A question about Homer ChIP-seq analysis

Question

[mikysyc2016]

Hi all, I use macs2 to do peak calling for my ChIP-seq results, and use narrowpeak file to run homer with bedgraph file. Please see below. $ annotatePeaks.pl '/home/Desktop/B6KOg/B6KOg_s_2_ACAGTG_peaks.narrowPeak' mm9 -size 400 -bedGraph '/home/Desktop/B6KOg/B6KOg_s_2_ACAGTG_treat_pileup.bdg' '/home/yachen/Desktop/B6KOg/B6KOg_s_2_ACAGTG_control_lambda.bdg' -raw > Desktop/outputB6KOg.txt Then i get a txt file and last two column show me (B6KOg_s_2_ACAGTG_treat_pileup.bdg bedGraph avg over 400 bp) and (B6KOg_s_2_ACAGTG_control_lambda.bdg bedGraph avg over 400 bp). do they means o get tag count for treat peaks and input peaks? or DO you know how to use macs2 to get tag count for samples directly? Thanks!

[James Ashmore]

Are you asking how to count the number of reads aligned within peak regions? If so, you can do this with Bedtools:

bedtools map -a peaks.bed -b coverage.bedgraph -c 4 -o mean

[mikysyc2016]

Thanks. I use two file from macs2 analysis (one is file:ko1_2.72_summits.bed, another is ko1_2.72_treat_pileup.bdg) as bed and bedgraph as you mentioned, but i get error as below: ERROR: chromomsome sort ordering for file ko1_0.01_q/ppara_ko1_2.72_treat_pileup.bedgraph is inconsistent with other files. Do you know why this happen?

[James Ashmore]

Bedtools expects both files to be sorted in the same order. Sort both files by chromosome and start position:

sort -k1,1 -k2,2n coverage.bedgraph > coverage_sorted.bedgraph
sort -k1,1 -k2,2n peaks.bed > peaks_sorted.bed

[mikysyc2016]

It works. Thank you.