I have been running bowtie2 on paired end sequences and noticed something funny when I tried to filter out reads that weren't properly paired. I seemed to be getting orphan reads left behind after filtering with
samtools view -f 2. After digging around, I noticed that after the initial alignment, I was getting reads that were flagged
NS500418:295:HWG3TBGXX:4:11405:20193:10248 163 chr1 1624577 255
NS500418:295:HWG3TBGXX:4:11405:20193:10248 113 chr1 1677965 255
samtools fixmate changes the flags to
81 , but that still leaves the bit 2 unaffected.
As I understand it, it seems like this means that one of the two is "read mapped in proper pair" as indicated by flag 2, but the other one isn't. Am I failing to understand something about what flag2 means?
Here is the bowtie command I ran:
bowtie2 -k1 -N1 -p16 -x hg19 -1 R1.fastq -2 R2.fastq -S file.sam
Any thoughts or help would be appreciated! Thanks!