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cd $SCRATCH/BDIB_breseq_lambda_mixed_pop module loadunload intel module load Rstats samtools breseq -j 48 -r lambda.gbk lambda_mixed_population.fastq &> log.txt & |
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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 SSAHA2), 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:
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This will finish very quickly (likely before you begin reading this) with a final line of "Creating index HTML table...". check this using the tail command. If you instead see this output:
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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 |
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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#DO NOT DO THIS IF BRESEQ COMPLETED SUCCESSFULLY
rm -r 0*
rm -r data
rm -r output
module unload samtools |
Looking at breseq predictions
breseq produced a lot of directories beginning 01_sequence_conversion
, 02_reference_alignment
, ... Each of these contains intermediate files that are used to 'pickup where it left off' if the run doesn't complete successfully. These can be deleted when the run completes, or explored if you are interested in the inner guts of what is going on. More importantly, breseq will also produce two directories called: data
and output
which contain files used to create .html output files and .html output files respectively. The most interesting files are the .html files which can't be viewed directly on lonestar. Therefore we first need to copy the output
directory back to your desktop computer. Go back to the first tutorial (BDIB_breseq_tutorial_1) and Use scp to transfer the contents of the output directory back to your local computer.
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To use To figure out the full path to your file, you can use the
Then you can then copy paste that information (in the correct position) into the scp command on the desktop's command line:
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- 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»
Examining breseq results
Exercise: Can you figure out how to archive all of the output directories and copy only those files (and not all of the very large intermediate files) back to your machine? - without deleting any files?
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You will want to use the tar command again, but you will need to use a wildcard to specify what goes into the compressed file, and only the output directories within each of the wildcard-matched directories.
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To use To figure out the full path to your file, you can use the
Then you can then copy paste that information (in the correct position) into the scp command on the desktop's command line:
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Click around in the results and see the different types of mutations you can detect.Return to the GVA2017 page