I have strand-specific reads from a paired-end RNAseq run. These are the SAM records for spot SRR1269551.1308177 after mapping:
SRR1269551.1308177 99 chrI 35 255 34M = 17 170 CACACCACACCACACCCACACACACACATCCTAA ?AAAAAAADEEEEDDDGGGGGGHHHHHHIIIIII NH:i:1 HI:i:1 AS:i:66 NM:i:0 MD:Z:34
SRR1269551.1308177 147 chrI 171 255 34M = 35 -170 CACTCCGAACCACCATCCATCCCTCTACTTACTA HHHEHHIHHHFCGGGFGGDDDDDDDDBBB??A?? NH:i:1 HI:i:1 AS:i:66 NM:i:0 MD:Z:34
samtools mpileup gives me following result:
[...]
chrI 65 C 1 . I
chrI 66 T 1 . I
chrI 67 A 1 . E
chrI 68 A 1 .$ A
chrI 171 C 1 ^~, E
chrI 172 A 1 , H
chrI 173 C 1 , H
chrI 174 T 1 , E
[...]
In the pileup the two reads map to different strands. This makes sense from a mapping point of view.
Looking at it from the transcript point of view, both come from the same transcript, thus the same (biological) DNA strand - it's just the technicalities of the paired-end sequencing that make the second read map to the opposite strand.
So what I actually would like to have is a pileup that flips the strand of the second read, so that I get
[...]
chrI 65 C 1 . I
chrI 66 T 1 . I
chrI 67 A 1 . E
chrI 68 A 1 .$ A
chrI 171 C 1 ^~. E
chrI 172 A 1 . H
chrI 173 C 1 . H
chrI 174 T 1 . E
[...]
I guess some hack like reverse complementing the second read before mapping would do, but to me this problem seems so basic that there must be a better way to do it.
Did I miss some obvious flags during mapping/pileup that could do this for me?
