opera error bwa mapping

[H1889]

Hi. When running an analysis with opera-ms an error is produced:

Error in during bwa mapping.

I hace checked /intermediate_files/polished_assembly/bwa.err, and this is shown:

[bam_sort] Use -T PREFIX / -o FILE to specify temporary and final output files
Usage: samtools sort [options...] [in.bam]
Options:
-l INT Set compression level, from 0 (uncompressed) to 9 (best)
-m INT Set maximum memory per thread; suffix K/M/G recognized [768M]
-n Sort by read name
-t TAG Sort by value of TAG. Uses position as secondary index (or read name if -n is set)
-o FILE Write final output to FILE rather than standard output
-T PREFIX Write temporary files to PREFIX.nnnn.bam
--no-PG do not add a PG line
--input-fmt-option OPT[=VAL]
Specify a single input file format option in the form
of OPTION or OPTION=VALUE
-O, --output-fmt FORMAT[,OPT[=VAL]]...
Specify output format (SAM, BAM, CRAM)
--output-fmt-option OPT[=VAL]
Specify a single output file format option in the form
of OPTION or OPTION=VALUE
--reference FILE
Reference sequence FASTA FILE [null]
-@, --threads INT
Number of additional threads to use [0]
--verbosity INT
Set level of verbosity
[M::main_mem] read 1117024 sequences (320000554 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 0, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[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 1117024 reads in 5923.134 CPU sec, 494.192 real sec

And the /intermediate_files/polished_assembly/bwa.out is empty

thank you in advance

[jsgounot]

Hi,

very interesting, it looks like it's similar to issue #67. I still don't know what's happening here. I will come back to you as soon as I know more. Please keep me updated if you find something on your own.

Could you please indicate to me which perl you're using?

regards,
jsgounot

[H1889]

Thanks for the fast reponse, my machine is Ubuntu 20.04.3 LTS (GNU/Linux 4.15.0-147-generic x86_64

and perl -V:
Summary of my perl5 (revision 5 version 30 subversion 0) configuration:

Platform:
osname=linux
osvers=4.19.0
archname=x86_64-linux-gnu-thread-multi
uname='linux localhost 4.19.0 #1 smp debian 4.19.0 x86_64 gnulinux '

best

[emblab-westlake]

Hi,
Has this problem been solved? I look forward to your sharing your experience.
best wishes

[jsgounot]

Hi, I'm slowly working on a global rewritting of the software and I didn't precisely find the origin of this issue. It looks like this is related to BWA version, which might fail now with some specific reads formatting. A potential fix would be to use a new BWA version (or minimap2) and link it to OPERA-MS by modifying the bwa path in the OPERA-MS directory (with a softlink for example). Sorry to not be able to provide a better solution right now.

[cyhofe]

Hi everyone,
I encountered the same problem and wanted to know if there is a solution to this problem by now?

[jsgounot]

Hi @cyhofe, I never found why this issue happens on some computer but not on others. Could you please share the tail of your bwa file? Otherwise, you can also run the polishing step (bwa-mem + pilon) and the result should be the same.

[Peterhhb]

Thanks for the fast reponse, my machine is Ubuntu 20.04.3 LTS (GNU/Linux 4.15.0-147-generic x86_64

and perl -V: Summary of my perl5 (revision 5 version 30 subversion 0) configuration:

Platform: osname=linux osvers=4.19.0 archname=x86_64-linux-gnu-thread-multi uname='linux localhost 4.19.0 #1 smp debian 4.19.0 x86_64 gnulinux '

best

hello is this problem solved？I want to know what method you use.

[jsgounot]

Hi Peter,

may I check you have the same type of error message in your /intermediate_files/polished_assembly/bwa.err file?

JS

[Peterhhb]

Hi Peter,

may I check you have the same type of error message in your /intermediate_files/polished_assembly/bwa.err file?

JS

ok this is bra.err .that's all.
[bam_sort] Use -T PREFIX / -o FILE to specify temporary and final output files
Usage: samtools sort [options...] [in.bam]
Options:
-l INT Set compression level, from 0 (uncompressed) to 9 (best)
-u Output uncompressed data (equivalent to -l 0)
-m INT Set maximum memory per thread; suffix K/M/G recognized [768M]
-M Use minimiser for clustering unaligned/unplaced reads
-R Do not use reverse strand (only compatible with -M)
-K INT Kmer size to use for minimiser [20]
-I FILE Order minimisers by their position in FILE FASTA
-w INT Window size for minimiser indexing via -I ref.fa [100]
-H Squash homopolymers when computing minimiser
-n Sort by read name (not compatible with samtools index command)
-t TAG Sort by value of TAG. Uses position as secondary index (or read name if -n is set)
-o FILE Write final output to FILE rather than standard output
-T PREFIX Write temporary files to PREFIX.nnnn.bam
--no-PG
Do not add a PG line
--template-coordinate
Sort by template-coordinate
--input-fmt-option OPT[=VAL]
Specify a single input file format option in the form
of OPTION or OPTION=VALUE
-O, --output-fmt FORMAT[,OPT[=VAL]]...
Specify output format (SAM, BAM, CRAM)
--output-fmt-option OPT[=VAL]
Specify a single output file format option in the form
of OPTION or OPTION=VALUE
--reference FILE
Reference sequence FASTA FILE [null]
-@, --threads INT
Number of additional threads to use [0]
--write-index
Automatically index the output files [off]
--verbosity INT
Set level of verbosity
[M::main_mem] read 200000 sequences (20000000 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (75, 7, 7, 84)
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (1960, 4311, 6718)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 16234)
[M::mem_pestat] mean and std.dev: (4312.21, 2900.45)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20992)
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (1473, 4043, 6264)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15846)
[M::mem_pestat] mean and std.dev: (4258.32, 2970.97)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20637)
[mem_sam_pe] paired reads have different names: "7001424:250:C9DJUANXX:1:1103:5561:1975", "7001424:250:C9DJUANXX:1:1103:3043:1993"

[mem_sam_pe] paired reads have different names: "7001424:250:C9DJUANXX:1:1103:2025:1997", "7001424:250:C9DJUANXX:1:1103:1169:1989"

[jsgounot]

Were you able to confirm your reads are properly paired?

[Peterhhb]

Were you able to confirm your reads are properly paired?

I use the test_files

[jsgounot]

It still seems that somehow you're using a different bwa or samtools version, which is very weird. If you don't mind, could you let me know:

1. If you edited the samtools or bwa in the tools_opera_ms directory
2. BWA and samtools versions should be 0.7.10-r789 and 0.1.19-44428cd respectively
3. If this issue comes when running the test command line in the main README or a different one
4. If you could run and share the result of this command line: grep SAMTOOLS_DIR intermediate_files/coverage_estimation/cov_estimate.config from the output directory.

Thanks.

[Peterhhb]

It still seems that somehow you're using a different bwa or samtools version, which is very weird. If you don't mind, could you let me know:

1. If you edited the samtools or bwa in the tools_opera_ms directory
2. BWA and samtools versions should be 0.7.10-r789 and 0.1.19-44428cd respectively
3. If this issue comes when running the test command line in the main README or a different one
4. If you could run and share the result of this command line: grep SAMTOOLS_DIR intermediate_files/coverage_estimation/cov_estimate.config from the output directory.

Thanks.

Thank you for your response
1.I have not edited Samtools and BWA. I also encountered this error after re downloading
2.The version of Samtool is 1.18, and the version of BWA is 0.7.10-r789
3.I’m sorry.I didn't understand what you meant
4.SAMTOOLS_DIR /home/peter/desk/nefu/huada/task/OPERA-MS/OPERA-MS/test_files/..// tools_opera_ms/