Are you using the latest version of samtools and HTSlib? If not, please specify.

Yes, 1.20.

Please describe your environment.

• OS: Linux 5.16.2-051602-generic
• machine architecture: x86_64
• compiler: gcc (Ubuntu 9.4.0-1ubuntu1~20.04.2) 9.4.0

Please specify the steps taken to generate the issue, the command you are running and the relevant output.

An interleaved FASTQ file was mapped using bwa mem -p -c 250 -M -t 9 hg19.fa in.bam mapped.bam. The following lines appear next to each other in the output, and the flags (73, 133) are correct, valid and complementary (sequences and quality scores removed for readability):

A01174:196:HGKFNDSX2:4:1651:6433:35336:TCA_TCA 73 chrM 8481 21 3S143M = 8481 0 NM:i:3 MD:Z:115A20A2C3 AS:i:131 XS:i:121 XA:Z:chr1,+569030,3S143M,5;

A01174:196:HGKFNDSX2:4:1651:6433:35336:TCA_TCA 133 chrM 8481 0 * = 8481 0 MC:Z:3S143M AS:i:0 XS:i:0

After running samtools collate -@ 1 -O -u mapped.bam 2> map.log | samtools fixmate -Mrcm - out.bam, only the first of these is retained (sequences match), and the SAM flag is now invalid (72: mate unmapped, first in pair, but paired bit not set):

A01174:196:HGKFNDSX2:4:1651:6433:35336:TCA_TCA 72 chrM 8481 21 3S143M = 8481 0 NM:i:3 MD:Z:115A20A2C3 AS:i:131 XS:i:121 XA:Z:chr1,+569030,3S143M,5; MC:Z:* ms:i:2872

This creates downstream problems with fgbio GroupReadsByUMI for me:

Exception in thread "main" htsjdk.samtools.SAMFormatException: SAM validation error: ERROR::INVALID_FLAG_MATE_UNMAPPED:Record 1248, Read name A01174:196:HGKFNDSX2:4:1651:6433:35336, Mate unmapped flag should not be set for unpaired read.

Picard complains about a 72 flag as well: https://broadinstitute.github.io/picard/explain-flags.html