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The optional tutorial from day 2 (Advanced variant calling tutorial (GVA14)) detailed the use of the breseq pipeline to call variants on clonal samples from an evolving E. Colicoli population. While this tutorial does not require completion of the previous tutorial, many of the finer points breseq are better covered there. This tutorial will focus on the use of the polymorphic mode of breseq to identify variants from a mixed population.

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All fastq files necessary for this tutorial can be found inside the $BI/gva_course/mixed_population folder folder. Copy all fastq files to a new folder named "fastq", and REL606.6.gbk to  to a new folder named "reference".

mkdir fastq
mkdir reference
cp $BI/gva_course/mixed_population/*.fastq fastq
cp $BI/gva_course/mixed_population/REL606.6.gbk reference

By default breseq preforms several statistical tests to rule out false positives. To make use of these tests, simply add a -p flag  flag to any breseq command. To highlight what breseq is normally doing by default we will run the same fastq files with and without several of the statistical tests. Specifically, base quality scores, polymorphism scores, polymorphism bias, and minimum strand coverage will be ignored. All 4 of these arguments can be found in the breseq -h output and their values should be set to 0. While running breseq in polymorphism mode is a fairly simple,  due to the complexity of the command with turning off all the additional options, it is recommended that you copy paste these commands into a commands file or an idev session.

breseq -j 6 -p -o breseq_output/with_stats -r reference/REL606.6.gbk fastq/REL964_TACAGCA_L003_R1_002.fastq fastq/REL964_TACAGCA_L003_R2_002.fastq >& Logs/with_stats.log.txt
breseq -j 6 -p -b 0 --polymorphism-score-cutoff 0 --polymorphism-bias-cutoff 0 --polymorphism-minimum-coverage-each-strand 0 -o breseq_output/without_stats -r reference/REL606.6.gbk fastq/REL964_TACAGCA_L003_R1_002.fastq fastq/REL964_TACAGCA_L003_R2_002.fastq >& Logs/without_stats.log.txt

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