Hi Group!
I'm dealing with quite the strange sequencing run. We don't normally do PE, so we don't have a standard operating procedure to fall back on, and are clearly making mistakes.
We did 101 read size paired end using nextera chemistry. We expected the distance between the ENDS of the forward and reverse reads to be ~135, but my investigation is telling me that we wound up with ~65 bp distance from STARTS of the forward and reverse.
So, I guess the reads start about 65 bp from each other, say hello on the way by and in the process, sequence past each of their respective starts by 35 bp.
I guess this in effect, creates basically single reads of ~135 long.
Further strangeness: the distance distribution for pairs is bimodal, with the biggest peak being the aforementioned one with ~65 bp distance between the STARTS of the forward and reverse reads. The second one, is much smaller then the first, but still quite pronounced and has a peak centered at a much more reasonable distance where there's 100 bp between the ENDS of the forward and reverse reads (closer to where we expected which was 135bp between ENDS).
This latter peak reflects a distance that is more what we were shooting for, but when I set distances using this, we break ~90% of our pairs.
Does anybody have any perspective on what is happening here? I'd be quite appreciative of any ideas at all..
Thanks for reading!
I'm dealing with quite the strange sequencing run. We don't normally do PE, so we don't have a standard operating procedure to fall back on, and are clearly making mistakes.
We did 101 read size paired end using nextera chemistry. We expected the distance between the ENDS of the forward and reverse reads to be ~135, but my investigation is telling me that we wound up with ~65 bp distance from STARTS of the forward and reverse.
So, I guess the reads start about 65 bp from each other, say hello on the way by and in the process, sequence past each of their respective starts by 35 bp.
I guess this in effect, creates basically single reads of ~135 long.
Further strangeness: the distance distribution for pairs is bimodal, with the biggest peak being the aforementioned one with ~65 bp distance between the STARTS of the forward and reverse reads. The second one, is much smaller then the first, but still quite pronounced and has a peak centered at a much more reasonable distance where there's 100 bp between the ENDS of the forward and reverse reads (closer to where we expected which was 135bp between ENDS).
This latter peak reflects a distance that is more what we were shooting for, but when I set distances using this, we break ~90% of our pairs.
Does anybody have any perspective on what is happening here? I'd be quite appreciative of any ideas at all..
Thanks for reading!
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