I am trying to extract unaligned reads from bam files and then realign them back to hg38. Here are the commands for these steps:
samtools view -@ 16 -b -F 2 $bamfile | samtools sort -@ 16 -n - | samtools fastq - -1 unaligned.fq1 -2 unaligned.fq2
bwa aln -k 3 -n 3 -t 16 -i 20 hg38.fa unaligned.fq1 > 1.sai
bwa aln -k 3 -n 3 -t 16 -i 20 hg38.fa unaligned.fq2 > 2.sai
bwa sampe hg38.fa 1.sai 2.sai unaligned.fq1 unaligned.fq2 | samtools view -b -@ 16 - | samtools sort -@ 16 - > realigned.bam
samtools index realigned.bam
In the last step where I tried to align unaligned.fq1 and unaligned.fq2 back to hg38, the output sam file keep having this error:
[W::sam_parse1] mapped query cannot have zero coordinate; treated as unmapped
I have tried to update to the latest version of bwa (0.7.17) but it didn't fix the problem. And the samtools I used is samtools/1.16.1.
Is this a problem with bwa or is this a problem with samtools? Or something else that I don't know about?