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6.4 years ago
mforde84
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1.4k
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?
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?
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? )
Great, thanks for the clarification.