Question: Explaining fragment distribution plots from ATAC-seq
1
gravatar for a.rex
20 days ago by
a.rex160
a.rex160 wrote:

I have just mapped my ATAC-seq fragments, resulting from paired-end sequencing using this command:

./samtools view file | awk '$9>0' | cut -f 9 | sort | uniq -c | sort -b -k2,2n | sed -e 's/^[ \t]*//' > frag.distribution

I see as expected, a large maximal peak at around 60bp, with most reads under 100bp, and then a second lower hump at around 160-200bp corresponding to the mononucleosome.

However, I also see spikes at 151bp and 139bp - consistent over replicates enter image description here. You can see similar 'spikes' in the fragment distribution of S. pombe in this paper: "Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions" Figure 3a.

Any idea what these spikes correspond to? Is this related to the fact that Tn5 is a dimer, that inserts two adapters 9bp distance apart?

atac-seq • 114 views
ADD COMMENTlink modified 20 days ago • written 20 days ago by a.rex160
1

Please post the pdf that this command produces (Picard tools from the Broad Institute), or alternatively the plot you made:

picard CollectInsertSizeMetrics I=in.bam O=out.txt H=out.pdf
ADD REPLYlink modified 20 days ago • written 20 days ago by ATpoint9.1k

I mapped to a reference genome - it is a species for which chrM is not known. I did trim the Nextera adapter.

ADD REPLYlink written 20 days ago by a.rex160
1

I cannot see anything suspicious on that distribution. These little spikes every 10bp correspond to the helical pitch of the DNA. I cannot explain why these two larger spikes are present, but I do not think that you should worry.

ADD REPLYlink written 20 days ago by ATpoint9.1k

Okay, thanks. If I had Nextera adapter, the peak would be way more shifted to the right? Those two peaks before the mononucleosome are present in the paper "Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions" Figure 3a. I was just wondering....

ADD REPLYlink written 20 days ago by a.rex160
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