BWA: paired end reads, wrong orientation but listed as properly paired

[rdeborja]

I have a set of BAM files for a targeted sequencing experiment. After reviewing things in IGV, I noticed that a pair of reads listed as orientation F2R1 are labelled as properly paired. I also noticed another pair listed as F1R2 that were also labelled as properly paired. From my understanding of paired-end sequencing, F1R2 is the correct orientation and F2R1 would be more consistent for mate-pair. The version of BWA used was 0.6.2 and Picard 1.107 for post-processing (conversion to BAM, sorting, indexing, read group addition). I confirmed alignment to the reference genome using BLAT and identified the reads in the original FASTQ files for read 1 and read 2. Any idea why it would list both orientations as properly paired?

I'm including the SAM entries below with modified read IDS:

17091 163 chrX 48649459 60 151M = 48649634 326 AAGGATTTCTGTGTCTGAGGACCCCTTCTGTCCTCGCAGGTTAATCCCCAGAGGCTCCAGGGAGTTCCCTGGCCTGGGGTCCCTGGGGACCTCAGAGCCCCTCCCCCAGTTTGTGGATCCTGCTCTGGTGTCCTCCACACCAGAATCAGGG @CCFFFFFHHHHHJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJJJJJHHHFFFFFECCEEDBBDDDDDDDDDDDDDDCDDDC@CDDDDDDDDDDDDDED?CBDDDDDCCCDDDDCDDDDDDDDDDDDDB?CADCCCD X0:i:1 X1:i:0 MD:Z:59T91 RG:Z:8932118E-CCA7-11E3-9394-35788F63E160 XG:i:0 AM:i:37 NM:i:1 SM:i:37 XM:i:1 XO:i:0 XT:A:U
49588 99 chrX 48649459 60 151M = 48649532 224 AAGGATTTCTGTGTCTGAGGACCCCTTCTGTCCTCGCAGGTTAATCCCCAGAGGCTCCAGGGAGTTCCCTGGCCTGGGGTCCCTGGGGACCTCAGAGCCCCTCCCCCAGTTTGTGGATCCTGCTCTGGTGTCCTCCACACCAGAATCAGGG CCCFFFFFHHHHHJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJHJJJHHJJJJJHHHFFFFFFEDEEDDDDDBDDDDDDDDDDDDDDDDCCDDDDDDDDDBDDDEDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDBDDDDDDD8 X0:i:1 X1:i:0 MD:Z:59T91 RG:Z:8932118E-CCA7-11E3-9394-35788F63E160 XG:i:0 AM:i:37 NM:i:1 SM:i:37 XM:i:1 XO:i:0 XT:A:U
49588 147 chrX 48649532 60 151M = 48649459 -224 CTGGGGTCCCTGGGGACCTCAGAGCCCCTCCCCCAGTTTGTGGATCCTGCTCTGGTGTCCTCCACACCAGAATCAGGGGTTTTCTTCCCCTCTGGGCCTGAGGGCTTGGATGCAGCAGCTTCCTCCACTGCCCCGAGCACAGCCACCGCTG DDDDDDDDDDDDDDCCDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDBBADDDDDDDDDDDDCADEDEDDDDDDDDDDDDDDDDDDDDDDDDFFFFHHHHJJIJJJJJJJJJJJJIJJJJJJJIIJJJJJJJJJJJHHHHHFFFFFCCC X0:i:1 X1:i:0 MD:Z:151 RG:Z:8932118E-CCA7-11E3-9394-35788F63E160 XG:i:0 AM:i:37 NM:i:0 SM:i:37 XM:i:0 XO:i:0 XT:A:U
17091 83 chrX 48649634 60 151M = 48649459 -326 GGCTTGGATGCAGCAGCTTCCTCCACTGCCCCGAGCACAGCCACCGCTGCAGCTGCGGCACTGGCCTACTACAGGGACGCTGAGGCCTACAGACACTCCCCAGGTAACTCCATTGAGTGGCTGTCTTGGCATTGGCTGAGTGCTGTTGGGG 8DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDBBDDDDDDDDDDDDDDDDDDDDDDEFEDDDDDDDDDDEFFFFFHHHHHHJIIHHJJIJJJJJJJIJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJHHHHHFFFFFCCC X0:i:1 X1:i:0 MD:Z:151 RG:Z:8932118E-CCA7-11E3-9394-35788F63E160 XG:i:0 AM:i:37 NM:i:0 SM:i:37 XM:i:0 XO:i:0 XT:A:U

[Brian Bushnell]

There's nothing wrong with F2R1 or F1R2. The strand of read 1 is random, and the strand of read 2 should be opposite. What's important is that the forward-mapped read is to the left of the reverse-mapped read (for insert sizes longer than read length), not which read maps forward and which maps reverse.

[dpryan]

Cross-posted on biostars, where I already answered (note, they should be listed as properly paired).

[rdeborja]

There's nothing wrong with F2R1 or F1R2. The strand of read 1 is random, and the strand of read 2 should be opposite. What's important is that the forward-mapped read is to the left of the reverse-mapped read (for insert sizes longer than read length), not which read maps forward and which maps reverse.

Thanks for the info. No clue why I didn't see that in the first place. After confirming the left most and right most reads, the orientation of F2R1 as properly paired clicked in. Thanks again!