My question is this: "mate reverse strand" (flag 16/0x10) or "read reverse strand" (flag 32/0x20) do not directly relate to the strandedness of the library in terms of library protocols (e.g., stranded vs. non-stranded); instead, they simply indicate the directionality of the read in relation to the reference genome—is that correct?
This is correct. I was conflating "reverse strand" with stranded library preparations.
That is, "mate reverse strand" means that a given alignment is considered "the mate" in a read pair and is the reverse complement of the sequence in the reference; "read reverse strand" means that a given alignment is considered "the read" in a read pair and is the reverse complement of the sequence in the reference. Is my understanding correct?
This is correct. More information is available at this Biostars post. Here is some important context from that post:
The paires of the read pair have the opposite direction. One was sequenced on the + strand and one on the - strand. But in the sam file all information are meant for the + strand and are going from 5'-end to 3'-end. The read whos information must be flipped, get a flag about it.
Here is what this means for 83/163 aligned read pairs.
This is how the two reads look in vivo:
(Read #1: FLAG 83) 3' <---------- 5'
5' -----------> 3' (Read #2: FLAG 163)
5' ------------------------------------------- 3' (Reference genome)
And this how the two reads look in the BAM file:
5' ----------> 3' (Read #1: FLAG 83)
5' -----------> 3' (Read #2: FLAG 163)
5' ------------------------------------------- 3' (Reference genome)
Here is what this means for 99/147 aligned read pairs.
This is how the two reads look in vivo:
(Read #2: FLAG 147) 3' <---------- 5'
5' -----------> 3' (Read #1: FLAG 99)
5' ------------------------------------------- 3' (Reference genome)
And this how the two reads look in the BAM file:
5' ----------> 3' (Read #2: FLAG 147)
5' -----------> 3' (Read #1: FLAG 99)
5' ------------------------------------------- 3' (Reference genome)
