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
•
written
by
richard.deborja
The reads are indeed properly paired. In one case you have:
read1---->
<---read2
and in the other
read2---->
<---read1
In both cases, the reads are pointing toward each other, meaning that they're in the appropriate orientation.
•
written
by
Devon Ryan
If sequencing is done 5' to 3', wouldn't the orientation be:
<--- read1
read2--->
In the cases where read1 is reverse complemented, it's 3' of read2.
Log in to answer this question.
Hello richard.deborja!
It appears that your post has been cross-posted to another site: http://seqanswers.com/forums/showthread.php?t=44081
This is typically not recommended as it runs the risk of annoying people in both communities.
Hello richard.deborja!
It appears that your post has been cross-posted to another site: Apparently cross posting is a bad idea, since I'm unable to delete/close the seqanswers post, closing this thread.
This is typically not recommended as it runs the risk of annoying people in both communities.
don't close the thread. That's ok. But in the future avoid to post on both sites within a short period or without linking to the cross-posts.