R1 R2 orientation

Question

[biotech]

Hi,

Will always:

R1 be mapped to forward strand and the R2 to the reverse strand?

Thanks, Bernardo

Answer 1

[Devon Ryan]

No, that will not always be the case.

[biotech]

So, how could I know if I had no reference genome to map?

[Devon Ryan]

You can't.

Answer 2

[seanvig]

This depends on how you generate your library. Usually, library generation involves some combination of fragmenting your genome and adding adaptors. If you are using some sort of kit, then your genome is probably fragmented at random, and adapters are added in any orientation. If you are doing Illumina sequencing you have the i5 and i7 adaptors. While one of these will always correspond to R1 (i5 I think, but don't hold me to that), you are never guaranteed that i5 is ligated upstream or downstream. Thus, R1 will always have a mixture of forward and reverse strands.

The only exception to this that I know of is if you are manually generating libraries through some sort of PCR amplification where you could always be sure that i5 and i7 are in a particular orientation with respect to your targets.