Error with working directories

[poddarharsh15]

Hi @kcleal Could please suggest me some ideas how to modify the command line to solve this directory errors!

cmd line:-

dysgu run \\
        -p ${task.cpus} \\     
        $fasta \\
        . \\
        $input_bam \\
        | bgzip ${args2} --threads ${task.cpus} --stdout > ${prefix}.vcf.gz
    tabix ${args3} ${prefix}.vcf.gz

2024-06-25 09:07:20,781 [INFO ] [dysgu-run] Version: 1.6.2
Traceback (most recent call last):
File "/usr/local/bin/dysgu", line 11, in
sys.exit(cli())
File "/usr/local/lib/python3.10/site-packages/click/core.py", line 1157, in call
return self.main(*args, **kwargs)
File "/usr/local/lib/python3.10/site-packages/click/core.py", line 1078, in main
rv = self.invoke(ctx)
File "/usr/local/lib/python3.10/site-packages/click/core.py", line 1688, in invoke
return _process_result(sub_ctx.command.invoke(sub_ctx))
File "/usr/local/lib/python3.10/site-packages/click/core.py", line 1434, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "/usr/local/lib/python3.10/site-packages/click/core.py", line 783, in invoke
return __callback(*args, **kwargs)
File "/usr/local/lib/python3.10/site-packages/click/decorators.py", line 33, in new_func
return f(get_current_context(), *args, **kwargs)
File "/usr/local/lib/python3.10/site-packages/dysgu/main.py", line 238, in run_pipeline
make_wd(kwargs)
File "/usr/local/lib/python3.10/site-packages/dysgu/main.py", line 125, in make_wd
raise ValueError("Working directory already exists. Add -x / --overwrite=True to proceed, "
ValueError: Working directory already exists. Add -x / --overwrite=True to proceed, or supply --ibam to re-use temp files in working directory

Thanks.

[kcleal]

Hi @poddarharsh15,
It looks like you are trying to use the current directory as the temp directory .. You will need to add -x to your command to overwrite any temp files

[poddarharsh15]

Thank you for quick reply can I modify command line like this:-

dysgu run \\
        -p ${task.cpus} \\     
        -x \\
        $fasta \\
        . \\
        $input_bam \\
        | bgzip ${args2} --threads ${task.cpus} --stdout > ${prefix}.vcf.gz
    tabix ${args3} ${prefix}.vcf.gz

Is this okay?

[kcleal]

Yes!

[poddarharsh15]

I am running the above mentioned command line, still I am not able to emit any outputs Could you please have a look at the log file, Thanks.

Input bam

2024-06-25 11:36:07,112 [INFO ] [dysgu-run] Version: 1.6.2
2024-06-25 11:36:07,112 [INFO ] run -p 2 -x genome.fasta . test.paired_end.recalibrated.sorted.bam
2024-06-25 11:36:07,112 [INFO ] Destination: .
2024-06-25 11:36:07,382 [INFO ] dysgu fetch test.paired_end.recalibrated.sorted.bam written to ./test.paired_end.recalibrated.sorted.dysgu_reads.bam, n=252, time=0:00:00 h:m:s
2024-06-25 11:36:07,382 [INFO ] Input file is: ./test.paired_end.recalibrated.sorted.dysgu_reads.bam
[W::hts_idx_load3] The index file is older than the data file: test.paired_end.recalibrated.sorted.bam.bai
2024-06-25 11:36:07,385 [INFO ] Sample name: normal
2024-06-25 11:36:07,385 [INFO ] Writing vcf to stdout
2024-06-25 11:36:07,385 [INFO ] Running pipeline
2024-06-25 11:36:07,552 [INFO ] Calculating insert size. Removed 0 outliers with insert size >= 1359.0
2024-06-25 11:36:07,563 [INFO ] Inferred read length 100.0, insert median 351, insert stdev 145
2024-06-25 11:36:07,564 [INFO ] Max clustering dist 1076
2024-06-25 11:36:07,564 [INFO ] Building graph with clustering 1076 bp
2024-06-25 11:36:07,566 [INFO ] Total input reads 252
2024-06-25 11:36:07,566 [INFO ] Graph constructed
2024-06-25 11:36:07,566 [INFO ] Minimum support 3
2024-06-25 11:36:07,602 [CRITICA] No events found
2024-06-25 11:36:07,602 [INFO ] dysgu run test.paired_end.recalibrated.sorted.bam complete, time=0:00:00 h:m:s

Input cram

2024-06-25 11:36:18,595 [INFO ] [dysgu-run] Version: 1.6.2
2024-06-25 11:36:18,595 [INFO ] run -p 2 -x genome.fasta . test.paired_end.recalibrated.sorted.cram
2024-06-25 11:36:18,595 [INFO ] Destination: .
2024-06-25 11:36:19,056 [INFO ] dysgu fetch test.paired_end.recalibrated.sorted.cram written to ./test.paired_end.recalibrated.sorted.dysgu_reads.bam, n=252, time=0:00:00 h:m:s
2024-06-25 11:36:19,057 [INFO ] Input file is: ./test.paired_end.recalibrated.sorted.dysgu_reads.bam
2024-06-25 11:36:19,058 [INFO ] Sample name: normal
2024-06-25 11:36:19,058 [INFO ] Writing vcf to stdout
2024-06-25 11:36:19,058 [INFO ] Running pipeline
2024-06-25 11:36:19,509 [INFO ] Calculating insert size. Removed 0 outliers with insert size >= 1359.0
2024-06-25 11:36:19,519 [INFO ] Inferred read length 100.0, insert median 351, insert stdev 145
2024-06-25 11:36:19,520 [INFO ] Max clustering dist 1076
2024-06-25 11:36:19,520 [INFO ] Building graph with clustering 1076 bp
2024-06-25 11:36:19,522 [INFO ] Total input reads 252
2024-06-25 11:36:19,522 [INFO ] Graph constructed
2024-06-25 11:36:19,522 [INFO ] Minimum support 3
2024-06-25 11:36:19,556 [CRITICA] No events found
2024-06-25 11:36:19,556 [INFO ] dysgu run test.paired_end.recalibrated.sorted.cram complete, time=0:00:00 h:m:s

[kcleal]

The log suggests only 252 reads were in your test.paired_end.recalibrated.sorted.cram file, is this correct?

[poddarharsh15]

252

Yes it is correct because these are the test_samples which are extremely small size. <300kb

[kcleal]

Perhaps there are no SVs present? Alternatively you can try adjusting the min-support parameter, it is set at 3 by default for paired-end reads.

[poddarharsh15]

Probably yes I was running on other test_samples which are a bit larger in size and dysgu was successfully able to detect SVs,
--min-support TEXT Minimum number of reads per SV [default: 3] I can change it to maybe 0?

dysgu run \
    -p ${cpus} \
    -x \
    --min-support 0
    ${fasta} \
    . \
    ${input_bam} \
    | bgzip ${args2} --threads ${cpus} --stdout > ${prefix}.vcf.gz

tabix ${args3} ${prefix}.vcf.gz

May I ask for running .cram files I need to add a specific parameters?

[kcleal]

There a no additional parameters to use a cram file. Min-support 0 or 1 will have the same effect, i.e. at least one bit of evidence for a SV call

[poddarharsh15]

I have tried both 0 and 1 parameters and still there is no change in results, I suppose there's no SV present in the test_data :(