samtool flagstat question

Question

[alex]

Hi,

So I have created a bam from a NIST sample. I did realignment and recalibration but the flagstat numbers don't change. Should I be worried? Thanks!

samtools flagstat NA12878-test1_S1_L001_R1_001.bam
55456561 + 0 in total (QC-passed reads + QC-failed reads)
5895280 + 0 duplicates
55320108 + 0 mapped (99.75%:-nan%)
55456561 + 0 paired in sequencing
27727985 + 0 read1
27728576 + 0 read2
55020320 + 0 properly paired (99.21%:-nan%)
55233536 + 0 with itself and mate mapped
86572 + 0 singletons (0.16%:-nan%)
67065 + 0 with mate mapped to a different chr
33459 + 0 with mate mapped to a different chr (mapQ>=5)

samtools flagstat NA12878-test1_S1_L001_R1_001.realigned.bam
55456561 + 0 in total (QC-passed reads + QC-failed reads)
5895280 + 0 duplicates
55320108 + 0 mapped (99.75%:-nan%)
55456561 + 0 paired in sequencing
27727985 + 0 read1
27728576 + 0 read2
55020320 + 0 properly paired (99.21%:-nan%)
55233536 + 0 with itself and mate mapped
86572 + 0 singletons (0.16%:-nan%)
67065 + 0 with mate mapped to a different chr
33459 + 0 with mate mapped to a different chr (mapQ>=5)

samtools flagstat NA12878-test1_S1_L001_R1_001.realigned.recalibrated.bam
55456561 + 0 in total (QC-passed reads + QC-failed reads)
5895280 + 0 duplicates
55320108 + 0 mapped (99.75%:-nan%)
55456561 + 0 paired in sequencing
27727985 + 0 read1
27728576 + 0 read2
55020320 + 0 properly paired (99.21%:-nan%)
55233536 + 0 with itself and mate mapped
86572 + 0 singletons (0.16%:-nan%)
67065 + 0 with mate mapped to a different chr
33459 + 0 with mate mapped to a different chr (mapQ>=5)

Thanks!

Answer 1

[Devon Ryan]

Realignment and such won't change these metrics. Realignment just locally realigns things and typically the assigned MAPQ values don't change (unmapped mates etc. also won't change). Recalibration typically affects only base qualities. I should note that recalibration is typically not needed anymore (the same goes for realignment if you're using something like GATK's haplotype caller).