Question: High Kmer Content in middle of the read
3
gravatar for Sam
3.0 years ago by
Sam2.2k
London
Sam2.2k wrote:

Hi there, we have now got the 12 sequencing samples back. However, when reading the FastQC report, we saw something weird: A large kmer content spike in the middle of the read.

Kmer Profile

There is slight amount of adapter leftover but they should inflate the kmer content at the start of the read. Has anyone seen something like that before? Is it something to be worried about?

The full report for a pair of the reads can be found here. Thank you very much!

rna-seq • 1.8k views
ADD COMMENTlink modified 4 weeks ago by jgarces0 • written 3.0 years ago by Sam2.2k
1
gravatar for Ian
3.0 years ago by
Ian5.3k
University of Manchester, UK
Ian5.3k wrote:

I was told that this represents mis-incorporation of Truseq adapters into the sequence.  But in my experience this only effects a minority of reads; tens of thousands out of millions.  Sorry, no reference for this.

ADD COMMENTlink written 3.0 years ago by Ian5.3k
1
gravatar for harold.smith.tarheel
3.0 years ago by
United States
harold.smith.tarheel4.2k wrote:

The over-represented sequences in your kmer reports (TATCTCGTATGCCGT and GTGGTCGCCGTATCAT) are perfect matches to segments of the TruSeq Index and Universal adapter primers, respectively. Without more details, it's difficult to know why only a portion of the adapters are present, and only at the positions indicated.

ADD COMMENTlink written 3.0 years ago by harold.smith.tarheel4.2k
0
gravatar for jgarces
4 weeks ago by
jgarces0
Spain
jgarces0 wrote:

Hi @Sam, I've got exactly the same problem with my samples... can you finally solve it?? I'd expect that adapter contamination or mis-incorporation were in the 5'/3'-ends but I don't understand why it's appearing in the middle...

Thanks a lot!

ADD COMMENTlink written 4 weeks ago by jgarces0

Unfortunately, that's like 3 years ago and I have no memory of that. I think @Ian's answer should most likely be what's happening

ADD REPLYlink written 4 weeks ago by Sam2.2k

Ok, thanks anyway... so I guess, according to Ian's response, that's useless try to do some kind of trimming, isn't it? Thanks again!

ADD REPLYlink written 4 weeks ago by jgarces0

Maybe just check the number of reads that show this kind of problem. If the number is small, just remove them

ADD REPLYlink written 4 weeks ago by Sam2.2k
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