MultiRepMacsChIPSeq - multirepchipseq
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multirepchipseq.pl
This is a multi-threaded pipeline for processing multiple-condition, multiple-replicate samples from ChIP-Seq, ATAC-Seq, Cut&Run, Cut&Tag, or any other chromatin-based assays for calling peaks, particularly for differential and/or comparative purposes.
At least two samples (conditions) are required. Single-sample, single-replicate analysis is not supported. If multiple replicates are available for only a single condition, then the replicates may be treated as separate samples for purposes of comparison.
Replicates are handled by two methods:
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Averaging the depth-normalized genomic coverage of the replicates into a mean coverage track and calling peaks on that. This smooths the signal and may rescue under-performing replicates. This is always enabled.
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Optionally call peaks on each replicate individually, then merge the replicate peak calls, requiring a minimum of two overlaps to accept as a sample peak. This is enabled with the –indedepent option.
All sample peak calls are merged into a final call set, which are re-scored and quantitated for comparison purposes. Multiple quality-control, comparitive, and analytical plots are generated. An HTML report including a subset of these plots is generated.
Multiple options are available for filtering and handling alignments, scoring, and thresholding of peak calls. Details on these options and when to choose them, along with numerous examples, are provided in online documentation. See the URL below for full usage and guide.
https://huntsmancancerinstitute.github.io/MultiRepMacsChIPSeq
Version: 21
Options:
Input files - repeat these options for each sample (condition)
--chip file1,file2... Comma-delimited paths to experiment files
--name text Name for the sample (condition)
--control file1,file2... Optional reference control (Input), comma-delimited
Output
--dir directory Directory for writing all files (./MultiRepPeakCall)
--out file basename Base filename for merged output files (all_peaks)
Genome size
--genome integer Specify effective mappable genome size
(default empirically determined)
Bam options
--pe Bam files are paired-end, default treat as single-end
Alignment filtering options
--mapq integer Minimum mapping quality, (0)
--chrskip "text" Chromosome skip regex (chrM|MT|alt|chrUn|random|EBV|Adapter|Lambda|PhiX)
--blacklist file Coordinate (bed) file of repeats or hotspots to avoid
Default determined empirically from control samples.
Specify 'none' for no filtering.
--min integer Minimum paired-end size allowed (50 bp)
--max integer Maximum paired-end size allowed (500 bp)
Duplication filtering
--nodedup Skip deduplication and use all primary, nondup alignments
--dupfrac float Target duplication rate for subsampling (0.05)
--maxdepth integer Maximum position alignment depth ()
set to 1 to remove all duplicates
--optdist integer Maximum distance for optical duplicates (0)
use 100 for HiSeq, 2500 for NovaSeq
--deduppair Run deduplication as paired-end, but coverage as single-end
Fragment coverage
--cutsite Set multiple options specific for ATACSeq cutsite analysis
--size integer Predicted fragment size. REQUIRED for single-end
--shift integer Shift the fragment in special situations
--fraction Record multiple-hit alignments as fraction of hits
--slocal integer Small local lambda size (1000 bp)
--llocal integer Large local lambda size (10000 bp)
--cbin integer ChIP fragment bin size (10 bp)
--slbin integer Small local lambda bin size (50 bp)
--llbin integer Large local lambda bin size (100 bp)
Chromosome-specific normalization
--chrnorm float Specific chromosome normalization factor
--chrapply "text" Apply factor to specified chromosomes via regex
Peak calling
--independent Call peaks independently for each replicate and merge
--cutoff number Threshold q-value for calling peaks (2)
Higher numbers are more significant, -1*log10(q)
--peaksize integer Minimum peak size to call (2 x fragment size)
Required for paired-end alignments.
--peakgap integer Maximum gap between peaks before merging (1 x size)
--broad Also perform broad (gapped) peak calling
--broadcut number Q-value cutoff for linking broad regions (0.5)
--broadgap integer Maximum link size between peaks in broad calls (4 x size bp)
--nolambda Skip lambda control, compare ChIP directly with control
--minpeakover integer Minimum number of overlapping replicate peaks to accept
in final when merging (default n-1, minimum 2)
--samedepth Use same target depth when calculating per sample
q-value enrichment for replicate-mean peaks
Peak scoring
--binsize integer Size of bins in 25 flanking peak bins for profile (100)
--targetdepth text Set target depth in Millions or method for setting:
median (default), mean, min
--rawcounts Use unscaled raw counts for re-scoring peaks
--noplot Do not plot figures of results
Job control
--cpu integer Number of CPUs to use per job (4)
--job integer Number of simultaneous jobs (2)
--dryrun Just print the commands without execution
--noorganize Do not organize files into subfolders when finished
--savebam Save de-duplicated bam files
--savebdg Save text bedGraph files
Application Paths
--bam2wig path (bam2wig.pl)
--bamdedup path (bam_partial_dedup.pl)
--bedtools path (bedtools)
--bw2bdg path (bigWigToBedGraph)
--data2wig path (data2wig.pl)
--getdata path (get_datasets.pl)
--getrel path (get_relative_data.pl)
--geteff path (get_chip_efficiency.pl)
--intersect path (intersect_peaks.pl)
--macs path (macs2)
--manwig path (manipulate_wig.pl)
--meanbdg path (generate_mean_bedGraph.pl)
--peak2bed path (peak2bed.pl)
--updatepeak path (update_peak_file.pl)
--pandoc path (pandoc)
--plotpeak path (plot_peak_figures.R)
--printchr path (print_chromosome_lengths.pl)
--reportmap path (report_mappable_space.pl)
--rscript path (Rscript)
--wig2bw path (wigToBigWig)