I have been reading some literature on read orientation and unfortunately I am still confused about how read orientation relates to the original fragment. Let's say that the following is one strand of the library fragment.
5' P7 <insert>AAAAAATTTGGGGGGGGGGGG</insert> P5 3'
From what I understand, before R1 is sequenced, this strand is hybridized to the surface of the flowcell by the 5' end only. So 5' P7 is stuck to the flowcell and 3' P5 is sticking out. When R1 is sequenced, the basecalls are the reverse complement, so that the following is written to the R1 FASTQ file: CCCCCCCCCCCCAAATTTTTT<adapter/> (obviously my insert is so short that the sequencer reads through the adapter).
If we are sequencing R2 as well, then the 3' P5 end of the above strand is hybridized to a 5' oligo on the flowcell surface and extended, and now we have a different strand:
5' P5 <insert>CCCCCCCCCCCCAAATTTTTT</insert> P7 3'
R2 is sequenced using this as a template strand, so now we get the following sequence written to the R2 file: AAAAAATTTGGGGGGGGGGGG<adapter/>
Perhaps this seems like a molecular biology question, but this is part of my understanding of how bioinformatics tools work with fastq files. So my overall question is, is my thinking correct? Have I missed anything?
