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snakemake-workflows/rna-seq-star-deseq2

RNA-seq workflow using STAR and DESeq2

Overview​

Topics: snakemake sciworkflows reproducibility gene-expression-analysis deseq2

Latest release: v2.1.2, last update: 2025-01-24

Linting: passed

Formatting: passed

Configuration​

General configuration

To configure this workflow, modify config/config.yaml according to your needs, following the explanations provided in the file.

DESeq2 differential expression analysis configuration​

To successfully run the differential expression analysis, you will need to tell DESeq2 which sample annotations to use (annotations are columns in the samples.tsv file described below). This is done in the config.yaml file with the entries under diffexp:. The comments for the entries should give all the necessary infos and linkouts. But if in doubt, please also consult the DESeq2 manual.

Sample and unit setup

The sample and unit setup is specified via tab-separated tabular files (.tsv). Missing values can be specified by empty columns or by writing NA.

sample sheet​

The default sample sheet is config/samples.tsv (as configured in config/config.yaml). Each sample refers to an actual physical sample, and replicates (both biological and technical) may be specified as separate samples. For each sample, you will always have to specify a sample_name. In addition, all variables_of_interest and batch_effects specified in the config/config.yaml under the diffexp: entry, will have to have corresponding columns in the config/samples.tsv. Finally, the sample sheet can contain any number of additional columns. So if in doubt about whether you might at some point need some metadata you already have at hand, just put it into the sample sheet already---your future self will thank you.

unit sheet​

The default unit sheet is config/units.tsv (as configured in config/config.yaml). For each sample, add one or more sequencing units (for example if you have several runs or lanes per sample).

.fastq file source​

For each unit, you will have to define a source for your .fastq files. This can be done via the columns fq1, fq2 and sra, with either of:

  1. A single .fastq file for single-end reads (fq1 column only; fq2 and sra columns present, but empty). The entry can be any path on your system, but we suggest something like a raw/ data directory within your analysis directory.
  2. Two .fastq files for paired-end reads (columns fq1 and fq2; column sra present, but empty). As for the fq1 column, the fq2 column can also point to anywhere on your system.
  3. A sequence read archive (SRA) accession number (sra column only; fq1 and fq2 columns present, but empty). The workflow will automatically download the corresponding .fastq data (currently assumed to be paired-end). The accession numbers usually start with SRR or ERR and you can find accession numbers for studies of interest with the SRA Run Selector. If both local files and an SRA accession are specified for the same unit, the local files will be used.

adapter trimming​

If you set trimming: activate: in the config/config.yaml to True, you will have to provide at least one cutadapt adapter argument for each unit in the adapters column of the units.tsv file. You will need to find out the adapters used in the sequencing protocol that generated a unit: from your sequencing provider, or for published data from the study's metadata (or its authors). Then, enter the adapter sequences into the adapters column of that unit, preceded by the correct cutadapt adapter argument.

strandedness of library preparation protocol​

To get the correct geneCounts from STAR output, you can provide information on the strandedness of the library preparation protocol used for a unit. STAR can produce counts for unstranded (none - this is the default), forward oriented (yes) and reverse oriented (reverse) protocols.
Enter the respective value into a strandedness column in the units.tsv file.