One of two mates listed as "properly paired" in Bowtie2

Question

[aredash]

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 163 and 113.

NS500418:295:HWG3TBGXX:4:11405:20193:10248 163 chr1 1624577 255

NS500418:295:HWG3TBGXX:4:11405:20193:10248 113 chr1 1677965 255

Running samtools fixmate changes the flags to 163 and 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!

Answer 1

[Devon Ryan]

Looks like you found a bug, please report it on github.

BTW, you used the "RNA-seq" tag, so I assume you're using this for that purpose. If so, please be aware that this is not an appropriate tool for mammalian RNAseq data. Please use STAR or another splice-aware aligner instead.

[aredash]

Thanks Devon. I also removed the rna-seq tag.