I did. Please see now. Thankyou
[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?
1 answer
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
Oh. Okay Thank you for providing this information.
and remove the 'tool' tag from the questions, that's not the appropriate use of it neither
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