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cd $SCRATCH/GVA_breseq_lambda_mixed_pop
breseq -j 48 -r lambda.gbk lambda_mixed_population.fastq &> log.txt &
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| title | Why do we have to unload samtools before running breseq?||||
As eluded to in the SNV tutorial, breseq uses (and includes) an older version of samtools that had slightly different options, so breseq will not run without the correct version of samtools. Speak to your instructors about changes to your .bashrc file and setting up breseq to be run in the best way if you expect to use it frequently. |
A bunch of progress messages will stream by during the breseq run which would clutter the screen if not for the redirection to the log.txt file. The & at the end of the line tells the system to run the previous command in the background which will enable you to still type and execute other commands while breseq runs. The output text details several steps in a pipeline that combines the steps of mapping (using SSAHA2bowtie2), variant calling, annotating mutations, etc. You can examine them by peeking in the log.txt file as your job runs using tail log.txt. While breseq is running lets look at what the different parts of the command are actually doing:
| part | puprose | ||
|---|---|---|---|
| -j 48 | Use 48 processors (the max available on lonestar5 nodes) | -r lambda.gbk | Use the lambda.gbk file as the reference to identify specific mutations |
| lambda_mixed_population.fastq | breseq assumes any argument not preceded by a - option to be an input fastq file to be used for mapping | ||
| &> log.txt | redirect the output and error to the file log.txt | ||
| & | run the preceding command in the background |
This will finish very quickly (likely before you begin reading this) with a final line of "Creating index HTML table...+++ SUCCESSFULLY COMPLETED". check this using the tail command. If you instead see this output:
| No Format |
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!!!!!!!!!!!!!!!!!!!!!!!> FATAL ERROR <!!!!!!!!!!!!!!!!!!!!!!!
Error running command:
[system] /opt/apps/samtools/1.3/bin/samtools sort ./03_candidate_junctions/best.unsorted.bam ./03_candidate_junctions/best
Result code: 256
FILE: libbreseq/common.h LINE: 1294
!!!!!!!!!!!!!!!!!!!!!!!> FATAL ERROR <!!!!!!!!!!!!!!!!!!!!!!!
breseq: libbreseq/common.h:92: void breseq::my_assertion_handler(bool, const char*, const char*, int, const string&): Assertion `false' failed.
Aborted
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It means that you tried to run breseq with the incorrect version of samtools loaded. Execute the following 4 commands and then retry running breseq.
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| language | bash |
|---|---|
| title | Commands IF breseq failed |
| collapse | true |
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something different, get my attention.
Looking at breseq predictions
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Click around through the different mutations and examine their evidence to see what kinds of mutations you can identify. Interact with your instructorsinstructor, and show us what different types of mutations you are able to identify, or ask us what mutations you don't understand. Additional information on analyzing the output can be found at the following reference:
- Deatherage, D.E., Barrick, J.E.. (2014) Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol. Biol. 1151:165-188. «PubMed»