Swapping first and second mates when pair aligned to reverse strand

[mikisvaz]

I noticed that Mutect2 filtered out a lot of variants based on orientation bias on a realigned pair of samples, which didn't happen when I used RevertSam instead. I seemed to have tracked the problem down to this:

A pair of samples might originally be

H0KU0ADXX130516:2:2112:19067:52017      163     1       30257   0       76M     =       30269   88      AAAAAGAGCATCATCAGTCCAAAGTCCAGCAGTTGTCCCTCCTGGAATCCGTTGGCTTGCCTCCGGCATTTTTGGC    CCDFCH<HHEBGDAHDHAHGEFEJBHGEJHCFBDJBHGGFHHGIIBGCHG@BDJHHFEJIGCIH@DGBCCEECGJF       MC:Z:76M        MD:Z:76 PG:Z:MarkDuplicates.1A  RG:Z:RG_H0KU0ADXX130516_2_R1    NM:i:0  MQ:i:0  AS:i:76 XS:i:76
H0KU0ADXX130516:2:2112:19067:52017      83      1       30269   0       76M     =       30257   -88     ATCAGTCCAAAGTCCAGCAGTTGTCCCTCCTGGAATCCGTTGGCTTGCCTCCGGCATTTTTGGCCCTTGCCTTTTA    BCHFFBIDBDFICDIGIIDDDDEBEIICHHBGHDCCH?GGDGHJEFJHHCH=GIHCEFFEEGHIHHFDHGEDDF?9       MC:Z:76M        MD:Z:76 PG:Z:MarkDuplicates.1A  RG:Z:RG_H0KU0ADXX130516_2_R1    NM:i:0  MQ:i:0  AS:i:76 XS:i:76

And after realignment become

H0KU0ADXX130516:2:2112:19067:52017      99      1       30257   0       76M     =       30269   88      AAAAAGAGCATCATCAGTCCAAAGTCCAGCAGTTGTCCCTCCTGGAATCCGTTGGCTTGCCTCCGGCATTTTTGGC    ABAFAF;GIEBHCAFCGAGGEFEIAIGEJHBDADIBGGFDHGEGGBFCIG@CDJGIFEJHFBGG@AEABBEDBDHE       MC:Z:76M        MD:Z:76 PG:Z:MarkDuplicates     RG:Z:H0KU0.2    NM:i:0  MQ:i:0  AS:i:76 XS:i:76
H0KU0ADXX130516:2:2112:19067:52017      147     1       30269   0       76M     =       30257   -88     ATCAGTCCAAAGTCCAGCAGTTGTCCCTCCTGGAATCCGTTGGCTTGCCTCCGGCATTTTTGGCCCTTGCCTTTTA    @CFEDBICCCEHBAIEHGDCDDDBDHICFGBGHDDCG@FFDGIIEFIGGCH>GIGCEFFDFGHHHGEEIFEDCA=9       MC:Z:76M        MD:Z:76 PG:Z:MarkDuplicates     RG:Z:H0KU0.2    NM:i:0  MQ:i:0  AS:i:76 XS:i:76

so the flags 163 and 83 become 99 and 147 all else being the same. In fact the first and second pairs come out of bazam swapped.