Sakshi Shekhar Mehta

Yes, the Invalid NM tag error you're seeing usually happens when the NM (edit distance) tag in your BAM file is missing, incorrect, or inconsistent with the alignment and reference. This tag is required by tools like GATK, which expect it to be properly calculated for downstream analysis.

By default, BWA-MEM2 does not always compute or validate all optional tags (like NM, MD) unless you explicitly request them or run a post-processing step.

You can fix this by recalculating NM (and MD) tags using samtools after alignment:

samtools calmd -bAr aligned_sorted.bam reference_genome.fa > aligned_sorted_fixed.bam

Then re-run gatk ValidateSamFile on aligned_sorted_fixed.bam.

You can also avoid this error from the start by not sorting within the BWA-MEM2 command, and instead do:

bwa-mem2 mem reference.fa reads.fq > aligned.sam

samtools view -Sb aligned.sam | samtools calmd -bAr - reference.fa > aligned.bam

samtools sort aligned.bam -o aligned_sorted.bam