bwaSampe command and the output error message

[febybaby]

Hi All,

I am trying to execute the bwaSampe stage. Please find the details below:

Command:

parallel -a /.lustre02/knome-pd/knome-isys/user-krinard/importTest/200000009/200000009/pair1.txt -a /.lustre02/knome-pd/knome-isys/user-krinard/importTest/200000009/200000009/pair2.txt -a /.lustre02/knome-pd/knome-isys/user-krinard/importTest/200000009/200000009/readGroups.txt -u "/knome-pd/babraham/isys/ksuite/software/bwa/bwa sampe -r \"@RGID:{3}SM:200000009LB:200000009PL:illumina\" /.lustre02/knome-pd/refdata/refgenome/hg19/human_hg19_full.fasta /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/sai/{1}.sai /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/sai/{2}.sai /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/splitFq/{1}.fq /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/splitFq/{2}.fq | samtools view -buhSo /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/ /bam/{1}.bam -"


Error:

[bwa_sai2sam_pe] malformated @RG line
[bwa_sai2sam_pe] malformated @RG line
[main_samview] fail to open "/bam/wo209sg2932_L1_R2_001-0000.bam" for reading.
[bwa_sai2sam_pe] malformated @RG line
[main_samview] fail to open "/bam/wo209sg2932_L1_R2_001-0002.bam" for reading.
[main_samview] fail to open "/bam/wo209sg2932_L1_R2_001-0003.bam" for reading.
[main_samview] fail to open "/bam/wo209sg2932_L1_R2_001-0001.bam" for reading.
[bwa_sai2sam_pe] malformated @RG line

I am trying to figure this out. Can someone help me to figure out the error.

[GenoMax]

Are these bam files available in "/bam" directory? Does your account have read permissions on that directory?

Also the read group argument may need single quotes instead of double.

In the argument listing:
-r STR Specify the read group in a format like ‘@RG\tID:foo\tSM:bar’. [null]

[febybaby]

Thank you for the reply

I have modified the command as below:

parallel -a /.lustre02/knome-pd/knome-isys/user-krinard/importTest/200000009/200000009/pair1.txt -a /.lustre02/knome-pd/knome-isys/user-krinard/importTest/200000009/200000009/pair2.txt -a /.lustre02/knome-pd/knome-isys/user-krinard/importTest/200000009/200000009/readGroups.txt -u "/knome-pd/babraham/isys/ksuite/software/bwa/bwa sampe -r '@RG\tID:{3}\tSM:200000009\tLB:200000009\tPL:illumina' /.lustre02/knome-pd/refdata/refgenome/hg19/human_hg19_full.fasta /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/sai/{1}.sai /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/sai/{2}.sai /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/splitFq/{1}.fq /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/splitFq/{2}.fq | samtools view -buhSo /knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/bam/{1}.bam -"

The bwa Sampe stage is progressing and I am getting an error like

[main_samview] fail to open "/knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/bam/wo209sg2932_L1_R2_001-0002.bam" for writing.

Can you please help me to figure this out. I am new to the bio-informatics domain. In one of the forum i have got an input to use -S along with "samtools view ".

http://seqanswers.com/forums/showthread.php?t=6270

I have modified this command. But still I am getting the same error.

output:

[bwa_read_seq] 3.3% bases are trimmed.
[bwa_read_seq] 4.2% bases are trimmed.
[bwa_read_seq] 4.8% bases are trimmed.
[bwa_read_seq] 7.0% bases are trimmed.
[bwa_read_seq] 2.4% bases are trimmed.
[bwa_sai2sam_pe_core] convert to sequence coordinate...
[bwa_read_seq] 3.6% bases are trimmed.
[bwa_sai2sam_pe_core] convert to sequence coordinate...
[bwa_read_seq] 2.7% bases are trimmed.
[bwa_sai2sam_pe_core] convert to sequence coordinate...
[bwa_read_seq] 3.8% bases are trimmed.
[bwa_sai2sam_pe_core] convert to sequence coordinate...
[infer_isize] (25, 50, 75) percentile: (158, 188, 228)
[infer_isize] low and high boundaries: 100 and 368 for estimating avg and std
[infer_isize] inferred external isize from 208735 pairs: 195.839 +/- 51.791
[infer_isize] skewness: 0.728; kurtosis: 0.210; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 556 (6.96 sigma)
[infer_isize] (25, 50, 75) percentile: (158, 189, 230)
[infer_isize] low and high boundaries: 100 and 374 for estimating avg and std
[infer_isize] inferred external isize from 207559 pairs: 197.094 +/- 52.652
[infer_isize] skewness: 0.734; kurtosis: 0.228; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 564 (6.96 sigma)
[infer_isize] (25, 50, 75) percentile: (156, 186, 225)
[infer_isize] low and high boundaries: 100 and 363 for estimating avg and std
[infer_isize] inferred external isize from 209165 pairs: 193.818 +/- 50.740
[infer_isize] skewness: 0.729; kurtosis: 0.229; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 547 (6.96 sigma)
[infer_isize] (25, 50, 75) percentile: (156, 185, 223)
[infer_isize] low and high boundaries: 100 and 357 for estimating avg and std
[infer_isize] inferred external isize from 206995 pairs: 192.334 +/- 49.533
[infer_isize] skewness: 0.716; kurtosis: 0.197; ap_prior: 1.00e-05
[infer_isize] inferred maximum insert size: 537 (6.96 sigma)
[bwa_sai2sam_pe_core] time elapses: 443.82 sec
[bwa_sai2sam_pe_core] changing coordinates of 6364 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_sai2sam_pe_core] time elapses: 444.28 sec
[bwa_sai2sam_pe_core] changing coordinates of 6300 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_sai2sam_pe_core] time elapses: 503.58 sec
[bwa_sai2sam_pe_core] changing coordinates of 6328 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_sai2sam_pe_core] time elapses: 471.52 sec
[bwa_sai2sam_pe_core] changing coordinates of 6550 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 4526 out of 6182 Q17 singletons are mated.
[bwa_paired_sw] 668 out of 9071 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 220.26 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 1.35 sec
[bwa_sai2sam_pe_core] print alignments... [samopen] SAM header is present: 93 sequences.
[main_samview] fail to open "/knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/bam/wo209sg2932_L1_R2_001-0000.bam" for writing.
[bwa_paired_sw] 3767 out of 5137 Q17 singletons are mated.
[bwa_paired_sw] 644 out of 8659 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 200.99 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 1.34 sec
[bwa_sai2sam_pe_core] print alignments... [samopen] SAM header is present: 93 sequences.
[main_samview] fail to open "/knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/bam/wo209sg2932_L1_R2_001-0003.bam" for writing.
[bwa_paired_sw] 6003 out of 7594 Q17 singletons are mated.
[bwa_paired_sw] 778 out of 7746 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 195.23 sec
[bwa_sai2sam_pe_core] refine gapped alignments... [bwa_paired_sw] 3760 out of 5077 Q17 singletons are mated.
[bwa_paired_sw] 688 out of 8219 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 240.79 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 1.39 sec
[bwa_sai2sam_pe_core] print alignments... [samopen] SAM header is present: 93 sequences.
[main_samview] fail to open "/knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/bam/wo209sg2932_L1_R2_001-0001.bam" for writing.
1.43 sec
[bwa_sai2sam_pe_core] print alignments... [samopen] SAM header is present: 93 sequences.
[main_samview] fail to open "/knome-pd/scratch/dev-ksuite-100000006-20130521-133318658-000-AlignURS/bam/wo209sg2932_L1_R2_001-0002.bam" for writing.

[bishwo]

Is output directory writable ?

[febybaby]

Now the error is fixed. I have run the command after creating the "bam" folder and the .bam files are created successfully.

Can I create the folder "bam" along with this command.?

[bishwo]

I do not think that it will create folder.