Hi, I recently got some problems with the output of NovaSeq 6000. I ran sample in paired mode, then found out that a portion of read 2 has poly-G (around 50bp) at the beginning. I understand that NovaSeq is a 2-color system, so the poly-G is likely signal lost, but I don't understand why it only appears at the beginning of the second read. I have considered:
- DNA strand break from sample: if this happen, then the read 1 won't have signal of the break at all
- DNA strand break from after fragmentation: if this happen, then the polyG will occur even at the tail of read 1.
- Reverse strand break at head: then how can the tail still have base? I think it will falls out of the adapter, am I right?
- Reverse strand break at tail: then polyG will occur at the tail, like many other reported case.
This phenomenon is really confusing and I haven't found any answer/explanation for it yet. I have tried it will new library prep kit but this still happens. I am using TruSeq DNA PCR-Free from Illumina
Any help/advice is warmly welcome!