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sequence in GenBank or GFF3 format (Ex: REL606.gbk)\n\n## Software\n* Adaptor filtering (Trimmomatic)\n * [Download Trimmomatic](http://www.usadellab.org/cms/?page=trimmomatic)\n * Use this [protocol](https://barricklab.org/twiki/bin/view/Lab/ProtocolsTrimmomaticCommands).\n* Read mapping (Bowtie2)\n  * [Download Bowtie2](http://bowtie-bio.sourceforge.net/bowtie2)\n  * Download an executable for your platform\n  * Add this directory to your $PATH or move bowtie, and bowtie-* star executable to your $PATH\n* Sequence conversion and runfule generation (_breseq_ and _gdtools_)\n  * [Download and install breseq](http://barricklab.org/breseq)\n* Read counting (htseq)\n  * Python3\n  * Install using pip\n  * Can install with bioconda (use 0.12.4 to avoid Pythion incompatibilities)\n* Differential gene expression (DEseq2)\n  * [Download and install R](http://cran.r-project.org/)\n  * Bioconductor R modules\n    * library(deseq2)\n* These _brnaseq_ scripts\n  * Put them into your `$PATH`.\n  * [Download from Github](https://github.com/barricklab/barricklab/blob/master/brnaseq).\n\n## Procedure\n\n### Create \u003ccode\u003egenomediff\u003c/code\u003e metadata files\n\nThe files should contain information about the reads and the references used in the analysis.\n\n```\nExample\n\n```\n\n### Trim adaptor sequences from reads\n\nUse this [protocol](https://barricklab.org/twiki/bin/view/Lab/ProtocolsTrimmomaticCommands).\n\n```\ngdtools RUNFILE --mode  trimmomatic-PE-unique --preserve-pairs\n```\n\n### Run brnaseq\n\n```\nbrnaseq\n```\n\n```\ngdtools RUNFILE --preserve-pairs --executable brnaseq --options \"-j 12 -k\"\n```\n\n### Analyze differential gene expression\n\n```\ndifferential_gene_expression.R\n```\n\nFor an explanation of the methods: [Manual and Instructions](http://bioconductor.org/packages/devel/bioc/html/DESeq.html)\n\n### Graph data for a specific gene\n\n```\ngraph_gene_counts.R\n```\n\n### Examine enrichment in certain gene categories\n\n* MetaCyc\n* [clusterProfiler R package](https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)\n\n### View reads in IGV\n\nYou should always inspect your data at the nucleotide level to see if there are\nindications that an analysis has gone awry. For example, are all of the forward\nreads on the correct strand? Are there the mutations you expect in a certain strain there?\n\nAnd convert to BAM format (assumes single-end data):\n```\nsamtools faidx REL606.fna\nsamtools import REL606.fna datasetX.sam datasetX.unsorted.bam\nsamtools sort --threads 8 -o datasetX.bam datasetX.unsorted.bam\nsamtools index datasetX.bam\n```\n\nNow you can use IGV to view them!\n\n### Plotting coverage\n\nExtract R1, sort and index; Index FASTA reference\n```\nsamtools faidx reference.fna\nsamtools view -hbf 64 aligned.paired.sam \u003e unsorted_R1.bam\nsamtools sort --threads 8 -o sorted_R1.bam unsorted_R1.bam\nsamtools index datasetX.bam\n```\n\nPlot to table file\n```\nbreseq BAM2COV -b sorted_R1.bam -f reference.fna -t -p 0 \u003cseq_id:start-end\u003e\n```\n\n## Citations\n\nIf you use this pipeline, you should cite:\n1. trimmomatic\n2. bowtie2\n3. htseq\n4. DESeq2\n","project_url":"https://awesome.ecosyste.ms/api/v1/projects/github.com%2Fbarricklab%2Fbrnaseq","html_url":"https://awesome.ecosyste.ms/projects/github.com%2Fbarricklab%2Fbrnaseq","lists_url":"https://awesome.ecosyste.ms/api/v1/projects/github.com%2Fbarricklab%2Fbrnaseq/lists"}