exomePeak2: Peak Calling and Differential Analysis of MeRIP-seq.

In ZhenWei10/exomePeak2: Peak Calling and differential analysis for MeRIP-Seq

Peak Calling and Differential Analysis of MeRIP-seq.

Description

exomePeak2 conducts peak calling and differential methylation analysis using BAM files of aligned MeRIP-seq reads.

Usage

exomePeak2(
  bam_ip = NULL,
  bam_input = NULL,
  bam_ip_treated = NULL,
  bam_input_treated = NULL,
  txdb = NULL,
  genome = NULL,
  gff = NULL,
  strandness = c("unstrand", "1st_strand", "2nd_strand"),
  fragment_length = 100,
  bin_size = 25,
  step_size = 25,
  test_method = c("Poisson", "DESeq2"),
  p_cutoff = 1e-10,
  diff_p_cutoff = 0.01,
  parallel = 1,
  plot_gc = TRUE,
  save_output = TRUE,
  save_dir = getwd(),
  experiment_name = "exomePeak2_output",
  mode = c("exon", "full_transcript", "whole_genome"),
  motif_based = FALSE,
  motif_sequence = "DRACH",
  absolute_diff = FALSE,
  confounding_factor = NULL
)

Arguments

bam_ip	a character vector for the BAM file directories of IP samples.
bam_input	a character vector for the BAM file directories of input samples.
bam_ip_treated	a character vector for the BAM file directories of treated IP samples.
bam_input_treated	a character vector for the BAM file directories of treated input samples. The arguments of BAM_ip and BAM_input are only required for differential methylation analysis.
txdb	a TxDb object for the transcript annotation.
genome	a character or a BSgenome for the reference genome. The character should be the UCSC genome name which is acceptable by getBSgenome or/and makeTxDbFromUCSC; example: "hg19".
gff	optional, a character which specifies the directory toward a gene annotation GFF/GTF file, it is applied when the TxDb object is not available; default = NULL.
strandness	a character specifying the strand protocol type of the RNA-seq library, can be one of c("unstrand", "1st_strand", "2nd_strand"); default = "unstrand". unstrand The randomly primed RNA-seq library type, i.e. both the strands generated during the first and the second strand sythesis are sequenced; example: Standard Illumina. 1st_strand The first strand-specific RNA-seq library, only the strand generated during the first strand sythesis is sequenced; examples: dUTP, NSR, NNSR. 2nd_strand The second strand-specific RNA-seq library, only the strand generated during the second strand sythesis is sequenced; examples: Ligation, Standard SOLiD.
fragment_length	a positive integer number for the expected fragment length (in bp); default = 100.
bin_size	a positive integer number for the width of the sliding window; default = 25.
step_size	a positive integer number for the step size of the sliding window; default = 25.
test_method	a character for the statistical testing method used in peak calling and differential analysis, can be one of c("Poisson", "DESeq2"); default = "Poisson" Poisson Wald test of Poisson GLM. DESeq2 Wald test of negative bionomial GLM with regularized estimation of over-dispersion parameters (implemented by DESeq2), Note that when using the test method of DESeq2, a larger p-value cut-off (e.g. 0.001) is often required. The cutoff can be set via the argument p_cutoff.
p_cutoff	a numeric value for the p value cutoff in peak calling; default = 1e-10.
diff_p_cutoff	a numeric value for the p value cutoff in differential analysis; default = 0.01.
parallel	a numeric value specifying the number of cores for parallel computing; default = 1.
plot_gc	a logical for saving the plots of bins' GC content v.s. bins' fitted coverage curves, which can be used as a diagnosis for GC content bias; default = TRUE.
save_output	a logical for saving the outcomes on disk; default = TRUE.
save_dir	a character for the output directory; default = getwd.
experiment_name	a character for the folder name generated in the output directory that contains all the results; default: ="exomePeak2_output"
mode	a character specifies the scope of peak calling on genome, can be one of c("exon", "full_transcript", "whole_genome"); default = "exon". exon generate sliding windows over exonic regions. full_transcript generate sliding windows over the full transcripts (include both introns and exons). whole_genome generate sliding windows over the whole genome (include introns, exons, and the intergenic regions). P.S. The full transcript mode and the whole genome mode demand big memory size (> 4GB) for large genomes.
motif_based	a logical for detecting (differential) modification over sites of motif; default = FALSE. If = TRUE, sliding windows will be replaced into the single based sites of the modification motif.
motif_sequence	a character for the motif sequence used for the reference sites, it is only applied when motif_based = TRUE; default = "DRACH".
absolute_diff	a logical for performing absolute differential modification without normalization over input control samples. If = TRUE, the regression design for differential modification test will be changed into comparing the direct changes of IP samples between treatment and control conditions; default = FALSE.
confounding_factor	A factor vector or a data.frame with factors as columns. The length of the factor vector or the number of rows (nrow) in the data.frame should match the total number of samples in IP and input. If supplied, Generalized Linear Models (GLMs) utilized for peak calling and differential methylation analysis will incorporate the specified factor(s) as covariates. This inclusion adjusts the computation of p-values and log fold change estimates by accounting for the confounding factors (e.g. experimental batches and library types); default = NULL.

Details

exomePeak2 call (differential) RNA modification peaks and calculate peak statistics from BAM files of a MeRIP-seq experiment.

The transcript annotation (from either the TxDb object or the GFF file) should be provided to perform analysis on exons.

The genome name or BSgenome object is required to perform the GC content bias correction. If the genome argument is not provided (= NULL), the analysis will proceed without GC correction.

If the BAM files in treated samples are provided at the arguments bam_ip_treated and bam_input_treated, the statistics of differential modification detection on peaks/sites will be reported.

Under the default setting, exomePeak2 will save the results of (differential) modification analysis under a folder named 'exomePeak2_output'. The results generated include a BED file, a RDS file, and a CSV table that stores the locations and statistics of the (differential) modified peaks/sites.

Value

a GRangesList object, the statistics and other annotations are saved in its metadata columns, which can be accessed through mcol(). If save_output = TRUE, exomePeak2 will output results both as BED, CSV, and RDS files on disk.

Examples

## Specify File Directories
GENE_ANNO_GTF = system.file("extdata", "example.gtf", package="exomePeak2")

f1 = system.file("extdata", "IP1.bam", package="exomePeak2")
f2 = system.file("extdata", "IP2.bam", package="exomePeak2")
f3 = system.file("extdata", "IP3.bam", package="exomePeak2")
f4 = system.file("extdata", "IP4.bam", package="exomePeak2")
IP_BAM = c(f1,f2,f3,f4)
f1 = system.file("extdata", "Input1.bam", package="exomePeak2")
f2 = system.file("extdata", "Input2.bam", package="exomePeak2")
f3 = system.file("extdata", "Input3.bam", package="exomePeak2")
INPUT_BAM = c(f1,f2,f3)

## Peak Calling
res <- exomePeak2(bam_ip = IP_BAM,
                  bam_input = INPUT_BAM,
                  gff = GENE_ANNO_GTF,
                  genome = "hg19")
res        ## Peak ranges
mcols(res) ## Peak statistics

## Differential Peak Detection (Comparison of Two Conditions)

f1 = system.file("extdata", "treated_IP1.bam", package="exomePeak2")
TREATED_IP_BAM = c(f1)
f1 = system.file("extdata", "treated_Input1.bam", package="exomePeak2")
TREATED_INPUT_BAM = c(f1)

res <- exomePeak2(bam_ip = IP_BAM,
                  bam_input = INPUT_BAM,
                  bam_ip_treated = TREATED_IP_BAM,
                  bam_input_treated = TREATED_INPUT_BAM,
                  gff = GENE_ANNO_GTF,
                  genome = "hg19")
res        ## Peak ranges
mcols(res) ## Peak statistics

ZhenWei10/exomePeak2 documentation built on March 26, 2024, 6:27 p.m.