MACS2_CALLPEAK(1)

📦
version 2.2.7.1 · 2 versions
, in ubuntu jammy

💖 NAME 💖

macs2_callpeak — Model-based Analysis for ChIP-Sequencing 🧬✨

(and yes bestie, this is the page for the MACS2_CALLPEAK(1) manual — the callpeak command, the moment, the icon 👑)

🌸 DESCRIPTION 🌸

okay so this is literally how you invoke it, no cap:

usage: macs2 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] [--buffer-size BUFFER_SIZE] [--to-large]
              [--ratio RATIO]

like, that's the whole lineup. take a breath, we're getting through all of it 💅

🫶 options: 🫶

  • -h, --help — shows this help message and dips. that's it, that's the whole job 🫡

🎀 Input files arguments: 🎀

  • -t TFILE [TFILE ...], --treatment TFILE [TFILE ...] — your ChIP-seq treatment file. and listen, if you hand it multiple files like -t A B C, bestie, they all get read and pooled together. and it's REQUIRED, so don't be out here forgetting it 😭
  • -c [CFILE ...], --control [CFILE ...] — the control file. give it multiple ones like -c A B C and they get pooled to estimate the ChIP-seq background noise. it's giving "know your baseline" energy 🌫️
  • -f {AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE,BEDPE}, --format {AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE,BEDPE} — the format of your tag file: "AUTO", "BED", "ELAND", "ELANDMULTI", "ELANDEXPORT", "SAM", "BAM", "BOWTIE", "BAMPE" or "BEDPE". the default AUTO option lets MACS decide which format the file is — except for BAMPE and BEDPE, which should be implicitly set. check the definition in README, fr. ⚠️ also note: if the format is set as BAMPE or BEDPE, MACS2 calls 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. and ALSO note that the BEDPE here only contains three columns, and is NOT the same BEDPE format used by BEDTOOLS. DEFAULT: "AUTO" ✨
  • -g GSIZE, --gsize GSIZE — effective genome size. can be 1.0e+9 or 1000000000, or use the shortcuts: 'hs' for human (2.7e9), 'mm' for mouse (1.87e9), 'ce' for C. elegans (9e7) and 'dm' for fruitfly (1.2e8). Default: hs 🌍
  • -s TSIZE, --tsize TSIZE — tag size / read length. this overrides the auto detected tag size. DEFAULT: Not set 🙈
  • --keep-dup KEEPDUPLICATES — controls the behavior towards duplicate tags at the exact same location — same coordination AND same strand, so like, actual twins. 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 tag. 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, MACS2 will still read them although the reads may be decided by MACS2 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 MACS2 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 get written to that directory. Default: the current working directory 📁
  • -n NAME, --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 — sets the verbose level of runtime messages. 0: only show critical message, 1: show additional warning message, 2: show process information, 3: show debug messages. DEFAULT: 2 🗣️
  • --trackline — tells MACS to include trackline with bedGraph files. including this trackline while displaying bedGraph at UCSC genome browser can show name and description of the file as well. however, the suggestion here is to convert bedGraph to bigWig, then show the smaller and faster binary bigWig file at UCSC genome browser, as well as downstream analysis. Require -B to be set. Default: Not 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 MACS2 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 that means shifting size = 100, so try setting 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! so don't even try it bestie) the arbitrary shift in bp. use discretion while setting it other than the 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 saying 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 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 the 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 to 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 using this automate fixation is a default behavior now. DEFAULT: False 🔧

🏔️ Peak calling arguments: 🏔️

  • -q QVALUE, --qvalue QVALUE — minimum FDR (q-value) cutoff for peak detection. DEFAULT: 0.05. -q and -p are mutually exclusive ⚔️
  • -p PVALUE, --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 using '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 — okay so this is the small nearby region in basepairs, used to calculate dynamic lambda. It's giving "capture the bias near the peak summit region" energy. 🦋 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, MACS2 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, MACS2 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 to filter out peaks with low fold-enrichment. Note, MACS2 use 1.0 as pseudocount while calculating fold-enrichment. DEFAULT: 1.0

🌈 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

💀 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. 💀

And that's the whole vibe check, bestie. This page is from macs2 callpeak 2.2.7.1, dated April 2022, and it's the manual for MACS2_CALLPEAK(1). Go slay those peaks. 💖✨🦋

📦 where this page comes from