Question: Identify RNA-seq reads containing polyA sequence
0
gravatar for goodez
8 months ago by
goodez470
United States
goodez470 wrote:

This may seem like a weird question, but we need to filter our RNAseq data for reads that contain polyA. The data is stranded RNA-seq, 50 bp reads. Would it be easier to find these reads before or after alignment? To be clear, the 50 bp read needs to contain a stretch of polyA, not just come from a transcript containing polyA. Has anyone done this type of analysis?

rna-seq • 243 views
ADD COMMENTlink modified 8 months ago by Buffo1.8k • written 8 months ago by goodez470
1
gravatar for genomax
8 months ago by
genomax76k
United States
genomax76k wrote:

Use bbduk.sh from BBMap suite in filter mode (don't specify ktrim= or qtrim= options) with literal=AAAAA to filter the reads out (adjust length of A's as needed). Use with original data.

ADD COMMENTlink modified 8 months ago • written 8 months ago by genomax76k

Thanks! I have some additional questions then. Since it is stranded RNA-seq, the polyA will actually be stretches of TTTTTT right?

Also I used grep to look for reads containing this, and many of the TTTTTT stretches are in the middle of a read. It doesn't seem possible that the polyA could be surrounded by other sequence on both ends.

ADD REPLYlink written 8 months ago by goodez470

If you are capturing second strand then yes. Past the TTTTT the sequence may be going into adapters. You can easily check that by trimming reads you filter and select.

ADD REPLYlink written 8 months ago by genomax76k
1
gravatar for Buffo
8 months ago by
Buffo1.8k
Buffo1.8k wrote:

If data is stranded, polyA tails will be always at the end of sequences, I have read a couple of papers where this information is important (to define UTRs mainly, if I remember the reference I will post it). They usually consider a 8-10 nucleotides as the minimum length.

ADD COMMENTlink written 8 months ago by Buffo1.8k

Thanks, that is good to know. Please do share the reference if you find it again!

ADD REPLYlink written 8 months ago by goodez470
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