ########################
{
########### mismatch delete function####
if (ConservationMethod==1) {
mismatch_del=function(score,start,end,seed_start,seed_end){
del=c()
mismatch_locat=c()
for (i in 1:length(score)) {
if (score[i]<ConservationThreshold) {
mismatch_locat=c(mismatch_locat,i)
}}
if (length(mismatch_locat)>2) {
del=TRUE
}
if (length(mismatch_locat)==2) {
if (any(mismatch_locat%in%start:end)==TRUE) {
del=TRUE}
}
if (length(mismatch_locat)==2) {
if (any(mismatch_locat%in%start:end)!=TRUE) {
del=FALSE}
}
if (length(mismatch_locat)==1) {
if (mismatch_locat%in%seed_start:seed_end==TRUE) {
del=TRUE}
}
if (length(mismatch_locat)==1) {
if (mismatch_locat%in%seed_start:seed_end!=TRUE) {
del=FALSE}
}
if (length(mismatch_locat)==0) {
del=FALSE
}
del
}
}
if (ConservationMethod==2) {
mismatch_del=function(score,start,end,seed_start,seed_end){
del=c()
if (start<=end & seed_start<=seed_end)
{ if (length(which(score<ConservationThreshold))<3) {
del=FALSE
}
else if (length(which(score<ConservationThreshold))>=3) {
del=TRUE
}}
del
}
}
###############################
############# revers compliment function########
revers_compliment=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
Rcompliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in length(seq):1) {
Rcompliment_sequence = c(Rcompliment_sequence, compliment(seq[i],
seq_type = type))
}
Rcompliment_sequence
}
# compliment function
compliment_seq=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
compliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in 1:length(seq)) {
compliment_sequence = c(compliment_sequence, compliment(seq[i],
seq_type = type))
}
compliment_sequence
}
# revers function
revers_function=function(v){
revers_v=c()
for (i in length(v):1) {
revers_v=c(revers_v,v[i])
}
revers_v
}
##########################
################## check internet function ###########
if (host_system=="Windows") {
havingIP <- function() {
if (.Platform$OS.type == "windows") {
ipmessage <- system("ipconfig", intern = TRUE)
} else {
ipmessage <- system("ifconfig", intern = TRUE)
}
validIP <- "((25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)[.]){3}(25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)"
any(grep(validIP, ipmessage))
}
}
if (host_system=="Linux") {
havingIP <- function() {
if (.Platform$OS.type == "unix") {
ipmessage <- system("ip r", intern = TRUE)
} else {
ipmessage <- system("ip r", intern = TRUE)
}
validIP <- "((25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)[.]){3}(25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)"
any(grep(validIP, ipmessage))
}
}
##################################
################ crRNA plot function for RNA sequence ##############
crRNA_plot=function(sequence,crstart,start,pos_mutatiopns,
type_SNP,main_plot,W,u_rich_number,
acc_flank_number,eficieny_number,cr_acc) {
sequence=toupper(sequence)
plot(NULL, xlim =c(1,length(sequence)), ylim =c(1,12),type = "n",
axes =F,main = main_plot ,ann = T,xlab = "sequence",ylab = "")
axis(side=1, at = 1:length(sequence), labels= sequence,cex.axis = 0.7,
font = 0.6,col="darkred",col.axis="darkred")
axis(side=1, at = crstart:(crstart+W-1), labels= sequence[crstart:(crstart+W-1)] ,
cex.axis = 0.7,col="darkblue", font = 1,col.axis="darkblue")
end=start+W
x=c(crstart,crstart,crstart+W-1,crstart+W-1)
y=c(2,4,4,2)
polygon(x,y,col = "pink3",border = "pink3")
text((x[4]+x[1])/2,(y[2]+y[1])/2,labels = " CRISPR RNA",cex = 1.4)
text(x[1],1,labels = start,cex = 0.8)
text(x[4],1,labels = end,cex = 0.8)
if (is.numeric(pos_mutatiopns)) {
for (mut in 1:length(pos_mutatiopns)) {
pos_mutatiopns1=pos_mutatiopns+crstart-1
M=pos_mutatiopns1[mut]
if (length(pos_mutatiopns)==1) {
segments(M,4,M,6)
points(M,6,pch = 16, cex = 1.7, col = "red")
text((M),6.5,type_SNP[mut],cex = 1.2)
}
if (length(pos_mutatiopns)==2) {
if (abs(pos_mutatiopns[1]-pos_mutatiopns[2])>1) {
segments(M,4,M,6)
points(M,6,pch = 16, cex = 1.7, col = "red")
text((M),6.5,type_SNP[mut],cex = 1.2)
}
else if (abs(pos_mutatiopns[1]-pos_mutatiopns[2])==1) {
segments(M,4,M,6)
points(M,6,pch = 16, cex = 1.7, col = "red")
text((M-0.1),6.5,type_SNP[mut],cex = 1.2,srt=90)
}
}
}
}
segments(crstart,9,crstart+W-1,9,lwd=1.5,col = "darkblue")
text(crstart+W-1,9+0.03,">",col = "darkblue")
text(crstart,9+0.03,"<",col = "darkblue")
text((x[1]+x[4])/2,9+2,paste(cr_acc,"Accessible",collapse = ""),cex = 0.9,col = "darkblue")
if (crstart>10) {
segments(1,(y[2]+y[1])/2,crstart,(y[2]+y[1])/2,col="darkred")
text(1,(y[2]+y[1])/2+0.03,"<",col="darkred")
text(x[1]/2,(y[2]+y[1])/2+1,u_rich_number,cex = 0.9,col="darkred")
text(x[1]/2,(y[2]+y[1])/2+2,paste(acc_flank_number,"Accessible",collapse = ""),cex = 0.9,col="darkred")
}
if (length(sequence)-(crstart+W)>10){
segments(crstart+W-1,(y[2]+y[1])/2,length(sequence),(y[2]+y[1])/2,col="darkred")
text(length(sequence),(y[2]+y[1])/2+0.03,">",col="darkred")
text((length(sequence)+x[4])/2,(y[2]+y[1])/2+1,u_rich_number,cex = 0.9,col="darkred")
text((length(sequence)+x[4])/2,(y[2]+y[1])/2+2,paste(acc_flank_number,"Accessible",collapse = ""),cex = 0.9,col="darkred")
}
}
###################################################
################# crRNA plot function for DNA sequence ##################
crRNA_plot_DNA=function(sequence,crstart,start,pos_mutatiopns,
type_SNP,main_plot,W,eficieny_number,
sign_strand,length_pam) {
sequence=toupper(sequence)
end=start+W
plot(NULL, xlim =c(1,length(sequence)), ylim =c(1,6),type = "n",
axes =F,main = main_plot ,ann = T,xlab = "sequence",ylab = "")
axis(side=1, at = 1:length(sequence), labels= sequence,
cex.axis = 0.7, font = 0.6,col="darkred",col.axis="darkred")
x=c(crstart,crstart,crstart+W-1,crstart+W-1)
y=c(2,3,3,2)
polygon(x,y,col = "pink3",border = "pink3")
text((x[4]+x[1])/2,(y[2]+y[1])/2,labels = " CRISPR RNA",cex = 1.4)
segments(1,1.3,length(sequence),1.3,col="darkred")
text(1:length(sequence),1.38,"--",srt = 90,col="darkred")
text(1:length(sequence),1.8,toupper(compliment_seq(tolower(sequence))),col="darkred",cex = 0.7)
if (sign_strand=="+") {
axis(side=1, at = crstart:(crstart+W-1), labels= sequence[crstart:(crstart+W-1)] ,
cex.axis = 0.7,col="darkblue", font = 1,col.axis="darkblue")
segments((crstart-length_pam),2.1,(crstart-1),2.1,col="black")
text(((crstart-length_pam)+(crstart-1))/2,2.2,"<",srt = 90,col="black")
text(((crstart-length_pam)+(crstart-1))/2,2.45,"------",srt = 90,col="black")
text(((crstart-length_pam)+(crstart-1))/2,3,"PAM",col="black")
text(x[1],1.05,labels = start,cex = 1)
text(x[4],1.05,labels = end,cex = 1)
segments((crstart-length_pam),1.3,(crstart-1),1.3,col="orange")
text((crstart-length_pam):(crstart-1),1.38,"--",srt = 90,col="orange")
text((crstart-length_pam):(crstart-1),1.8,
toupper(compliment_seq(tolower(sequence)))[(crstart-length_pam):(crstart-1)],
col="orange",cex = 0.7)
}
if (sign_strand=="-") {
segments(crstart,1.3,(crstart+W-1),1.3,col="darkblue")
text(crstart:(crstart+W-1),1.38,"--",srt = 90,col="darkblue")
text(crstart:(crstart+W-1),1.8,
toupper(compliment_seq(tolower(sequence)))[crstart:(crstart+W-1)],
col="darkblue",cex = 0.7)
axis(side=1, at = (crstart+W):(crstart+W+length_pam-1), labels= sequence[(crstart+W):(crstart+W+length_pam-1)] ,
cex.axis = 0.7,col="orange", font = 1,col.axis="orange")
segments((crstart+W),2.1,(crstart+W+length_pam-1),2.1,col="black")
text(((crstart+W)+(crstart+W+length_pam-1))/2,2.2,"<",srt = 90,col="black")
text(((crstart+W)+(crstart+W+length_pam-1))/2,2.45,"------",srt = 90,col="black")
text(((crstart+W)+(crstart+W+length_pam-1))/2,3,"PAM",cex = 1.1,col="black")
text(x[1],1.05,labels =end ,cex = 1)
text(x[4],1.05,labels = start,cex = 1)
}
if (is.numeric(pos_mutatiopns)) {
for (mut in 1:length(pos_mutatiopns)) {
pos_mutatiopns1=pos_mutatiopns+crstart-1
M=pos_mutatiopns1[mut]
segments(M,3,M,4)
points(M,4,pch = 16, cex = 1.7, col = "red")
text((M-0.1),4.5,type_SNP[mut],cex = 1.1,srt = 90)
}
}
}
####################################################
############### my Dot plot function ################
my_Dot_Plot=function(sequence1,sequence2, wsize = 1 , wstep = 1 , nmatch = 1 ,
table_col = "goldenrod1",points_col = "red", xlab = "sequence1", ylab = "sequence2")
{
#Split the Elements of strin
if (length(sequence1)==1){
x <- strsplit(sequence1,split = "")[[1]]}
if (length(sequence2)==1){
y <- strsplit(sequence2,split = "")[[1]]}
if (length(sequence1)!=1){
x <-sequence1}
if (length(sequence2)!=1){
y <- sequence2}
if (nchar(x[1]) > 1)
stop("seq1 should be provided as a vector of single chars")
if (nchar(y[1]) > 1)
stop("seq2 should be provided as a vector of single chars")
if (wsize < 1)
stop("non allowed value for wsize")
if (wstep < 1)
stop("non allowed value for wstep")
if (nmatch < 1)
stop("non allowed value for nmatch")
if (nmatch > wsize)
stop("nmatch > wsize is not allowed")
mkwin <- function(seq, wsize, wstep) {
sapply(seq(from = 1, to = length(seq) - wsize + 1, by = wstep),
function(i) c2s(seq[i:(i + wsize - 1)]))
}
wseq1 <- mkwin(x, wsize, wstep)
wseq2 <- mkwin(y, wsize, wstep)
if (nmatch == wsize) {
xy <- outer(wseq1, wseq2, "==")
}
else {
"%==%" <- function(x, y) colSums(sapply(x, s2c) == sapply(y,
s2c)) >= nmatch
xy <- outer(wseq1, wseq2, "%==%")
}
x=toupper(x)
y=toupper(y)
a=matrix(0,length(x),length(y))
# Naming the rows and columns of the formed matrix
row.names(a)=x
colnames(a)=y
for (i in 1:nrow(xy)) {
for (j in 1:ncol(xy)){
if (xy[i,j]==T) {
a[i,j]=1
}
}
}
h_line=seq(0+((seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])/2),max(length(x),length(y)),
by=seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])
v_line=seq(1+((seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])/2),max(length(x),length(y)),
by=seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])
aaaa=0.04*max(length(x),length(y))
plot(0:max(length(x),length(y)),0:max(length(x),length(y)),type = "n",
axes =F,frame.plot=TRUE,main ="" ,
ann = T,xlab = xlab , ylab = ylab)
aa=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb=c(h_line[length(h_line)],
max(length(x),length(y))+aaaa,
max(length(x),length(y))+aaaa,
h_line[length(h_line)])
polygon(aa,bb,col = table_col)
aa1=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb1=c(max(length(x),length(y))-aaaa,
max(length(x),length(y))+0.5,
max(length(x),length(y))+0.5,
max(length(x),length(y))-aaaa)
polygon(bb1-max(length(x),length(y)),aa1,col = table_col)
# y axes
axis(side=2, at = seq(0,max(length(x),length(y))-1,l=length(x)),
labels= length(x):1,cex.axis = 0.7, font = 2)
abline(h=h_line,col="black",lwd=1, lty=1)
text((aa1[2]+h_line[1])/2,
seq(0,max(length(x),length(y))-1,l=length(x)),
rev(x),col = "black",cex = 1,pch = 16,font = 2)
#x axes
axis(side=3, at = seq(1,max(length(x),length(y)),
l=length(y)), labels= 1:length(y),cex.axis = 0.7, font = 2)
abline(v=v_line, col="black",lwd=1, lty=1)
abline(v=0.5,col="black",lwd=1, lty=1)
text(seq(1,max(length(x),length(y)),l=length(y))
,(bb[2]+bb[1])/2,
y,col = "black",cex = 1,pch = 16,font = 2)
for (i in 1:length(x)){
for (j in 1:length(y))
if (a[i,j]==1)
points(seq(1,max(length(x),length(y)),l=length(y))[j],seq(0,max(length(x),length(y))-1,l=length(x))[length(x)-i+1],cex=1.5,col=points_col,pch = 16)
}
}
#####################################################
############### prediction structure of sequence ###############
pridict_structure=function(seq,RNAfold_location)
{
seq_split=strsplit(seq,split = "")[[1]]
write.fasta(seq_split,"seq_split","seq_split.fasta")
structure_of_seq=system(paste(RNAfold_location," ","seq_split.fasta" ),intern=T)
file.remove("seq_split.fasta")
file.remove("seq_split_ss.ps")
structure_of_seq_final=strsplit(structure_of_seq[3],split = "")[[1]][1:length(seq_split)]
free_anergy_seq=as.numeric(paste(strsplit(structure_of_seq[3],split = "")[[1]][(length(seq_split)+3):(length(strsplit(structure_of_seq[3],split = "")[[1]])-1)],collapse = ""))
list( "structure"=structure_of_seq_final, "free_anergy_seq"= free_anergy_seq)
}
################################################################
############### secondary structure plot of backbone and spacer ######################
sec_structure_plot=function(seq,sructure_seq,first_backbone,backbone_name,length_backbone,main,free_energy)
{
length_seq=length(strsplit(seq,split = "")[[1]])
ct=makeCt(paste(sructure_seq,collapse = ""),paste(seq,collapse = ""))
coord=ct2coord(ct)
if (first_backbone==T) {
RNAPlot(coord,labTF=T,pointSize=1.8,lineWd=0.5,modspec=TRUE,ranges=data.frame(min=c(1,length_backbone+1),
max=c(length_backbone,length_seq),
col=c(2,5),desc=c(backbone_name,"Spacer")),main=main)
RNAPlot(coord,nt=TRUE,add=TRUE,dp=0.7,tsize=1)
}
else if (first_backbone==F) {
RNAPlot(coord,labTF=T,pointSize=1.8,lineWd=0.5,modspec=TRUE,ranges=data.frame(min=c(1,length_seq-length_backbone+1),
max=c(length_seq-length_backbone,length_seq),
col=c(5,2),desc=c("Spacer",backbone_name)),main=main)
RNAPlot(coord,nt=TRUE,add=TRUE,dp=0.7,tsize=1)
}
else if (first_backbone=="N") {
RNAPlot(coord,labTF=T,pointSize=1.8,lineWd=0.5,modspec=TRUE,ranges=data.frame(min=1,
max=length_seq,
col=5,desc= paste0("Spacer ","(Free Energy = ",free_energy,")")),main=main)
RNAPlot(coord,nt=TRUE,add=TRUE,dp=0.7,tsize=1)
}
}
######################################################################################
}
##############################################################
for (CRISPR_Type in CrisprTypes) {
if (CRISPR_Type=="casVI_A") {
W=28
Nucleotide_rich="t"
backbone="GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC"
local_rich_type="Local_U_Rich"
Normal_rich_type="Normalized_U_Rich"
start=8
end=27
seed_start=13
seed_end=24
blast_database="refseq_rna"
length_pam_cas=0
first_backbone=T
blast_strand="plus"
ID_code="VI_A"
bacteria="Leptotrichia wadei"
cas_protein_type="Cas13a/C2c2"
max_free_energy=-57
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casVI_D") {
W=22
Nucleotide_rich="t"
backbone="TACCCCTACCAACTGGTCGGGGTTTGAAAC"
local_rich_type="Local_U_Rich"
Normal_rich_type="Normalized_U_Rich"
start=3
end=20
seed_start=2
seed_end=8
blast_database="refseq_rna"
length_pam_cas=0
first_backbone=T
blast_strand="plus"
ID_code="VI_D"
bacteria="Ruminococcus flavefaciens"
cas_protein_type="Cas13d"
max_free_energy=-45
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casVI_B") {
W=30
Nucleotide_rich="a"
backbone="GTTGTGGAAGGTCCAGTTTTGAGGGGCTATTACAAC"
local_rich_type="Local_A_Rich"
Normal_rich_type="Normalized_A_Rich"
start=12
end=29
seed_start=12
seed_end=26
blast_database="refseq_rna"
length_pam_cas=2
first_backbone=F
blast_strand="plus"
ID_code="VI_B"
bacteria="Prevotella sp."
cas_protein_type="Cas13b/C2c6"
max_free_energy=-59
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casV_B") {
W=20
backbone="GTCTAGAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGCTTCTCAAATCTGAGAAGTGGCAC"
start=0
end=0
seed_start=0
seed_end=0
blast_database="nr"
length_pam_cas=3
first_backbone=T
blast_strand="both"
ID_code="V_B"
bacteria="Alicyclobacillus acidiphilus"
cas_protein_type="Cas12b/C2c1"
max_free_energy=-102
direct_repeat="Scaffold"
}
if (CRISPR_Type=="casV_A") {
W=20
backbone="TAATTTCTACTAAGTGTAGAT"
start=1
end=6
seed_start=1
seed_end=6
blast_database="nr"
length_pam_cas=4
first_backbone=T
blast_strand="both"
ID_code="V_A"
bacteria="Lachnospiraceae bacterium"
cas_protein_type="Cas12a/Cpf1"
max_free_energy=-34
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casV_F1") {
W=20
backbone="TGCAGAACCCGAATAGACGAATGAAGGAATGCAAC"
start=9
end=16
seed_start=9
seed_end=16
blast_database="nr"
length_pam_cas=0
first_backbone=T
blast_strand="both"
ID_code="V_F1"
bacteria="Uncultured archaeon"
cas_protein_type="Cas14a/Cas12f1"
max_free_energy=-34
direct_repeat="Direct Repeat"
}
if (length(final_str_frags)<W+105) {
message(paste("Your gene is too short, so casilico can not design guides RNA for",CRISPR_Type))
}
if (length(final_str_frags)>=W+105) {
backbone_structure_and_anergy=pridict_structure(backbone,RNAfold_location = RNAfold_path)
backbone_structure=backbone_structure_and_anergy$structure
backbone_free_enargy=backbone_structure_and_anergy$free_anergy_seq
############### create dir for final result ############
setwd(first_main_dir)
name_of_fasta_file_genes=ResultsFolder
result_folder_name=paste0(CRISPR_Type,"-","ConservationMethod_",ConservationMethod,"-","threshold_",ConservationThreshold*100,"-result_",name_of_fasta_file_genes)
plots_folder_name="Plots"
sec_plots_folder_name="Secondary Structure"
MSA_folder="Multiple Sequenc Alignment"
first_dir=paste0(first_main_dir,"/",result_folder_name)
if (dir.exists(result_folder_name)) {
unlink(paste0("./",result_folder_name), recursive = TRUE)
}
dir.create(result_folder_name)
file.copy("final_fasta_file.aln",first_dir)
#file.remove("final_fasta_file.aln")
setwd(first_dir)
dir.create(plots_folder_name)
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"| CRISPR_Type=="casV_F1") {
dir.create(sec_plots_folder_name)
}
dir.create(MSA_folder)
file.copy("final_fasta_file.aln",MSA_folder)
file.remove("final_fasta_file.aln")
setwd(MSA_folder)
file.rename("final_fasta_file.aln","Multiple Sequence Alignment.aln")
write.fasta(final_str_frags,"Consensus Sequence","consensus sequence.fasta")
setwd(first_dir)
#####################################################
#################### first empty value ##############
crRNA_seq=c()
vv=list()
normalized_U_rich=list()
local_U_rich=list()
start_pos=list()
mismatch_pos=list()
mismatch_num=list()
efficiency=list()
type_if_SNP=list()
accessible_side=list()
accessible_flank_side=list()
rank=list()
prtospaser=list()
strand=list()
crRNA_free_energy=list()
Guide_RNA_ID=list()
link_plots=list()
GC_content=list()
score_Gaide=list()
#####################################################
###################### secondary structure ################
if (CRISPR_Type=="casVI_D"|
CRISPR_Type=="casVI_A"|
CRISPR_Type=="casVI_B"|
CRISPR_Type=="casV_F1"){
if (CRISPR_Type=="casVI_D"|
CRISPR_Type=="casVI_A"|
CRISPR_Type=="casVI_B") {
u_rich_in_whole=sum(final_score1[which(final_str_frags==Nucleotide_rich)])/sum(final_score1)
}
if (length(final_str_frags)<=2000){
write.fasta(final_str_frags,"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:length(final_str_frags)]
RNAplot_file_name=paste0(">","final_structure")
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags,collapse = ""),
paste(RNAfols_structure_format,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structure_ss.svg",plots_folder_name)
file.remove("final_structure_ss.svg")
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structure_ss.ps",plots_folder_name)
file.remove("final_structure_ss.ps")
}
}
if (length(final_str_frags)>2000) {
# start
write.fasta(final_str_frags[1:2000],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format_start=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur","_1_to_2000")
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags[1:2000],collapse = ""),
paste(RNAfols_structure_format_start,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_1_to_2000_ss.svg",plots_folder_name)
file.remove("final_structur_1_to_2000_ss.svg")
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_1_to_2000_ss.ps",plots_folder_name)
file.remove("final_structur_1_to_2000_ss.ps")
}
# end
write.fasta(final_str_frags[(length(final_str_frags)-2000+1):length(final_str_frags)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format_end=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur","_Last_2000")
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags[(length(final_str_frags)-2000+1):length(final_str_frags)],collapse = "")
,paste(RNAfols_structure_format_end,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_Last_2000_ss.svg",plots_folder_name)
file.remove("final_structur_Last_2000_ss.svg")
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_Last_2000_ss.ps",plots_folder_name)
file.remove("final_structur_Last_2000_ss.ps")
}
}}
###########################################################
###################### for casVI_A type & casVI_D type ######################
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A") {
for (i in 1:(length(final_str_frags)-W+1)) {
candidate=final_str_frags[i:(i+W-1)]
candidate_scores=c(final_score1[i:(i+W-1)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
candidate_U_rich=(sum(final_score1[which(final_str_frags[i:(i+W-1)]==Nucleotide_rich)]))
if (mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
select_candidate=paste(revers_compliment(candidate),collapse = "")
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
crRNA_seq=c(crRNA_seq,select_candidate)
vv[[select_candidate]]=round(revers_function(candidate_scores),digits = 4)
start_pos[[select_candidate]]=i
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
mismatch_pos[[select_candidate]]=which(revers_function(candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
pos_spacer_in_sec_stracture=i
if (length(final_str_frags)>2000){
if (i<=1000) {
RNAfols_structure_format=RNAfols_structure_format_start
pos_spacer_in_sec_stracture=i
}
else if (i>1000 & i<length(final_str_frags)-1000+1) {
write.fasta(final_str_frags[(i-1000):(i+1000-1)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur_",(i-1000),"_to_",(i+1000-1))
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags[(i-1000):(i+1000-1)],collapse = ""),
paste(RNAfols_structure_format,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"))
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"))
}
pos_spacer_in_sec_stracture=1000
}
else if (i>=length(final_str_frags)-1000) {
RNAfols_structure_format=RNAfols_structure_format_end
pos_spacer_in_sec_stracture=2000-(length(final_str_frags)-i)
}
}
if (i<=100) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[1:(i+W+100-1)]==Nucleotide_rich))]))-candidate_U_rich)/
(sum(final_score1[1:(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>100 & i<length(final_str_frags)-100-W+1) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100):(i+W+100-1)]==Nucleotide_rich))+i-100-1]))-candidate_U_rich)/
(sum(final_score1[(i-100):(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]=
round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>=length(final_str_frags)-100-W) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100+1):length(final_str_frags)]==Nucleotide_rich))+i-100]))-candidate_U_rich)/
((sum(final_score1[(i-100+1):length(final_str_frags)]))-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)), digits=4)
}
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags[i+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & ")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=
c(paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot=paste0("Information plot for ","(ID=",Guide_RNA_ID[[select_candidate]],") candidate ")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[1:(i+W+20-1)],i,start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",Normal_rich_type)
,paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name, "/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & length(final_str_frags)-20-W+1) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],title_for_crRNA_plot,W,
paste0(local_U_rich[[select_candidate]]," ",local_rich_type," , ",
normalized_U_rich[[select_candidate]]," ",Normal_rich_type),
paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name, "/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags)-20-W) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20+1):length(final_str_frags)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,
paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",
normalized_U_rich[[select_candidate]]," ",
Normal_rich_type)
,paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name, "/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*2*local_U_rich[[select_candidate]])
,(2.5*accessible_side[[select_candidate]])
,(2.5*accessible_flank_side[[select_candidate]])
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
blast_query=prtospaser
}
#############################################################################
############################# for casVI_B type ##########################
if (CRISPR_Type=="casVI_B") {
prtospaseraa=list()
prtospasergg=list()
prtospasertt=list()
prtospaserag=list()
prtospaserga=list()
prtospaserat=list()
prtospaserta=list()
prtospasergt=list()
prtospasertg=list()
for (i in 3:(length(final_str_frags)-W+1)) {
candidate=final_str_frags[i:(i+W-1)]
candidate_scores=c(final_score1[i:(i+W-1)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
candidate_U_rich=(sum(final_score1[which(final_str_frags[i:(i+W-1)]==Nucleotide_rich)]))
if (final_str_frags[i-1] != "c" & final_str_frags[i-2] != "c"){
if (mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
select_candidate=paste(revers_compliment(candidate),collapse = "")
backbone_spacer=paste0(select_candidate,backbone,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[(W+1):length(backbone_spacer_structure$structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[(W+1):length(backbone_spacer_structure$structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
prtospaseraa[[select_candidate]]=c("a","a",candidate)
prtospasergg[[select_candidate]]=c("g","g",candidate)
prtospasertt[[select_candidate]]=c("t","t",candidate)
prtospaserag[[select_candidate]]=c("a","g",candidate)
prtospaserga[[select_candidate]]=c("g","a",candidate)
prtospaserat[[select_candidate]]=c("a","t",candidate)
prtospaserta[[select_candidate]]=c("t","a",candidate)
prtospasergt[[select_candidate]]=c("g","t",candidate)
prtospasertg[[select_candidate]]=c("t","g",candidate)
crRNA_seq=c(crRNA_seq,select_candidate)
vv[[select_candidate]]=round(revers_function(candidate_scores),digits = 4)
start_pos[[select_candidate]]=i
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
mismatch_pos[[select_candidate]]=which(revers_function(candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
pos_spacer_in_sec_stracture=i
if (length(final_str_frags)>2000){
if (i<=1000) {
RNAfols_structure_format=RNAfols_structure_format_start
pos_spacer_in_sec_stracture=i
}
else if (i>1000 & i<length(final_str_frags)-1000+1) {
write.fasta(final_str_frags[(i-1000):(i+1000-1)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur_",(i-1000),"_to_",(i+1000-1))
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,paste(final_str_frags[(i-1000):(i+1000-1)],collapse = ""),
paste(RNAfols_structure_format_start,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"))
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"))
}
pos_spacer_in_sec_stracture=1000
}
else if (i>=length(final_str_frags)-1000) {
RNAfols_structure_format=RNAfols_structure_format_end
pos_spacer_in_sec_stracture=2000-(length(final_str_frags)-i)
}
}
if (i<=100) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[1:(i+W+100-1)]==Nucleotide_rich))]))-candidate_U_rich)/
(sum(final_score1[1:(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[i:(i+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((1:i-1),(i+W):(i+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=round(length(a_flank_side)/
length(c((1:i-1),(i+W):(i+W+100-1))), digits=4)
}
else if (i>100 & i<length(final_str_frags)-100-W+1) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100):(i+W+100-1)]==Nucleotide_rich)+i-100)]))-candidate_U_rich)/
(sum(final_score1[(i-100):(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]=round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[i:(i+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((i-100):(i-1),(i+W):(i+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((i-100):(i-1),(i+W):(i+W+100-1))), digits=4)
}
else if (i>=length(final_str_frags)-100-W) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100+1):length(final_str_frags)]==Nucleotide_rich)+i-100)]))-candidate_U_rich)/
((sum(final_score1[(i-100+1):length(final_str_frags)]))-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[i:(i+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((i-100):(i-1),(i+W-1):length(final_str_frags))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((i-100):(i-1),(i+W-1):length(final_str_frags))), digits=4)
}
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags[i+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]],collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=
c(paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", "),collapse = " & ",sep=""),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", "),collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot=paste0("Information plot for ","(ID=",Guide_RNA_ID[[select_candidate]],") candidate ")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[1:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",
Normal_rich_type),paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & length(final_str_frags)-20-W+1) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",
Normal_rich_type),paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags)-20-W) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20+1):length(final_str_frags)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",
Normal_rich_type),paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*2*local_U_rich[[select_candidate]])
,(2.5*accessible_side[[select_candidate]])
,(2.5*accessible_flank_side[[select_candidate]])
,(10*(1-(final_free_anergy/max_free_energy)))))
}}
}
blast_query=c(prtospaseraa,
prtospasergg,
prtospasertt,
prtospaserag,
prtospaserga,
prtospaserat,
prtospaserta,
prtospasergt,
prtospasertg)
}
#########################################################################
############################# for casV_B type ###########################
if (CRISPR_Type=="casV_B") {
final_str_frags_plus_vb=c("l","l","l",final_str_frags,"l","l","l")
prtospasertta_pos=list()
prtospaserttg_pos=list()
prtospaserttt_pos=list()
prtospaserttc_pos=list()
prtospasertta_neg=list()
prtospaserttg_neg=list()
prtospaserttt_neg=list()
prtospaserttc_neg=list()
for (i in 4:(length(final_str_frags_plus_vb)-W+1-6)) {
candidate=final_str_frags_plus_vb[i:(i+W-1)]
candidate_scores1=c(final_score1[(i-3):(i+W-1-3)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
if ((final_str_frags_plus_vb[i-2] == "t" & final_str_frags_plus_vb[i-3] == "t")|
(final_str_frags_plus_vb[i+W+1] == "a" & final_str_frags_plus_vb[i+W+2] == "a")){
if (mismatch_del(candidate_scores1,start,end,seed_start,seed_end)!=TRUE) {
if (final_str_frags_plus_vb[i+W+2] == "a" &
final_str_frags_plus_vb[i+W+1] == "a") {
select_candidate=paste(revers_compliment(candidate),collapse = "")
candidate_scores=revers_function(candidate_scores1)
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=revers_compliment(candidate)
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
vv[[select_candidate]]=round((candidate_scores),digits = 4)
start_pos[[select_candidate]]=(length(final_str_frags_plus_vb)-6)-((i-3)+W-1)+1
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="-"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospasertta_neg[[select_candidate]]=c("t","t","a",revers_compliment(candidate))
prtospaserttg_neg[[select_candidate]]=c("t","t","g",revers_compliment(candidate))
prtospaserttt_neg[[select_candidate]]=c("t","t","t",revers_compliment(candidate))
prtospaserttc_neg[[select_candidate]]=c("t","t","c",revers_compliment(candidate))
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags_plus_vb[(i)+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(revers_compliment(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", " )[[1]]),sep = "",collapse = " & ")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_vb[(i)+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_vb[(i)+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(revers_compliment(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", " )[[1]]),sep = "",collapse = " & "),
paste(revers_compliment(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", " )[[1]]),sep = "",collapse = " & "))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
title_for_crRNA_Dotplot=paste0("Dotplot for ", Guide_RNA_ID[[select_candidate]]," crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_vb)-20-W+1-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_vb)-20-W-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(length(final_str_frags_plus_vb)-3)],21,
start_pos[[select_candidate]],mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}
if (final_str_frags_plus_vb[i-2] == "t" &
final_str_frags_plus_vb[i-3] == "t") {
select_candidate=paste(candidate,collapse = "")
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
candidate_scores=candidate_scores1
prtospaser[[select_candidate]]=candidate
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
vv[[select_candidate]]=round((candidate_scores),digits = 4)
start_pos[[select_candidate]]=i-3
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="+"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospasertta_pos[[select_candidate]]=c("t","t","a",candidate)
prtospaserttg_pos[[select_candidate]]=c("t","t","g",candidate)
prtospaserttt_pos[[select_candidate]]=c("t","t","t",candidate)
prtospaserttc_pos[[select_candidate]]=c("t","t","c",candidate)
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(mismatch_pos[[select_candidate]]-1)]]
sssnp[which(names(sssnp)==final_str_frags_plus_vb[(i)+(mismatch_pos[[select_candidate]]-1)])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & ")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(mismatch_pos[[select_candidate]][1]-1)]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_vb[(i)+(mismatch_pos[[select_candidate]][1]-1)])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(mismatch_pos[[select_candidate]][2]-1)]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_vb[(i)+(mismatch_pos[[select_candidate]][2])-1])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_vb)-20-W+1-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_vb)-20-W-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(length(final_str_frags_plus_vb)-3)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
}
}
blast_query=c(prtospasertta_pos,
prtospaserttg_pos,
prtospaserttt_pos,
prtospaserttc_pos,
prtospasertta_neg,
prtospaserttg_neg,
prtospaserttt_neg,
prtospaserttc_neg)
}
#########################################################################
############################# for casV_A type ###########################
if (CRISPR_Type=="casV_A") {
final_str_frags_plus_va=c("l","l","l","l",final_str_frags,"l","l","l","l")
prtospaserttta_pos=list()
prtospasertttg_pos=list()
prtospasertttc_pos=list()
prtospaserttta_neg=list()
prtospasertttg_neg=list()
prtospasertttc_neg=list()
for (i in 5:(length(final_str_frags_plus_va)-W+1-8)) {
candidate=final_str_frags_plus_va[i:(i+W-1)]
candidate_scores1=final_score1[(i-4):(i+W-1-4)]
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
if (final_str_frags_plus_va[i+W] != "a" &
final_str_frags_plus_va[i+W+1] == "a" &
final_str_frags_plus_va[i+W+2] == "a" &
final_str_frags_plus_va[i+W+3] == "a") {
select_candidate=paste(revers_compliment(candidate),collapse = "")
candidate_scores=revers_function(candidate_scores1)
if(mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=revers_compliment(candidate)
vv[[select_candidate]]=round((candidate_scores),digits = 4)
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
start_pos[[select_candidate]]=(length(final_str_frags_plus_va)-8)-((i-4)+W-1)+1
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="-"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospaserttta_neg[[select_candidate]]=c("t","t","t","a",revers_compliment(candidate))
prtospasertttg_neg[[select_candidate]]=c("t","t","t","g",revers_compliment(candidate))
prtospasertttc_neg[[select_candidate]]=c("t","t","t","c",revers_compliment(candidate))
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags_plus_va[(i)+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(revers_compliment(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]]),collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_va[(i)+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_va[(i)+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(revers_compliment(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", ")[[1]]),collapse = " & ",sep=""),
paste(revers_compliment(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", ")[[1]]),collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
title_for_crRNA_Dotplot=paste0("Dotplot for ", Guide_RNA_ID[[select_candidate]]," crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_va)-20-W+1-4) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_va)-20-W-4) {
setwd(plots_folder_name)
png(filename2, width = 1200,height = 800)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(length(final_str_frags_plus_va)-4)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}}
if (final_str_frags_plus_va[i-4] == "t" &
final_str_frags_plus_va[i-3] == "t" &
final_str_frags_plus_va[i-2] == "t" &
final_str_frags_plus_va[i-1] != "t") {
select_candidate=paste(candidate,collapse = "")
candidate_scores=candidate_scores1
if(mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE){
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
vv[[select_candidate]]=round((candidate_scores),digits = 4)
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
start_pos[[select_candidate]]=i-4
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=mismatch_num[[select_candidate]]+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="+"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospaserttta_pos[[select_candidate]]=c("t","t","t","a",candidate)
prtospasertttg_pos[[select_candidate]]=c("t","t","t","g",candidate)
prtospasertttc_pos[[select_candidate]]=c("t","t","t","c",candidate)
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(mismatch_pos[[select_candidate]]-1)]]
sssnp[which(names(sssnp)==final_str_frags_plus_va[(i)+(mismatch_pos[[select_candidate]]-1)])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]],collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(mismatch_pos[[select_candidate]][1]-1)]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_va[(i)+(mismatch_pos[[select_candidate]][1]-1)])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(mismatch_pos[[select_candidate]][2]-1)]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_va[(i)+(mismatch_pos[[select_candidate]][2])-1])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", ")[[1]],sep = "" ,collapse = " & "),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", ")[[1]],collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
title_for_crRNA_Dotplot=paste0("Dotplot for ", Guide_RNA_ID[[select_candidate]]," crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_va)-20-W+1-4) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main="Spacer self complementary",
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),"crRNA self complementary")
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_va)-20-W-4) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(length(final_str_frags_plus_va)-4)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
}
blast_query=c(prtospaserttta_pos,
prtospasertttg_pos,
prtospasertttc_pos,
prtospaserttta_neg,
prtospasertttg_neg,
prtospasertttc_neg)
}
#########################################################################
############################# for casV_F1 type ##########################
if (CRISPR_Type=="casV_F1") {
for (i in 1:(length(final_str_frags)-W+1)) {
candidate=final_str_frags[i:(i+W-1)]
candidate_scores=c(final_score1[i:(i+W-1)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
if (mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
select_candidate=paste(revers_compliment(candidate),collapse = "")
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
crRNA_seq=c(crRNA_seq,select_candidate)
vv[[select_candidate]]=round(revers_function(candidate_scores),digits = 4)
start_pos[[select_candidate]]=i
mismatch_pos[[select_candidate]]=which(revers_function(candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
pos_spacer_in_sec_stracture=i
if (length(final_str_frags)>2000){
if (i<=1000) {
RNAfols_structure_format=RNAfols_structure_format_start
pos_spacer_in_sec_stracture=i
}
else if (i>1000 & i<length(final_str_frags)-1000+1) {
write.fasta(final_str_frags[(i-1000):(i+1000-1)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur_",(i-1000),"_to_",(i+1000-1))
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,paste(final_str_frags[(i-1000):(i+1000-1)],collapse = ""),
paste(RNAfols_structure_format_start,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"))
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"))
}
pos_spacer_in_sec_stracture=1000
}
else if (i>=length(final_str_frags)-1000) {
RNAfols_structure_format=RNAfols_structure_format_end
pos_spacer_in_sec_stracture=2000-(length(final_str_frags)-i)
}
}
if (i<=100) {
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>100 & i<length(final_str_frags)-100-W+1) {
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>=length(final_str_frags)-100-W) {
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)), digits=4)
}
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags[i+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]],collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", ")[[1]],collapse = " & ",sep=""),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", ")[[1]],collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[1:(i+W+20-1)],
i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,"",paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & length(final_str_frags)-20-W+1) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,"",
paste(accessible_flank_side[[select_candidate]]),paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags)-20-W) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20+1):length(final_str_frags)],21,
start_pos[[select_candidate]],mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],title_for_crRNA_plot,W,"",
paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(2.5*accessible_side[[select_candidate]])
,(2.5*accessible_flank_side[[select_candidate]])
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
blast_query=prtospaser
}
#########################################################################
############ replace sec plots to Secondary structure folder ############
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"|CRISPR_Type=="casV_F1") {
current_folder <- paste0(first_dir,"/Plots")
new_folder <- paste0(first_dir,"/Secondary Structure")
if (host_system=="Windows") {
list_of_files <- list.files(current_folder, ".svg")
}
if (host_system=="Linux") {
list_of_files <- list.files(current_folder, ".ps")
}
file.copy(file.path(current_folder,list_of_files), new_folder)
file.remove(file.path(current_folder,list_of_files))
}
#########################################################################
################# View data (data.frame construction) ############
if (length(crRNA_seq)>0) {
data=as.data.frame(crRNA_seq)
data$Guide_RNA_ID=Guide_RNA_ID
names(data)[names(data) == "crRNA_seq"] <- "Guide_RNA"
if (length(topoTypes_names)>1) {
data$Major_Allele_Frequency=vv
}
data$Start=start_pos
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data$Strand=strand
}
data$GC_content=GC_content
if (length(topoTypes_names)>1) {
data$Mismatch_Number=mismatch_num
data$Mismatch_Position=mismatch_pos
data$Type_Of_SNP=type_if_SNP
data$Efficiency=efficiency
names(data)[names(data) == "Efficiency"] <- "Conservation Efficiency"
}
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"){
data$local_U_rich=local_U_rich
names(data)[names(data) == "local_U_rich"] <- local_rich_type
data$Normalized_U_rich=normalized_U_rich
names(data)[names(data) == "Normalized_U_rich"] <- Normal_rich_type
}
if (CRISPR_Type=="casVI_D"| CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"|CRISPR_Type=="casV_F1"){
data$Protospacer_Accessibility_Score=accessible_side
data$Local_Accessibility_Score=accessible_flank_side
}
data$Rank=rank
data$Self_Complementarity_Free_Energy=crRNA_free_energy
data$Score=unlist(score_Gaide)
###################################################################
###################### DT options #####################
my.options <- list(autoWidth = T,
searching = T,
ordering = T,
filter = 'none',
lengthChange = T,
lengthMenu = c(5,10,50,100,1000),
paging = T,
info = T,
pageLength = 5,
searchHighlight = TRUE,
searchable=F)
header.style <- "th { font-family: 'Times New Roman'; font-weight: bold; color: white ; background-color: #000000;}"
########################################################
###### checking internet connection and blast with rules (off_target_prediction) ######
setwd(first_dir)
if (OffTarget==T) {
if (LocalOff!=T & havingIP()==TRUE){
if (is.null(Organism)!=T) {
if (OffAsk) {
min_approximate_blast_time=length(blast_query)*0.3*length(Organism)
max_approximate_blast_time=length(blast_query)*1*length(Organism)
question <- readline(paste0("off-Targets Prediction for ",ID_code," CRISPR-Cas System ","may be take long time",
" (About ",
min_approximate_blast_time," to ",
max_approximate_blast_time,
" minutes) ",
"would you like to continue or no? (Y/N)"))
}
else if (OffAsk==F) {
question="y"
}
}
else{
message('If you want to do online mode for off-Target Prediction,
please Ensure you introduced the "Organism" parameter.')
question="n"
}
}
if (LocalOff == T) {
if (all(!(LocalFasta %in% c("path1", "path2")))) {
if (length(LocalName) == length(LocalFasta)) {
Organism = LocalName
}
else if (length(LocalName) != length(LocalFasta)) {
Organism = paste0("User_Sequence_",
1:length(LocalFasta))
}
if (OffAsk) {
question <- readline("Depending on the size of your genomes, this step may be time consuming, Do you want to continue?(Y/N)")
}
else if (OffAsk == F) {
question = "y"
}
}
else {
message("please Ensure you introduced the \"LocalFasta\" parameter",
"\n and check the \"LocalOff\" parameter be true.")
question = "n"
}
}
if(question== 'y'| question== 'Y'){
Guide_RNA=data$Guide_RNA
len_list=rep(0,length(Guide_RNA))
count_mm0=as.list(len_list)
count_mm1=as.list(len_list)
count_mm2=as.list(len_list)
count_mm3=as.list(len_list)
count_mm4=as.list(len_list)
data_off_target=as.data.frame(Guide_RNA)
data_off_target$Guide_RNA_ID=data$Guide_RNA_ID
for (org in Organism) {
list_blast_mm_0=list()
list_blast_mm_1=list()
list_blast_mm_2=list()
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
list_blast_mm_3=list()
list_blast_mm_4=list()
}
write.fasta( blast_query,names(blast_query), file.out = "blast_query.FASTA")
# online blast
if (LocalOff!=T){
entrez_query=paste(org,' [organism]',sep = "")
entrez_query <- paste0('"',entrez_query,'"')
blast_command=paste(Blast_location,
' -task blastn -db ', blast_database ,
' -entrez_query ' , entrez_query,
' -word_size 7 -evalue 10 -query blast_query.FASTA -out blast_out.txt ' ,
' -strand ', blast_strand,
' -outfmt "10 delim=@ qseqid sacc qseq sseq length qstart qend sstart send gapopen mismatch " -num_alignments 5000 -remote',sep = '' ,collapse = "" )
lapply(blast_command, system)
}
#offline blast
if (LocalOff==T){
number_of_genome_file=which(org==Organism)
file.copy(LocalFasta[number_of_genome_file],first_dir)
neme_genome=basename(LocalFasta[number_of_genome_file])
file.rename(neme_genome,"genome_name.fasta")
makeblastdb_command=paste( makeblastdb_location ,
' -in genome_name.fasta -out genome_name_index -dbtype nucl -parse_seqids')
lapply(makeblastdb_command, system)
blast_command=paste(Blast_location,
' -task blastn -db genome_name_index ' ,
' -word_size 7 -evalue 10 -query blast_query.FASTA -out blast_out.txt ' ,
' -strand ', blast_strand,' -num_threads ', Threads,
' -outfmt "10 delim=@ qseqid sacc qseq sseq length qstart qend sstart send gapopen mismatch " -num_alignments 5000 ',
sep = '' ,collapse = "" )
lapply(blast_command, system)
removes_file=list.files(first_dir,pattern = "genome_name*")
file.remove(removes_file)
}
info_blast = file.info("blast_out.txt")
if (info_blast$size!=0) {
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casV_F1"){
blast_result=read.table("blast_out.txt",sep = "@")
blast_result=rbind(blast_result[blast_result$V5==((W+length_pam_cas)-2) & blast_result$V11==0 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-1) & blast_result$V11<=1 & blast_result$V10==0,],
blast_result[blast_result$V5==(W+length_pam_cas) & blast_result$V11<=2 & blast_result$V10==0,])
}
else if (CRISPR_Type=="casVI_B"){
blast_result=read.table("blast_out.txt",sep = "@")
blast_result=rbind(blast_result[blast_result$V5==((W+length_pam_cas)-2) & blast_result$V6==1 & blast_result$V11==0 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-1) & blast_result$V6==1 & blast_result$V11<=1 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)) & blast_result$V6==1 & blast_result$V11<=2 & blast_result$V10==0,])
}
else if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
blast_result=read.table("blast_out.txt",sep = "@")
blast_result=rbind(blast_result[blast_result$V5==((W+length_pam_cas)-4) & blast_result$V6==1 & blast_result$V11==0 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-3) & blast_result$V6==1 & blast_result$V11<=1 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-2) & blast_result$V6==1 & blast_result$V11<=2 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-1) & blast_result$V6==1 & blast_result$V11<=3 & blast_result$V10==0,],
blast_result[blast_result$V5==(W+length_pam_cas) & blast_result$V6==1 & blast_result$V11<=4 & blast_result$V10==0,])
}
if (length(blast_result$V2)!=0) {
for (crRNA_predict in 1:length(data$Guide_RNA)) {
list_blast_mm_0[[crRNA_predict]]="No_Hit"
list_blast_mm_1[[crRNA_predict]]="No_Hit"
list_blast_mm_2[[crRNA_predict]]="No_Hit"
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
list_blast_mm_3[[crRNA_predict]]="No_Hit"
list_blast_mm_4[[crRNA_predict]]="No_Hit"
}
genes_mm0=c()
genes_mm1=c()
genes_mm2=c()
genes_mm3=c()
genes_mm4=c()
for (blast_crRNA_predict in 1:length(unique(blast_result$V1))){
if (data$Guide_RNA[crRNA_predict]==unique(blast_result$V1)[blast_crRNA_predict]) {
s_blast_result=blast_result[blast_result$V1==unique(blast_result$V1)[blast_crRNA_predict],]
for (gene in 1:length(s_blast_result$V2)) {
s_blast_result_gene=s_blast_result[gene,]
pos_mismach_blast_crRNA_gene=which(strsplit(s_blast_result_gene$V3,split = "")[[1]]!=strsplit(s_blast_result_gene$V4,split = "")[[1]])
#pam mismatch checked
if (any(pos_mismach_blast_crRNA_gene%in%0:length_pam_cas)!=TRUE) {
if (CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_B"|CRISPR_Type=="casV_F1"){
if ((W+length_pam_cas)-s_blast_result_gene$V5==1) {
if (s_blast_result_gene$V6==1) {
pos_mismach_blast_crRNA_gene=c(pos_mismach_blast_crRNA_gene,(W+length_pam_cas))
}
else if (s_blast_result_gene$V6!=1) {
pos_mismach_blast_crRNA_gene=c(1,pos_mismach_blast_crRNA_gene)
}
}
if ((W+length_pam_cas)-s_blast_result_gene$V5==2) {
if (s_blast_result_gene$V6==1) {
pos_mismach_blast_crRNA_gene=c(pos_mismach_blast_crRNA_gene,(W+length_pam_cas)-1,(W+length_pam_cas))
}
else if (s_blast_result_gene$V6==2) {
pos_mismach_blast_crRNA_gene=c(1,pos_mismach_blast_crRNA_gene,(W+length_pam_cas))
}
else if (s_blast_result_gene$V6==3) {
pos_mismach_blast_crRNA_gene=c(1,2,pos_mismach_blast_crRNA_gene)
}
}
seq_gene=rep(1,(W+length_pam_cas))
seq_gene[pos_mismach_blast_crRNA_gene]=0
if (mismatch_del(seq_gene[(length_pam_cas+1):length(seq_gene)],start,end,seed_start,seed_end)!=TRUE) {
gene_off_target=paste0(s_blast_result_gene$V2,"_pos",":",s_blast_result_gene$V8,"-",s_blast_result_gene$V9)
#mm0
if (length(pos_mismach_blast_crRNA_gene)==0) {
genes_mm0=c(genes_mm0,gene_off_target)
}
#mm1
else if (length(pos_mismach_blast_crRNA_gene)==1) {
genes_mm1=c(genes_mm1,gene_off_target)
}
#mm2
else if (length(pos_mismach_blast_crRNA_gene)==2) {
genes_mm2=c(genes_mm2,gene_off_target)
}
}}
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
number_mismach_blast_crRNA_gene=(((W+length_pam_cas)-s_blast_result_gene$V5)+s_blast_result_gene$V11)
gene_off_target=paste0(s_blast_result_gene$V2,"_pos",":",s_blast_result_gene$V8,"-",s_blast_result_gene$V9)
#mm0
if (number_mismach_blast_crRNA_gene==0) {
genes_mm0=c(genes_mm0,gene_off_target)
}
#mm1
else if (number_mismach_blast_crRNA_gene==1) {
genes_mm1=c(number_mismach_blast_crRNA_gene)
}
#mm2
else if (number_mismach_blast_crRNA_gene==2) {
genes_mm2=c(genes_mm2,gene_off_target)
}
#mm3
else if (number_mismach_blast_crRNA_gene==3) {
genes_mm3=c(genes_mm3,gene_off_target)
}
#mm4
else if (number_mismach_blast_crRNA_gene==4) {
genes_mm4=c(genes_mm4,gene_off_target)
}
}
}
}
#mm0
if (length(genes_mm0)>0) {
list_blast_mm_0[[crRNA_predict]]=genes_mm0
count_mm0[[crRNA_predict]]=count_mm0[[crRNA_predict]]+length(genes_mm0)
}
#mm1
if (length(genes_mm1)>0) {
list_blast_mm_1[[crRNA_predict]]=genes_mm1
count_mm1[[crRNA_predict]]=count_mm1[[crRNA_predict]]+length(genes_mm1)
}
#mm2
if (length(genes_mm2)>0) {
list_blast_mm_2[[crRNA_predict]]=genes_mm2
count_mm2[[crRNA_predict]]=count_mm2[[crRNA_predict]]+length(genes_mm2)
}
#mm3
if (length(genes_mm3)>0) {
list_blast_mm_3[[crRNA_predict]]=genes_mm3
count_mm3[[crRNA_predict]]=count_mm3[[crRNA_predict]]+length(genes_mm3)
}
#mm4
if (length(genes_mm4)>0) {
list_blast_mm_4[[crRNA_predict]]=genes_mm4
count_mm4[[crRNA_predict]]=count_mm4[[crRNA_predict]]+length(genes_mm4)
}
break
}
}
}
data_off_target$blast_mm_0=list_blast_mm_0
data_off_target$blast_mm_1=list_blast_mm_1
data_off_target$blast_mm_2=list_blast_mm_2
names(data_off_target)[names(data_off_target) == "blast_mm_0"] <- paste("No mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_1"] <- paste("1 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_2"] <- paste("2 mismatched off Targets in",org)
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data_off_target$blast_mm_3=list_blast_mm_3
data_off_target$blast_mm_4=list_blast_mm_4
names(data_off_target)[names(data_off_target) == "blast_mm_3"] <- paste("3 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_4"] <- paste("4 mismatched off targets in",org)
}
}
if (length(blast_result$V2)==0) {
data_off_target$blast_mm_0="No_Hit"
data_off_target$blast_mm_1="No_Hit"
data_off_target$blast_mm_2="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_0"] <- paste("No mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_1"] <- paste("1 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_2"] <- paste("2 mismatched off targets in",org)
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data_off_target$blast_mm_3="No_Hit"
data_off_target$blast_mm_4="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_3"] <- paste("3 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_4"] <- paste("4 mismatched off targets in",org)
}
}
}
if (info_blast$size==0) {
data_off_target$blast_mm_0="No_Hit"
data_off_target$blast_mm_1="No_Hit"
data_off_target$blast_mm_2="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_0"] <- paste("No mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_1"] <- paste("1 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_2"] <- paste("2 mismatched off targets in",org)
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data_off_target$blast_mm_3="No_Hit"
data_off_target$blast_mm_4="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_3"] <- paste("3 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_4"] <- paste("4 mismatched off targets in",org)
}
}
}
data$No_Mismatch_off_Target=count_mm0
data$One_Mismatch_off_Targets=count_mm1
data$Two_Mismatch_off_Targets=count_mm2
col_number=3
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
data$Three_Mismatch_off_Targets=count_mm3
data$Four_Mismatch_off_Targets=count_mm4
col_number=col_number+2
}
setwd(first_dir)
html_result_folder="Off_Target-result.html"
dir.create( html_result_folder)
number_of_link_for_off=0
for (mm_type in (length(data)-(col_number-1)):length(data)) {
for ( guide in 1:length(data$Guide_RNA)) {
if (data[guide,mm_type]>0) {
number_of_link_for_off=number_of_link_for_off+1
zz=c("NO_mismatch","1_mismatch","2_mismatches","3_mismatches","4_mismatches")
type_of_mm=which(mm_type==(length(data)-(col_number-1)):length(data))
number_of_organism=length(Organism)
if (number_of_organism==1) {
show_col=c(1,2,type_of_mm+2)
}
if (number_of_organism>1) {
show_col_1_plus=(type_of_mm+2)+1:(number_of_organism-1)*col_number
show_col=c(1,2,type_of_mm+2,show_col_1_plus)
}
header.names <- c("",names(data_off_target[guide,][,show_col]))
sketch_for_each_gene <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
)
)
))
rr=as.data.frame(lapply(data_off_target[guide,][,show_col], function(x) {gsub(",", "\n", x)}))
cc=as.data.frame(lapply(rr, function(x) {gsub("c\\(", "", x)}))
bb=as.data.frame(lapply(cc, function(x) {gsub("\\)", "", x)}))
data_each_spacer_off_target=as.data.frame(lapply(bb, function(x) {gsub('"', '', x)}))
result_of_guide= datatable(data_each_spacer_off_target, filter = 'none',
options =my.options,
container = sketch_for_each_gene,
rownames = TRUE,
caption = paste0('Results Table for ',CRISPR_Type,' (Guide RNA=',data_off_target[guide,1],')'))
result_of_guide <- formatStyle(result_of_guide,
columns = " ",
backgroundColor = "#000000",
borderBottomColor = "#000000",
borderBottomStyle = "solid",
borderBottomWidth = "1px",
borderCollapse = "collapse",
borderRightColor = "#000000",
borderRightStyle = "solid",
borderRightWidth = "1px",
color = "rgb(255, 255, 255)",
fontFamily = "Times New Roman",
fontSize = "13px",
fontWeight = "bold",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle",
)
result_of_guide <- formatStyle(result_of_guide,
columns = 1:(length(Organism)+2),
fontFamily = "Times New Roman",
fontSize = "13px",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle")
name_html_Guide=paste0(zz[type_of_mm],"_",data[[guide,2]],".html",collapse = "")
setwd(html_result_folder)
htmlwidgets::saveWidget(result_of_guide, name_html_Guide )
setwd(first_dir)
Guide_html_Link = tibble( file = dir( file.path( '.', html_result_folder ), pattern= name_html_Guide ) ) %>%
mutate( path = paste0('file:', html_result_folder,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', data[guide,mm_type],'</a>' ) )
data[guide,mm_type]=Guide_html_Link$link
}
}
}
if (number_of_link_for_off==0 ) {
if (dir.exists(html_result_folder)) {
unlink(paste0("./",html_result_folder), recursive = TRUE)
}
}
############# create sketch for DT package ###########
zz=c("NO_mismatch","1_mismatch","2_mismatches","3_mismatches","4_mismatches")
name_subcolumn_off_target=zz[1: col_number]
header.names <- c("",names(data_off_target))
# The container parameter allows us to design the header of the table
# using CSS
sketch_off_Target <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names[1:3], th,rowspan = 2, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white"),
lapply(Organism, th,colspan = col_number, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
),
tr(
rep(lapply(name_subcolumn_off_target, th),length(Organism))
)
)
))
######################################################
rr=as.data.frame(lapply(data_off_target, function(x) {gsub(",", "\n", x)}))
cc=as.data.frame(lapply(rr, function(x) {gsub("c\\(", "", x)}))
bb=as.data.frame(lapply(cc, function(x) {gsub("\\)", "", x)}))
data_off_target=as.data.frame(lapply(bb, function(x) {gsub('"', '', x)}))
result_off_targets= datatable(data_off_target, filter = 'none', options = my.options,rownames = TRUE,container = sketch_off_Target)
result_off_targets <- formatStyle(result_off_targets,
columns = " ",
backgroundColor = "#000000",
borderBottomColor = "#000000",
borderBottomStyle = "solid",
borderBottomWidth = "1px",
borderCollapse = "collapse",
borderRightColor = "#000000",
borderRightStyle = "solid",
borderRightWidth = "1px",
color = "rgb(255, 255, 255)",
fontFamily = "Times New Roman",
fontSize = "13px",
fontWeight = "bold",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle",
)
result_off_targets <- formatStyle(result_off_targets,
columns = 1:length(data_off_target),
fontFamily = "Times New Roman",
fontSize = "13px",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle")
htmlwidgets::saveWidget(result_off_targets, "result2.html")
html_result=read_file("result2.html")
line_heder=paste0('<header><h1 style="color:blue;font-size:30px;" align="middle">',
paste0('Off_Targets Results Table for ',ID_code,' CRISPR-Cas System'),'</h1>')
line_img_tehran=paste('<img src= "https://drive.google.com/thumbnail?id=1NrT3Zw15mdetkgB4xLZ7GlZnDj-uAeNw"','width= 300 height= 130',' align="left" >')
line_img_MRBLAB=paste('<img src= "https://drive.google.com/thumbnail?id=1BWDyB9RcBdes4dRNRh5ff3lUxMaUwdME" ','width= 100 height= 100',' align="left" >')
file_Conaction<-file("Off_Targets_Information.Html")
writeLines(c(line_heder,line_img_tehran,html_result,line_img_MRBLAB), file_Conaction)
close(file_Conaction)
file.remove("result2.html")
file.remove("blast_out.txt")
file.remove("blast_query.FASTA")
}
if(question == 'n'| question == 'N'){
message("Running continue without Off_Target prediction" )
}
if (havingIP()!=TRUE) {
message("Problem in Internet connection ",
"continue without off_Target result")
}
}
#######################################################################################
######################### assign link for figure and sort data ##################
setwd(first_dir)
data$Plot=link_plots
data=data[order(-data$Score),]
data$Score=NULL
#################################################################################
################### create sketch for DT package ################################
if (OffTarget==T) {
if (question== 'y'| question== 'Y') {
name_subcolumn_off_target=zz[1: col_number]
header.names <- c("",names(data)[1:(length(names(data))-(col_number+1))])
# The container parameter allows us to design the header of the table
# using CSS
sketch_Specer_informatin <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th,rowspan = 2, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white"),
lapply("Off_Target", th,colspan = col_number, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white"),
th(rowspan = 2,"Plots",style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
),
tr(
lapply(name_subcolumn_off_target, th)
)
)
))
}
else if (question== 'n'| question== 'N') {
header.names <- c("",names(data))
sketch_Specer_informatin <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
)
)
))
}
}
if (OffTarget==F) {
header.names <- c("",names(data))
sketch_Specer_informatin <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
)
)
))
}
#################################################################################
################# final result html report and save ###############
result_spacers= datatable(data, filter = 'none', options = my.options,rownames = TRUE,container = sketch_Specer_informatin ,
caption = paste0(direct_repeat,' from ',bacteria,' (',backbone,")"))
result_spacers <- formatStyle(result_spacers,
columns = " ",
backgroundColor = "#000000",
borderBottomColor = "#000000",
borderBottomStyle = "solid",
borderBottomWidth = "1px",
borderCollapse = "collapse",
borderRightColor = "#000000",
borderRightStyle = "solid",
borderRightWidth = "1px",
color = "rgb(255, 255, 255)",
fontFamily = "Times New Roman",
fontSize = "13px",
fontWeight = "bold",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle",
)
result_spacers <- formatStyle(result_spacers,
columns = 1:length(data),
fontFamily = "Times New Roman",
fontSize = "13px",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle")
setwd(first_dir)
htmlwidgets::saveWidget(result_spacers, "result1.html")
#####################################################################
#edit HTML result
html_result=read_file("result1.html")
line_heder=paste0('<header><h1 style="color:blue;font-size:30px;" align="middle">',
paste0('Results Table for ',ID_code,' CRISPR-Cas System'),'</h1>')
line_img_tehran=paste('<img src= "https://drive.google.com/thumbnail?id=1NrT3Zw15mdetkgB4xLZ7GlZnDj-uAeNw"','width= 20% height= 20%',' align="left" >')
line_img_MRBLAB=paste('<img src= "https://drive.google.com/thumbnail?id=1BWDyB9RcBdes4dRNRh5ff3lUxMaUwdME"','width= 12% height= 20%',' align="left" >')
line_help='<p>more detailed description of columns is available in the <a href="https://sadegh65v.github.io/casilico/Casilico%20Guide.html" rel="nofollow noreferrer">HELP</a> section</p>'
file_Conaction<-file("Spacers_Information.Html")
writeLines(c(line_heder,line_img_tehran,html_result,line_help,line_img_MRBLAB), file_Conaction)
close(file_Conaction)
file.remove("result1.html")
setwd(first_main_dir)
}
if (length(crRNA_seq)==0 ) {
message("No candidate result pridicted for ",CRISPR_Type,
" please decrease mismatch threshold or change the ConservationMethod used ")
setwd(first_main_dir)
unlink(paste0("./",result_folder_name), recursive = TRUE)
}
}
}
file.remove("final_fasta_file.aln")
return("Design_complte")
options(warn = defaultW)
}
#' MAFFT (Multiple Sequence Alignment) in R
#' @param TargetFasta Full path to the target sequence(s); Example: TargetFasta="/usr/mrb/casilico/genome.fasta"
#' @param mafft_Options Options to run mafft; Example: MafftOptions="--auto --inputorder"
#' @return msa file (output of mafft), always output format is Clustal and name is final_fasta_file.aln
#' @section Further details: mafftR reports output as a cluster in final_fasta_file.aln file.
#' details for Options at https://mafft.cbrc.jp/alignment/software/manual/manual.html
#' @export mafftR
#'
#'
#'
mafftR=function(TargetFasta, MafftOptions = "--auto --inputorder"){
fasta_file=read.fasta(TargetFasta)
host_system=Sys.info()[['sysname']]
if (host_system %in% c("Windows","Linux")==F) {
stop("Currently CaSilico only supports Windows and Linux systems")
}
package_location<-system.file(package = "CaSilico")
if (host_system=="Windows") {
MAFFT_location=paste0(package_location,"/","dependency_win/mafft-win")
}
############## change mod sotware ###########
if (host_system=="Linux") {
MAFFT_location=paste0(package_location,"/dependency_linux/mafft-linux64")
lapply(paste0("chmod -R 777 ",MAFFT_location), system)
}
final_fasta_file=fasta_file
write.fasta(final_fasta_file,names(final_fasta_file),"final_fasta_file.fasta")
file.copy("final_fasta_file.fasta",MAFFT_location)
file.remove("final_fasta_file.fasta")
first_dir=getwd()
setwd(MAFFT_location)
MAFFT_list<- paste("mafft --clustalout",MafftOptions," final_fasta_file.fasta > final_fasta_file.aln")
lapply(MAFFT_list, system) ###run MAFFT for all input .txt files
file.copy("final_fasta_file.aln",first_dir)
file.remove("final_fasta_file.fasta")
file.remove("final_fasta_file.aln")
setwd(first_dir)
}
#' Dot plot for short sequence
#' @param sequence1 First sequence
#' @param sequence2 Second sequence
#' @param wsize the Size in chars of the moving window.
#' @param wstep the Size in chars for the steps of the moving window. Use wstep == wsize for non-overlapping windows.
#' @param nmatch If the number of match per window is greater than or equal to nmatch, then a dot is produced.
#' @param table_col Table color by default table_col = "goldenrod1".
#' @param points_col Point color by default points_col = "red".
#' @param xlab Label for x axis.
#' @param ylab Label for y axis.
#' @return plots.
#' @section References:
#' ...
#' @section Further details:In order to visualize this function better, short sequences are recommended.
#' ...
#' @export VDotplot
#'
#'
VDotplot=function(sequence1,sequence2, wsize = 1 , wstep = 1 , nmatch = 1 ,
table_col = "goldenrod1",points_col = "red", xlab = "sequence1", ylab = "sequence2")
{
#Split the Elements of strin
if (length(sequence1)==1){
x <- strsplit(sequence1,split = "")[[1]]}
if (length(sequence2)==1){
y <- strsplit(sequence2,split = "")[[1]]}
if (length(sequence1)!=1){
x <-sequence1}
if (length(sequence2)!=1){
y <- sequence2}
if (nchar(x[1]) > 1)
stop("seq1 should be provided as a vector of single chars")
if (nchar(y[1]) > 1)
stop("seq2 should be provided as a vector of single chars")
if (wsize < 1)
stop("non allowed value for wsize")
if (wstep < 1)
stop("non allowed value for wstep")
if (nmatch < 1)
stop("non allowed value for nmatch")
if (nmatch > wsize)
stop("nmatch > wsize is not allowed")
mkwin <- function(seq, wsize, wstep) {
sapply(seq(from = 1, to = length(seq) - wsize + 1, by = wstep),
function(i) c2s(seq[i:(i + wsize - 1)]))
}
wseq1 <- mkwin(x, wsize, wstep)
wseq2 <- mkwin(y, wsize, wstep)
if (nmatch == wsize) {
xy <- outer(wseq1, wseq2, "==")
}
else {
"%==%" <- function(x, y) colSums(sapply(x, s2c) == sapply(y,
s2c)) >= nmatch
xy <- outer(wseq1, wseq2, "%==%")
}
x=toupper(x)
y=toupper(y)
a=matrix(0,length(x),length(y))
# Naming the rows and columns of the formed matrix
row.names(a)=x
colnames(a)=y
for (i in 1:nrow(xy)) {
for (j in 1:ncol(xy)){
if (xy[i,j]==T) {
a[i,j]=1
}
}
}
h_line=seq(0+((seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])/2),max(length(x),length(y)),
by=seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])
v_line=seq(1+((seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])/2),max(length(x),length(y)),
by=seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])
aaaa=0.04*max(length(x),length(y))
plot(0:max(length(x),length(y)),0:max(length(x),length(y)),type = "n",
axes =F,frame.plot=TRUE,main ="" ,
ann = T,xlab = xlab , ylab = ylab)
aa=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb=c(h_line[length(h_line)],
max(length(x),length(y))+aaaa,
max(length(x),length(y))+aaaa,
h_line[length(h_line)])
polygon(aa,bb,col = table_col)
aa1=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb1=c(max(length(x),length(y))-aaaa,
max(length(x),length(y))+0.5,
max(length(x),length(y))+0.5,
max(length(x),length(y))-aaaa)
polygon(bb1-max(length(x),length(y)),aa1,col = table_col)
# y axes
axis(side=2, at = seq(0,max(length(x),length(y))-1,l=length(x)),
labels= length(x):1,cex.axis = 0.7, font = 2)
abline(h=h_line,col="black",lwd=1, lty=1)
text((aa1[2]+h_line[1])/2,
seq(0,max(length(x),length(y))-1,l=length(x)),
rev(x),col = "black",cex = 1,pch = 16,font = 2)
#x axes
axis(side=3, at = seq(1,max(length(x),length(y)),
l=length(y)), labels= 1:length(y),cex.axis = 0.7, font = 2)
abline(v=v_line, col="black",lwd=1, lty=1)
abline(v=0.5,col="black",lwd=1, lty=1)
text(seq(1,max(length(x),length(y)),l=length(y))
,(bb[2]+bb[1])/2,
y,col = "black",cex = 1,pch = 16,font = 2)
for (i in 1:length(x)){
for (j in 1:length(y))
if (a[i,j]==1)
points(seq(1,max(length(x),length(y)),l=length(y))[j],seq(0,max(length(x),length(y))-1,l=length(x))[length(x)-i+1],cex=1.5,col=points_col,pch = 16)
}
}
#' Compute and return revers compliment of a sequence
#' @param seq Sequence of DNA or RNA.
#' @return Reverse compliment of sequence .
#' @section References:
#' ...
#' @section Further details:
#' ...
#' @export Rcompliment
#'
#'
Rcompliment=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
Rcompliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in length(seq):1) {
Rcompliment_sequence = c(Rcompliment_sequence, compliment(seq[i],
seq_type = type))
}
Rcompliment_sequence
}
#' Compute and return compliment of a sequence.
#' @param seq Sequence of DNA or RNA
#' @return Compliment of sequence .
#' @section References:
#' ...
#' @section Further details:
#' ...
#' @export compliment
#'
#'
compliment=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
compliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in 1:length(seq)) {
compliment_sequence = c(compliment_sequence, compliment(seq[i],
seq_type = type))
}
compliment_sequence
}
mrb20045/CaSilico documentation built on Nov. 1, 2023, 10:34 a.m.
R Package Documentation
Browse R Packages
We want your feedback!
Note that we can't provide technical support on individual packages. You should contact the package authors for that.
Defines functions VDotplot mafftR CaSilico
Documented in CaSilico mafftR VDotplot
#' In silico crRNA design for CRISPR-Cas12 (A, B and F1 subtypes) and CRISPR-Cas13 (A, B and D subtypes).
#'
#'
#' @param ResultsFolder Name of the folder to save the final results; Default: ReaultsFolder="CaSilico_output".
#' @param TargetFasta Full path to the target sequence(s) to design crRNA for; Example: TargetFasta="/usr/mrb/casilico/genome.fasta"
#' @param TargetAccession Genome/gene/transcript accession numbers(s)(RefSeq or ENSEMBL ID)for the target sequence(s) to design crRNA for; Example: TargetAccession=c("U15717", "U15718").
#' @param TargetCoordinate Genomic coordinate(s)for the target sequence(s) to design crRNA for. CaSilico automatically retrieves the sequences based on the coordinates and their species; Example: TargetCoordinate=list(chromosome=c("1","5"), start=c("2000","12000"), end=c("2150","15500"), strand=c("-","-"), species = c("bos_taurus","bos_taurus")).
#' @param CrisprTypes A vector of CRSPR subtypes to design crRNAs based on their characteristics. One or more subtypes can be selected; Example: CrisprTypes=c("casVI_A","casVI_B","casVI_D","casV_A","casV_B","casV_F1").
#' @param ConservationMethod Desired method (1 or 2) to detect the conserved regions when more than one target sequence is submitted. See detailes for more information; Default: 1.
#' @param ConservationThreshold Desired threshold (between 0 and 1) to detect the conserved regions when more than one target sequence is submitted; Default: 0.95.
#' @param OffTarget If TRUE, CaSilico search for off-targets in the sequences specified by the Organism or LocalOff argument. This analysis may take a long time based on the number of subtypes in CrisprTypes argument and number of the selected sequences to be searched (Organism or LocalFasta arguments); Default: FALSE.
#' @param OffAsk If TRUE, in online mode before starting the off target analysis for each subtype, CaSilico ask from the user if he/she wants to continue or not; Default: FALSE.
#' @param Organism A vector of desired organisms for online off target analysis. CaSilico automatically search the identified crRNAs on the sequences(s)(NR or Refseq database, based on the CRISPR-Cas subtype) of the specified organismes; Example: Organism=c("Homo sapiens","Bos taurus",...).
#' @param LocalOff If TRUE, CaSilico search for off targets in local mode and Organism argument dose not works. LocalFile have to be set. Default: FALSE.
#' @param LocalFasta Full path to the fasta file sequence(s) to be used for local off target analysis. One or more fasta file can be addressed; Example: LocalFasta=c("/yourpath1/filename1.fasta","/yourpath2/filename2.fasta").
#' @param LocalName A vector including desired names related to the LocalFasta argument to determine the name of the results. If it is'nt specified by the user, Casilico considers sequence1, sequence2 and etc based on the number of fasta files in LocalFasta argument; Example: LocalName=c("Name1", "Name2").
#' @param Threads Number of threads to use, default value is 4.; Example: Threads=8..
#' @return Casilico presents the results in HTML and PDF files that can be found in ReaultsFolder.
#' @section References:
#' ...
#' @section Further details:
#' Casilico accepts three kinds of input formats as target sequence to be scanned for all potential target sites: 1) direct sequence(s) (TargetFasta argument); 2) Accession nember (TargetAccession argument) and 3) genomic coordinate (TargetCoordinate argument). One of these arguments have to be determined by the user as TargetFasta is recommended. Also, there are two different approaches to identify conserved regions (using ConservationMethod argument): 1) Most or all positions in the fragments (with length of w) have to be conserved and ≤2 positions can be polymorphic (based on the defined conservation score), as these positions can be occurred in the specific locations of the window, 2) Most or all positions in the fragments (with length of w) have to be conserved and ≤2 positions can be polymorphic (based on the defined conservation score) in everywhere of the fragment (with length of w).
#' @export
#' @import seqinr DT RRNA ape htmltools httr tibble dplyr readr
#' @examples
#
#'
#'############# (1) #############
#'# Run with TargetFasta and without off-target analysis
#'
#'# Full path of target sequences
#'data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")
#'
#'# Run CaSilico for subtype VI_A with ConservationThreshold = 0.98 and ConservationMethod = 1
#'CaSilico(ResultsFolder="Example",
#' TargetFasta=data,
#' TargetAccession=NULL,
#' CrisprTypes=c("casVI_A"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = F,
#' OffAsk = F)
#'##############################
#'#
#'#
#'#
#'############# (2) #############
#'
#'# Run with TargetFasta and offline off-target analysis
#'
#'# Full path of target sequences
#'data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")
#'
#'# Full path to fasta file sequences for local off target analysis
#'genome_dir <- paste0(system.file(package = "CaSilico"),"/data/genomes_off_target")
#'Local_Fasta <- file.path(genome_dir,list.files(genome_dir))
#'
#'
#'# Names related to the LocalFasta argument to determine the name of the results. (optional)
#'Local_Name <- c("Genome1","Genome2","Genome3")
#'
#'# Run CaSilico for subtypes V_A and V_B with ConservationThreshold = 0.98 and ConservationMethod = 1
#'CaSilico(ResultsFolder="Example1",
#' TargetFasta=data,
#' TargetAccession=NULL,
#' CrisprTypes=c("casV_A","casV_B"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = T,
#' OffAsk = F,
#' Organism=NULL,
#' LocalOff=T,
#' LocalFasta=Local_Fasta,
#' LocalName=Local_Name)
#'##############################
#'#
#'#
#'#
#'############# (3) #############
#'# Run with TargetFasta and online off-target analysis
#'
#'# Full path of target sequences
#'data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")
#'
#'# Run CaSilico for subtype VI_A with ConservationThreshold = 0.98 and ConservationMethod = 1
#'# and online off-target analysis for Homo sapiens
#'CaSilico(ResultsFolder="Example2",
#' TargetFasta=data ,
#' TargetAccession=NULL,
#' CrisprTypes=c("casVI_A"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = T,
#' OffAsk = F,
#' Organism="Homo sapiens",
#' LocalOff=F)
#'##############################
#'#
#'#
#'#
#'############# (4) #############
#'# Run with accession numbers
#'
#'# Run CaSilico for subtypes VI_A and VI_B with ConservationThreshold = 0.98 and ConservationMethod = 1
#'# The accession numbers used are: "U15717" and "U15718"
#' CaSilico(ResultsFolder="Example3",
#' TargetAccession=c("U15717", "U15718"),
#' CrisprTypes=c("casVI_A","casVI_B"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = F,
#' OffAsk = F)
#'##############################
#'#
#'#
#'#
#'############# (5) #############
#' # Run with sequence coordinate
#'
#' # Run CaSilico for subtypes VI_A and VI_B with ConservationThreshold = 0.98 and ConservationMethod = 1
#' # The desired coordinates are:
#' # (1) chromosome = 1, start = 2000, end = 2150, strand = "-", species = "bos_taurus"
#' # (2) chromosome = 1, start = 2000, end = 2150, strand = "-", species = "bos_taurus"
#' CaSilico(ResultsFolder="Example4",
#' TargetCoordinate=list(chromosome=c("1","1"),
#' start=c("2000","2000"),
#' end=c("2150","2150"),
#' strand=c("-","-"),
#' species = c("bos_taurus","bos_taurus")),
#' CrisprTypes=c("casVI_B","casVI_A"),
#' ConservationMethod = 1,
#' ConservationThreshold=0.98,
#' OffTarget = F,
#' OffAsk = F)
#'##############################
#'
CaSilico=function(ResultsFolder="CaSilico_output",
TargetFasta=NULL,
TargetAccession=NULL,
TargetCoordinate=list(chromosome=NA, start=NA, end=NA, strand=NA, species = NA ),
CrisprTypes,
ConservationMethod = 1,
ConservationThreshold=0.95,
OffTarget = F,
OffAsk=F,
Organism=NULL,
LocalOff=F,
LocalFasta=c("Path1","Path2"),
LocalName=c("Organism1","Organism2"),
Threads=4){
library("seqinr")
library("DT")
library("RRNA")
library("ape")
library("htmltools")
library("tibble")
library("dplyr")
library("readr")
defaultW <- getOption("warn")
options(warn = -1)
host_system=Sys.info()[['sysname']]
if (host_system %in% c("Windows","Linux")==F) {
stop("Currently CaSilico only supports Windows and Linux systems")
}
first_main_dir=getwd()
package_location<-system.file(package = "CaSilico")
if (host_system=="Windows") {
MAFFT_location=paste0(package_location,"/","dependency_win/mafft-win")
RNAfold_path = paste0(package_location,"/","dependency_win/Blastn/RNAfold.exe")
Blast_location=paste0(package_location,"/","dependency_win/Blastn/blastn.exe")
makeblastdb_location=paste0(package_location,"/","dependency_win/Blastn/makeblastdb.exe")
RNAplot_location=paste0(package_location,"/","dependency_win/Blastn/RNAplot.exe")
}
############## change mod sotware ###########
if (host_system=="Linux") {
MAFFT_location=paste0(package_location,"/dependency_linux/mafft-linux64")
RNAfold_path = paste0(package_location,"/dependency_linux/Blastn/RNAfold")
Blast_location=paste0(package_location,"/dependency_linux/Blastn/blastn")
makeblastdb_location=paste0(package_location,"/dependency_linux/Blastn/makeblastdb")
RNAplot_location=paste0(package_location,"/dependency_linux/Blastn/RNAfold")
path_software=c(RNAfold_path,
Blast_location,
makeblastdb_location,
RNAplot_location,
paste0(MAFFT_location,"/mafft.bat",collapse = ""))
for (path in path_software) {
chmod_command=paste0("chmod 777 ","'",path,"'")
lapply(chmod_command, system)
}
lapply(paste0("chmod -R 777 ",MAFFT_location), system)
}
#############################################
is_empty=function(X){
length(X)==0
}
# input seq is path
if (is_empty(TargetFasta)==FALSE) {
fasta_file=read.fasta(TargetFasta)
if (exists("fasta_file")==F | length(fasta_file)==0){
stop("There is a problem in your file or path of file. please check it.")
}
TargetAccession=NULL
TargetCoordinate=NULL
}
# input seq is accession number
if (length(TargetAccession)>=1) {
input_genes_fasta=read.GenBank(TargetAccession)
if (is_empty(input_genes_fasta)==T) {
stop("The access number entered is incorrect. please check it")
}
write.dna(input_genes_fasta,
file ="Input_Genes.fasta",
format = "fasta",
append = FALSE,
nbcol = 6,
colsep = "",
colw = 10)
fasta_file=read.fasta("Input_Genes.fasta")
file.remove("Input_Genes.fasta")
TargetFasta=NULL
TargetCoordinate=NULL
}
# input seq is coordinate
if (all(is.na(TargetCoordinate))==F) {
library("httr")
getseq=function(chromosome, start, end, strand, species = "Homo_sapiens"){
url = paste0("https://useast.ensembl.org/", species, "/Export/Output/Location?db=core;flank3_display=0;flank5_display=0;output=fasta;r=",
chromosome, ":", start, "-", end, ";strand=", strand,
";utr5=yes;cdna=yes;intron=yes;utr3=yes;peptide=yes;coding=yes;genomic=unmasked;exon=yes;_format=Text")
r=GET(url)
w=content(r, "text")
s=strsplit(w,split = "\r\n")[[1]]
paste0(s[-c(1,length(s))],collapse = "")
}
seqs_of_cordinate=c()
for (no_cor in 1:length(TargetCoordinate$chromosome)) {
seqs_of_cordinate=c(seqs_of_cordinate,
paste0(TargetCoordinate$chromosome[no_cor],"@",
TargetCoordinate$start[no_cor],"@",
TargetCoordinate$end[no_cor],"@",
TargetCoordinate$strand[no_cor],"@",
TargetCoordinate$species[no_cor],"@"))
}
input_coordinate_seq=c()
for (cordinate_seq in 1:length(seqs_of_cordinate)) {
temp_seq=seqs_of_cordinate[cordinate_seq]
temp_seq=strsplit(temp_seq,split = "@")[[1]]
temp_sequence="ddd"
iter=0
while(all(iter<=150 , temp_sequence=="ddd")){
try({
temp_sequence=getseq(temp_seq[1],temp_seq[2],temp_seq[3],temp_seq[4],temp_seq[5])
},silent = T)
iter=iter+1
}
input_coordinate_seq=c(input_coordinate_seq,temp_sequence)
}
if (any(input_coordinate_seq=="ddd")) {
stop("CaSilico is unable to download your coordinate sequences due to a problem on the Ensembl site.
Please try again later.")
}
write_line_in=c()
for (i in 1:length(input_coordinate_seq)) {
write_line_in=c(write_line_in,paste0(">",i),input_coordinate_seq[i])
}
file_Conaction<-file("Input_coordinate.fasta")
writeLines(write_line_in, file_Conaction)
close(file_Conaction)
fasta_file=read.fasta("Input_coordinate.fasta")
file.remove("Input_coordinate.fasta")
TargetFasta=NULL
TargetCoordinate=NULL
}
final_file_list=list()
# calculated total nucleotide for mafft limitation (mafft limitation > 1500 sequences * 30kb)
total_Nucleotide=0
for (h in 1:length(fasta_file)) {
total_Nucleotide=total_Nucleotide+length(fasta_file[[h]])
empty_pos=which(fasta_file[[h]]==" ")
if (length(empty_pos)>0) {
final_file_list[[h]]=fasta_file[[h]][-empty_pos]
}
else {final_file_list[[h]]=fasta_file[[h]]}
}
if ( total_Nucleotide > 40000000 ) {
print("Restrictions on Multiple Alignment")
}
if (total_Nucleotide <= 40000000) {
if (length(final_file_list)==1) {
write.fasta(list(final_file_list[[1]],final_file_list[[1]]), c(names(fasta_file),names(fasta_file)), "final_fasta_file.fasta")
}else{
write.fasta(final_file_list, names(fasta_file), "final_fasta_file.fasta")
}
setwd(MAFFT_location)
if (file.exists("final_fasta_file.fasta")) {
file.remove("final_fasta_file.fasta")
}
if (file.exists("final_fasta_file.aln")) {
file.remove("final_fasta_file.aln")
}
setwd(first_main_dir)
file.copy("final_fasta_file.fasta",MAFFT_location)
file.remove("final_fasta_file.fasta")
}
# Run mafft
setwd(MAFFT_location)
if (host_system=="Linux") {
MAFFT_list<- paste0("./mafft.bat --auto "," --thread ",Threads, " --inputorder final_fasta_file.fasta > final_fasta_file.aln")
}
if (host_system=="Windows") {
MAFFT_list<- paste0("mafft --auto "," --thread ",Threads, " --inputorder final_fasta_file.fasta > final_fasta_file.aln")
}
lapply(MAFFT_list, system) ###run MAFFT for all input .txt files
FMDValn <- read.fasta(file = "final_fasta_file.aln", as.string = T)
file.copy("final_fasta_file.aln",first_main_dir)
file.remove("final_fasta_file.fasta")
file.remove("final_fasta_file.aln")
setwd(first_main_dir)
topoTypes_sequences <- FMDValn
topoTypes_names <- names(FMDValn)
################# calculate frequency matrix for each base in sequences #########
score_matrix=matrix(0,5,length(strsplit(topoTypes_sequences[[1]],split = "")[[1]]))
rownames(score_matrix)=c("g","t","c","a","-")
for (i in 1:length(topoTypes_sequences)) {
seq=strsplit(topoTypes_sequences[[i]],split = "")[[1]]
for (j in 1:length(seq)) {
# one Nucleotide symbols
if(seq[j]=="g" | seq[j]=="G"){
score_matrix[1,j]= score_matrix[1,j]+1}
else if(seq[j]=="t" | seq[j]=="T" | seq[j]=="U" | seq[j]=="u"){
score_matrix[2,j]= score_matrix[2,j]+1}
else if(seq[j]=="c" | seq[j]=="C"){
score_matrix[3,j]= score_matrix[3,j]+1}
else if(seq[j]=="a" |seq[j]=="A"){
score_matrix[4,j]= score_matrix[4,j]+1}
else if(seq[j]=="-"){
score_matrix[5,j]= score_matrix[5,j]+1}
# four Nucleotide symbols
else if(seq[j]=="n" | seq[j]=="N" ){
score_matrix[1:4,j]= score_matrix[1:4,j]+0.25}
# two Nucleotide symbols
else if(seq[j]=="r" | seq[j]=="R" ){
score_matrix[c(1,4),j]= score_matrix[c(1,4),j]+0.5}
else if(seq[j]=="y" | seq[j]=="Y" ){
score_matrix[c(2,3),j]= score_matrix[c(2,3),j]+0.5}
else if(seq[j]=="k" | seq[j]=="K" ){
score_matrix[1:2,j]= score_matrix[1:2,j]+0.5}
else if(seq[j]=="m" | seq[j]=="M" ){
score_matrix[3:4,j]= score_matrix[3:4,j]+0.5}
else if(seq[j]=="s" | seq[j]=="S" ){
score_matrix[c(1,3),j]= score_matrix[c(1,3),j]+0.5}
else if(seq[j]=="w" | seq[j]=="W" ){
score_matrix[c(2,4),j]= score_matrix[c(2,4),j]+0.5}
# three Nucleotide symbols
else if(seq[j]=="b" | seq[j]=="B" ){
score_matrix[1:2,j]= score_matrix[1:2,j]+(0.33)
score_matrix[3,j]= score_matrix[3,j]+(0.34)}
else if(seq[j]=="d" | seq[j]=="D" ){
score_matrix[1:2,j]= score_matrix[1:2,j]+(0.33)
score_matrix[4,j]= score_matrix[4,j]+(0.34)}
else if(seq[j]=="h" | seq[j]=="H" ){
score_matrix[2:3,j]= score_matrix[2:3,j]+(0.33)
score_matrix[4,j]= score_matrix[4,j]+(0.34)}
else if(seq[j]=="v" | seq[j]=="V" ){
score_matrix[3:4,j]= score_matrix[3:4,j]+(0.33)
score_matrix[1,j]= score_matrix[1,j]+(0.34)}
}
}
#################################################################################
################### score matrix normalization ####################
normal_score_matrix=score_matrix/length(topoTypes_sequences)
normal_score_matrix1=normal_score_matrix
row_name_matrix=c("g","t","c","a","Del")
rownames(normal_score_matrix1)=row_name_matrix
####################################################################
######################### creATE msa sequence #####################
extract_higher_frequency=function(x){
for (g in 1:nrow(normal_score_matrix)) {
if(normal_score_matrix[g,x]==max(normal_score_matrix[,x]))
break
}
normal_score_matrix[g,x]
}
final_score=c()
for (k in 1:ncol(normal_score_matrix)) {
final_score=c(final_score,extract_higher_frequency(k))
}
final_str=toString(names(final_score),)
scores=as.data.frame(final_score)[[1]]
str_frags=strsplit(final_str,split = ", ")[[1]]
final_str_frags=c()
final_score1=c()
pso_del=c()
for (i in 1:length(str_frags)) {
if (str_frags[i]!="-") {
final_str_frags=c(final_str_frags,str_frags[i])
final_score1=c(final_score1,scores[i])
}
else if (str_frags[i]=="-") {
pso_del=c(pso_del,i)
}
}
if (length(pso_del)==0) {
original_pos_in_matrix=1:length(str_frags)
}
if (length(pso_del)!=0) {
original_pos_in_matrix=setdiff(1:length(str_frags),pso_del)
}
###############################################################
########################### Functions ########################
{
########### mismatch delete function####
if (ConservationMethod==1) {
mismatch_del=function(score,start,end,seed_start,seed_end){
del=c()
mismatch_locat=c()
for (i in 1:length(score)) {
if (score[i]<ConservationThreshold) {
mismatch_locat=c(mismatch_locat,i)
}}
if (length(mismatch_locat)>2) {
del=TRUE
}
if (length(mismatch_locat)==2) {
if (any(mismatch_locat%in%start:end)==TRUE) {
del=TRUE}
}
if (length(mismatch_locat)==2) {
if (any(mismatch_locat%in%start:end)!=TRUE) {
del=FALSE}
}
if (length(mismatch_locat)==1) {
if (mismatch_locat%in%seed_start:seed_end==TRUE) {
del=TRUE}
}
if (length(mismatch_locat)==1) {
if (mismatch_locat%in%seed_start:seed_end!=TRUE) {
del=FALSE}
}
if (length(mismatch_locat)==0) {
del=FALSE
}
del
}
}
if (ConservationMethod==2) {
mismatch_del=function(score,start,end,seed_start,seed_end){
del=c()
if (start<=end & seed_start<=seed_end)
{ if (length(which(score<ConservationThreshold))<3) {
del=FALSE
}
else if (length(which(score<ConservationThreshold))>=3) {
del=TRUE
}}
del
}
}
###############################
############# revers compliment function########
revers_compliment=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
Rcompliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in length(seq):1) {
Rcompliment_sequence = c(Rcompliment_sequence, compliment(seq[i],
seq_type = type))
}
Rcompliment_sequence
}
# compliment function
compliment_seq=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
compliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in 1:length(seq)) {
compliment_sequence = c(compliment_sequence, compliment(seq[i],
seq_type = type))
}
compliment_sequence
}
# revers function
revers_function=function(v){
revers_v=c()
for (i in length(v):1) {
revers_v=c(revers_v,v[i])
}
revers_v
}
##########################
################## check internet function ###########
if (host_system=="Windows") {
havingIP <- function() {
if (.Platform$OS.type == "windows") {
ipmessage <- system("ipconfig", intern = TRUE)
} else {
ipmessage <- system("ifconfig", intern = TRUE)
}
validIP <- "((25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)[.]){3}(25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)"
any(grep(validIP, ipmessage))
}
}
if (host_system=="Linux") {
havingIP <- function() {
if (.Platform$OS.type == "unix") {
ipmessage <- system("ip r", intern = TRUE)
} else {
ipmessage <- system("ip r", intern = TRUE)
}
validIP <- "((25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)[.]){3}(25[0-5]|2[0-4][0-9]|[01]?[0-9][0-9]?)"
any(grep(validIP, ipmessage))
}
}
##################################
################ crRNA plot function for RNA sequence ##############
crRNA_plot=function(sequence,crstart,start,pos_mutatiopns,
type_SNP,main_plot,W,u_rich_number,
acc_flank_number,eficieny_number,cr_acc) {
sequence=toupper(sequence)
plot(NULL, xlim =c(1,length(sequence)), ylim =c(1,12),type = "n",
axes =F,main = main_plot ,ann = T,xlab = "sequence",ylab = "")
axis(side=1, at = 1:length(sequence), labels= sequence,cex.axis = 0.7,
font = 0.6,col="darkred",col.axis="darkred")
axis(side=1, at = crstart:(crstart+W-1), labels= sequence[crstart:(crstart+W-1)] ,
cex.axis = 0.7,col="darkblue", font = 1,col.axis="darkblue")
end=start+W
x=c(crstart,crstart,crstart+W-1,crstart+W-1)
y=c(2,4,4,2)
polygon(x,y,col = "pink3",border = "pink3")
text((x[4]+x[1])/2,(y[2]+y[1])/2,labels = " CRISPR RNA",cex = 1.4)
text(x[1],1,labels = start,cex = 0.8)
text(x[4],1,labels = end,cex = 0.8)
if (is.numeric(pos_mutatiopns)) {
for (mut in 1:length(pos_mutatiopns)) {
pos_mutatiopns1=pos_mutatiopns+crstart-1
M=pos_mutatiopns1[mut]
if (length(pos_mutatiopns)==1) {
segments(M,4,M,6)
points(M,6,pch = 16, cex = 1.7, col = "red")
text((M),6.5,type_SNP[mut],cex = 1.2)
}
if (length(pos_mutatiopns)==2) {
if (abs(pos_mutatiopns[1]-pos_mutatiopns[2])>1) {
segments(M,4,M,6)
points(M,6,pch = 16, cex = 1.7, col = "red")
text((M),6.5,type_SNP[mut],cex = 1.2)
}
else if (abs(pos_mutatiopns[1]-pos_mutatiopns[2])==1) {
segments(M,4,M,6)
points(M,6,pch = 16, cex = 1.7, col = "red")
text((M-0.1),6.5,type_SNP[mut],cex = 1.2,srt=90)
}
}
}
}
segments(crstart,9,crstart+W-1,9,lwd=1.5,col = "darkblue")
text(crstart+W-1,9+0.03,">",col = "darkblue")
text(crstart,9+0.03,"<",col = "darkblue")
text((x[1]+x[4])/2,9+2,paste(cr_acc,"Accessible",collapse = ""),cex = 0.9,col = "darkblue")
if (crstart>10) {
segments(1,(y[2]+y[1])/2,crstart,(y[2]+y[1])/2,col="darkred")
text(1,(y[2]+y[1])/2+0.03,"<",col="darkred")
text(x[1]/2,(y[2]+y[1])/2+1,u_rich_number,cex = 0.9,col="darkred")
text(x[1]/2,(y[2]+y[1])/2+2,paste(acc_flank_number,"Accessible",collapse = ""),cex = 0.9,col="darkred")
}
if (length(sequence)-(crstart+W)>10){
segments(crstart+W-1,(y[2]+y[1])/2,length(sequence),(y[2]+y[1])/2,col="darkred")
text(length(sequence),(y[2]+y[1])/2+0.03,">",col="darkred")
text((length(sequence)+x[4])/2,(y[2]+y[1])/2+1,u_rich_number,cex = 0.9,col="darkred")
text((length(sequence)+x[4])/2,(y[2]+y[1])/2+2,paste(acc_flank_number,"Accessible",collapse = ""),cex = 0.9,col="darkred")
}
}
###################################################
################# crRNA plot function for DNA sequence ##################
crRNA_plot_DNA=function(sequence,crstart,start,pos_mutatiopns,
type_SNP,main_plot,W,eficieny_number,
sign_strand,length_pam) {
sequence=toupper(sequence)
end=start+W
plot(NULL, xlim =c(1,length(sequence)), ylim =c(1,6),type = "n",
axes =F,main = main_plot ,ann = T,xlab = "sequence",ylab = "")
axis(side=1, at = 1:length(sequence), labels= sequence,
cex.axis = 0.7, font = 0.6,col="darkred",col.axis="darkred")
x=c(crstart,crstart,crstart+W-1,crstart+W-1)
y=c(2,3,3,2)
polygon(x,y,col = "pink3",border = "pink3")
text((x[4]+x[1])/2,(y[2]+y[1])/2,labels = " CRISPR RNA",cex = 1.4)
segments(1,1.3,length(sequence),1.3,col="darkred")
text(1:length(sequence),1.38,"--",srt = 90,col="darkred")
text(1:length(sequence),1.8,toupper(compliment_seq(tolower(sequence))),col="darkred",cex = 0.7)
if (sign_strand=="+") {
axis(side=1, at = crstart:(crstart+W-1), labels= sequence[crstart:(crstart+W-1)] ,
cex.axis = 0.7,col="darkblue", font = 1,col.axis="darkblue")
segments((crstart-length_pam),2.1,(crstart-1),2.1,col="black")
text(((crstart-length_pam)+(crstart-1))/2,2.2,"<",srt = 90,col="black")
text(((crstart-length_pam)+(crstart-1))/2,2.45,"------",srt = 90,col="black")
text(((crstart-length_pam)+(crstart-1))/2,3,"PAM",col="black")
text(x[1],1.05,labels = start,cex = 1)
text(x[4],1.05,labels = end,cex = 1)
segments((crstart-length_pam),1.3,(crstart-1),1.3,col="orange")
text((crstart-length_pam):(crstart-1),1.38,"--",srt = 90,col="orange")
text((crstart-length_pam):(crstart-1),1.8,
toupper(compliment_seq(tolower(sequence)))[(crstart-length_pam):(crstart-1)],
col="orange",cex = 0.7)
}
if (sign_strand=="-") {
segments(crstart,1.3,(crstart+W-1),1.3,col="darkblue")
text(crstart:(crstart+W-1),1.38,"--",srt = 90,col="darkblue")
text(crstart:(crstart+W-1),1.8,
toupper(compliment_seq(tolower(sequence)))[crstart:(crstart+W-1)],
col="darkblue",cex = 0.7)
axis(side=1, at = (crstart+W):(crstart+W+length_pam-1), labels= sequence[(crstart+W):(crstart+W+length_pam-1)] ,
cex.axis = 0.7,col="orange", font = 1,col.axis="orange")
segments((crstart+W),2.1,(crstart+W+length_pam-1),2.1,col="black")
text(((crstart+W)+(crstart+W+length_pam-1))/2,2.2,"<",srt = 90,col="black")
text(((crstart+W)+(crstart+W+length_pam-1))/2,2.45,"------",srt = 90,col="black")
text(((crstart+W)+(crstart+W+length_pam-1))/2,3,"PAM",cex = 1.1,col="black")
text(x[1],1.05,labels =end ,cex = 1)
text(x[4],1.05,labels = start,cex = 1)
}
if (is.numeric(pos_mutatiopns)) {
for (mut in 1:length(pos_mutatiopns)) {
pos_mutatiopns1=pos_mutatiopns+crstart-1
M=pos_mutatiopns1[mut]
segments(M,3,M,4)
points(M,4,pch = 16, cex = 1.7, col = "red")
text((M-0.1),4.5,type_SNP[mut],cex = 1.1,srt = 90)
}
}
}
####################################################
############### my Dot plot function ################
my_Dot_Plot=function(sequence1,sequence2, wsize = 1 , wstep = 1 , nmatch = 1 ,
table_col = "goldenrod1",points_col = "red", xlab = "sequence1", ylab = "sequence2")
{
#Split the Elements of strin
if (length(sequence1)==1){
x <- strsplit(sequence1,split = "")[[1]]}
if (length(sequence2)==1){
y <- strsplit(sequence2,split = "")[[1]]}
if (length(sequence1)!=1){
x <-sequence1}
if (length(sequence2)!=1){
y <- sequence2}
if (nchar(x[1]) > 1)
stop("seq1 should be provided as a vector of single chars")
if (nchar(y[1]) > 1)
stop("seq2 should be provided as a vector of single chars")
if (wsize < 1)
stop("non allowed value for wsize")
if (wstep < 1)
stop("non allowed value for wstep")
if (nmatch < 1)
stop("non allowed value for nmatch")
if (nmatch > wsize)
stop("nmatch > wsize is not allowed")
mkwin <- function(seq, wsize, wstep) {
sapply(seq(from = 1, to = length(seq) - wsize + 1, by = wstep),
function(i) c2s(seq[i:(i + wsize - 1)]))
}
wseq1 <- mkwin(x, wsize, wstep)
wseq2 <- mkwin(y, wsize, wstep)
if (nmatch == wsize) {
xy <- outer(wseq1, wseq2, "==")
}
else {
"%==%" <- function(x, y) colSums(sapply(x, s2c) == sapply(y,
s2c)) >= nmatch
xy <- outer(wseq1, wseq2, "%==%")
}
x=toupper(x)
y=toupper(y)
a=matrix(0,length(x),length(y))
# Naming the rows and columns of the formed matrix
row.names(a)=x
colnames(a)=y
for (i in 1:nrow(xy)) {
for (j in 1:ncol(xy)){
if (xy[i,j]==T) {
a[i,j]=1
}
}
}
h_line=seq(0+((seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])/2),max(length(x),length(y)),
by=seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])
v_line=seq(1+((seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])/2),max(length(x),length(y)),
by=seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])
aaaa=0.04*max(length(x),length(y))
plot(0:max(length(x),length(y)),0:max(length(x),length(y)),type = "n",
axes =F,frame.plot=TRUE,main ="" ,
ann = T,xlab = xlab , ylab = ylab)
aa=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb=c(h_line[length(h_line)],
max(length(x),length(y))+aaaa,
max(length(x),length(y))+aaaa,
h_line[length(h_line)])
polygon(aa,bb,col = table_col)
aa1=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb1=c(max(length(x),length(y))-aaaa,
max(length(x),length(y))+0.5,
max(length(x),length(y))+0.5,
max(length(x),length(y))-aaaa)
polygon(bb1-max(length(x),length(y)),aa1,col = table_col)
# y axes
axis(side=2, at = seq(0,max(length(x),length(y))-1,l=length(x)),
labels= length(x):1,cex.axis = 0.7, font = 2)
abline(h=h_line,col="black",lwd=1, lty=1)
text((aa1[2]+h_line[1])/2,
seq(0,max(length(x),length(y))-1,l=length(x)),
rev(x),col = "black",cex = 1,pch = 16,font = 2)
#x axes
axis(side=3, at = seq(1,max(length(x),length(y)),
l=length(y)), labels= 1:length(y),cex.axis = 0.7, font = 2)
abline(v=v_line, col="black",lwd=1, lty=1)
abline(v=0.5,col="black",lwd=1, lty=1)
text(seq(1,max(length(x),length(y)),l=length(y))
,(bb[2]+bb[1])/2,
y,col = "black",cex = 1,pch = 16,font = 2)
for (i in 1:length(x)){
for (j in 1:length(y))
if (a[i,j]==1)
points(seq(1,max(length(x),length(y)),l=length(y))[j],seq(0,max(length(x),length(y))-1,l=length(x))[length(x)-i+1],cex=1.5,col=points_col,pch = 16)
}
}
#####################################################
############### prediction structure of sequence ###############
pridict_structure=function(seq,RNAfold_location)
{
seq_split=strsplit(seq,split = "")[[1]]
write.fasta(seq_split,"seq_split","seq_split.fasta")
structure_of_seq=system(paste(RNAfold_location," ","seq_split.fasta" ),intern=T)
file.remove("seq_split.fasta")
file.remove("seq_split_ss.ps")
structure_of_seq_final=strsplit(structure_of_seq[3],split = "")[[1]][1:length(seq_split)]
free_anergy_seq=as.numeric(paste(strsplit(structure_of_seq[3],split = "")[[1]][(length(seq_split)+3):(length(strsplit(structure_of_seq[3],split = "")[[1]])-1)],collapse = ""))
list( "structure"=structure_of_seq_final, "free_anergy_seq"= free_anergy_seq)
}
################################################################
############### secondary structure plot of backbone and spacer ######################
sec_structure_plot=function(seq,sructure_seq,first_backbone,backbone_name,length_backbone,main,free_energy)
{
length_seq=length(strsplit(seq,split = "")[[1]])
ct=makeCt(paste(sructure_seq,collapse = ""),paste(seq,collapse = ""))
coord=ct2coord(ct)
if (first_backbone==T) {
RNAPlot(coord,labTF=T,pointSize=1.8,lineWd=0.5,modspec=TRUE,ranges=data.frame(min=c(1,length_backbone+1),
max=c(length_backbone,length_seq),
col=c(2,5),desc=c(backbone_name,"Spacer")),main=main)
RNAPlot(coord,nt=TRUE,add=TRUE,dp=0.7,tsize=1)
}
else if (first_backbone==F) {
RNAPlot(coord,labTF=T,pointSize=1.8,lineWd=0.5,modspec=TRUE,ranges=data.frame(min=c(1,length_seq-length_backbone+1),
max=c(length_seq-length_backbone,length_seq),
col=c(5,2),desc=c("Spacer",backbone_name)),main=main)
RNAPlot(coord,nt=TRUE,add=TRUE,dp=0.7,tsize=1)
}
else if (first_backbone=="N") {
RNAPlot(coord,labTF=T,pointSize=1.8,lineWd=0.5,modspec=TRUE,ranges=data.frame(min=1,
max=length_seq,
col=5,desc= paste0("Spacer ","(Free Energy = ",free_energy,")")),main=main)
RNAPlot(coord,nt=TRUE,add=TRUE,dp=0.7,tsize=1)
}
}
######################################################################################
}
##############################################################
for (CRISPR_Type in CrisprTypes) {
if (CRISPR_Type=="casVI_A") {
W=28
Nucleotide_rich="t"
backbone="GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC"
local_rich_type="Local_U_Rich"
Normal_rich_type="Normalized_U_Rich"
start=8
end=27
seed_start=13
seed_end=24
blast_database="refseq_rna"
length_pam_cas=0
first_backbone=T
blast_strand="plus"
ID_code="VI_A"
bacteria="Leptotrichia wadei"
cas_protein_type="Cas13a/C2c2"
max_free_energy=-57
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casVI_D") {
W=22
Nucleotide_rich="t"
backbone="TACCCCTACCAACTGGTCGGGGTTTGAAAC"
local_rich_type="Local_U_Rich"
Normal_rich_type="Normalized_U_Rich"
start=3
end=20
seed_start=2
seed_end=8
blast_database="refseq_rna"
length_pam_cas=0
first_backbone=T
blast_strand="plus"
ID_code="VI_D"
bacteria="Ruminococcus flavefaciens"
cas_protein_type="Cas13d"
max_free_energy=-45
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casVI_B") {
W=30
Nucleotide_rich="a"
backbone="GTTGTGGAAGGTCCAGTTTTGAGGGGCTATTACAAC"
local_rich_type="Local_A_Rich"
Normal_rich_type="Normalized_A_Rich"
start=12
end=29
seed_start=12
seed_end=26
blast_database="refseq_rna"
length_pam_cas=2
first_backbone=F
blast_strand="plus"
ID_code="VI_B"
bacteria="Prevotella sp."
cas_protein_type="Cas13b/C2c6"
max_free_energy=-59
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casV_B") {
W=20
backbone="GTCTAGAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGCTTCTCAAATCTGAGAAGTGGCAC"
start=0
end=0
seed_start=0
seed_end=0
blast_database="nr"
length_pam_cas=3
first_backbone=T
blast_strand="both"
ID_code="V_B"
bacteria="Alicyclobacillus acidiphilus"
cas_protein_type="Cas12b/C2c1"
max_free_energy=-102
direct_repeat="Scaffold"
}
if (CRISPR_Type=="casV_A") {
W=20
backbone="TAATTTCTACTAAGTGTAGAT"
start=1
end=6
seed_start=1
seed_end=6
blast_database="nr"
length_pam_cas=4
first_backbone=T
blast_strand="both"
ID_code="V_A"
bacteria="Lachnospiraceae bacterium"
cas_protein_type="Cas12a/Cpf1"
max_free_energy=-34
direct_repeat="Direct Repeat"
}
if (CRISPR_Type=="casV_F1") {
W=20
backbone="TGCAGAACCCGAATAGACGAATGAAGGAATGCAAC"
start=9
end=16
seed_start=9
seed_end=16
blast_database="nr"
length_pam_cas=0
first_backbone=T
blast_strand="both"
ID_code="V_F1"
bacteria="Uncultured archaeon"
cas_protein_type="Cas14a/Cas12f1"
max_free_energy=-34
direct_repeat="Direct Repeat"
}
if (length(final_str_frags)<W+105) {
message(paste("Your gene is too short, so casilico can not design guides RNA for",CRISPR_Type))
}
if (length(final_str_frags)>=W+105) {
backbone_structure_and_anergy=pridict_structure(backbone,RNAfold_location = RNAfold_path)
backbone_structure=backbone_structure_and_anergy$structure
backbone_free_enargy=backbone_structure_and_anergy$free_anergy_seq
############### create dir for final result ############
setwd(first_main_dir)
name_of_fasta_file_genes=ResultsFolder
result_folder_name=paste0(CRISPR_Type,"-","ConservationMethod_",ConservationMethod,"-","threshold_",ConservationThreshold*100,"-result_",name_of_fasta_file_genes)
plots_folder_name="Plots"
sec_plots_folder_name="Secondary Structure"
MSA_folder="Multiple Sequenc Alignment"
first_dir=paste0(first_main_dir,"/",result_folder_name)
if (dir.exists(result_folder_name)) {
unlink(paste0("./",result_folder_name), recursive = TRUE)
}
dir.create(result_folder_name)
file.copy("final_fasta_file.aln",first_dir)
#file.remove("final_fasta_file.aln")
setwd(first_dir)
dir.create(plots_folder_name)
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"| CRISPR_Type=="casV_F1") {
dir.create(sec_plots_folder_name)
}
dir.create(MSA_folder)
file.copy("final_fasta_file.aln",MSA_folder)
file.remove("final_fasta_file.aln")
setwd(MSA_folder)
file.rename("final_fasta_file.aln","Multiple Sequence Alignment.aln")
write.fasta(final_str_frags,"Consensus Sequence","consensus sequence.fasta")
setwd(first_dir)
#####################################################
#################### first empty value ##############
crRNA_seq=c()
vv=list()
normalized_U_rich=list()
local_U_rich=list()
start_pos=list()
mismatch_pos=list()
mismatch_num=list()
efficiency=list()
type_if_SNP=list()
accessible_side=list()
accessible_flank_side=list()
rank=list()
prtospaser=list()
strand=list()
crRNA_free_energy=list()
Guide_RNA_ID=list()
link_plots=list()
GC_content=list()
score_Gaide=list()
#####################################################
###################### secondary structure ################
if (CRISPR_Type=="casVI_D"|
CRISPR_Type=="casVI_A"|
CRISPR_Type=="casVI_B"|
CRISPR_Type=="casV_F1"){
if (CRISPR_Type=="casVI_D"|
CRISPR_Type=="casVI_A"|
CRISPR_Type=="casVI_B") {
u_rich_in_whole=sum(final_score1[which(final_str_frags==Nucleotide_rich)])/sum(final_score1)
}
if (length(final_str_frags)<=2000){
write.fasta(final_str_frags,"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:length(final_str_frags)]
RNAplot_file_name=paste0(">","final_structure")
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags,collapse = ""),
paste(RNAfols_structure_format,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structure_ss.svg",plots_folder_name)
file.remove("final_structure_ss.svg")
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structure_ss.ps",plots_folder_name)
file.remove("final_structure_ss.ps")
}
}
if (length(final_str_frags)>2000) {
# start
write.fasta(final_str_frags[1:2000],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format_start=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur","_1_to_2000")
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags[1:2000],collapse = ""),
paste(RNAfols_structure_format_start,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_1_to_2000_ss.svg",plots_folder_name)
file.remove("final_structur_1_to_2000_ss.svg")
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_1_to_2000_ss.ps",plots_folder_name)
file.remove("final_structur_1_to_2000_ss.ps")
}
# end
write.fasta(final_str_frags[(length(final_str_frags)-2000+1):length(final_str_frags)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format_end=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur","_Last_2000")
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags[(length(final_str_frags)-2000+1):length(final_str_frags)],collapse = "")
,paste(RNAfols_structure_format_end,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_Last_2000_ss.svg",plots_folder_name)
file.remove("final_structur_Last_2000_ss.svg")
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy("final_structur_Last_2000_ss.ps",plots_folder_name)
file.remove("final_structur_Last_2000_ss.ps")
}
}}
###########################################################
###################### for casVI_A type & casVI_D type ######################
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A") {
for (i in 1:(length(final_str_frags)-W+1)) {
candidate=final_str_frags[i:(i+W-1)]
candidate_scores=c(final_score1[i:(i+W-1)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
candidate_U_rich=(sum(final_score1[which(final_str_frags[i:(i+W-1)]==Nucleotide_rich)]))
if (mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
select_candidate=paste(revers_compliment(candidate),collapse = "")
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
crRNA_seq=c(crRNA_seq,select_candidate)
vv[[select_candidate]]=round(revers_function(candidate_scores),digits = 4)
start_pos[[select_candidate]]=i
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
mismatch_pos[[select_candidate]]=which(revers_function(candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
pos_spacer_in_sec_stracture=i
if (length(final_str_frags)>2000){
if (i<=1000) {
RNAfols_structure_format=RNAfols_structure_format_start
pos_spacer_in_sec_stracture=i
}
else if (i>1000 & i<length(final_str_frags)-1000+1) {
write.fasta(final_str_frags[(i-1000):(i+1000-1)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur_",(i-1000),"_to_",(i+1000-1))
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,
paste(final_str_frags[(i-1000):(i+1000-1)],collapse = ""),
paste(RNAfols_structure_format,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"))
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"))
}
pos_spacer_in_sec_stracture=1000
}
else if (i>=length(final_str_frags)-1000) {
RNAfols_structure_format=RNAfols_structure_format_end
pos_spacer_in_sec_stracture=2000-(length(final_str_frags)-i)
}
}
if (i<=100) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[1:(i+W+100-1)]==Nucleotide_rich))]))-candidate_U_rich)/
(sum(final_score1[1:(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>100 & i<length(final_str_frags)-100-W+1) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100):(i+W+100-1)]==Nucleotide_rich))+i-100-1]))-candidate_U_rich)/
(sum(final_score1[(i-100):(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]=
round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>=length(final_str_frags)-100-W) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100+1):length(final_str_frags)]==Nucleotide_rich))+i-100]))-candidate_U_rich)/
((sum(final_score1[(i-100+1):length(final_str_frags)]))-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)), digits=4)
}
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags[i+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & ")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=
c(paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot=paste0("Information plot for ","(ID=",Guide_RNA_ID[[select_candidate]],") candidate ")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[1:(i+W+20-1)],i,start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",Normal_rich_type)
,paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name, "/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & length(final_str_frags)-20-W+1) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],title_for_crRNA_plot,W,
paste0(local_U_rich[[select_candidate]]," ",local_rich_type," , ",
normalized_U_rich[[select_candidate]]," ",Normal_rich_type),
paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name, "/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags)-20-W) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20+1):length(final_str_frags)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,
paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",
normalized_U_rich[[select_candidate]]," ",
Normal_rich_type)
,paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name, "/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*2*local_U_rich[[select_candidate]])
,(2.5*accessible_side[[select_candidate]])
,(2.5*accessible_flank_side[[select_candidate]])
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
blast_query=prtospaser
}
#############################################################################
############################# for casVI_B type ##########################
if (CRISPR_Type=="casVI_B") {
prtospaseraa=list()
prtospasergg=list()
prtospasertt=list()
prtospaserag=list()
prtospaserga=list()
prtospaserat=list()
prtospaserta=list()
prtospasergt=list()
prtospasertg=list()
for (i in 3:(length(final_str_frags)-W+1)) {
candidate=final_str_frags[i:(i+W-1)]
candidate_scores=c(final_score1[i:(i+W-1)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
candidate_U_rich=(sum(final_score1[which(final_str_frags[i:(i+W-1)]==Nucleotide_rich)]))
if (final_str_frags[i-1] != "c" & final_str_frags[i-2] != "c"){
if (mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
select_candidate=paste(revers_compliment(candidate),collapse = "")
backbone_spacer=paste0(select_candidate,backbone,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[(W+1):length(backbone_spacer_structure$structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[(W+1):length(backbone_spacer_structure$structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
prtospaseraa[[select_candidate]]=c("a","a",candidate)
prtospasergg[[select_candidate]]=c("g","g",candidate)
prtospasertt[[select_candidate]]=c("t","t",candidate)
prtospaserag[[select_candidate]]=c("a","g",candidate)
prtospaserga[[select_candidate]]=c("g","a",candidate)
prtospaserat[[select_candidate]]=c("a","t",candidate)
prtospaserta[[select_candidate]]=c("t","a",candidate)
prtospasergt[[select_candidate]]=c("g","t",candidate)
prtospasertg[[select_candidate]]=c("t","g",candidate)
crRNA_seq=c(crRNA_seq,select_candidate)
vv[[select_candidate]]=round(revers_function(candidate_scores),digits = 4)
start_pos[[select_candidate]]=i
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
mismatch_pos[[select_candidate]]=which(revers_function(candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
pos_spacer_in_sec_stracture=i
if (length(final_str_frags)>2000){
if (i<=1000) {
RNAfols_structure_format=RNAfols_structure_format_start
pos_spacer_in_sec_stracture=i
}
else if (i>1000 & i<length(final_str_frags)-1000+1) {
write.fasta(final_str_frags[(i-1000):(i+1000-1)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur_",(i-1000),"_to_",(i+1000-1))
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,paste(final_str_frags[(i-1000):(i+1000-1)],collapse = ""),
paste(RNAfols_structure_format_start,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"))
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"))
}
pos_spacer_in_sec_stracture=1000
}
else if (i>=length(final_str_frags)-1000) {
RNAfols_structure_format=RNAfols_structure_format_end
pos_spacer_in_sec_stracture=2000-(length(final_str_frags)-i)
}
}
if (i<=100) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[1:(i+W+100-1)]==Nucleotide_rich))]))-candidate_U_rich)/
(sum(final_score1[1:(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[i:(i+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((1:i-1),(i+W):(i+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=round(length(a_flank_side)/
length(c((1:i-1),(i+W):(i+W+100-1))), digits=4)
}
else if (i>100 & i<length(final_str_frags)-100-W+1) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100):(i+W+100-1)]==Nucleotide_rich)+i-100)]))-candidate_U_rich)/
(sum(final_score1[(i-100):(i+W+100-1)])-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]=round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[i:(i+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((i-100):(i-1),(i+W):(i+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((i-100):(i-1),(i+W):(i+W+100-1))), digits=4)
}
else if (i>=length(final_str_frags)-100-W) {
local_U_rich[[select_candidate]]=
round(((sum(final_score1[(which(final_str_frags[(i-100+1):length(final_str_frags)]==Nucleotide_rich)+i-100)]))-candidate_U_rich)/
((sum(final_score1[(i-100+1):length(final_str_frags)]))-sum(candidate_scores)),digits = 4)
normalized_U_rich[[select_candidate]]= round(local_U_rich[[select_candidate]]/u_rich_in_whole,digits = 4)
a_side=which(RNAfols_structure_format[i:(i+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((i-100):(i-1),(i+W-1):length(final_str_frags))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((i-100):(i-1),(i+W-1):length(final_str_frags))), digits=4)
}
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags[i+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]],collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=
c(paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", "),collapse = " & ",sep=""),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", "),collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot=paste0("Information plot for ","(ID=",Guide_RNA_ID[[select_candidate]],") candidate ")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[1:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",
Normal_rich_type),paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & length(final_str_frags)-20-W+1) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",
Normal_rich_type),paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags)-20-W) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20+1):length(final_str_frags)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,paste0(local_U_rich[[select_candidate]]," ",
local_rich_type," , ",normalized_U_rich[[select_candidate]]," ",
Normal_rich_type),paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*2*local_U_rich[[select_candidate]])
,(2.5*accessible_side[[select_candidate]])
,(2.5*accessible_flank_side[[select_candidate]])
,(10*(1-(final_free_anergy/max_free_energy)))))
}}
}
blast_query=c(prtospaseraa,
prtospasergg,
prtospasertt,
prtospaserag,
prtospaserga,
prtospaserat,
prtospaserta,
prtospasergt,
prtospasertg)
}
#########################################################################
############################# for casV_B type ###########################
if (CRISPR_Type=="casV_B") {
final_str_frags_plus_vb=c("l","l","l",final_str_frags,"l","l","l")
prtospasertta_pos=list()
prtospaserttg_pos=list()
prtospaserttt_pos=list()
prtospaserttc_pos=list()
prtospasertta_neg=list()
prtospaserttg_neg=list()
prtospaserttt_neg=list()
prtospaserttc_neg=list()
for (i in 4:(length(final_str_frags_plus_vb)-W+1-6)) {
candidate=final_str_frags_plus_vb[i:(i+W-1)]
candidate_scores1=c(final_score1[(i-3):(i+W-1-3)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
if ((final_str_frags_plus_vb[i-2] == "t" & final_str_frags_plus_vb[i-3] == "t")|
(final_str_frags_plus_vb[i+W+1] == "a" & final_str_frags_plus_vb[i+W+2] == "a")){
if (mismatch_del(candidate_scores1,start,end,seed_start,seed_end)!=TRUE) {
if (final_str_frags_plus_vb[i+W+2] == "a" &
final_str_frags_plus_vb[i+W+1] == "a") {
select_candidate=paste(revers_compliment(candidate),collapse = "")
candidate_scores=revers_function(candidate_scores1)
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=revers_compliment(candidate)
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
vv[[select_candidate]]=round((candidate_scores),digits = 4)
start_pos[[select_candidate]]=(length(final_str_frags_plus_vb)-6)-((i-3)+W-1)+1
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="-"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospasertta_neg[[select_candidate]]=c("t","t","a",revers_compliment(candidate))
prtospaserttg_neg[[select_candidate]]=c("t","t","g",revers_compliment(candidate))
prtospaserttt_neg[[select_candidate]]=c("t","t","t",revers_compliment(candidate))
prtospaserttc_neg[[select_candidate]]=c("t","t","c",revers_compliment(candidate))
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags_plus_vb[(i)+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(revers_compliment(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", " )[[1]]),sep = "",collapse = " & ")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_vb[(i)+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_vb[(i)+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(revers_compliment(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", " )[[1]]),sep = "",collapse = " & "),
paste(revers_compliment(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", " )[[1]]),sep = "",collapse = " & "))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
title_for_crRNA_Dotplot=paste0("Dotplot for ", Guide_RNA_ID[[select_candidate]]," crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_vb)-20-W+1-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_vb)-20-W-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(length(final_str_frags_plus_vb)-3)],21,
start_pos[[select_candidate]],mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}
if (final_str_frags_plus_vb[i-2] == "t" &
final_str_frags_plus_vb[i-3] == "t") {
select_candidate=paste(candidate,collapse = "")
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
candidate_scores=candidate_scores1
prtospaser[[select_candidate]]=candidate
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
vv[[select_candidate]]=round((candidate_scores),digits = 4)
start_pos[[select_candidate]]=i-3
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="+"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospasertta_pos[[select_candidate]]=c("t","t","a",candidate)
prtospaserttg_pos[[select_candidate]]=c("t","t","g",candidate)
prtospaserttt_pos[[select_candidate]]=c("t","t","t",candidate)
prtospaserttc_pos[[select_candidate]]=c("t","t","c",candidate)
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(mismatch_pos[[select_candidate]]-1)]]
sssnp[which(names(sssnp)==final_str_frags_plus_vb[(i)+(mismatch_pos[[select_candidate]]-1)])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & ")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(mismatch_pos[[select_candidate]][1]-1)]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_vb[(i)+(mismatch_pos[[select_candidate]][1]-1)])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-3)+(mismatch_pos[[select_candidate]][2]-1)]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_vb[(i)+(mismatch_pos[[select_candidate]][2])-1])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", " )[[1]],sep = "",collapse = " & "))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_vb)-20-W+1-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_vb)-20-W-3) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_vb[(i-20):(length(final_str_frags_plus_vb)-3)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],3)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/" , sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
}
}
blast_query=c(prtospasertta_pos,
prtospaserttg_pos,
prtospaserttt_pos,
prtospaserttc_pos,
prtospasertta_neg,
prtospaserttg_neg,
prtospaserttt_neg,
prtospaserttc_neg)
}
#########################################################################
############################# for casV_A type ###########################
if (CRISPR_Type=="casV_A") {
final_str_frags_plus_va=c("l","l","l","l",final_str_frags,"l","l","l","l")
prtospaserttta_pos=list()
prtospasertttg_pos=list()
prtospasertttc_pos=list()
prtospaserttta_neg=list()
prtospasertttg_neg=list()
prtospasertttc_neg=list()
for (i in 5:(length(final_str_frags_plus_va)-W+1-8)) {
candidate=final_str_frags_plus_va[i:(i+W-1)]
candidate_scores1=final_score1[(i-4):(i+W-1-4)]
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
if (final_str_frags_plus_va[i+W] != "a" &
final_str_frags_plus_va[i+W+1] == "a" &
final_str_frags_plus_va[i+W+2] == "a" &
final_str_frags_plus_va[i+W+3] == "a") {
select_candidate=paste(revers_compliment(candidate),collapse = "")
candidate_scores=revers_function(candidate_scores1)
if(mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=revers_compliment(candidate)
vv[[select_candidate]]=round((candidate_scores),digits = 4)
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
start_pos[[select_candidate]]=(length(final_str_frags_plus_va)-8)-((i-4)+W-1)+1
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="-"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospaserttta_neg[[select_candidate]]=c("t","t","t","a",revers_compliment(candidate))
prtospasertttg_neg[[select_candidate]]=c("t","t","t","g",revers_compliment(candidate))
prtospasertttc_neg[[select_candidate]]=c("t","t","t","c",revers_compliment(candidate))
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags_plus_va[(i)+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(revers_compliment(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]]),collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_va[(i)+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_va[(i)+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(revers_compliment(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", ")[[1]]),collapse = " & ",sep=""),
paste(revers_compliment(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", ")[[1]]),collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
title_for_crRNA_Dotplot=paste0("Dotplot for ", Guide_RNA_ID[[select_candidate]]," crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_va)-20-W+1-4) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_va)-20-W-4) {
setwd(plots_folder_name)
png(filename2, width = 1200,height = 800)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(length(final_str_frags_plus_va)-4)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}}
if (final_str_frags_plus_va[i-4] == "t" &
final_str_frags_plus_va[i-3] == "t" &
final_str_frags_plus_va[i-2] == "t" &
final_str_frags_plus_va[i-1] != "t") {
select_candidate=paste(candidate,collapse = "")
candidate_scores=candidate_scores1
if(mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE){
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
vv[[select_candidate]]=round((candidate_scores),digits = 4)
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
start_pos[[select_candidate]]=i-4
mismatch_pos[[select_candidate]]=which((candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=mismatch_num[[select_candidate]]+1
efficiency[[select_candidate]]=mean(candidate_scores)
strand[[select_candidate]]="+"
crRNA_seq=c(crRNA_seq,select_candidate)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
prtospaserttta_pos[[select_candidate]]=c("t","t","t","a",candidate)
prtospasertttg_pos[[select_candidate]]=c("t","t","t","g",candidate)
prtospasertttc_pos[[select_candidate]]=c("t","t","t","c",candidate)
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(mismatch_pos[[select_candidate]]-1)]]
sssnp[which(names(sssnp)==final_str_frags_plus_va[(i)+(mismatch_pos[[select_candidate]]-1)])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]],collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(mismatch_pos[[select_candidate]][1]-1)]]
sssnp1[which(names(sssnp1)==final_str_frags_plus_va[(i)+(mismatch_pos[[select_candidate]][1]-1)])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[(i-4)+(mismatch_pos[[select_candidate]][2]-1)]]
sssnp2[which(names(sssnp2)==final_str_frags_plus_va[(i)+(mismatch_pos[[select_candidate]][2])-1])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", ")[[1]],sep = "" ,collapse = " & "),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", ")[[1]],collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot_DNA=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
title_for_crRNA_Dotplot=paste0("Dotplot for ", Guide_RNA_ID[[select_candidate]]," crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[4:(i+W+20-1)],i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & i<length(final_str_frags_plus_va)-20-W+1-4) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main="Spacer self complementary",
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),"crRNA self complementary")
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags_plus_va)-20-W-4) {
setwd(plots_folder_name)
png(filename2, width = 1500,height = 866)
layout(matrix(c(2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
2,2,3,3,4,4,
1,1,1,1,1,1,
1,1,1,1,1,1,
1,1,1,1,1,1), 8,6, byrow = TRUE))
crRNA_plot_DNA(final_str_frags_plus_va[(i-20):(length(final_str_frags_plus_va)-4)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot_DNA,W,
paste(efficiency[[select_candidate]],"Efficiency"),
strand[[select_candidate]],4)
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
}
blast_query=c(prtospaserttta_pos,
prtospasertttg_pos,
prtospasertttc_pos,
prtospaserttta_neg,
prtospasertttg_neg,
prtospasertttc_neg)
}
#########################################################################
############################# for casV_F1 type ##########################
if (CRISPR_Type=="casV_F1") {
for (i in 1:(length(final_str_frags)-W+1)) {
candidate=final_str_frags[i:(i+W-1)]
candidate_scores=c(final_score1[i:(i+W-1)])
GC_condidate=round(length(which(candidate=="g"| candidate=="c"))/length(candidate)*100,digits = 2)
if (mismatch_del(candidate_scores,start,end,seed_start,seed_end)!=TRUE) {
select_candidate=paste(revers_compliment(candidate),collapse = "")
backbone_spacer=paste0(backbone,select_candidate,collapse = "")
backbone_spacer_structure=pridict_structure(backbone_spacer,RNAfold_location = RNAfold_path)
spaser_structure=pridict_structure(select_candidate,RNAfold_path)
if (paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")==
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq-backbone_free_enargy
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Natural structure of DR")
back_name=paste("Direct Repeat (Natural structure) ")
}
if(paste(backbone_spacer_structure$structure[1:length(backbone_structure)],collapse = "")!=
paste(backbone_structure,collapse = "")){
final_free_anergy=backbone_spacer_structure$free_anergy_seq
crRNA_free_energy[[select_candidate]]=paste(round(final_free_anergy,digits = 4),"Unnatural structure of DR")
back_name=paste("Direct Repeat (Unnatural structure) ")
}
prtospaser[[select_candidate]]=candidate
GC_content[[select_candidate]]=paste0(GC_condidate,"%")
crRNA_seq=c(crRNA_seq,select_candidate)
vv[[select_candidate]]=round(revers_function(candidate_scores),digits = 4)
start_pos[[select_candidate]]=i
mismatch_pos[[select_candidate]]=which(revers_function(candidate_scores)<ConservationThreshold)
mismatch_num[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))
rank[[select_candidate]]=length(which(candidate_scores<ConservationThreshold))+1
efficiency[[select_candidate]]=mean(candidate_scores)
Guide_RNA_ID[[select_candidate]]=paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate))
pos_spacer_in_sec_stracture=i
if (length(final_str_frags)>2000){
if (i<=1000) {
RNAfols_structure_format=RNAfols_structure_format_start
pos_spacer_in_sec_stracture=i
}
else if (i>1000 & i<length(final_str_frags)-1000+1) {
write.fasta(final_str_frags[(i-1000):(i+1000-1)],"final_str_frags","final_str_frags.fasta")
structure_of_RNAfold=system(paste(RNAfold_path," ","final_str_frags.fasta" ),intern=T)
file.remove("final_str_frags.fasta")
file.remove("final_str_frags_ss.ps")
RNAfols_structure_format=strsplit(structure_of_RNAfold[3],split = "")[[1]][1:2000]
RNAplot_file_name=paste0(">","final_structur_",(i-1000),"_to_",(i+1000-1))
fileConn<-file("sec_stracuter.txt")
writeLines(c(RNAplot_file_name,paste(final_str_frags[(i-1000):(i+1000-1)],collapse = ""),
paste(RNAfols_structure_format_start,collapse = "")), fileConn)
close(fileConn)
if (host_system=="Windows") {
RNAPlot_command_line=paste(RNAplot_location," -o svg ", " sec_stracuter.txt")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.svg"))
}
if (host_system=="Linux") {
RNAPlot_command_line=paste(RNAplot_location, " sec_stracuter.txt ")
lapply(RNAPlot_command_line, system)
file.remove("sec_stracuter.txt")
file.copy(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"),plots_folder_name)
file.remove(paste0("final_structur_",(i-1000),"_to_",(i+1000-1),"_ss.ps"))
}
pos_spacer_in_sec_stracture=1000
}
else if (i>=length(final_str_frags)-1000) {
RNAfols_structure_format=RNAfols_structure_format_end
pos_spacer_in_sec_stracture=2000-(length(final_str_frags)-i)
}
}
if (i<=100) {
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((1:pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>100 & i<length(final_str_frags)-100-W+1) {
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):(pos_spacer_in_sec_stracture+W+100-1))), digits=4)
}
else if (i>=length(final_str_frags)-100-W) {
a_side=which(RNAfols_structure_format[pos_spacer_in_sec_stracture:(pos_spacer_in_sec_stracture+W-1)]==".")
a_flank_side=which(RNAfols_structure_format[c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)]==".")
accessible_side[[select_candidate]]=round(length(a_side)/W, digits=4)
accessible_flank_side[[select_candidate]]=
round(length(a_flank_side)/length(c((pos_spacer_in_sec_stracture-100):(pos_spacer_in_sec_stracture-1),
(pos_spacer_in_sec_stracture+W):2000)), digits=4)
}
if (mismatch_num[[select_candidate]]>0){
if (mismatch_num[[select_candidate]]==1) {
sssnp=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]])]]
sssnp[which(names(sssnp)==final_str_frags[i+(W-mismatch_pos[[select_candidate]])])]=0
SNP=sssnp>0
type_if_SNP[[select_candidate]]=
paste(strsplit(toString(names(SNP[SNP==TRUE]),),split = ", ")[[1]],collapse = " & ",sep="")
}
else if (mismatch_num[[select_candidate]]==2) {
type_if_SNP[[select_candidate]]=c()
sssnp1=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][1])]]
sssnp1[which(names(sssnp1)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][1])])]=0
sssnp2=normal_score_matrix1[,original_pos_in_matrix[i+(W-mismatch_pos[[select_candidate]][2])]]
sssnp2[which(names(sssnp2)==final_str_frags[i+(W-mismatch_pos[[select_candidate]][2])])]=0
SNP1=sssnp1>0
SNP2=sssnp2>0
type_if_SNP[[select_candidate]]=c(
paste(strsplit(toString(names(SNP1[SNP1==TRUE]),),split = ", ")[[1]],collapse = " & ",sep=""),
paste(strsplit(toString(names(SNP2[SNP2==TRUE]),),split = ", ")[[1]],collapse = " & ",sep=""))
}}
if (mismatch_num[[select_candidate]]==0) {
type_if_SNP[[select_candidate]]="No_SNP"
mismatch_pos[[select_candidate]]="-"
}
###File name for plot result
title_for_crRNA_plot=paste0("Information plot for ",Guide_RNA_ID[[select_candidate]], " crRNA candidate")
filename2=paste0("ID-",paste0(ID_code,"_",ConservationMethod,"_",which(crRNA_seq==select_candidate)),".png",collapse = "")
if (i<=20) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[1:(i+W+20-1)],
i,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,"",paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>20 & length(final_str_frags)-20-W+1) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20):(i+W+20-1)],21,
start_pos[[select_candidate]],
mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],
title_for_crRNA_plot,W,"",
paste(accessible_flank_side[[select_candidate]]),paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
if (i>=length(final_str_frags)-20-W) {
setwd(plots_folder_name)
png(filename2, width = 1300,height = 750)
layout(matrix(c(2,2,3,3,4,4,2,2,3,3,4,4,1,1,1,1,1,1), 3, 6, byrow = TRUE))
crRNA_plot(final_str_frags[(i-20+1):length(final_str_frags)],21,
start_pos[[select_candidate]],mismatch_pos[[select_candidate]],
type_if_SNP[[select_candidate]],title_for_crRNA_plot,W,"",
paste(accessible_flank_side[[select_candidate]]),
paste(efficiency[[select_candidate]],"efficiency"),
paste(accessible_side[[select_candidate]]))
my_Dot_Plot(select_candidate,
select_candidate,
points_col = "cyan3",
xlab = "Specer Seq",
ylab = "Specer Seq")
sec_structure_plot(select_candidate,
spaser_structure$structure,
first_backbone = "N",
main=paste0("Spacer Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"),
free_energy = spaser_structure$free_anergy_seq)
sec_structure_plot(backbone_spacer,
backbone_spacer_structure$structure,
first_backbone,
back_name,
length(backbone_structure),
paste0("crRNA Self complementarity ","(ID=",Guide_RNA_ID[[select_candidate]],")"))
dev.off()
setwd(first_dir)
link= tibble( file = dir( file.path( '.', plots_folder_name ), pattern=filename2 ) ) %>%
mutate( path = paste0('file:', plots_folder_name,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', file,'</a>' ) )
link_plots[[select_candidate]]=link$link
}
score_Gaide[[select_candidate]]=sum(c((75-(mismatch_num[[select_candidate]]*5))
,(2.5*accessible_side[[select_candidate]])
,(2.5*accessible_flank_side[[select_candidate]])
,(10*(1-(final_free_anergy/max_free_energy)))))
}
}
blast_query=prtospaser
}
#########################################################################
############ replace sec plots to Secondary structure folder ############
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"|CRISPR_Type=="casV_F1") {
current_folder <- paste0(first_dir,"/Plots")
new_folder <- paste0(first_dir,"/Secondary Structure")
if (host_system=="Windows") {
list_of_files <- list.files(current_folder, ".svg")
}
if (host_system=="Linux") {
list_of_files <- list.files(current_folder, ".ps")
}
file.copy(file.path(current_folder,list_of_files), new_folder)
file.remove(file.path(current_folder,list_of_files))
}
#########################################################################
################# View data (data.frame construction) ############
if (length(crRNA_seq)>0) {
data=as.data.frame(crRNA_seq)
data$Guide_RNA_ID=Guide_RNA_ID
names(data)[names(data) == "crRNA_seq"] <- "Guide_RNA"
if (length(topoTypes_names)>1) {
data$Major_Allele_Frequency=vv
}
data$Start=start_pos
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data$Strand=strand
}
data$GC_content=GC_content
if (length(topoTypes_names)>1) {
data$Mismatch_Number=mismatch_num
data$Mismatch_Position=mismatch_pos
data$Type_Of_SNP=type_if_SNP
data$Efficiency=efficiency
names(data)[names(data) == "Efficiency"] <- "Conservation Efficiency"
}
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"){
data$local_U_rich=local_U_rich
names(data)[names(data) == "local_U_rich"] <- local_rich_type
data$Normalized_U_rich=normalized_U_rich
names(data)[names(data) == "Normalized_U_rich"] <- Normal_rich_type
}
if (CRISPR_Type=="casVI_D"| CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_B"|CRISPR_Type=="casV_F1"){
data$Protospacer_Accessibility_Score=accessible_side
data$Local_Accessibility_Score=accessible_flank_side
}
data$Rank=rank
data$Self_Complementarity_Free_Energy=crRNA_free_energy
data$Score=unlist(score_Gaide)
###################################################################
###################### DT options #####################
my.options <- list(autoWidth = T,
searching = T,
ordering = T,
filter = 'none',
lengthChange = T,
lengthMenu = c(5,10,50,100,1000),
paging = T,
info = T,
pageLength = 5,
searchHighlight = TRUE,
searchable=F)
header.style <- "th { font-family: 'Times New Roman'; font-weight: bold; color: white ; background-color: #000000;}"
########################################################
###### checking internet connection and blast with rules (off_target_prediction) ######
setwd(first_dir)
if (OffTarget==T) {
if (LocalOff!=T & havingIP()==TRUE){
if (is.null(Organism)!=T) {
if (OffAsk) {
min_approximate_blast_time=length(blast_query)*0.3*length(Organism)
max_approximate_blast_time=length(blast_query)*1*length(Organism)
question <- readline(paste0("off-Targets Prediction for ",ID_code," CRISPR-Cas System ","may be take long time",
" (About ",
min_approximate_blast_time," to ",
max_approximate_blast_time,
" minutes) ",
"would you like to continue or no? (Y/N)"))
}
else if (OffAsk==F) {
question="y"
}
}
else{
message('If you want to do online mode for off-Target Prediction,
please Ensure you introduced the "Organism" parameter.')
question="n"
}
}
if (LocalOff == T) {
if (all(!(LocalFasta %in% c("path1", "path2")))) {
if (length(LocalName) == length(LocalFasta)) {
Organism = LocalName
}
else if (length(LocalName) != length(LocalFasta)) {
Organism = paste0("User_Sequence_",
1:length(LocalFasta))
}
if (OffAsk) {
question <- readline("Depending on the size of your genomes, this step may be time consuming, Do you want to continue?(Y/N)")
}
else if (OffAsk == F) {
question = "y"
}
}
else {
message("please Ensure you introduced the \"LocalFasta\" parameter",
"\n and check the \"LocalOff\" parameter be true.")
question = "n"
}
}
if(question== 'y'| question== 'Y'){
Guide_RNA=data$Guide_RNA
len_list=rep(0,length(Guide_RNA))
count_mm0=as.list(len_list)
count_mm1=as.list(len_list)
count_mm2=as.list(len_list)
count_mm3=as.list(len_list)
count_mm4=as.list(len_list)
data_off_target=as.data.frame(Guide_RNA)
data_off_target$Guide_RNA_ID=data$Guide_RNA_ID
for (org in Organism) {
list_blast_mm_0=list()
list_blast_mm_1=list()
list_blast_mm_2=list()
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
list_blast_mm_3=list()
list_blast_mm_4=list()
}
write.fasta( blast_query,names(blast_query), file.out = "blast_query.FASTA")
# online blast
if (LocalOff!=T){
entrez_query=paste(org,' [organism]',sep = "")
entrez_query <- paste0('"',entrez_query,'"')
blast_command=paste(Blast_location,
' -task blastn -db ', blast_database ,
' -entrez_query ' , entrez_query,
' -word_size 7 -evalue 10 -query blast_query.FASTA -out blast_out.txt ' ,
' -strand ', blast_strand,
' -outfmt "10 delim=@ qseqid sacc qseq sseq length qstart qend sstart send gapopen mismatch " -num_alignments 5000 -remote',sep = '' ,collapse = "" )
lapply(blast_command, system)
}
#offline blast
if (LocalOff==T){
number_of_genome_file=which(org==Organism)
file.copy(LocalFasta[number_of_genome_file],first_dir)
neme_genome=basename(LocalFasta[number_of_genome_file])
file.rename(neme_genome,"genome_name.fasta")
makeblastdb_command=paste( makeblastdb_location ,
' -in genome_name.fasta -out genome_name_index -dbtype nucl -parse_seqids')
lapply(makeblastdb_command, system)
blast_command=paste(Blast_location,
' -task blastn -db genome_name_index ' ,
' -word_size 7 -evalue 10 -query blast_query.FASTA -out blast_out.txt ' ,
' -strand ', blast_strand,' -num_threads ', Threads,
' -outfmt "10 delim=@ qseqid sacc qseq sseq length qstart qend sstart send gapopen mismatch " -num_alignments 5000 ',
sep = '' ,collapse = "" )
lapply(blast_command, system)
removes_file=list.files(first_dir,pattern = "genome_name*")
file.remove(removes_file)
}
info_blast = file.info("blast_out.txt")
if (info_blast$size!=0) {
if (CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_A"|CRISPR_Type=="casV_F1"){
blast_result=read.table("blast_out.txt",sep = "@")
blast_result=rbind(blast_result[blast_result$V5==((W+length_pam_cas)-2) & blast_result$V11==0 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-1) & blast_result$V11<=1 & blast_result$V10==0,],
blast_result[blast_result$V5==(W+length_pam_cas) & blast_result$V11<=2 & blast_result$V10==0,])
}
else if (CRISPR_Type=="casVI_B"){
blast_result=read.table("blast_out.txt",sep = "@")
blast_result=rbind(blast_result[blast_result$V5==((W+length_pam_cas)-2) & blast_result$V6==1 & blast_result$V11==0 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-1) & blast_result$V6==1 & blast_result$V11<=1 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)) & blast_result$V6==1 & blast_result$V11<=2 & blast_result$V10==0,])
}
else if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
blast_result=read.table("blast_out.txt",sep = "@")
blast_result=rbind(blast_result[blast_result$V5==((W+length_pam_cas)-4) & blast_result$V6==1 & blast_result$V11==0 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-3) & blast_result$V6==1 & blast_result$V11<=1 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-2) & blast_result$V6==1 & blast_result$V11<=2 & blast_result$V10==0,],
blast_result[blast_result$V5==((W+length_pam_cas)-1) & blast_result$V6==1 & blast_result$V11<=3 & blast_result$V10==0,],
blast_result[blast_result$V5==(W+length_pam_cas) & blast_result$V6==1 & blast_result$V11<=4 & blast_result$V10==0,])
}
if (length(blast_result$V2)!=0) {
for (crRNA_predict in 1:length(data$Guide_RNA)) {
list_blast_mm_0[[crRNA_predict]]="No_Hit"
list_blast_mm_1[[crRNA_predict]]="No_Hit"
list_blast_mm_2[[crRNA_predict]]="No_Hit"
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
list_blast_mm_3[[crRNA_predict]]="No_Hit"
list_blast_mm_4[[crRNA_predict]]="No_Hit"
}
genes_mm0=c()
genes_mm1=c()
genes_mm2=c()
genes_mm3=c()
genes_mm4=c()
for (blast_crRNA_predict in 1:length(unique(blast_result$V1))){
if (data$Guide_RNA[crRNA_predict]==unique(blast_result$V1)[blast_crRNA_predict]) {
s_blast_result=blast_result[blast_result$V1==unique(blast_result$V1)[blast_crRNA_predict],]
for (gene in 1:length(s_blast_result$V2)) {
s_blast_result_gene=s_blast_result[gene,]
pos_mismach_blast_crRNA_gene=which(strsplit(s_blast_result_gene$V3,split = "")[[1]]!=strsplit(s_blast_result_gene$V4,split = "")[[1]])
#pam mismatch checked
if (any(pos_mismach_blast_crRNA_gene%in%0:length_pam_cas)!=TRUE) {
if (CRISPR_Type=="casVI_A"|CRISPR_Type=="casVI_D"|CRISPR_Type=="casVI_B"|CRISPR_Type=="casV_F1"){
if ((W+length_pam_cas)-s_blast_result_gene$V5==1) {
if (s_blast_result_gene$V6==1) {
pos_mismach_blast_crRNA_gene=c(pos_mismach_blast_crRNA_gene,(W+length_pam_cas))
}
else if (s_blast_result_gene$V6!=1) {
pos_mismach_blast_crRNA_gene=c(1,pos_mismach_blast_crRNA_gene)
}
}
if ((W+length_pam_cas)-s_blast_result_gene$V5==2) {
if (s_blast_result_gene$V6==1) {
pos_mismach_blast_crRNA_gene=c(pos_mismach_blast_crRNA_gene,(W+length_pam_cas)-1,(W+length_pam_cas))
}
else if (s_blast_result_gene$V6==2) {
pos_mismach_blast_crRNA_gene=c(1,pos_mismach_blast_crRNA_gene,(W+length_pam_cas))
}
else if (s_blast_result_gene$V6==3) {
pos_mismach_blast_crRNA_gene=c(1,2,pos_mismach_blast_crRNA_gene)
}
}
seq_gene=rep(1,(W+length_pam_cas))
seq_gene[pos_mismach_blast_crRNA_gene]=0
if (mismatch_del(seq_gene[(length_pam_cas+1):length(seq_gene)],start,end,seed_start,seed_end)!=TRUE) {
gene_off_target=paste0(s_blast_result_gene$V2,"_pos",":",s_blast_result_gene$V8,"-",s_blast_result_gene$V9)
#mm0
if (length(pos_mismach_blast_crRNA_gene)==0) {
genes_mm0=c(genes_mm0,gene_off_target)
}
#mm1
else if (length(pos_mismach_blast_crRNA_gene)==1) {
genes_mm1=c(genes_mm1,gene_off_target)
}
#mm2
else if (length(pos_mismach_blast_crRNA_gene)==2) {
genes_mm2=c(genes_mm2,gene_off_target)
}
}}
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
number_mismach_blast_crRNA_gene=(((W+length_pam_cas)-s_blast_result_gene$V5)+s_blast_result_gene$V11)
gene_off_target=paste0(s_blast_result_gene$V2,"_pos",":",s_blast_result_gene$V8,"-",s_blast_result_gene$V9)
#mm0
if (number_mismach_blast_crRNA_gene==0) {
genes_mm0=c(genes_mm0,gene_off_target)
}
#mm1
else if (number_mismach_blast_crRNA_gene==1) {
genes_mm1=c(number_mismach_blast_crRNA_gene)
}
#mm2
else if (number_mismach_blast_crRNA_gene==2) {
genes_mm2=c(genes_mm2,gene_off_target)
}
#mm3
else if (number_mismach_blast_crRNA_gene==3) {
genes_mm3=c(genes_mm3,gene_off_target)
}
#mm4
else if (number_mismach_blast_crRNA_gene==4) {
genes_mm4=c(genes_mm4,gene_off_target)
}
}
}
}
#mm0
if (length(genes_mm0)>0) {
list_blast_mm_0[[crRNA_predict]]=genes_mm0
count_mm0[[crRNA_predict]]=count_mm0[[crRNA_predict]]+length(genes_mm0)
}
#mm1
if (length(genes_mm1)>0) {
list_blast_mm_1[[crRNA_predict]]=genes_mm1
count_mm1[[crRNA_predict]]=count_mm1[[crRNA_predict]]+length(genes_mm1)
}
#mm2
if (length(genes_mm2)>0) {
list_blast_mm_2[[crRNA_predict]]=genes_mm2
count_mm2[[crRNA_predict]]=count_mm2[[crRNA_predict]]+length(genes_mm2)
}
#mm3
if (length(genes_mm3)>0) {
list_blast_mm_3[[crRNA_predict]]=genes_mm3
count_mm3[[crRNA_predict]]=count_mm3[[crRNA_predict]]+length(genes_mm3)
}
#mm4
if (length(genes_mm4)>0) {
list_blast_mm_4[[crRNA_predict]]=genes_mm4
count_mm4[[crRNA_predict]]=count_mm4[[crRNA_predict]]+length(genes_mm4)
}
break
}
}
}
data_off_target$blast_mm_0=list_blast_mm_0
data_off_target$blast_mm_1=list_blast_mm_1
data_off_target$blast_mm_2=list_blast_mm_2
names(data_off_target)[names(data_off_target) == "blast_mm_0"] <- paste("No mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_1"] <- paste("1 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_2"] <- paste("2 mismatched off Targets in",org)
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data_off_target$blast_mm_3=list_blast_mm_3
data_off_target$blast_mm_4=list_blast_mm_4
names(data_off_target)[names(data_off_target) == "blast_mm_3"] <- paste("3 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_4"] <- paste("4 mismatched off targets in",org)
}
}
if (length(blast_result$V2)==0) {
data_off_target$blast_mm_0="No_Hit"
data_off_target$blast_mm_1="No_Hit"
data_off_target$blast_mm_2="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_0"] <- paste("No mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_1"] <- paste("1 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_2"] <- paste("2 mismatched off targets in",org)
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data_off_target$blast_mm_3="No_Hit"
data_off_target$blast_mm_4="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_3"] <- paste("3 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_4"] <- paste("4 mismatched off targets in",org)
}
}
}
if (info_blast$size==0) {
data_off_target$blast_mm_0="No_Hit"
data_off_target$blast_mm_1="No_Hit"
data_off_target$blast_mm_2="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_0"] <- paste("No mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_1"] <- paste("1 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_2"] <- paste("2 mismatched off targets in",org)
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A"){
data_off_target$blast_mm_3="No_Hit"
data_off_target$blast_mm_4="No_Hit"
names(data_off_target)[names(data_off_target) == "blast_mm_3"] <- paste("3 mismatched off targets in",org)
names(data_off_target)[names(data_off_target) == "blast_mm_4"] <- paste("4 mismatched off targets in",org)
}
}
}
data$No_Mismatch_off_Target=count_mm0
data$One_Mismatch_off_Targets=count_mm1
data$Two_Mismatch_off_Targets=count_mm2
col_number=3
if (CRISPR_Type=="casV_B"|CRISPR_Type=="casV_A") {
data$Three_Mismatch_off_Targets=count_mm3
data$Four_Mismatch_off_Targets=count_mm4
col_number=col_number+2
}
setwd(first_dir)
html_result_folder="Off_Target-result.html"
dir.create( html_result_folder)
number_of_link_for_off=0
for (mm_type in (length(data)-(col_number-1)):length(data)) {
for ( guide in 1:length(data$Guide_RNA)) {
if (data[guide,mm_type]>0) {
number_of_link_for_off=number_of_link_for_off+1
zz=c("NO_mismatch","1_mismatch","2_mismatches","3_mismatches","4_mismatches")
type_of_mm=which(mm_type==(length(data)-(col_number-1)):length(data))
number_of_organism=length(Organism)
if (number_of_organism==1) {
show_col=c(1,2,type_of_mm+2)
}
if (number_of_organism>1) {
show_col_1_plus=(type_of_mm+2)+1:(number_of_organism-1)*col_number
show_col=c(1,2,type_of_mm+2,show_col_1_plus)
}
header.names <- c("",names(data_off_target[guide,][,show_col]))
sketch_for_each_gene <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
)
)
))
rr=as.data.frame(lapply(data_off_target[guide,][,show_col], function(x) {gsub(",", "\n", x)}))
cc=as.data.frame(lapply(rr, function(x) {gsub("c\\(", "", x)}))
bb=as.data.frame(lapply(cc, function(x) {gsub("\\)", "", x)}))
data_each_spacer_off_target=as.data.frame(lapply(bb, function(x) {gsub('"', '', x)}))
result_of_guide= datatable(data_each_spacer_off_target, filter = 'none',
options =my.options,
container = sketch_for_each_gene,
rownames = TRUE,
caption = paste0('Results Table for ',CRISPR_Type,' (Guide RNA=',data_off_target[guide,1],')'))
result_of_guide <- formatStyle(result_of_guide,
columns = " ",
backgroundColor = "#000000",
borderBottomColor = "#000000",
borderBottomStyle = "solid",
borderBottomWidth = "1px",
borderCollapse = "collapse",
borderRightColor = "#000000",
borderRightStyle = "solid",
borderRightWidth = "1px",
color = "rgb(255, 255, 255)",
fontFamily = "Times New Roman",
fontSize = "13px",
fontWeight = "bold",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle",
)
result_of_guide <- formatStyle(result_of_guide,
columns = 1:(length(Organism)+2),
fontFamily = "Times New Roman",
fontSize = "13px",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle")
name_html_Guide=paste0(zz[type_of_mm],"_",data[[guide,2]],".html",collapse = "")
setwd(html_result_folder)
htmlwidgets::saveWidget(result_of_guide, name_html_Guide )
setwd(first_dir)
Guide_html_Link = tibble( file = dir( file.path( '.', html_result_folder ), pattern= name_html_Guide ) ) %>%
mutate( path = paste0('file:', html_result_folder,"/", sort(file ))
, path = stringr::str_replace_all( path, ' ', '%20')
, link = paste0('<a target=_blank href=', path, '>', data[guide,mm_type],'</a>' ) )
data[guide,mm_type]=Guide_html_Link$link
}
}
}
if (number_of_link_for_off==0 ) {
if (dir.exists(html_result_folder)) {
unlink(paste0("./",html_result_folder), recursive = TRUE)
}
}
############# create sketch for DT package ###########
zz=c("NO_mismatch","1_mismatch","2_mismatches","3_mismatches","4_mismatches")
name_subcolumn_off_target=zz[1: col_number]
header.names <- c("",names(data_off_target))
# The container parameter allows us to design the header of the table
# using CSS
sketch_off_Target <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names[1:3], th,rowspan = 2, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white"),
lapply(Organism, th,colspan = col_number, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
),
tr(
rep(lapply(name_subcolumn_off_target, th),length(Organism))
)
)
))
######################################################
rr=as.data.frame(lapply(data_off_target, function(x) {gsub(",", "\n", x)}))
cc=as.data.frame(lapply(rr, function(x) {gsub("c\\(", "", x)}))
bb=as.data.frame(lapply(cc, function(x) {gsub("\\)", "", x)}))
data_off_target=as.data.frame(lapply(bb, function(x) {gsub('"', '', x)}))
result_off_targets= datatable(data_off_target, filter = 'none', options = my.options,rownames = TRUE,container = sketch_off_Target)
result_off_targets <- formatStyle(result_off_targets,
columns = " ",
backgroundColor = "#000000",
borderBottomColor = "#000000",
borderBottomStyle = "solid",
borderBottomWidth = "1px",
borderCollapse = "collapse",
borderRightColor = "#000000",
borderRightStyle = "solid",
borderRightWidth = "1px",
color = "rgb(255, 255, 255)",
fontFamily = "Times New Roman",
fontSize = "13px",
fontWeight = "bold",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle",
)
result_off_targets <- formatStyle(result_off_targets,
columns = 1:length(data_off_target),
fontFamily = "Times New Roman",
fontSize = "13px",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle")
htmlwidgets::saveWidget(result_off_targets, "result2.html")
html_result=read_file("result2.html")
line_heder=paste0('<header><h1 style="color:blue;font-size:30px;" align="middle">',
paste0('Off_Targets Results Table for ',ID_code,' CRISPR-Cas System'),'</h1>')
line_img_tehran=paste('<img src= "https://drive.google.com/thumbnail?id=1NrT3Zw15mdetkgB4xLZ7GlZnDj-uAeNw"','width= 300 height= 130',' align="left" >')
line_img_MRBLAB=paste('<img src= "https://drive.google.com/thumbnail?id=1BWDyB9RcBdes4dRNRh5ff3lUxMaUwdME" ','width= 100 height= 100',' align="left" >')
file_Conaction<-file("Off_Targets_Information.Html")
writeLines(c(line_heder,line_img_tehran,html_result,line_img_MRBLAB), file_Conaction)
close(file_Conaction)
file.remove("result2.html")
file.remove("blast_out.txt")
file.remove("blast_query.FASTA")
}
if(question == 'n'| question == 'N'){
message("Running continue without Off_Target prediction" )
}
if (havingIP()!=TRUE) {
message("Problem in Internet connection ",
"continue without off_Target result")
}
}
#######################################################################################
######################### assign link for figure and sort data ##################
setwd(first_dir)
data$Plot=link_plots
data=data[order(-data$Score),]
data$Score=NULL
#################################################################################
################### create sketch for DT package ################################
if (OffTarget==T) {
if (question== 'y'| question== 'Y') {
name_subcolumn_off_target=zz[1: col_number]
header.names <- c("",names(data)[1:(length(names(data))-(col_number+1))])
# The container parameter allows us to design the header of the table
# using CSS
sketch_Specer_informatin <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th,rowspan = 2, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white"),
lapply("Off_Target", th,colspan = col_number, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white"),
th(rowspan = 2,"Plots",style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
),
tr(
lapply(name_subcolumn_off_target, th)
)
)
))
}
else if (question== 'n'| question== 'N') {
header.names <- c("",names(data))
sketch_Specer_informatin <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
)
)
))
}
}
if (OffTarget==F) {
header.names <- c("",names(data))
sketch_Specer_informatin <- withTags(table(
style(type = "text/css", header.style),
thead(
tr(
lapply(header.names, th, style = "text-align: center ; border-right-width: 1px;border-right-style: solid; border-right-color: white; border-bottom-width: 1px; border-bottom-style: solid; border-bottom-color: white")
)
)
))
}
#################################################################################
################# final result html report and save ###############
result_spacers= datatable(data, filter = 'none', options = my.options,rownames = TRUE,container = sketch_Specer_informatin ,
caption = paste0(direct_repeat,' from ',bacteria,' (',backbone,")"))
result_spacers <- formatStyle(result_spacers,
columns = " ",
backgroundColor = "#000000",
borderBottomColor = "#000000",
borderBottomStyle = "solid",
borderBottomWidth = "1px",
borderCollapse = "collapse",
borderRightColor = "#000000",
borderRightStyle = "solid",
borderRightWidth = "1px",
color = "rgb(255, 255, 255)",
fontFamily = "Times New Roman",
fontSize = "13px",
fontWeight = "bold",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle",
)
result_spacers <- formatStyle(result_spacers,
columns = 1:length(data),
fontFamily = "Times New Roman",
fontSize = "13px",
lineHeight = "normal",
paddingBottom = "2.6px",
paddingLeft = "5.2px",
paddingRight = "5.2px",
paddingTop = "2.6px",
textAlign = "center",
verticalAlign = "middle")
setwd(first_dir)
htmlwidgets::saveWidget(result_spacers, "result1.html")
#####################################################################
#edit HTML result
html_result=read_file("result1.html")
line_heder=paste0('<header><h1 style="color:blue;font-size:30px;" align="middle">',
paste0('Results Table for ',ID_code,' CRISPR-Cas System'),'</h1>')
line_img_tehran=paste('<img src= "https://drive.google.com/thumbnail?id=1NrT3Zw15mdetkgB4xLZ7GlZnDj-uAeNw"','width= 20% height= 20%',' align="left" >')
line_img_MRBLAB=paste('<img src= "https://drive.google.com/thumbnail?id=1BWDyB9RcBdes4dRNRh5ff3lUxMaUwdME"','width= 12% height= 20%',' align="left" >')
line_help='<p>more detailed description of columns is available in the <a href="https://sadegh65v.github.io/casilico/Casilico%20Guide.html" rel="nofollow noreferrer">HELP</a> section</p>'
file_Conaction<-file("Spacers_Information.Html")
writeLines(c(line_heder,line_img_tehran,html_result,line_help,line_img_MRBLAB), file_Conaction)
close(file_Conaction)
file.remove("result1.html")
setwd(first_main_dir)
}
if (length(crRNA_seq)==0 ) {
message("No candidate result pridicted for ",CRISPR_Type,
" please decrease mismatch threshold or change the ConservationMethod used ")
setwd(first_main_dir)
unlink(paste0("./",result_folder_name), recursive = TRUE)
}
}
}
file.remove("final_fasta_file.aln")
return("Design_complte")
options(warn = defaultW)
}
#' MAFFT (Multiple Sequence Alignment) in R
#' @param TargetFasta Full path to the target sequence(s); Example: TargetFasta="/usr/mrb/casilico/genome.fasta"
#' @param mafft_Options Options to run mafft; Example: MafftOptions="--auto --inputorder"
#' @return msa file (output of mafft), always output format is Clustal and name is final_fasta_file.aln
#' @section Further details: mafftR reports output as a cluster in final_fasta_file.aln file.
#' details for Options at https://mafft.cbrc.jp/alignment/software/manual/manual.html
#' @export mafftR
#'
#'
#'
mafftR=function(TargetFasta, MafftOptions = "--auto --inputorder"){
fasta_file=read.fasta(TargetFasta)
host_system=Sys.info()[['sysname']]
if (host_system %in% c("Windows","Linux")==F) {
stop("Currently CaSilico only supports Windows and Linux systems")
}
package_location<-system.file(package = "CaSilico")
if (host_system=="Windows") {
MAFFT_location=paste0(package_location,"/","dependency_win/mafft-win")
}
############## change mod sotware ###########
if (host_system=="Linux") {
MAFFT_location=paste0(package_location,"/dependency_linux/mafft-linux64")
lapply(paste0("chmod -R 777 ",MAFFT_location), system)
}
final_fasta_file=fasta_file
write.fasta(final_fasta_file,names(final_fasta_file),"final_fasta_file.fasta")
file.copy("final_fasta_file.fasta",MAFFT_location)
file.remove("final_fasta_file.fasta")
first_dir=getwd()
setwd(MAFFT_location)
MAFFT_list<- paste("mafft --clustalout",MafftOptions," final_fasta_file.fasta > final_fasta_file.aln")
lapply(MAFFT_list, system) ###run MAFFT for all input .txt files
file.copy("final_fasta_file.aln",first_dir)
file.remove("final_fasta_file.fasta")
file.remove("final_fasta_file.aln")
setwd(first_dir)
}
#' Dot plot for short sequence
#' @param sequence1 First sequence
#' @param sequence2 Second sequence
#' @param wsize the Size in chars of the moving window.
#' @param wstep the Size in chars for the steps of the moving window. Use wstep == wsize for non-overlapping windows.
#' @param nmatch If the number of match per window is greater than or equal to nmatch, then a dot is produced.
#' @param table_col Table color by default table_col = "goldenrod1".
#' @param points_col Point color by default points_col = "red".
#' @param xlab Label for x axis.
#' @param ylab Label for y axis.
#' @return plots.
#' @section References:
#' ...
#' @section Further details:In order to visualize this function better, short sequences are recommended.
#' ...
#' @export VDotplot
#'
#'
VDotplot=function(sequence1,sequence2, wsize = 1 , wstep = 1 , nmatch = 1 ,
table_col = "goldenrod1",points_col = "red", xlab = "sequence1", ylab = "sequence2")
{
#Split the Elements of strin
if (length(sequence1)==1){
x <- strsplit(sequence1,split = "")[[1]]}
if (length(sequence2)==1){
y <- strsplit(sequence2,split = "")[[1]]}
if (length(sequence1)!=1){
x <-sequence1}
if (length(sequence2)!=1){
y <- sequence2}
if (nchar(x[1]) > 1)
stop("seq1 should be provided as a vector of single chars")
if (nchar(y[1]) > 1)
stop("seq2 should be provided as a vector of single chars")
if (wsize < 1)
stop("non allowed value for wsize")
if (wstep < 1)
stop("non allowed value for wstep")
if (nmatch < 1)
stop("non allowed value for nmatch")
if (nmatch > wsize)
stop("nmatch > wsize is not allowed")
mkwin <- function(seq, wsize, wstep) {
sapply(seq(from = 1, to = length(seq) - wsize + 1, by = wstep),
function(i) c2s(seq[i:(i + wsize - 1)]))
}
wseq1 <- mkwin(x, wsize, wstep)
wseq2 <- mkwin(y, wsize, wstep)
if (nmatch == wsize) {
xy <- outer(wseq1, wseq2, "==")
}
else {
"%==%" <- function(x, y) colSums(sapply(x, s2c) == sapply(y,
s2c)) >= nmatch
xy <- outer(wseq1, wseq2, "%==%")
}
x=toupper(x)
y=toupper(y)
a=matrix(0,length(x),length(y))
# Naming the rows and columns of the formed matrix
row.names(a)=x
colnames(a)=y
for (i in 1:nrow(xy)) {
for (j in 1:ncol(xy)){
if (xy[i,j]==T) {
a[i,j]=1
}
}
}
h_line=seq(0+((seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])/2),max(length(x),length(y)),
by=seq(0,max(length(x),length(y))-1,
l=length(x))[2]-seq(0,max(length(x),length(y))-1,
l=length(x))[1])
v_line=seq(1+((seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])/2),max(length(x),length(y)),
by=seq(1,max(length(x),length(y)),
l=length(y))[2]-seq(1,max(length(x),length(y)),
l=length(y))[1])
aaaa=0.04*max(length(x),length(y))
plot(0:max(length(x),length(y)),0:max(length(x),length(y)),type = "n",
axes =F,frame.plot=TRUE,main ="" ,
ann = T,xlab = xlab , ylab = ylab)
aa=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb=c(h_line[length(h_line)],
max(length(x),length(y))+aaaa,
max(length(x),length(y))+aaaa,
h_line[length(h_line)])
polygon(aa,bb,col = table_col)
aa1=c(-aaaa,-aaaa,max(length(x),length(y))+aaaa,max(length(x),length(y))+aaaa)
bb1=c(max(length(x),length(y))-aaaa,
max(length(x),length(y))+0.5,
max(length(x),length(y))+0.5,
max(length(x),length(y))-aaaa)
polygon(bb1-max(length(x),length(y)),aa1,col = table_col)
# y axes
axis(side=2, at = seq(0,max(length(x),length(y))-1,l=length(x)),
labels= length(x):1,cex.axis = 0.7, font = 2)
abline(h=h_line,col="black",lwd=1, lty=1)
text((aa1[2]+h_line[1])/2,
seq(0,max(length(x),length(y))-1,l=length(x)),
rev(x),col = "black",cex = 1,pch = 16,font = 2)
#x axes
axis(side=3, at = seq(1,max(length(x),length(y)),
l=length(y)), labels= 1:length(y),cex.axis = 0.7, font = 2)
abline(v=v_line, col="black",lwd=1, lty=1)
abline(v=0.5,col="black",lwd=1, lty=1)
text(seq(1,max(length(x),length(y)),l=length(y))
,(bb[2]+bb[1])/2,
y,col = "black",cex = 1,pch = 16,font = 2)
for (i in 1:length(x)){
for (j in 1:length(y))
if (a[i,j]==1)
points(seq(1,max(length(x),length(y)),l=length(y))[j],seq(0,max(length(x),length(y))-1,l=length(x))[length(x)-i+1],cex=1.5,col=points_col,pch = 16)
}
}
#' Compute and return revers compliment of a sequence
#' @param seq Sequence of DNA or RNA.
#' @return Reverse compliment of sequence .
#' @section References:
#' ...
#' @section Further details:
#' ...
#' @export Rcompliment
#'
#'
Rcompliment=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
Rcompliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in length(seq):1) {
Rcompliment_sequence = c(Rcompliment_sequence, compliment(seq[i],
seq_type = type))
}
Rcompliment_sequence
}
#' Compute and return compliment of a sequence.
#' @param seq Sequence of DNA or RNA
#' @return Compliment of sequence .
#' @section References:
#' ...
#' @section Further details:
#' ...
#' @export compliment
#'
#'
compliment=function (seq)
{
compliment = function(n, seq_type) {
if (seq_type == "D") {
if (n == "a") {
return("t")
}
if (n == "A") {
return("T")
}
}
if (seq_type == "R") {
if (n == "a") {
return("u")
}
if (n == "A") {
return("U")
}
}
if (n == "t") {
return("a")
}
if (n == "c") {
return("g")
}
if (n == "g") {
return("c")
}
if (n == "C") {
return("G")
}
if (n == "G") {
return("C")
}
if (n == "T") {
return("A")
}
if (n == "u") {
return("a")
}
if (n == "U") {
return("A")
}
if (n != "U" & n != "A" & n != "T" &
n != "G" & n != "C" & n != "u" &
n != "a" & n != "t" & n != "g" &
n != "c") {
return(n)
}
}
compliment_sequence = c()
if (length(seq) > 1) {
seq = seq
}
if (length(seq) == 1) {
seq = strsplit(seq, split = "")[[1]]
}
type = "D"
if (any(seq == "u") | any(seq == "U")) {
type = "R"
}
for (i in 1:length(seq)) {
compliment_sequence = c(compliment_sequence, compliment(seq[i],
seq_type = type))
}
compliment_sequence
}
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