Changes between Version 8 and Version 9 of SOPs/chip_seq_peaks


Ignore:
Timestamp:
05/14/14 16:03:50 (11 years ago)
Author:
ibarrasa
Comment:

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  • SOPs/chip_seq_peaks

    v8 v9  
    1313
    1414 * See the [[http://barcwiki.wi.mit.edu/wiki/SOPs/mapping|mapping SOP]] for more details.
    15 
    16 === Step 2: Call peaks (bound regions) ===
     15=== Step 2: Strand cross correlation analysis ===
     16 * The goal of this step is to asses the quality of the IP and to estimate the fragment size of the DNA immunoprecipitated.
     17 * For a detailed explanation on strand cross-correlation analysis see box 2 of this paper ([[http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1003326|Practical Guidelines for the Comprehensive Analysis of ChIP-seq Data ]]).
     18{{{
     19/nfs/BaRC_Public/phantompeakqualtools/run_spp.R -c=TreatmentIP.bam -savp -out=TreatmentIP.run_spp.out
     20}}}
     21
     22  * After this analysis a good ChIP-seq experiment will have a second peak (reflecting the fragment size) at least as tall as the first peak (reflecting read length). This is how the graph should look: ([[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431496/figure/F4/|Fig4E ]]). If the second peak is smaller that the first, ([[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431496/figure/F4/|like the example shown in Fig4G Marginal ]]),  macs will not estimate fragment size correctly. In that case we recommend running macs with parameters "--nomodel" and "--shiftsize=half_of_the_fragment_size". The fragment size is detected on the strand cross correlation analysis.
     23 
     24=== Step 3: Call peaks (bound regions) ===
    1725Some of the parameters to consider when comparing programs are:
    1826  * Adjustment of sequence tags to better represent the original DNA fragment (by shifting tags in the 3′ direction or by extending tags to the estimated length of the original fragments)
    … …  
    2432Based on our ChIP-Seq bake off and on a published review ([[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011471|Evaluation of ChIP-Seq performance]]), MACs and SISSRs are good programs to try.
    2533
    26 ==== MACS ====
     34==== MACS14 ====
    2735  * For MACS to work the header of the sequences has to have no spaces.
    2836  * macs points to macs14 on WIBR local machines
    … …  
    3240}}}
    3341
     42
    3443[[http://liulab.dfci.harvard.edu/MACS/|MACS]] ([[http://liulab.dfci.harvard.edu/MACS/00README.html|README]]) ([[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Search&db=pubmed&term=18798982|Zhang et al., 2008]])[[br]][[br]]
    3544
    … …  
    3746
    3847Sample commands to run MACS (current version as of March 5 2012): 1.4.2 using mapped reads in map or sam format:
    39 
    4048{{{
    4149macs -t IP_mapped.map -c Control_mapped.map -g 1.87e9 --name=outputName --format=BOWTIE --tsize=36 --wig --space=25  --mfold=10,30
    4250macs -t IP_mapped.sam -c Control_mapped.sam -g 1.87e9 --name=outputName --format=SAM    --tsize=36 --wig --space=25  --mfold=10,30
    43 }}}
     51macs -t IP_mapped.sam -c Control_mapped.sam -g 1.87e9 --name=outputName --format=SAM    --tsize=36 --wig --space=25  --nomodel --shiftsize=100
     52}}}
     53
    4454
    4555The parameters used on the command are:
    … …  
    5262  * --mfold=MFOLD      Select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit. DEFAULT:10,30
    5363  * -g 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
    54   * --keep-dup=1   Controls the MACS behavior towards duplicate tags at the exact same location.  DEFAULT: 1 in MACS 1.4; auto in MACS2. 
     64  * --keep-dup=1   Controls the MACS behavior towards duplicate tags at the exact same location.  DEFAULT: 1 in MACS 1.4; auto in MACS2.   
     65  * --nomodel  whether or not to build the shifting model. If True, MACS will not build model. by default it means shifting size = 100.                     
     66  * --shiftsize The arbitrary shift size in bp. When nomodel is true, MACS will use this value as 1/2 of fragment size. DEFAULT: 100.
    5567
    5668{{{
    … …  
    5971}}}
    6072
    61 '''Note:''' The wig files that macs14 generates are not normalized.
     73
     74''Note'': The wig files that macs14 generates are not normalized.
     75
     76
     77==== MACS2 ====
     78
     79  * MACS2 is appropriate for both proteins like transcription factors that may have narrow peaks, as well as histone modifications that may affect broader regions. For broader peaks we recommend using --nomodel, --nolambda (if there's no control), and using the fragment size calculated on the strand cross correlation analysis.  We recommend using macs2 rather than macs14 for broad peaks.
     80{{{
     81macs2  callpeak -t IP_reads.mapped_only.bam  -c Control_reads.mapped_only.bam -f BAM -g mm  -n Name --nomodel   -B
     82}}}
     83  * --nomodel  whether or not to build the shifting model. If True, MACS will not build model. by default it means shifting size = 100.                     
     84  * -f Input format
     85  * -B create bedgraph output files
    6286==== SISSRs ====
    6387[[http://dir.nhlbi.nih.gov/papers/lmi/epigenomes/sissrs/|SiSSRs]] ([[http://nar.oxfordjournals.org/cgi/content/full/36/16/5221|Reference]]) ([[http://dir.nhlbi.nih.gov/papers/lmi/epigenomes/sissrs/SISSRs-Manual.pdf|Manual]])
    … …  
    96120
    97121
    98 === Step 3: Linking bound regions to genes ===
     122=== Step 4: Linking bound regions to genes ===
    99123Both MACS and SISSRs provide bed files with the set of peaks, presumably indicating bound regions.
    100124
    … …  
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