I had a similar question about this last week but wasn't super clear about what I was asking. I have some old bam files that I don't have the original fastq files for. I tried using picard Samtofastq but I am having an issues when I realign the reads. I have been using picard to revert the old bam files back to fastq files, bwa to align the reads, and picard to sort and deduplicate. I used samtools flagstat to check out what was going on with my new bam files and this is what I got
473763117 + 0 in total (QC-passed reads + QC-failed reads) 1618429 + 0 secondary 0 + 0 supplementary 24690025 + 0 duplicates 473247331 + 0 mapped (99.89% : N/A) 472144688 + 0 paired in sequencing 236072344 + 0 read1 236072344 + 0 read2 466910892 + 0 properly paired (98.89% : N/A) 471430496 + 0 with itself and mate mapped 198406 + 0 singletons (0.04% : N/A) 3805718 + 0 with mate mapped to a different chr 2458472 + 0 with mate mapped to a different chr (mapQ>=5)
I have checked out my files and it doesn't look like it is an issue with bwa or picard sort/deduplicate. The only thing I can think of is that there is some issue with picard samtofastq. So the issue is that samtools
mpileup isn't calling any variants for those particular samples. All the variants are "no calls" or ./. and I am not sure why this is happening? I know it's not an issue with samtools
mpileup because it works fine with all my other samples, it is just those old files that are giving me an issue. I know it is just some minor issue but I am just not sure where to look.
This is the command I use
java -jar picard.jar SamToFastq I=C01767.bam FASTQ=C01767.R1.fastq SECOND_END_FASTQ=C01767.R2.fastq
Am I doing something wrong here?
I know GATK has a way to do this, but their method only give you one files here kind of want both of the fastq files