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Old 11-21-2012, 12:02 AM   #1
stylz2k
Junior Member
 
Location: Germany

Join Date: Nov 2012
Posts: 8
Default Trouble with bwa sampe

Hi guys,

i use bwa to align Whole Human Genome Data (Paired End Data from Ilumina). I have 2 Datasets. One is working one isnt.

I used bwa 0.6.1 and 0.6.2 but get stucked at the same point.

Here is what I have done so far:

Index:

bwa index -a bwtsw -p hg19 hg19.fa

Alignment:

bwa aln -t 4 hg19 S2_R1.fastq > S2_R1.sai
bwa aln -t 4 hg19 S2_R2.fastq > S2_R2.sai

Now i wanted to use bwa sampe to generate a sam file of the two Alignments:

bwa sampe hg19 S2_R1.sai S2_R2.sai S2_R1.fastq S2_R2.fastq > S2_alignment.sam

And here is my Problem:

bwa runs for about 6 or 7 hours and then gets stucked at a certain point

Code:
...
[bwa_paired_sw] 18708 out of 20824 Q17 singletons are mated.
[bwa_paired_sw] 94 out of 2476 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 5.62 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.46 sec
[bwa_sai2sam_pe_core] print alignments... 1.32 sec
[bwa_sai2sam_pe_core] 38273024 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 249)
[infer_isize] low and high boundaries: 97 and 389 for estimating avg and std
[infer_isize] inferred external isize from 231530 pairs: 217.078 +/- 51.705
[infer_isize] skewness: 0.648; kurtosis: 0.139; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 576 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.44 sec
[bwa_sai2sam_pe_core] changing coordinates of 997 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 17247 out of 19212 Q17 singletons are mated.
[bwa_paired_sw] 97 out of 2585 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 5.30 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.47 sec
[bwa_sai2sam_pe_core] print alignments... 1.33 sec
[bwa_sai2sam_pe_core] 38535168 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 249)
[infer_isize] low and high boundaries: 97 and 389 for estimating avg and std
[infer_isize] inferred external isize from 233040 pairs: 217.053 +/- 51.725
[infer_isize] skewness: 0.657; kurtosis: 0.157; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 577 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.24 sec
[bwa_sai2sam_pe_core] changing coordinates of 1043 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 15045 out of 17028 Q17 singletons are mated.
[bwa_paired_sw] 111 out of 2590 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 4.85 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.45 sec
[bwa_sai2sam_pe_core] print alignments... 1.32 sec
[bwa_sai2sam_pe_core] 38797312 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 249)
[infer_isize] low and high boundaries: 97 and 389 for estimating avg and std
[infer_isize] inferred external isize from 234891 pairs: 216.851 +/- 51.611
[infer_isize] skewness: 0.650; kurtosis: 0.153; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 576 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.59 sec
[bwa_sai2sam_pe_core] changing coordinates of 1105 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 14812 out of 16736 Q17 singletons are mated.
[bwa_paired_sw] 110 out of 2525 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 4.73 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.46 sec
[bwa_sai2sam_pe_core] print alignments... 1.33 sec
[bwa_sai2sam_pe_core] 39059456 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 249)
[infer_isize] low and high boundaries: 97 and 389 for estimating avg and std
[infer_isize] inferred external isize from 234091 pairs: 216.650 +/- 51.588
[infer_isize] skewness: 0.652; kurtosis: 0.144; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 575 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.37 sec
[bwa_sai2sam_pe_core] changing coordinates of 1071 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 15456 out of 17430 Q17 singletons are mated.
[bwa_paired_sw] 108 out of 2569 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 4.92 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.46 sec
[bwa_sai2sam_pe_core] print alignments... 1.33 sec
[bwa_sai2sam_pe_core] 39321600 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 249)
[infer_isize] low and high boundaries: 97 and 389 for estimating avg and std
[infer_isize] inferred external isize from 231644 pairs: 216.603 +/- 51.586
[infer_isize] skewness: 0.661; kurtosis: 0.171; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 575 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.49 sec
[bwa_sai2sam_pe_core] changing coordinates of 1105 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 17707 out of 19720 Q17 singletons are mated.
[bwa_paired_sw] 105 out of 2476 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 5.36 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.46 sec
[bwa_sai2sam_pe_core] print alignments... 1.33 sec
[bwa_sai2sam_pe_core] 39583744 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 249)
[infer_isize] low and high boundaries: 97 and 389 for estimating avg and std
[infer_isize] inferred external isize from 231864 pairs: 216.571 +/- 51.411
[infer_isize] skewness: 0.658; kurtosis: 0.166; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 574 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.47 sec
[bwa_sai2sam_pe_core] changing coordinates of 1109 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 17254 out of 19247 Q17 singletons are mated.
[bwa_paired_sw] 104 out of 2568 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 5.32 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.45 sec
[bwa_sai2sam_pe_core] print alignments... 1.35 sec
[bwa_sai2sam_pe_core] 39845888 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (179, 210, 248)
[infer_isize] low and high boundaries: 97 and 386 for estimating avg and std
[infer_isize] inferred external isize from 134785 pairs: 216.212 +/- 51.171
[infer_isize] skewness: 0.645; kurtosis: 0.146; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 572 (6.95 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.53 sec
[bwa_sai2sam_pe_core] changing coordinates of 574 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 11908 out of 21661 Q17 singletons are mated.
[bwa_paired_sw] 71 out of 100413 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 32.24 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.45 sec
[bwa_sai2sam_pe_core] print alignments... 1.26 sec
[bwa_sai2sam_pe_core] 40108032 sequences have been processed.
[bwa_sai2sam_pe_core] convert to sequence coordinate... 
[infer_isize] (25, 50, 75) percentile: (26201, 48608, 61867)
[infer_isize] low and high boundaries: 97 and 133199 for estimating avg and std
[infer_isize] inferred external isize from 48 pairs: 46035.354 +/- 27108.104
[infer_isize] skewness: 0.172; kurtosis: -0.844; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 210582 (6.07 sigma)
[bwa_sai2sam_pe_core] time elapses: 8.41 sec
[bwa_sai2sam_pe_core] changing coordinates of 5 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
Here i have a 100 % CPU usage on one core and 13.7 memory usage and nothing happens anymore. The generated samfile is about 27.9 gb big. This output is from bwa-0.6.2 but i get the same on 0.6.1 and the samfile has the same size.

Any suggestions ?
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Old 11-21-2012, 03:00 AM   #2
xied75
Senior Member
 
Location: Oxford

Join Date: Feb 2012
Posts: 129
Default

You are really very patient to wait 6,7 hours for an error.

If you look the last 8 lines, you'll notice the inferred insert size is wrong. I guess your fastq is combined from several small files, somewhere after 40 million rows, the pair no longer match.

If you can open the fastq and have a look around that region, see if you can spot anything strange, like an empty line, corrupt line, etc.

And if you want test again, just cut 1 million rows from each fastq after 40108032 and ALN and SAMPE on those again.

Best,

dong
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Old 11-21-2012, 03:12 AM   #3
stylz2k
Junior Member
 
Location: Germany

Join Date: Nov 2012
Posts: 8
Default

Thanks again xied75!

You are really awesome

I had an mistake in combining my fastq files...

I Think this will solve my problems, for now

Thank you very much!

Best,

stylz2k
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