Changes between Version 2 and Version 3 of SOPs/InProgressChipSeq


Ignore:
Timestamp:
05/14/14 12:21:14 (11 years ago)
Author:
ibarrasa
Comment:

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  • SOPs/InProgressChipSeq

    v2 v3  
    1414 * See the [[http://barcwiki.wi.mit.edu/wiki/SOPs/mapping|mapping SOP]] for more details.
    1515=== Step 2: Calculate strand cross correlation ===
     16 * 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 ]]).
    1617{{{
    1718Rscript /nfs/BaRC_Public/phantompeakqualtools/run_spp.R -c=TreatmentIP.bam -savp -out=TreatmentIP.run_spp.out
    1819}}}
    1920
    20 
     21  * After this analysis a good ChIP-seq experiment will have a second peak (reflecting the fragment size) as tall or taller than the first peak (reflecting read length). If the second peak is smaller that the first one 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.
     22 
    2123=== Step 3: Call peaks (bound regions) ===
    2224Some of the parameters to consider when comparing programs are:
    … …  
    2931Based 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.
    3032
    31 ==== MACS ====
     33==== MACS14 ====
    3234  * For MACS to work the header of the sequences has to have no spaces.
    3335  * macs points to macs14 on WIBR local machines
    … …  
    4648macs -t ./s7_mouse_mm9.k1.n2.l36.best.map -c ./s8_control_mouse_mm9.k1.n2.l36.best.map -g 1.87e9 --name=outputName --format=BOWTIE --tsize=36 --wig --space=25  --mfold=10,30
    4749macs -t ./s7_mouse_mm9.k1.n2.l36.best.sam -c ./s8_control_mouse_mm9.k1.n2.l36.best.sam -g 1.87e9 --name=outputName --format=SAM --tsize=36 --wig --space=25  --mfold=10,30
     50macs -t ./s7_mouse_mm9.k1.n2.l36.best.sam -c ./s8_control_mouse_mm9.k1.n2.l36.best.sam -g 1.87e9 --name=outputName --format=SAM --tsize=36 --wig --space=25 --nomodel --shiftsize=100
     51
    4852}}}
    4953
    … …  
    5761  * --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
    5862  * -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
    59   * --keep-dup=1   Controls the MACS behavior towards duplicate tags at the exact same location.  DEFAULT: 1 in MACS 1.4; auto in MACS2. 
     63  * --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  * --nomodel  whether or not to build the shifting model. If True, MACS will not build model. by default it means shifting size = 100.                     
     65  * --shiftsize The arbitrary shift size in bp. When nomodel is true, MACS will use this value as 1/2 of fragment size. DEFAULT: 100.
    6066
    6167{{{
    … …  
    6470}}}
    6571
    66 '''Note:''' The wig files that macs14 generates are not normalized.
     72''Note'': The wig files that macs14 generates are not normalized.
     73
     74
     75==== MACS2 ====
     76{{{
     77macs2  callpeak -t IP_reads.mapped_only.bam  -c Control_reads.mapped_only.bam -f BAM -g mm  -n Name --nomodel   -B
     78}}}
     79  * --nomodel  whether or not to build the shifting model. If True, MACS will not build model. by default it means shifting size = 100.                     
     80  * -f Input format
     81  * -B create bedgraph output files
    6782==== SISSRs ====
    6883[[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]])
    … …  
    101116
    102117
    103 === Step 3: Linking bound regions to genes ===
     118=== Step 4: Linking bound regions to genes ===
    104119Both MACS and SISSRs provide bed files with the set of peaks, presumably indicating bound regions.
    105120