Question: Explaining fragment distribution plots from ATAC-seq
1
gravatar for a.rex
3 months ago by
a.rex170
a.rex170 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 • 318 views
ADD COMMENTlink modified 25 days ago by Biostar ♦♦ 20 • written 3 months ago by a.rex170
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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 3 months ago • written 3 months ago by ATpoint12k

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 3 months ago by a.rex170
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 3 months ago by ATpoint12k

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 3 months ago by a.rex170

The spikes at 140bp and 150bp correspond to the size of the core nucleosome particle in conjunction with the 10bp periodicity of the DNA helix mentioned by @ATpoint. These sizes were first reported by biochemists Axel, van Holde, Kornberg, and others in the 1970s, using micrococcal nuclease digestion.

ADD REPLYlink written 24 days ago by harold.smith.tarheel4.2k
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