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jianhong / ATACseqQC

ATAC-seq Quality Control
https://jianhong.github.io/ATACseqQC/articles/ATACseqQC.html
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estimated library size of NucleosomeFree.bam is unexpectedly small #59

Open cparsania opened 4 months ago

cparsania commented 4 months ago

Hi,

After shift and splitting bams according to fragment size, I see amongst all bam the largest bam is NulceosomeFree.bam which is expected. However, when I run estLibSize (from ChIPpeakAnno package), the output is <1000 which is smaller than the output of mononucleosome.bam which is in millions although the size of bam is smaller than NuclesomeFree.bam. can you tell me what's wrong here. ?? Attached here is the plot showing library size

image

jianhong commented 4 months ago

Could you try to run 

samtools flagstat NulceosomeFree.bam

and share the results?

cparsania commented 4 months ago

Hi,

Thanks for your reply. I managed to solve the issue. The problem was due to large portion of the paired-end reads were aligned discordantly. This happened because I did fix 15 bp trimming from both the reads prior to alignment. This made ATAC-seq shorter fragments even shorter and hence increase multi-mapping and mostly mates aligned far from each other. These alignments were marked as discordant alignments by bowtie2. Although all these reads were part of the NucleosomeFree.bam most of them were considered coming from different fragment, hence smaller library size. So downstream analysis on NucleosomeFree.bam failed. I redo alignment without hard trimming and to my surprise % of concordant alignments increased to 95% from 20%. I hope this will resolve the issue of downstream analysis.

Cheers