Changes between Version 41 and Version 42 of SOPs/chip_seq_peaks
- Timestamp:
- 09/27/17 15:37:15 (7 years ago)
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SOPs/chip_seq_peaks
v41 v42 4 4 === Basic Approach === 5 5 * For each sample map the reads to the appropriate genome (e.g. with Bowtie2). 6 * Call peaks (eg. MACS) . Peaks can be called without control, however, it's highly recommended a control was used in the experimental design, eg. IgG or input, input is preferred over IgG.6 * Call peaks (eg. MACS) or use another method to analyze enrichment and/or presumed binding. For ChIP-seq experiments profiling transcription factors (with discrete binding sites), binding site identification (via peak calling) is typically recommended. Peaks can be called without control; however, it's highly recommended to include a control sample (e.g. IgG or input, with input generally preferred over IgG). For ChIP-seq experiments profiling epigenetic (such as histone) modifications, however, modeling ChIP enrichment as peaks may not accurately describe the actual data, and some other (such as sliding window) quantification may be more relevant. 7 7 * Note that quality control is important after read mapping and after peak calling. The ENCODE consortium recommends some [[https://genome.ucsc.edu/ENCODE/qualityMetrics.html|quality metrics]]. 8 8 … … 30 30 * 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 than 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", or "--nomodel" and "--extsize=fragment_size". The fragment size is detected on the strand cross correlation analysis. 31 31 32 === Step 3: Call peaks ( bound regions)===32 === Step 3: Call peaks (presumed bound regions), especially for transcription factors === 33 33 Some of the parameters to consider when comparing programs are: 34 34 * 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) … … 147 147 148 148 149 === Step 4 [for experiments with replication]: Identify reproducible peaks ===149 === Step 4 [for peak-calling experiments with replication]: Identify reproducible peaks === 150 150 151 151 For more information about the method, see the main [[https://sites.google.com/site/anshulkundaje/projects/idr| IDR page]]. … … 200 200 }}} 201 201 202 === Step 5: Link bound regions to genes===202 === Step 5: Link "bound" (or other interesting) regions to genomic features (genes, promoters, enhancers, etc.) === 203 203 Both MACS and SISSRs provide bed files with the set of peaks, presumably indicating bound regions. 204 204