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Skip orientation RF

Hi, I'm using BWA to align some exome sequences:

/home/Programas/bwa-0.7.10/bwa index -p genome -a bwtsw /local/Referencias/Homo_sapiens/UCSC/hg19/Sequence/BWAIndex/version0.7.10-r789/genome.fa
/home/Programas/bwa-0.7.10/bwa mem -t 10 /local/Referencias/Homo_sapiens/UCSC/hg19/Sequence/BWAIndex/version0.7.10-r789/genome /local/CLL/Raw_data/MONONUCLEARCells_R1.fastq.gz /local/CLL/Raw_data/MONONUCLEARCells_R2.fastq.gz > /local/CLL/Processing/FGO/Alignment/FGO_20070115_MONONUCLEARCells.sam

These are the "warnings" shown by bwa.

[M::main_mem] read 1315790 sequences (100000040 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (65, 570757, 14, 19)
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (80, 109, 176)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 368)
[M::mem_pestat] mean and std.dev: (127.23, 64.83)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 464)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (166, 200, 248)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (2, 412)
[M::mem_pestat] mean and std.dev: (210.69, 60.88)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 494)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (79, 194, 389)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1009)
[M::mem_pestat] mean and std.dev: (242.92, 225.64)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 1319)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (104, 150, 224)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 464)
[M::mem_pestat] mean and std.dev: (157.95, 63.90)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 584)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR

And

[M::mem_pestat] low and high boundaries for proper pairs: (1, 499)
[M::mem_pestat] skip orientation RF as there are not enough pairs

Should I be worried (about the skip orientation...). This is the first time I have seen this output.

Thanks one more time.

skip rf bwa orientation exome

2 answers

This is normal. The Illumina reads orientation is FR. You got majority FR and skipped other three.

If there is an inversion, might that be interpreted by the aligner as, say, FF orientation, and should therefore be included in SAM/BAM rather than being skipped?

Unless you have mate pairs, they shouldn't face away from each other (aka, have an RF orientation). This is expected with illumina sequencing.

I do have illumina data.

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