Strange Fastqc Report

Question

[Farhat]

I obtained sequenced reads (using Illumina) from a ChIP-seq run that seems to have gone bad according to the FASTQC output. The results of the FASTQC output are shared here http://dl.dropbox.com/u/17931758/Run18_s_4_sequence_fastqc/fastqc_report.html Does anyone have any ideas on what could have gone wrong? I asked them to prepare the libraries again and resequence but the results are similar.

[User 59]

What is it that particularly concerns you and why? There doesn't seem to be a technical problem with the sequencing, your quality scores look good across the read. You do understand that ChIP-Seq is going to enrich for certain sequences in the result set don't you? Hence you will expect to get a certain level of duplication and that will potentially affect overall base composition, Kmer composition and overall GC content.

[Farhat]

I would expect the GC-content and kmer composition to get affected. What I wasn't expecting is that the GC content would vary so strongly from base to base along the read. I would expect G-C content along the read to be relatively constant even if different from the content of the underlying genome. Also, on aligning I found only a small fraction of the reads aligning to the target genome (~1%).

[User 59]

That information would have been useful in the question. Do your non-aligning reads map to anything else?

[Farhat]

Well, the alignment was underway when I put the question but yes it is useful information. I will be checking next if it maps to anything else.

Answer 1

[Gareth Palidwor]

Are you sure you're mapping to the right genome? I BLAST-ed a bunch of the over-represented sequences against the nr db and they all map to "Schistosoma japonicum" mRNA. Is this expected? Without more data or explanation there's not much that can be done to diagnose your problem...

[Farhat]

That's certainly unexpected. These reads are supposed to be from Bombyx mori (honeybee).