== Get reproducible peaks from multiple ChIP-seq replicates with IDR (after using macs for peak calling) ==

For more information about the method, see the main [[https://sites.google.com/site/anshulkundaje/projects/idr IDR page]]

=== Run macs2 (macs version 2, not version 1) to call ChIP-seq peaks.  Adjust fragment length with --nomodel --shiftsize if NSC/RSC plots are not good ===

The IDR analysis requires that you call lots of peaks, including all "good" ones and some that are just noise. 

{{{
# -B => create bedgraph output files
# -p 1e -3 => Set p-value cutoff to 1e-3 (which is more relaxed than the default setting)

bsub macs2 callpeak -t IP_1.bam -c control_1.bam -f BAM -g hs -n IP.1_vs_control.1 -B -p 1e-3
bsub macs2 callpeak -t IP_2.bam -c control_2.bam -f BAM -g hs -n IP.2_vs_control.2 -B -p 1e-3
}}}

=== Sort peaks in .narrowPeak files (created by macs2) ===

Sort from best to worst using the -log10(p-value) column (column 8), and only keep the top 100k peaks (at most).

{{{ 
bsub "sort -k 8,8nr IP.1_vs_control.1_peaks.narrowPeak | head -n 100000 | gzip -c >| IP.1_vs_control.1.regionPeak.gz"
bsub "sort -k 8,8nr IP.2_vs_control.2_peaks.narrowPeak | head -n 100000 | gzip -c >| IP.2_vs_control.2.regionPeak.gz"
}}}

=== Estimate Irreproducibility Discovery Rate (IDR) between replicates ===

The interface of this command has been slightly modified by BaRC, but the main method remains exactly the same.

The command requires names and lengths of all chromosomes in a file (ex: chromInfo) of format chromosome<tab>size.

{{{
# USAGE: batch-consistency-analysis.r [peakfile1] [peakfile2] [peak.half.width] [outfile.prefix] [min.overlap.ratio] [is.broadpeak] [ranking.measure]\
#
# peak.half.width => set the peak.half.width to the reported peak width in the peak files (-1 (minus one) is recommended)
# outfile.prefix => the one-word name of your analysis
# min.overlap.ratio => fractional bp overlap (0 to 1) between peaks in replicates to be considered as overlapping peaks. (0 is recommended)  
# is.broadpeak => Is the peak file format broadPeak (or else is narrowPeak)? (F for false)
# ranking.measure => p.value is recommended
#

batch-consistency-analysis.r IP.1_vs_control.1.regionPeak.gz IP.2_vs_control.2.regionPeak.gz -1 rep1_vs_rep2_IDR 0 F p.value chromInfo.txt 
}}}

=== Create the IDR plots ===
{{{
# USAGE: batch-consistency-plot.r [npairs] [output.prefix] [input.file.prefix1] [input.file.prefix2] [input.file.prefix3] ....
# This method can plot 1 or more pairs of replicates

batch-consistency-plot.r 1 rep1_vs_rep2_IDR_plot rep1_vs_rep2_IDR
}}}

=== Generate a conservative and an optimal final set of peak calls === 
{{{
# Use an IDR cutoff of 0.01 to 0.05, depending on the number of pre-IDR peaks and size of the genome:
# IDR <= 0.05 for < 100K pre-IDR peaks for large genomes (human/mouse)
# IDR <= 0.01 or 0.02 for ~15K to 40K peaks in smaller genomes such as worm

awk '{ if($NF < 0.05) print $0 }' rep1_vs_rep2_IDR-overlapped-peaks.txt > rep1_vs_rep2_conserved_peaks_by_IDR.txt
}}}