there are not enough pairs message on screen when running BWA MEM

Question

[S AR]

[M::mem_pestat] low and high boundaries for proper pairs: (150, 353)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_process_seqs] Processed 99010 reads in 5.077 CPU sec, 4.721 real sec
[M::process] read 99010 sequences (10000010 bp)...
[M::process] read 99010 sequences (10000010 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (5, 46755, 5, 4)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (236, 252, 266)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (176, 326)
[M::mem_pestat] mean and std.dev: (252.49, 25.15)
[M::mem_pestat] low and high boundaries for proper pairs: (146, 356)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 99010 reads in 5.145 CPU sec, 4.968 real sec
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (10, 46811, 3, 2)
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (71, 126, 140)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 278)
[M::mem_pestat] mean and std.dev: (113.80, 56.09)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 347)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (236, 252, 266)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (176, 326)
[M::mem_pestat] mean and std.dev: (252.50, 25.34)
[M::mem_pestat] low and high boundaries for proper pairs: (146, 356)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_process_seqs] Processed 99010 reads in 5.736 CPU sec, 5.375 real sec
[M::process] read 78246 sequences (7902846 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (2, 36852, 8, 2)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (236, 252, 266)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (176, 326)
[M::mem_pestat] mean and std.dev: (252.51, 25.14)
[M::mem_pestat] low and high boundaries for proper pairs: (146, 356)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 78246 reads in 3.975 CPU sec, 3.678 real sec
[main] Version: 0.7.15-r1140
[main] CMD: bwa mem -M -R @RG\tID:HWI-ST610\tLB:D0DELACXX\tPL:ILLUMINA\tPM:HISEQ2000\tSM:Pool22 ../Reference/MTB.fa M_tuberculosis__Pool22_X60_1048__L6_ACTTGA_L006_R1.fastq M_tuberculosis__Pool22_X60_1048__L6_ACTTGA_L006_R2.fastq
[main] Real time: 926.964 sec; CPU: 905.573 sec

All of my reads having this message while aligning that there are not enough pairs. However, I have checked it everything is fine in the files. I ran the following command:

bwa mem \
  -M \
  -R '@RG\tID:HWI-ST610\tLB:D0DELACXX\tPL:ILLUMINA\tPM:HISEQ2000\tSM:Pool22' \
  ../Reference/MTB.fa M_tuberculosis__Pool22_X60_1048__L6_ACTTGA_L006_R1.fastq \
  M_tuberculosis__Pool22_X60_1048__L6_ACTTGA_L006_R2.fastq >  Pool22_X60_1048__L6.sam

Following is the output of the sam generated:

HWI-ST610:135:D0DELACXX:6:1101:2078:1958        99      AL123456.3      636003  60      101M
HWI-ST610:135:D0DELACXX:6:1101:2078:1958        147     AL123456.3      636141  60      101M
HWI-ST610:135:D0DELACXX:6:1101:2723:1941        77      *       0       0       *       *
HWI-ST610:135:D0DELACXX:6:1101:2723:1941        141     *       0       0       *       *
HWI-ST610:135:D0DELACXX:6:1101:2962:1941        83      AL123456.3      3869093 60      101M
HWI-ST610:135:D0DELACXX:6:1101:2962:1941        163     AL123456.3      3868962 60      101M
HWI-ST610:135:D0DELACXX:6:1101:3858:1995        77      *       0       0       *       *
HWI-ST610:135:D0DELACXX:6:1101:3858:1995        141     *       0       0       *       *
HWI-ST610:135:D0DELACXX:6:1101:4561:1958        83      AL123456.3      705624  60      101M
HWI-ST610:135:D0DELACXX:6:1101:4561:1958        163     AL123456.3      705476  40      81M20S
HWI-ST610:135:D0DELACXX:6:1101:4968:1941        99      AL123456.3      2427899 60      101M
HWI-ST610:135:D0DELACXX:6:1101:4968:1941        147     AL123456.3      2427966 60      101M
HWI-ST610:135:D0DELACXX:6:1101:5737:1941        99      AL123456.3      1394131 60      101M
HWI-ST610:135:D0DELACXX:6:1101:5737:1941        147     AL123456.3      1394270 60      101M
HWI-ST610:135:D0DELACXX:6:1101:5603:1944        99      AL123456.3      6566    60      101M

Should I be worried? as I think sam output is fine?

[Bastien Hervé]

Note that the "tool section" is dedicated to highlight a new tool not for asking question about one

Answer 1

[Bastien Hervé]

Please try to format your post with appropriate code sample and blockquote, this is quite unreadable.

About your question, it seems like these are log messages not error messages, so you should be fine with your sam output

See also Pierre's answer here

[lieven.sterck]

and remove the 'tool' tag from the questions, that's not the appropriate use of it neither

[S AR]

I did. Please see now. Thankyou

[lieven.sterck]

As Bastien Hervé says you will be fine with the output you got.

It merrily reports that any of the 3 alternative read pair orientations are not really present in you dataset (FR is the expected one) , so this is actually good news, given that you are analyzing a normal paired end dataset

[S AR]

Oh. Okay Thank you for providing this information.