Question: fastqc of chipseq dataset - two peaks in GC content
1
gravatar for Assa Yeroslaviz
22 months ago by
Assa Yeroslaviz1.4k
Munich
Assa Yeroslaviz1.4k 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 21 months ago by Biostar ♦♦ 20 • written 22 months ago by Assa Yeroslaviz1.4k

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

ADD REPLYlink written 22 months ago by GenoMax94k

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 22 months ago • written 22 months ago by Assa Yeroslaviz1.4k
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 22 months ago • written 22 months ago by GenoMax94k

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 22 months ago by Assa Yeroslaviz1.4k

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 22 months ago by GenoMax94k
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