Question: Best way to trim PolyA in RNA Seq reads?
0
gravatar for DVA
6 months ago by
DVA470
United States
DVA470 wrote:

Hello,

I am trying to learn what my options are if I would like to trim polyA from my reads. (FastqC repo see previous discussion: https://www.biostars.org/p/302411/#302507) We are quite certain we need to trim adapter, which we would use bbduk, but we also see polyA showing in many reads, and would like to trim that too.

Since polyA shows in different reads with different length, it does not make sense to trim by length. I hope to trim all the way till the nucleotide is not A. Any suggestions?

(Or maybe I should just go ahead with alignment (Tophat) and let the aligner ignore polyA in the reads?)

Thank you very much!

rna-seq • 432 views
ADD COMMENTlink modified 6 months ago by WouterDeCoster32k • written 6 months ago by DVA470
1

(Or maybe I should just go ahead with alignment (Tophat) and let the aligner ignore polyA in the reads?)

You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

ADD REPLYlink written 6 months ago by WouterDeCoster32k

Thank you so much! I was wondering about that recently lol and this confirms it. Appreciate it!

ADD REPLYlink written 6 months ago by DVA470

By the way, in your experience, do you think these aligners can handle 10-20bps polyA in the reads?

ADD REPLYlink written 6 months ago by DVA470

I usually trim my reads, but I don't know if it's necessary. Haven't evaluated that.

ADD REPLYlink written 6 months ago by WouterDeCoster32k
1

You can use bbduk.sh to remove poly-A tails as well. with literal=AAAAA and adjusting value of k= as needed to something small.

ADD REPLYlink modified 6 months ago • written 6 months ago by genomax55k

Thank you very much for following up with my questions. Can this be done in one command, together with trimming the adaptor? I assume not - because k for adaptor will be around 20, while this one should be something like 2-3 right?

ADD REPLYlink written 6 months ago by DVA470
1

You can use unix pipes for multiple bbduk.sh passes. Something like (example for SE reads, adjust for PE reads with in1= in2= etc):

bbduk.sh in=seq.fq out=stdout.fq ref=adapters.fa k=20 ktrim=r | bbduk.sh in=stdin.fq out=final.fq literal=AAAAA k=2 ktrim=r
ADD REPLYlink modified 6 months ago • written 6 months ago by genomax55k

Thank you. One more question - For adaptor trimming, is there a particular reason not to just do force-trimming? (Simply trim # number of bps from one end)

ADD REPLYlink written 6 months ago by DVA470
1

You could (forcetrimright= or forcetrimleft=) but the the problem is your adapter contamination may not always be in the same spot (unless you are doing some special library prep and expect it to be so).

ADD REPLYlink modified 6 months ago • written 6 months ago by genomax55k

I thought the adaptor is always showing up at the left of the reads... why they will not be at the same spot please? Sorry for all these questions, but I really want to understand it better. Thank you.

ADD REPLYlink written 6 months ago by DVA470

Adapter should always be towards the right end of the read. See this document for clarification on how the libraries are constructed. Since your insert size varies all fragments will not have the adapter in the same location (assuming you have short inserts and have run of real DNA to sequence).

ADD REPLYlink written 6 months ago by genomax55k
1
gravatar for WouterDeCoster
6 months ago by
Belgium
WouterDeCoster32k wrote:

You could use prinseq or fastp for trimming polyA tails

ADD COMMENTlink modified 6 months ago • written 6 months ago by WouterDeCoster32k

Thank you very much!

ADD REPLYlink written 6 months ago by DVA470
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