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Old 11-17-2020, 09:57 AM   #1
ajb_seq
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Default Per base sequence content, Illumina

Hello,
I'm fairly new to analyzing sequencing data like this. I've noticed this pattern appear several times (both when I look at full runs, or individual samples within a single run).

Sequencer: Illumina NextSeq
Kit: 71 x 10 x 10

I've processed untrimmed reads (though I've also tried trimming, and the same overall pattern appears). At ~38bp, I see a favoring(?) of "A" over "C", "G", or "T". This favoring continues, more-or-less, throughout the remainder of the read.

I'm not quite sure what to make of this pattern. Whenever I run FASTQC on my data, this metric shows up as a "WARN" or "FAIL" every time. I'm not sure what this might be due to. The material we are attempting to use is DNA (from lysate, plant material); not sure if that has an impact or not. Thanks in advance for any advice you all might have!
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Old 11-18-2020, 03:52 AM   #2
GenoMax
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It is difficult to say what is happening. It may be fine to use the data. If you have a reference then you could try aligning and see.

There was no known problem with this run correct?
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Old 11-18-2020, 06:23 AM   #3
ajb_seq
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No sequencing problems that I'm aware of. We've run ~5 sequencing runs, and I've noticed that, on all 5, this same pattern appears. When I map these raw reads to an ampli-ome, only ~50% of the reads map (and when I map them to the genome, the same amount map), so I don't think I'm dealing with off-target amplification. My guess right now is that we are dealing with a lot of dimers soaking up reads on the Illumina flowcell. But the fact that the Per-Base-Sequence-Content was consistently giving me this pattern made me think that maybe I'm dealing with something else. But, I'm not very sure. I was curious if anyone else had seen a pattern like this.
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Old 11-18-2020, 09:59 AM   #4
microgirl123
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I think you're right about adapter dimers - after you sequence through the adapter and hit the flow cell you see overcalling of either A or G.
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Old 11-18-2020, 10:02 AM   #5
ajb_seq
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Ah, thank you!
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