BooK
- https://github.com/Shicheng-Guo/HowtoBook/tree/master/APP/macs2/ChIP-Seq-Slides-Shicheng-Guo-2020-MACS2.pdf
- https://github.com/Shicheng-Guo/HowtoBook/tree/master/APP/macs2/ENCODE 3 - ChIPSeq Pipeline - Google Docs.pdf
usage: macs2 callpeak [-h] -t TFILE [TFILE ...] [-c [CFILE [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}] [--ratio RATIO]
[--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]
optional arguments:
-h, --help show this help message and exit
Input files arguments:
-t TFILE [TFILE ...], --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 [CFILE [CFILE ...]], --control [CFILE [CFILE ...]]
Control file. If multiple files are given as '-c A B
C', they will be pooled to estimate ChIP-seq
background noise.
-f {AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE,BEDPE}, --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,
MACS2 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 GSIZE
Effective genome size. It can be 1.0e+9 or 1000000000,
or 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 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,
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 will be 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 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 Tells MACS to include trackline with bedGraph files.
To include this trackline while displaying bedGraph at
UCSC genome browser, can show name and description of
the file as well. However my suggestion 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 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 MFOLD MFOLD
Select the regions within MFOLD range of high-
confidence 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 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'.
--ratio RATIO When set, use a custom scaling ratio of ChIP/control
(e.g. calculated using NCIS) for linear scaling.
DEFAULT: ingore
--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, 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.
