This file will provide detailed instruction on setting up an automated RNA-seq pipeline. Following tools are required for this analysis. These must be installed and in path.
- Quality control - fastQC
- Alignment of reads - STAR
- Differential expression analysis - DeSEQ2
- Download compressed readFiles from designated website/server.
- Construct a text file with list of samples/folder. This file will serve as the input for generation of an automation script.
- Run the following command:
perl star_salmon_bash_generator.pl folder_list.txt
This will generate an automation script RNA_seq_STAR_SALMON_soham.sh
sh RNA_seq_STAR_SALMON_soham.sh
This will perform quality control (QC) using fastQC, followed by reads alignment. Genome index has to be generated using STAR command as instructed in manual.
All gene counts file generated as a result of running STAR, has to be concatenated based on each replicate of each sample.
Contruct a 3-column table with Folder-name, sample-name and group (replicates) information:
| Folder-name | sample-name | group |
|---|---|---|
| STAR_AlignmentWN1 | WN1 | WT_untreated |
| STAR_AlignmentWN2 | WN2 | WT_untreated |
| STAR_AlignmentWN3 | WN3 | WT_untreated |
This file will serve as a input to concatenate gene counts.
perl star2deseq_strand-specific_paired-end.pl
This generates two files to be used as input files for DeSEQ2.