Question: fastqc of chipseq dataset - two peaks in GC content
1
gravatar for Assa Yeroslaviz
6 months ago by
Assa Yeroslaviz1.2k
Munich
Assa Yeroslaviz1.2k wrote:

We are running a ChIP-Seq experiment of multiple samples on one flow cell. when looking at the fastqc results of the samples, they all look strange.

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both overrepresented adapter and adapter content are in the green and sow no accumulation.

Can anyone explain this kind of behavior? Are these contaminations?

thanks

ADD COMMENTlink modified 6 months ago by Biostar ♦♦ 20 • written 6 months ago by Assa Yeroslaviz1.2k

Have you scanned/trimmed this data? Is it from a 2-color chemistry sequencer (e.g. NovaSeq/NextSeq).

ADD REPLYlink written 6 months ago by genomax73k

I haven't trimmed it yet. the sequencing was done a a nextseq with dual indexing.

About the trimming - do I just trim the first bases of the read (hard trim) or should I look for the barcodes (soft trim)?

ADD REPLYlink modified 6 months ago • written 6 months ago by Assa Yeroslaviz1.2k
2

Looks like you have a significant number of reads that may just be poly-G (certainly start with G, no signal = G) and those should be removed. Before you do any directed trimming why not just look for adapter sequence and see what happens. Any scan/trim program should work. Post plots after trimming.

ADD REPLYlink modified 6 months ago • written 6 months ago by genomax73k

This I have also considered, but why don't I see it also in the overrepresented sequences section? Is it possible that the adapter are so GC (or more G)-rich, that it skew the results so much?

ADD REPLYlink written 6 months ago by Assa Yeroslaviz1.2k

Can we see what we get post-trimming? FastQC samples data for some of the features it looks at and does not include the entire dataset.

ADD REPLYlink written 6 months ago by genomax73k
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