I am trying to understand why my peaks look to be noisy as compared to some references from ENCODE. Any thoughts from your side would be highly appreciated.
I have a group of treated (n = 7) vs untreated (n = 7) samples that were precipitated by H3K4me3 antibody and sequenced on an Illumina HiSeq machine. I used bowtie to map pair-end reads to the hg19 genome and using
samtools I created
.bam files. For peak calling I used
MACS2 . To make bigwig files I used both
deeptools and UCSC
# macs2 # ___peak calling macs2 callpeak -t sample1.bam \ -c input_sample1.bam input_sample3.bam input_sample7.bam \ #since there are no input per samples, all inputs included -f BAMPE \ -g hs --SPMR --keep-dup auto --outdir ./outputs -n sample1_macs2_call \ -B -q 0.01 --trackline --nomodel --extsize 147 #___ converting bdg to bigwig macs2 bdgcmp -t sample1_treat_pileup.bdg \ -c sample1_control_lambda.bdg \ -m FE \ -o sample1_FE.bdg #_ and macs2 bdgcmp -t sample1_treat_pileup.bdg \ -c sample1_control_lambda.bdg \ -m logLR -p 0.00001 \ -o sample1_logLR.bdg #_finally sort -k1,1 -k2,2n sample1_FE.bdg > sample1_FE_sorted.bdg sort -k1,1 -k2,2n sample1_logLR.bdg > sample1_logLR_sorted.bdg bedGraphToBigWig sample1_FE_sorted.bdg hg19.sizes sample1_FE.bw bedGraphToBigWig sample1_logLR_sorted.bdg hg19.sizes sample1_logLR.bw #___ making bigwig by deeptools bamCoverage -b sample1.bam \ -o sample1.bw \ --binSize 20 \ --normalizeUsing BPM \ --smoothLength 60 \ --extendReads 150 \ --centerReads \ -p 12
Here is a screenshot from IGV to show how the peaks look like. For the sake of comparison, two samples one from ENCODE and another from GSE120339 were added. As you can see there are several peaks(noise?) in the first three tracks. I indicated some of them by the red arrows. Also regarding on-target peaks (here those overlapped with promoters) peaks in the first three tracks (my sample) are look different than the control track (last two tracks).
Check this link to see a bigger version of the image.
Please let me know if I am doing something wrong... Thanks