brnaseq

Basic analysis of bacterial RNAseq data for differential gene expression.

Materials

• Read files
• RNA-Seq data in FASTQ format (Ex: dataset1.fastq, dataset2.fastq)
• Reference files
• Genome sequence in GenBank or GFF3 format (Ex: REL606.gbk)

Software

• Adaptor filtering (Trimmomatic)
• Download Trimmomatic
• Use this protocol.
• Read mapping (Bowtie2)
  • Download Bowtie2
  • Download an executable for your platform
  • Add this directory to your $PATH or move bowtie, and bowtie-* star executable to your $PATH
• Sequence conversion and runfule generation (breseq and gdtools)
  • Download and install breseq
• Read counting (htseq)
  • Python3
  • Install using pip
  • Can install with bioconda (use 0.12.4 to avoid Pythion incompatibilities)
• Differential gene expression (DEseq2)
  • Download and install R
  • Bioconductor R modules
    • library(deseq2)
• These brnaseq scripts
  • Put them into your $PATH.
  • Download from Github.

Procedure

Create genomediff metadata files

The files should contain information about the reads and the references used in the analysis.

Example

Trim adaptor sequences from reads

Use this protocol.

gdtools RUNFILE --mode  trimmomatic-PE-unique --preserve-pairs

Run brnaseq

brnaseq

gdtools RUNFILE --preserve-pairs --executable brnaseq --options "-j 12 -k"

Analyze differential gene expression

differential_gene_expression.R

For an explanation of the methods: Manual and Instructions

Graph data for a specific gene

graph_gene_counts.R

Examine enrichment in certain gene categories

• MetaCyc
• clusterProfiler R package

View reads in IGV

You should always inspect your data at the nucleotide level to see if there are indications that an analysis has gone awry. For example, are all of the forward reads on the correct strand? Are there the mutations you expect in a certain strain there?

And convert to BAM format (assumes single-end data):

samtools faidx REL606.fna
samtools import REL606.fna datasetX.sam datasetX.unsorted.bam
samtools sort --threads 8 -o datasetX.bam datasetX.unsorted.bam
samtools index datasetX.bam

Now you can use IGV to view them!

Plotting coverage

Extract R1, sort and index; Index FASTA reference

samtools faidx reference.fna
samtools view -hbf 64 aligned.paired.sam > unsorted_R1.bam
samtools sort --threads 8 -o sorted_R1.bam unsorted_R1.bam
samtools index datasetX.bam

Plot to table file

breseq BAM2COV -b sorted_R1.bam -f reference.fna -t -p 0 <seq_id:start-end>

Citations

If you use this pipeline, you should cite:

1. trimmomatic
2. bowtie2
3. htseq
4. DESeq2