High Mapping but No Reads Properly Paired

Question

[pld]

After mapping PE reads with bwa-mem, samtools flagstat gives me this output:

53681238 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
779896 + 0 supplementary
0 + 0 duplicates
48364704 + 0 mapped (90.10% : N/A)
52901342 + 0 paired in sequencing
26450671 + 0 read1
26450671 + 0 read2
0 + 0 properly paired (0.00% : N/A)
47584808 + 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)

Viewing this in IGV shows all reads mapping in FF or RR.

I've never seen something like this before, any suggestions?

[GenoMax]

Any chance the data files are mislabeled? Do the headers look right?

[pld]

I don't think so. I'll double check.

[Pierre Lindenbaum]

did you reverse-complement one of your fastq files ?

[pld]

Not to my knowledge. Any chance this could have happened pulling the data off the machine?

[GenoMax]

No. It will have to be done consciously.

[Dan D]

Can you show us your bwa command line?

[pld]

I'll just save myself the embarrassment: I supplied the forward read file twice.

I've been sitting here scratching my head and checking everything but the command I ran. Today is not my day.

[Dan D]

Take comfort that each of us reading this thread will have a "not my day" day soon.

[rohenriques]

Hi! Can I resurrect this thread? I'm having the same problem, and I didn't supply the same file twice... Also, everything worked for the other populations I'm using, just one failed...

BWA command line: bwa mem -R '@RG\tID:pop1\tSM:PCN\tLB:library1' referenceP30 PCN_R1_30G.fastqsanger PCN_R2_30G.fastqsanger -a -t 16 -T 10 > PCN30.sam