Macs2 操作手册与介绍.ppt

1、ChIP-seq analysis with MACS2Tips and tricks,Sami Heikkinen, PhD Docent in Molecular Bioinformatics Institute of Biomedicine, UEF,Schmidt et al, Methods, 2009,ChIP-Seq simplified,Where?,Park, Nat Rev Genetics, 2009,From binding to binding sites,Typically millions of reads per sample,Park, Nat Rev Gen

2、etics, 2009,ChIP-seq,200 bp,36-50 bp,Control sample: “Input” or “IgG” Input: sonicated chromatin without immunoprecipitation IgG: “unspecific” IP,MACS2,Model-based Analysis of ChIP-Seq Original version published by Yong Zhang and Tao Liu from the lab of X. Shirley Liu at the Dana-Farber Cancer Insti

3、tute, Boston Genome Biology 2008, 9:R137 now at version 2.1.0.20140616, developed and maintained by Tao Liu at Package of command line programs to call peaks in ChIP-seq data Much improved since v1.x!,diffpeak,bdgdiff,bdgcmp,bdgbroadcall,MACS2 program(s),peaks.narrowPeak,callpeak,summits.bed,peaks.x

4、ls,model.r,model.pdf,INPUT DATA: aligned sequence reads,OUTPUT FILEs,treat_pileup.bdg,control_lambda.bdg,refinepeaks,refinepeak.bed,randsample,filterdup,predictd,pileup,pileup.bdg,bdgpeakcall,OUTPUT,callpeak - Options,Various options to indicate/control input, output, peak modelling and peak calling

5、 macs2 callpeak usage: macs2 callpeak -h -t TFILE TFILE . -c CFILE CFILE . -f AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE, BAMPE -g GSIZE -keep-dup KEEPDUPLICATES -buffer-size BUFFER_SIZE -outdir OUTDIR -n NAME -B -verbose VERBOSE -trackline -SPMR -s TSIZE -bw BW -m MFOLD MFOLD -fix-bimodal

6、 -nomodel -shift SHIFT -extsize EXTSIZE -q QVALUE -p PVALUE -to-large -ratio RATIO -down-sample -seed SEED -nolambda -slocal SMALLLOCAL -llocal LARGELOCAL -broad -broad-cutoff BROADCUTOFF -call-summits -t/-treatment FILENAME This is the only REQUIRED parameter for MACS.,Using MACS connect to server,

7、Open the SSH client at Win All programs SSH Secure shell Secure shell client “Quick connect” connection : intron.uef.fi username : password: ,Unix 101,pwd show Present Working Directory cd Change Directory e.g. cd /home/work/public to get to the folder we use today (from wherever you are) or, to get

8、 back to your home directory: cd $HOME or, back one step cd ., or two steps cd ././ Usage tip: use up/down arrow keys to move in command history ls LiSt files in directory e.g. ls -l to show file and folder names AND other info (Long format) head / tail show first/last lines of a (text) file e.g. he

9、ad -20 ref_hg19.txt Usage tip: use the TAB key to fill in available file/folder names,Using MACS - setup,cd /home/work/public mkdir macsout_ : e.g. spheikki for me each student MUST have their own folder! to avoid overlapping MACS outputs checks on seq files ls l seq head seq/* check that macs2 work

10、s macs2 callpeak,callpeak - Options,Various options to indicate/control input, output, peak modelling and peak calling macs2 callpeak usage: macs2 callpeak -h -t TFILE TFILE . -c CFILE CFILE . -f AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE, BAMPE -g GSIZE -keep-dup KEEPDUPLICATES -buffer-si

11、ze BUFFER_SIZE -outdir OUTDIR -n NAME -B -verbose VERBOSE -trackline -SPMR -s TSIZE -bw BW -m MFOLD MFOLD -fix-bimodal -nomodel -shift SHIFT -extsize EXTSIZE -q QVALUE -p PVALUE -to-large -ratio RATIO -down-sample -seed SEED -nolambda -slocal SMALLLOCAL -llocal LARGELOCAL -broad -broad-cutoff BROADC

12、UTOFF -call-summits,callpeak Options - Input,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 combined. REQUIRED. -c CFILE CFILE ., -control CFILE CFILE . Control file. If multiple files

13、 are given as -c A B C, then they will all be read and combined. -f AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE, -format AUTO,BAM,SAM,BED,ELAND,ELANDMULTI,ELANDEXPORT,BOWTIE,BAMPE Format of tag file, AUTO, BED or ELAND or ELANDMULTI or ELANDEXPORT or SAM or BAM or BOWTIE or BAMPE. The

14、 default AUTO option will let MACS decide which format the file is. Please check the definition in README file if you choose ELAND/ELANDMULTI/ELANDEXPORT/SAM/BAM/BOWTIE. 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

15、for mouse (1.87e9), ce for C. elegans (9e7) and dm for fruitfly (1.2e8), Default:hs -keep-dup KEEPDUPLICATES It controls the MACS 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

16、 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. The default is to keep one tag at the same location. Default: 1 -buffer-size BUFFER_SIZE Buffer size f

17、or incrementally increasing internal array size to store reads alignment information. In most cases, you dont have to change this parameter. However, if there are large number of chromosomes/contigs/scaffolds in your alignment, its recommended to specify a smaller buffer size in order to decrease me

18、mory 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 * 2 Bytes. DEFAULT: 100000,callpeak Options - Output,Output arguments: -outdir OUTDIR If specified all output files will be written to that

19、 directory. Default: the present 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 -verbos

20、e 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 UCS

21、C 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

22、, MACS will save signal per million reads for fragment pileup profiles. Require -B to be set. Default: False,Using MACS test different settings,Run 1: Using default settings Run 2: Call summits Run 3: Adjust model band width Run 4: Adjust mfold limits macs2 callpeak -t seq/treat_chr3.sam -c seq/inpu

23、t_chr3.sam -outdir macsout_ -n defaults,Using MACS test different settings,Run 1: Using default settings Run 2: Call summits Run 3: Adjust model band width Run 4: Adjust mfold limits macs2 callpeak -t seq/treat_chr3.sam -c seq/input_chr3.sam -outdir macsout_ -n defaults ls l macsout_ head -40 macsou

24、t_/*,callpeak Options Peak calling 1,Peak calling arguments 2: -nolambda If True (=set), 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 potential chromatin structure. -slocal SMALLLOCAL Th

25、e 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. T

26、he final local bias should be the maximum of the lambda value from d, slocal, and llocal size windows. 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 calc

27、ulation. *Note* that MACS will always perform a d-size local lambda calculation. The final local bias should be the maximum of the lambda value from d, slocal, and llocal size windows. DEFAULT: 10000. -broad If set, MACS will try to call broad peaks by linking nearby highly enriched regions. The lin

28、king region is controlled by another cutoff through -linking-cutoff. The maximum linking region length is 4 times of d from MACS. 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, its

29、 a qvalue cutoff. DEFAULT: 0.1 -call-summits If set, MACS will use a more sophisticated signal processing approach to find subpeak summits in each enriched peak region. DEFAULT: False,-call-summits,Using MACS test different settings,Run 1: Using default settings Run 2: Call summits Run 3: Adjust mod

30、el band width Run 4: Adjust mfold limits From command history, find the previous macs2 command and edit the red parts: macs2 callpeak -t seq/treat_chr3.sam -c seq/input_chr3.sam -outdir macsout_ -call-summits -n cs.defaults,callpeak Options Peak calling 2,Peak calling arguments 1: -q QVALUE, -qvalue

31、 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

32、 final .xls file. -to-large When set, scale the small sample up to the bigger sample. By default, the bigger dataset will be scaled down towards the smaller dataset, which will lead to smaller p/qvalues and more specific results. Keep in mind that scaling down will bring down background noise more.

33、DEFAULT: False -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

34、 result unstable and irreproducible since each time, random reads would be selected. Consider to use randsample script instead. If used together with SPMR, 1 million unique reads will be randomly picked. Caution: due to the implementation, the final number of selected reads may not be as you expecte

35、d! 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,callpeak Options The Model,Shifting model arguments: -s TSIZE, -tsize TSIZE Tag size (=read length). This will overide the auto detected tag size. DEFA

36、ULT: Not set -bw BW Band width for picking regions to compute fragment size. This value is only used while building the shifting model. DEFAULT: 300 -m MFOLD MFOLD, -mfold MFOLD MFOLD Select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. Fold-en

37、richment in regions must be lower than upper limit, and higher than the lower limit. Use as -m 10 30. 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 tow

38、ards 3 direction. Not to use this automate fixation is a default behavior now. DEFAULT: False -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. DEFAULT: False -shift SHIFT (NOT the legacy

39、 -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 towar

40、d 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 cant set values other than 0 if format is BAMPE for paired-end data. DEFAULT: 0. -extsiz

41、e 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. Its 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