Mate negative strand flag does not match read negative strand flag of mate

Question

[user230613]

I get this error when running picard ValidateSamFile in a RNA-Seq bam file produced by hisat2:

ERROR: Record 52, Read name K00103:277:HL7HMBBXX:6:2109:23551:26054, Mate negative strand flag does not match read negative strand flag of mate

This is the read in the bam file:

samtools view -@ 12 hisat2_sort.bam | grep "K00103:277:HL7HMBBXX:6:2109:23551:26054"

K00103:277:HL7HMBBXX:6:2109:23551:26054 69  1   3747256 0   *   =   3747256 0   CTGGCTACACAAACAGGACCCAACATTCTGCTGCTTACAGGAAACCCATCTCAGGGAAAAAGACAGACACTACCT AAFFFJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJFJJFJJJJJJJJJJJJJJJFJJJJJJJJ YT:Z:UP RG:Z:F72_tumor_R1
K00103:277:HL7HMBBXX:6:2109:23551:26054 153 1   3747256 60  75M =   3747256 0   ATTGACCATATAATTGGTCACAAAATAGGCCTCAACAGAAACAAAAATATTGAAATTGTCCCATGTATCCTATCA JFJFJJJAFJJ<JFA7F<F<F<FJ-<AAA<-<7FJ<A-J-J<JJJF<FJFJJJJJJJJJJJJJJJJJJJFFFFA< AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:75 YT:Z:UP NH:i:1  RG:Z:F72_tumor_R1

I don't know what's going on and I need to fix this.

Any help would be appreciated.

:)

Answer 1

[Santosh Anand]

Sam specification says that If a SAM record has the negative strand flag (0x10) set, then it's mate should have the mate negative strand flag (0x20) set. In your case, flag 153 has 0x10 set, but flag 69 doesn't contain 0x20.