Man page - macs3_callpeak(1)

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MAC3_CALLPEAK

NAME
DESCRIPTION
options:
Input files arguments:
Output arguments:
Shifting model arguments:
Peak calling arguments:
Post-processing options:
Obsolete options:
Other options:
EXAMPLES
1. Peak calling for regular TF ChIP-seq:
2. Broad peak calling on Histone Mark ChIP-seq:
3. Peak calling on ATAC-seq (paired-end mode):
4. Peak calling on ATAC-seq ( focusing on insertion sites, and usingsingle-end mode):

NAME

mac3_callpeak - Model-based Analysis for ChIP-Sequencing

DESCRIPTION

usage: macs3 callpeak [-h] -t TFILE [TFILE ...] [-c [CFILE ...]]
[-f {AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE,BEDPE}]

[-g GSIZE] [-s TSIZE] [--keep-dup KEEPDUPLICATES] [--outdir OUTDIR] [-n NAME] [-B] [--verbose VERBOSE] [--trackline] [--SPMR] [--nomodel] [--shift SHIFT] [--extsize EXTSIZE] [--bw BW] [--d-min D_MIN] [-m MFOLD MFOLD] [--fix-bimodal] [-q QVALUE | -p PVALUE] [--scale-to {large,small}] [--down-sample] [--seed SEED] [--tempdir TEMPDIR] [--nolambda] [--slocal SMALLLOCAL] [--llocal LARGELOCAL] [--max-gap MAXGAP] [--min-length MINLEN] [--broad] [--broad-cutoff BROADCUTOFF] [--cutoff-analysis] [--call-summits] [--fe-cutoff FECUTOFF] [--to-large] [--ratio RATIO] [--buffer-size BUFFER_SIZE]

options:

-h , --help

show this help message and exit

Input files arguments:

-t , --treatment TFILE [TFILE ...]

ChIP-seq treatment file. If multiple files are given as ’-t A B C’, then they will all be read and pooled together. REQUIRED.

-c , --control [CFILE ...]

Control file. If multiple files are given as ’-c A B C’, they will be pooled to estimate ChIP-seq background noise.

-f , --format
{AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE,BEDPE}

Format of tag file, "AUTO", "BED" or "ELAND" or "ELANDMULTI" or "ELANDEXPORT" or "SAM" or "BAM" or "BOWTIE" or "BAMPE" or "BEDPE". The default AUTO option will let MACS decide which format (except for BAMPE and BEDPE which should be implicitly set) the file is. Please check the definition in README. Please note that if the format is set as BAMPE or BEDPE, MACS3 will call its special Paired-end mode to call peaks by piling up the actual ChIPed fragments defined by both aligned ends, instead of predicting the fragment size first and extending reads. Also please note that the BEDPE only contains three columns, and is NOT the same BEDPE format used by BEDTOOLS. DEFAULT: "AUTO"

-g , --gsize GSIZE

Effective genome size. It can be 1.0e+9 or 1000000000, or shortcuts:’hs’ for human (2,913,022,398), ’mm’ for mouse (2,652,783,500), ’ce’ for C. elegans (100,286,401) and ’dm’ for fruitfly (142,573,017), Default:hs. The effective genome size numbers for the above four species are collected from Deeptools https: //deeptools.readthedocs.io/en/develop/content/feature/ effectiveGenomeSize.html Please refer to deeptools to define the best genome size you plan to use.

-s , --tsize TSIZE

Tag size/read length. This will override the auto detected tag size. DEFAULT: Not set

--keep-dup KEEPDUPLICATES

It controls the behavior towards duplicate tags at the exact same location -- the same coordination and the same strand. The ’auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff; and the ’all’ option keeps every tags. If an integer is given, at most this number of tags will be kept at the same location. Note, if you’ve used samtools or picard to flag reads as ’PCR/Optical duplicate’ in bit 1024, MACS3 will still read them although the reads may be decided by MACS3 as duplicate later. If you plan to rely on samtools/picard/any other tool to filter duplicates, please remove those duplicate reads and save a new alignment file then ask MACS3 to keep all by ’--keep-dup all’. The default is to keep one tag at the same location. Default: 1

Output arguments:

--outdir OUTDIR

If specified all output files will be written to that directory. Default: the current working directory

-n , --name NAME

Experiment name, which will be used to generate output file names. DEFAULT: "NA"

-B , --bdg

Whether or not to save extended fragment pileup, and local lambda tracks (two files) at every bp into a bedGraph file. DEFAULT: False

--verbose VERBOSE

Set verbose level of runtime message. 0: only show critical message, 1: show additional warning message, 2: show process information, 3: show debug messages. DEFAULT:2

--trackline

Instruct MACS to include trackline in the header of output files, including the bedGraph, narrowPeak, gappedPeak, BED format files. To include this trackline is necessary while uploading them to the UCSC genome browser. You can also mannually add these trackline to corresponding output files. For example, in order to upload narrowPeak file to UCSC browser, add this to as the first line -- ‘track type=narrowPeak name="my_peaks" description="my peaks"‘. Default: Not to include trackline.

--SPMR

If True, MACS will SAVE signal per million reads for fragment pileup profiles. It won’t interfere with computing pvalue/qvalue during peak calling, since internally MACS3 keeps using the raw pileup and scaling factors between larger and smaller dataset to calculate statistics measurements. If you plan to use the signal output in bedGraph to call peaks using bdgcmp and bdgpeakcall, you shouldn’t use this option because you will end up with different results. However, this option is recommended for displaying normalized pileup tracks across many datasets. Require -B to be set. Default: False

Shifting model arguments:

--nomodel

Whether or not to build the shifting model. If True, MACS will not build model. by default it means shifting size = 100, try to set extsize to change it. It’s highly recommended that while you have many datasets to process and you plan to compare different conditions, aka differential calling, use both ’nomodel’ and ’extsize’ to make signal files from different datasets comparable. DEFAULT: False

--shift SHIFT

(NOT the legacy --shiftsize option!) The arbitrary shift in bp. Use discretion while setting it other than default value. When NOMODEL is set, MACS will use this value to move cutting ends (5’) towards 5’->3’ direction then apply EXTSIZE to extend them to fragments. When this value is negative, ends will be moved toward 3’->5’ direction. Recommended to keep it as default 0 for ChIP-Seq datasets, or -1 * half of EXTSIZE together with EXTSIZE option for detecting enriched cutting loci such as certain DNAseI-Seq datasets. Note, you can’t set values other than 0 if format is BAMPE or BEDPE for paired-end data. DEFAULT: 0.

--extsize EXTSIZE

The arbitrary extension size in bp. When nomodel is true, MACS will use this value as fragment size to extend each read towards 3’ end, then pile them up. It’s exactly twice the number of obsolete SHIFTSIZE. In previous language, each read is moved 5’->3’ direction to middle of fragment by 1/2 d, then extended to both direction with 1/2 d. This is equivalent to say each read is extended towards 5’->3’ into a d size fragment. DEFAULT: 200. EXTSIZE and SHIFT can be combined when necessary. Check SHIFT option.

--bw BW

Band width for picking regions to compute fragment size. This value is only used while building the shifting model. Tweaking this is not recommended. DEFAULT: 300

--d-min D_MIN

Minimum fragment size in basepair. Any predicted fragment size less than this will be excluded. DEFAULT: 20

-m , --mfold MFOLD MFOLD

Select the regions within MFOLD range of highconfidence enrichment ratio against background to build model. Fold-enrichment in regions must be lower than upper limit, and higher than the lower limit. Use as "-m 10 30". This setting is only used while building the shifting model. Tweaking it is not recommended. DEFAULT:5 50

--fix-bimodal

Whether turn on the auto pair model process. If set, when MACS failed to build paired model, it will use the nomodel settings, the --exsize parameter to extend each tags towards 3’ direction. Not to use this automate fixation is a default behavior now. DEFAULT: False

Peak calling arguments:

-q , --qvalue QVALUE

Minimum FDR (q-value) cutoff for peak detection. DEFAULT: 0.05. -q , and -p are mutually exclusive.

-p , --pvalue PVALUE

Pvalue cutoff for peak detection. DEFAULT: not set. -q , and -p are mutually exclusive. If pvalue cutoff is set, qvalue will not be calculated and reported as -1 in the final .xls file.

--scale-to {large,small}

When set to ’small’, scale the larger sample up to the smaller sample. When set to ’larger’, scale the smaller sample up to the bigger sample. By default, scale to ’small’. This option replaces the obsolete ’ --to-large ’ option. The default behavior is recommended since it will lead to less significant p/q-values in general but more specific results. Keep in mind that scaling down will influence control/input sample more. DEFAULT: ’small’, the choice is either ’small’ or ’large’.

--down-sample

When set, random sampling method will scale down the bigger sample. By default, MACS uses linear scaling. Warning: This option will make your result unstable and irreproducible since each time, random reads would be selected. Consider to use ’randsample’ script instead. <not implmented>If used together with --SPMR , 1 million unique reads will be randomly picked.</not implemented> Caution: due to the implementation, the final number of selected reads may not be as you expected! DEFAULT: False

--seed SEED

Set the random seed while down sampling data. Must be a non-negative integer in order to be effective. DEFAULT: not set

--tempdir TEMPDIR

Optional directory to store temp files. DEFAULT: /tmp

--nolambda

If True, MACS will use fixed background lambda as local lambda for every peak region. Normally, MACS calculates a dynamic local lambda to reflect the local bias due to the potential chromatin accessibility.

--slocal SMALLLOCAL

The small nearby region in basepairs to calculate dynamic lambda. This is used to capture the bias near the peak summit region. Invalid if there is no control data. If you set this to 0, MACS will skip slocal lambda calculation. *Note* that MACS will always perform a d-size local lambda calculation while the control data is available. The final local bias would be the maximum of the lambda value from d, slocal, and llocal size windows. While control is not available, d and slocal lambda won’t be considered. DEFAULT: 1000

--llocal LARGELOCAL

The large nearby region in basepairs to calculate dynamic lambda. This is used to capture the surround bias. If you set this to 0, MACS will skip llocal lambda calculation. *Note* that MACS will always perform a d-size local lambda calculation while the control data is available. The final local bias would be the maximum of the lambda value from d, slocal, and llocal size windows. While control is not available, d and slocal lambda won’t be considered. DEFAULT: 10000.

--max-gap MAXGAP

Maximum gap between significant sites to cluster them together. The DEFAULT value is the detected read length/tag size.

--min-length MINLEN

Minimum length of a peak. The DEFAULT value is the predicted fragment size d. Note, if you set a value smaller than the fragment size, it may have NO effect on the result. For BROAD peak calling, try to set a large value such as 500bps. You can also use ’-- cutoff-analysis’ option with default setting, and check the column ’avelpeak’ under different cutoff values to decide a reasonable minlen value.

--broad

If set, MACS will try to call broad peaks using the --broad-cutoff setting. Please tweak ’--broad-cutoff’ setting to control the peak calling behavior. At the meantime, either -q or -p cutoff will be used to define regions with ’stronger enrichment’ inside of broad peaks. The maximum gap is expanded to 4 * MAXGAP ( --max-gap parameter). As a result, MACS will output a ’gappedPeak’ and a ’broadPeak’ file instead of ’narrowPeak’ file. Note, a broad peak will be reported even if there is no ’stronger enrichment’ inside. DEFAULT: False

--broad-cutoff BROADCUTOFF

Cutoff for broad region. This option is not available unless --broad is set. If -p is set, this is a pvalue cutoff, otherwise, it’s a qvalue cutoff. Please note that in broad peakcalling mode, MACS3 uses this setting to control the overall peak calling behavior, then uses -q or -p setting to define regions inside broad region as ’stronger’ enrichment. DEFAULT: 0.1

--cutoff-analysis

While set, MACS3 will analyze number or total length of peaks that can be called by different p-value cutoff then output a summary table to help user decide a better cutoff. The table will be saved in NAME_cutoff_analysis.txt file. Note, minlen and maxgap may affect the results. WARNING: May take ˜30 folds longer time to finish. The result can be useful for users to decide a reasonable cutoff value. DEFAULT: False

Post-processing options:

--call-summits

If set, MACS will use a more sophisticated signal processing approach to find subpeak summits in each enriched peak region. DEFAULT: False

--fe-cutoff FECUTOFF

When set, the value will be used as the minimum requirement to filter out peaks with low foldenrichment. Note, MACS3 adds one as pseudocount while calculating fold-enrichment. By default, it is set as 1 so there is no filtering. DEFAULT: 1.0

Obsolete options:

--to-large

Obsolete option. Please use ’--scale-to large’ instead.

--ratio RATIO

Obsolete option. Originally designed to normalize treatment and control with customized ratio, now it won’t have any effect.

Other options:

--buffer-size BUFFER_SIZE

Buffer size for incrementally increasing internal array size to store reads alignment information. In most cases, you don’t have to change this parameter. However, if there are large number of chromosomes/contigs/scaffolds in your alignment, it’s recommended to specify a smaller buffer size in order to decrease memory usage (but it will take longer time to read alignment files). Minimum memory requested for reading an alignment file is about # of CHROMOSOME * BUFFER_SIZE * 8 Bytes. DEFAULT: 100000

EXAMPLES

1. Peak calling for regular TF ChIP-seq:

$ macs3 callpeak -t ChIP.bam -c Control.bam -f BAM -g hs -n test -B -q 0.01

2. Broad peak calling on Histone Mark ChIP-seq:

$ macs3 callpeak -t ChIP.bam -c Control.bam --broad -g hs --broad-cutoff 0.1

3. Peak calling on ATAC-seq (paired-end mode):

$ macs3 callpeak -f BAMPE -t ATAC.bam -g hs -n test -B -q 0.01

4. Peak calling on ATAC-seq ( focusing on insertion sites, and usingsingle-end mode):

$ macs3 callpeak -f BAM -t ATAC.bam -g hs -n test -B -q 0.01 --shift -50 --extension 100