Question: Should I merge bacterial RNA-seq paired-end data?
1
gravatar for biotech
4.8 years ago by
biotech520
United States
biotech520 wrote:

Hi,

I have bacterial RNA-seq data from paired-end reads (2x75). I'm interested in sense/antisense differential expression.

I would like to know if makes sense to merge R1 and R2 into one read (if overlaps) or maybe work only with 75 bp reads (R2 ones).

Thanks, Bernardo

rna-seq bacteria paired-end • 2.1k views
ADD COMMENTlink modified 4.8 years ago by Devon Ryan89k • written 4.8 years ago by biotech520

If your experiment was conducted so that the fragment size allowed pairs to overlap, merging them would make a lot of sense. So, finding out your fragment size would be the first thing..

ADD REPLYlink written 4.8 years ago by 5heikki8.4k

200 bp but maybe shorter. The thing is that I need to convert paired-end to single-end data anyway in order to quantify antisense transcription too.

ADD REPLYlink modified 4.8 years ago • written 4.8 years ago by biotech520
1
gravatar for Devon Ryan
4.8 years ago by
Devon Ryan89k
Freiburg, Germany
Devon Ryan89k wrote:

Why bother? Presuming that you used a stranded protocol when making the libraries, you can just align things as pairs and tell whichever program you use to do counting to observe strandedness of pairs. Both htseq-count and featureCounts can do that.

To get at the antisense transcription, one could just run the counters again and just tell them to use the other read as the strand reference (e.g., running htseq-count -s yes ... and then htseq-count -s reverse ...).

ADD COMMENTlink modified 4.8 years ago • written 4.8 years ago by Devon Ryan89k

I did like you describe when working with single-end data. Is the same with paired-end data? I though that because one of the pairs aligns to reverse strand would count as antisense when is not. Also, I though longer reads would map better.

ADD REPLYlink modified 4.8 years ago • written 4.8 years ago by biotech520

Any decent program that does counting will count pairs as a single unit, rather than individually, so yes it is the same.

ADD REPLYlink written 4.8 years ago by Devon Ryan89k
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