Pysam not finding mapped reads in BAM file

Question

[taraeicher]

I have a BAM file with the following stats from samtools flagstat:

8022676 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
1336910 + 0 duplicates
8022676 + 0 mapped (100.00% : N/A)
8022676 + 0 paired in sequencing
4011338 + 0 read1
4011338 + 0 read2
8022676 + 0 properly paired (100.00% : N/A)
8022676 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

However, when I run the following code interactively in python:

import pysam
samfile = pysam.AlignmentFile("mybam.bam", "rb")
samfile.mapped

it's printing out 0. Interestingly, it does the same if I try to print the unmapped reads. Is there something that pysam looks for in a BAM file that is different from what samtools flagstat does? If so, is there any way I can convert my BAM file into a format that is compatible with pysam?

Answer 1

[taraeicher]

After some investigating, I found that it was the index to the BAM file that was causing the problem. I generated this index using bamtools index. Given that this happened with several of my files which were indexed in this way, it looks like there may be a compatibility issue between my version of bamtools (2.2.2) and pysam.

I'm still not sure exactly where the bug is, but I was able to run the following instead of bamtools index as a workaround:

import pysam
samfile = pysam.index("mybam.bam")