Dear all, I have samples sequenced with Illumina TruSight One panel on a MiSeq sequencer. We are interested in variants on SHOX gene that is located on the pseudo-autosomic region on chrX and chrY. I used BWA mem for the alignments and the reference is the hg19 downloaded from UCSC:

bwa mem -t 2 -M hg19 R1.fastq.gz R2.fastq.gz | samblaster | samtools view -Sb - | samtools sort - -T sample.tmp -o sample_sorted.bam

I indexed the genome using only the canonical chromosomes and I verified that the pseudo-autosomic region on chrY is masked in the fasta file with N. I expected to have only alignments on chrX, but I have alignments on both chromosome and all of them are with mapping quality 0. As consequence I do not have variant calls on the SHOX gene. As test I excluded the chrY from the genome, re-indexed the genome and performed again the alignments. As result I have reads correctly mapped on SHOX chrX with good quality score.

So my questions are: Why is it happening if the region is masked? Is there a specific parameter that I have to use with bwa? Have anyone of you the same problem? Am I doing something wrong?

My apologies if the question was already asked, but I searched a lot and I didn't find something that fitted with my problem.

Any suggestion is appreciated. Thanks, Stefania