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-   -   Weird trace in Next-Seq run (http://seqanswers.com/forums/showthread.php?t=93287)

Audrey_Sa 03-05-2020 01:09 PM

Weird trace in Next-Seq run
 
Hi,

I have an issue with a recent Next-Seq run and I'm wondering if someone might have any insight. In this run, we pooled together two libraries that were prepared as a customization of the 10x 3' protocol.
The first 25 base pairs seem to be okay, but then there is a stretch of about 75bp of T's. This is not something we were expecting in our library.
I was thinking maybe the adaptor ligation for one of these libraries didn't work, and that possibly messes up the sequencing run? Any other idea would be greatly appreciated since at this point we're not sure what went wrong.

luc 03-05-2020 03:40 PM

This is expected. You are sequencing through the poly-A tail. You can ignore this part of the data, and sequence only ~30 (?) cycles for the forward read next time.

jdk787 03-05-2020 04:02 PM

Quote:

Originally Posted by luc (Post 231481)
This is expected. You are sequencing through the poly-A tail. You can ignore this part of the data, and sequence only ~30 (?) cycles for the forward read next time.

Correct, I think 10X says to do 28 cycles for R1, then 8 for i7, then 90 or however many cycles you have left for the insert read in R2


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