README.md

In GrahamHamilton/pipelineTools: Pipeline Tools for NGS Sequence Analysis Pipelines

pipelineTools

The goal of pipelineTools is to streamline the NGS analysis pipelines and result reporting within RStudio. PipelineTools provides packages to run standard open source NGS tools

Installation

Can be installed using devtools directly from GitHub using the following commands.

# Install devtools
install.packages("devtools")
library("devtools")
#Install package from GitHub
install_github("GrahamHamilton/pipelineTools")

Example

Load the required libraries ```{r load libraries} library("pipelineTools") library("biomaRt") library("tximport")

Set the paths to the software installed on the system
```{r software paths, echo = FALSE}
fastq.screen.path <- "/software/fastq_screen_v0.13.0/fastq_screen"
fastp.path <- "/software/bin/fastp"
kallisto.path <- "/software/kallisto_v0.45.1/kallisto"
hisat.path <- "/software/hisat_v2-2.1.0/hisat2"
multiqc.path <- "/usr/local/bin/multiqc"
samtools.path <- "/software/samtools_v1.9/samtools"
stringtie.path <- "/software/stringtie-1.3.6/stringtie"

Version numbers for the software used ```{r versions, warning=FALSE, echo = FALSE} fastqscreen.version <- run_fastq_screen(fastq_screen = fastq.screen.path, version = TRUE) fastp.version <- run_fastp(fastp = fastp.path, version = TRUE) kallisto.version <- run_kallisto(kallisto = kallisto.path, version = TRUE) hisat.version <- run_hisat2(hisat2 = hisat.path, version = TRUE) multiqc.version <- run_multiqc(multiqc = multiqc.path, version = TRUE) samtools.version <- run_samtools(samtools = samtools.path, version = TRUE) stringtie.version <- run_stringtie(stringtie = stringtie.path, version = TRUE) r.version <- getRversion() pipelineTools.version <- packageDescription("pipelineTools")$Version deseq2.version <- packageDescription("DESeq2")$Version

Create the results directories
```{r results directories}
# Trimmed reads directory
trimmed.reads.dir <- "trimmed_reads"
#Create the directory for the trimmed reads
dir.create(trimmed.reads.dir, showWarnings = FALSE)

# FastQScreen results directory
fastq.screen.dir <- "Screen"
# Create the directory for the fastq screen results
dir.create(fastq.screen.dir, showWarnings = FALSE)

# FastP results directory
fastp.results.dir <- "FastpQC"
# Create the directory for the FastP results
dir.create(fastp.results.dir, showWarnings = FALSE)

# Kallisto results directory
kalisto.results.dir <- "kallisto"
#Create the directory for the Kallisto results
dir.create(kalisto.results.dir, showWarnings = FALSE)

# Hisat2 alignment results directory
hisat2.alignments.dir <- "hisat2_alignments"
#Create the directory for the HiSat2 results
dir.create(hisat2.alignments.dir, showWarnings = FALSE)

# Stringtie results directory
stringtie.dir <- "stringtie"
#Create the directory for the HiSat2 results
dir.create(stringtie.dir, showWarnings = FALSE)

Read in the fastq file paths to lists and then create sample names lists and trimmed read file paths ```{r setup files} reads.path <- "raw_reads"

reads.patt.1 <- "_S\d{1,2}\_L001_R1_001.fastq.gz$" reads.patt.2 <- "_S\d{1,2}\_L001_R2_001.fastq.gz$"

sample.dataframe <- prepare_samples(reads.path, c(reads.patt.1,reads.patt.2),trimmed.reads.dir)

mate1 <- as.character(sample.dataframe$reads.path.1) mate1.trim <- as.character(sample.dataframe$trimmed.reads.path.1)

For paired end sequence

mate2 <- as.character(sample.dataframe$reads.path.2) mate2.trim <- as.character(sample.dataframe$trimmed.reads.path.2)

sample.names <- as.character(sample.dataframe$sample.names)

Sequence adapters
```{r sequence adapters}
adapter1 <- "AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC"
adapter2 <- "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT"

The full paths to the reference genome/transcriptome indexes and annotation files ```{r references}

Path to the reference transcriptome

transcriptome <- "/path/to/kallisto/index"

Path to the reference genome

genome <- "/path/to/hisat2/index"

Path to the gtf file

gtf <- "/path/to/gtf/file"

### Fastq Screen
Results are stored in the Screen folder
```{r fastqscreen, echo = FALSE, eval = eval}
fastq.screen.conf <- "/export/jessie3/gmh5n/PipelineTest/fastq_screen.conf"
fastq.screen.cmds <- run_fastq_screen(fq.files = mate1,
                                      out.dir = fastq.screen.dir,
                                      conf = fastq.screen.conf,
                                      fastq_screen = fastq.screen.path)
write.table(fastq.screen.cmds,"FastqScreen_commands.sh", quote = FALSE, row.names = FALSE, col.names = FALSE)

FastP

Adapter and quality trimmed reads are stored in the r trimmed.reads.dir directory, QC files are stored in the FastpQC folder ```{r fastp, echo = FALSE, eval = eval} fastp.cmds <- run_fastp(mate1 = mate1, mate2 = mate2, mate1.out = mate1.out, mate2.out = mate2.out, adapter1 = adapter1, adapter2 = adapter2, sample.name = sample.names, out.dir = fastp.results.dir, fastp = fastp.path)

write.table(fastp.cmds,"FastP_commands.sh", quote = FALSE, row.names = FALSE, col.names = FALSE)

### Kallisto
Pseudo align the reads to the reference transcriptome with Kallisto
```{r kallisto, echo = FALSE, eval = eval}
strandedness <- "second"
# Paired end
kallisto.cmds <- run_kallisto(mate1 = mate1,
                              mate2 = mate2,
                              index = transcriptome,
                              sample.name = sample.names,
                              strandedness = strandedness,
                              out.dir = kalisto.results.dir,
                              kallisto = kallisto.path)

write.table(kallisto.cmds,"Kallisto_commands.sh", quote = FALSE, row.names = FALSE, col.names = FALSE)

TXImport for Kallisto data

```{r include=FALSE} mart<-"ensembl" db<-"mmusculus_gene_ensembl" # Change to organism in study filt<-"ensembl_gene_id"

Create the biomaRt object

ensembl = useEnsembl(biomart=mart, dataset=db)

Get all the transcript ids and corresponding gene ids from BiomaRt

att<-c("ensembl_transcript_id","ensembl_gene_id") txTable<-getBM(attributes=att,mart=ensembl)

Set the column names for the transcript to gene table

colnames(txTable)<-c("tx_id","gene_id")

Read in the experimental design file, tab seperated

sampleTable <- read.csv("SampleDescription.txt", sep="\t", row.names=1)

Read in the file names form the kallisto results directory

dir <- getwd() files <- file.path(dir,kalisto.results.dir,row.names(sampleTable),"abundance.h5", fsep = .Platform$file.sep) names(files)<-row.names(sampleTable)

txi <- tximport(files, type = "kallisto", tx2gene = txTable,ignoreTxVersion = TRUE, ignoreAfterBar = TRUE) ```

GrahamHamilton/pipelineTools documentation built on March 5, 2024, 12:23 p.m.