Question: Reasons for polyA in RNA Seq Reads
0
gravatar for DVA
12 months ago by
DVA500
United States
DVA500 wrote:

In my poly A capture RNA sequencing fastq output, I noticed that about 20% of the reads contain poly A in the middle (or even close to the front). I would like to understand more on this, because with DNA fragments mostly >300bps and read length 100 bps, we were not expecting to see poly A show up that frequently in reads. I would appreciate your thinking.

To my understanding, even it is a poly A capture sequencing, during the library preparation, the mRNA tail fragments (with polyA) are the only fragments will be selected. In this selection, adapter contains ~15bps poly T can bind anywhere on the polyA tail, which can be >200bps long. My theory is that, if it binds towards the 3' end of polyA, then that literally allows major part of the polyA tail gets amplified in PCR, and can potentially pass size selection, but the coding region in this case can be short at 5'.

E.g.

mRNA-coding-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(...)AAAAAAAAAAAAAAAAAAAAAAA

and

TTTTTTTTTTTTTTTTT-AdapterSeq

Say the Ts-Adapter start binding at the highlighted A, then the last 2-3 A will be gone after first round of pcr, but the rest of As will remain till sequencing.

Any advise is appreciated. Thank you.

rna-seq • 484 views
ADD COMMENTlink modified 12 months ago • written 12 months ago by DVA500

You should ask questions like this on SeqAnswers.com which is more geared towards experimental aspects of NGS.

ADD REPLYlink modified 12 months ago • written 12 months ago by genomax64k

okay will do. Thank you for all the comments you provided to my questions recently.

ADD REPLYlink written 12 months ago by DVA500

You are welcome.

ADD REPLYlink written 12 months ago by genomax64k
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