I wish to call SNPs using SAMtools mpileup function. I first map my Illumina paired end reads to the reference using Bowtie and then I have custom scripts that filter out non-uniquely mapping reads. To do this I rely on the absence of XS to indicate a uniquely mapped read.
However, this creates a potential problem as the SAM flags are often no longer meaningful. Eg. Originally I have two reads that are paired and both map Forward read flag 83 Reverse read flag 163 If Forward read is not uniquely mapping then I remove this from the sam file, leaving Reverse read intact. The flag of Reverse read is not changed and therefore now falsely indicates it has a mapped pair. This isn't a problem if my downstream programs do not use the flag. Therefore, I wanted to check, does the mpileup function read/use the flags in the sam file? If so, is there another way to only use uniquely mapping reads to call SNPs. Thanks very much
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