Weird kmer distribution (using fastqc)

feralBiologist · 07-12-2013, 10:12 AM

I ran FatsQC on a set of 4 Illumina PE exomes and got really weird graphs for the kmer distribution. I've never seen this pattern before - does anyone know what is going on and what to do about it?

fkrueger · 07-12-2013, 02:25 PM

I am not entirely sure about what is going on since I don't have the full length Illumina adapter sequences handy, but my guess is that the plot shows an adapter or primer dimer of some sort. Seeing that GGAAG is part of the start of the Illumina adapter (GATCGGAAGAGC..., wihtout the A-tail at the start) and the spacing is always the same this might simply be full-length adapter dimers. Such sequences should not align anywhere, so you probably don't need to worry about them. This doesn't mean that you don't have to trim your sample at all, this is difficult to say without seeing the rest of the plots.

feralBiologist · 07-14-2013, 05:07 AM

@fkrueger: I found that two TruSeq adaptors were listed among the over-represented sequences in the fastqc report so I removed them for one of the samples and re-ran fastqc. The graph changed somewhat but there are still non-random peaks (below). Any ideas?

fkrueger · 07-14-2013, 05:20 AM

I don't know how you removed the adapters, but the k-mers could still result from adapter dimers. Trim Galore for example only removes the entire adapter sequence AGATCGGAAGAGC but only if it starts with the A from the A-tailing process. In your case the sequence seems to start with GATC(GGAAG) and thus wouldn't be affected by adapter trimming.

The CACAC and ACACA is often a highly occurring sequence in MeDIP-Seq or the like. I wouldn't worry too much about the plot for the moment, and only come back to it if you find that you've got an unusually low mapping efficiency or similar.

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07-12-2013, 10:12 AM	#1
feralBiologist Member Location: UK Join Date: Jun 2011 Posts: 61	Weird kmer distribution (using fastqc) I ran FatsQC on a set of 4 Illumina PE exomes and got really weird graphs for the kmer distribution. I've never seen this pattern before - does anyone know what is going on and what to do about it?

07-14-2013, 05:07 AM	#3
feralBiologist Member Location: UK Join Date: Jun 2011 Posts: 61	removing over-represented sequences does not help @fkrueger: I found that two TruSeq adaptors were listed among the over-represented sequences in the fastqc report so I removed them for one of the samples and re-ran fastqc. The graph changed somewhat but there are still non-random peaks (below). Any ideas?

07-12-2013, 02:25 PM	#2
fkrueger Senior Member Location: Cambridge, UK Join Date: Sep 2009 Posts: 625	I am not entirely sure about what is going on since I don't have the full length Illumina adapter sequences handy, but my guess is that the plot shows an adapter or primer dimer of some sort. Seeing that GGAAG is part of the start of the Illumina adapter (GATCGGAAGAGC..., wihtout the A-tail at the start) and the spacing is always the same this might simply be full-length adapter dimers. Such sequences should not align anywhere, so you probably don't need to worry about them. This doesn't mean that you don't have to trim your sample at all, this is difficult to say without seeing the rest of the plots.

07-14-2013, 05:20 AM	#4
fkrueger Senior Member Location: Cambridge, UK Join Date: Sep 2009 Posts: 625	I don't know how you removed the adapters, but the k-mers could still result from adapter dimers. Trim Galore for example only removes the entire adapter sequence AGATCGGAAGAGC but only if it starts with the A from the A-tailing process. In your case the sequence seems to start with GATC(GGAAG) and thus wouldn't be affected by adapter trimming. The CACAC and ACACA is often a highly occurring sequence in MeDIP-Seq or the like. I wouldn't worry too much about the plot for the moment, and only come back to it if you find that you've got an unusually low mapping efficiency or similar.