Question: Slight confusion about trimming paired end reads in RNAseq experiment
0
gravatar for mforde84
3 days ago by
mforde841.0k
mforde841.0k wrote:

We have paired end RNAseq reads which were trimmed 15bp and 10bp at the beginning and end of each respective read. My question is if this is appropriate considering it seems that each read will now have an overhang compared to it's paired read. Wouldn't this make the paired reads mismatching simply because of the shift in read coverage on the respective strands?

rnaseq • 128 views
ADD COMMENTlink modified 3 days ago • written 3 days ago by mforde841.0k
2
gravatar for genomax
3 days ago by
genomax39k
United States
genomax39k wrote:

How so? Unless they were perfectly (100%) overlapping to begin with that should not happen.

ADD COMMENTlink modified 3 days ago • written 3 days ago by genomax39k

Maybe I'm simply misunderstanding how library generation works. The inserted fragment should be size selected, my assumption was that the insert size was the expected size of the read? So if I have a 100bp read that means I'm sequencing the same 100bp fragment but from both directions.

Or is it more like one read is an anchor for the other which is further downstream or upstream of that insert? Eg., say the insert is 1kb, and I'm paired end sequencing 100bp, that would mean each paired read is either ~900bp upstream or downstream of the other read?

ADD REPLYlink modified 3 days ago • written 3 days ago by mforde841.0k
3

No for your first question. Sort of to second. Sequencing with a 100% overlap would be a waste of sequencing (unless you had some specific need).

See if this figure helps you visualize (A: What is the different between Read and Fragment in RNA-seq? )

ADD REPLYlink modified 3 days ago • written 3 days ago by genomax39k

Great, thanks for the clarification.

ADD REPLYlink written 3 days ago by mforde841.0k
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