RNA-seq quantification pipeline

Quickstart

The --directory option will configure where the pipeline will both read needed input files (e.g. genomes) and write intermediate and output files.

$ cd quantify-RNA-pipeline/
$ cp samples.example.tsv path/to/working/directory/samples.tsv
$ nano path/to/working/directory/samples.tsv
$ conda env create -f environment.yml
$ conda activate quantify-RNA-pipeline
(quantify-RNA-pipeline) $ snakemake --directory path/to/working/directory

The default workflow profile is designed to run on a 12 core machine with 64 GB of memory. You may add --workflow-profile profiles/X to use the X snakemake workflow profile instead of the default.

samples.tsv

sample_id       species genotype        library_layout  library_selection       reads_location
SRR8848655      Oryza sativa    Nipponbare      paired-end      random  sra
SRR8848654      Oryza sativa    Nipponbare      paired-end      random  sra

If location is sra, then the sample_id is assumed to be an SRA accession ID and the reads will be downloaded directly from the SRA.

If location is local then the data/reads directory in the working directory will be searched for a directory named after the sample_id. For single-read layout samples, there should be a sample_id.fq.gz file in this folder. (e.g. data/reads/SRR8848655/SRR8848655.fq.gz) For paired-end samples, there should be sample_id_1.fq.gz and sample_id_2.fq.gz files in this folder.

library_selection should be either random or polyA (for 3' RNA-seq).

The reference genome will be searched for in the working directory defined by --directory. It will be expecting a bgziped assembly at data/genomes/species/genotype/assembly.fa.gz, and it has to be indexed with samtools faidx (e.g. Oryza_sativa/Nipponbare/assembly.fa.gz). Spaces in the species name must be replaced with underscores in the path names.

An annotation should also exist at data/genomes/species/genotype/annotation.gff.gz.

Generate report

$ snakemake --directory path/to/working/directory --report report.html

This will generate a report.html file in your working directory.