Merge BAM files of replicates prior to peak calling
Merging BAM files of technical and/or biological replicates can improve
the sensitivity of the peak calling by increasing the depth of read
mergeBamByFactor function merges BAM files based on grouping information
specified by a
factor, here the
Factor column of the imported targets file. It
also returns an updated
SYSargs object containing the paths to the
merged BAM files as well as to any unmerged files without replicates.
This step can be skipped if merging of BAM files is not desired.
args <- systemArgs(sysma=NULL, mytargets="targets_bam.txt") args_merge <- mergeBamByFactor(args, overwrite=TRUE) writeTargetsout(x=args_merge, file="targets_mergeBamByFactor.txt", overwrite=TRUE)
Peak calling without input/reference sample
MACS2 can perform peak calling on ChIP-Seq data with and without input
samples (Zhang et al., 2008). The following performs peak calling without
input on all samples specified in the corresponding
args object. Note, due to
the small size of the sample data, MACS2 needs to be run here with the
nomodel setting. For real data sets, users want to remove this parameter
in the corresponding
args <- systemArgs(sysma="param/macs2_noinput.param", mytargets="targets_mergeBamByFactor.txt") sysargs(args) # Command-line parameters for first FASTQ file runCommandline(args) file.exists(outpaths(args)) writeTargetsout(x=args, file="targets_macs.txt", overwrite=TRUE)
Peak calling with input/reference sample
To perform peak calling with input samples, they can be most
conveniently specified in the
SampleReference column of the initial
targets file. The
writeTargetsRef function uses this information to create a
file intermediate for running MACS2 with the corresponding input samples.
writeTargetsRef(infile="targets_mergeBamByFactor.txt", outfile="targets_bam_ref.txt", silent=FALSE, overwrite=TRUE) args_input <- systemArgs(sysma="param/macs2.param", mytargets="targets_bam_ref.txt") sysargs(args_input) # Command-line parameters for first FASTQ file runCommandline(args_input) file.exists(outpaths(args_input)) writeTargetsout(x=args_input, file="targets_macs_input.txt", overwrite=TRUE)
The peak calling results from MACS2 are written for each sample to
separate files in the
results directory. They are named after the corresponding
files with extensions used by MACS2.
Identify consensus peaks
The following example shows how one can identify consensus preaks among two peak sets sharing either a minimum absolute overlap and/or
minimum relative overlap using the
olRanges functions, respectively. Note, the latter is
a custom function imported below by sourcing it.
source("http://faculty.ucr.edu/~tgirke/Documents/R_BioCond/My_R_Scripts/rangeoverlapper.R") peak_M1A <- outpaths(args)["M1A"] peak_M1A <- as(read.delim(peak_M1A, comment="#")[,1:3], "GRanges") peak_A1A <- outpaths(args)["A1A"] peak_A1A <- as(read.delim(peak_A1A, comment="#")[,1:3], "GRanges") (myol1 <- subsetByOverlaps(peak_M1A, peak_A1A, minoverlap=1)) # Returns any overlap myol2 <- olRanges(query=peak_M1A, subject=peak_A1A, output="gr") # Returns any overlap with OL length information myol2[values(myol2)["OLpercQ"][,1]>=50] # Returns only query peaks with a minimum overlap of 50%