Skip orientation RF

Question

[Folder40g]

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.

Answer 1

[wzchang]

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

[bdolin]

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?

Answer 2

[Devon Ryan]

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

[Folder40g]

I do have illumina data.