The simplest and fastest RNA-seq analysis pipeline for Pseudomonas aeruginosa (or any bacterium with a reference genome) using Rockhopper system. This software was developed by Brian Tjaden at the Department of Computer Science, Wellesley College.
The example provided here is done using Pseudomonas aeruginosa strain UCBPP-PA14. For any other bacteria that already has a reference genome, you can simply modify the -g argument with your reference genome. Rockhopper can run on any platform, but herein I providing an example using UNIX because I had many files. Rockhopper needs Java to run, so make sure you have it first. Then download the JAR file of Rockhopper and run it.
You can use either FASTQ or FASTA as input. Here I have 2 experiemntal groups, control and treatment, and in each group I have 3 replicates. Notice that for each sample there are 2 files (one ending with R1 and the other with R2) because they resulted from paired-end sequencing. You need to separate each pair of files with "%", the samples with ",", and the 2 groups with a " ".
java -Xmx4096m -cp ~/Rockhopper.jar Rockhopper -o output/ -g genomes/Pseudomonas_aeruginosa_UCBPP_PA14/ C1_R1.fastq%C1_R2.fastq,C2_R1.fastq%C2_R2.fastq,C3_R1.fastq%C3_R2.fastq T1_R1.fastq%T1_R2.fastq,T2_R1.fastq%T2_R2.fastq,T3_R1.fastq%T3_R2.fastq -v true -L control,treatment
The main output file is a CSV file that includes, raw count, normalized count for each gene, p-value and q-value (corrected p-value). The other file "summary" will show you the percentage of reads that were aligned to each strand, protein-coding, non-coding regions, and ribosomal RNA.