Nextflow RNA-seq pipeline

Initial setup

Install nextflow e.g. in an conda environment:

conda create -n nextflow -c bioconda nextflow

Activate the conda environment with nextflow:

conda activate nextflow

Configuration

Pipeline config

Override the configuration (called nextflow.config) using an own config file and using the -C flag on the nextflow run (see docs).

Pipeline parameters

Parameters are either passed in as command line arguments (with double-dashed arguments --)

nextflow run ... --input samplesheet.csv --genome genome.fa --annotation_gtf annotation.gtf

or in a yaml (or json) file via the -params-file argument.

input: "path/to/samplesheet.csv"
genome: "path/to/genome.fasta"
annotation_gtf: "path/to/gene_annotation.gtf"

nextflow run NBorthLab/nf-rnaseq -params-file parameters.yml

The example below shows how such a sample sheet CSV file might look like (visually aligned for easier redability).

 sample,                    fastq_1,                    fastq_2, strandedness
sample1,   /data/sample1_1.fastq.gz,   /data/sample1_2.fastq.gz,         auto
sample2,     /data/sample2.fastq.gz,                           ,         auto
sample3, /data/sample3.1_1.fastq.gz, /data/sample3.1_2.fastq.gz,         auto
sample3, /data/sample3.2_1.fastq.gz, /data/sample3.2_2.fastq.gz,         auto

First column is the sample name; multiple mentions are allowed and will be treated as technical replicates. Second column is the first, third column the (optional) second fastq file. Fourth column is the 'strandedness'. If unknown set to auto.

sample1 is an example of a paired-end sequenced sample. sample2 is single-end. sample3 is paired-end and has technical replicates.

Notifications (optional)

Set up an account at ntfy.sh and set up a topic for yourself. Add the URL of the ntfy topic as a secret to get notified on pipeline runs.

nextflow secrets set NTFY_URL http://ntfy.sh/<YOUR-TOPIC>

Test pipeline

nextflow run NBorthLab/nf-rnaseq -profile test

Run pipeline

nextflow run NBorthLab/nf-rnaseq

# or, for subsequent runs
nextflow run NBorthLab/nf-rnaseq -resume

# RECOMMENDED: Use a specific version of the pipeline with the -r option
nextflow run NBorthLab/nf-rnaseq -r 2.2.1

Output

Pipeline results can bee retrieved from the results directory.

results/
├── aligned_reads/          # Aligned reads
├── counts/                 # Counts of individual samles
│   └── all_counts.tsv      # (!!!) Combined counts of all samples
├── fastqc/                 # FASTQC of raw reads
├── genome_index/           # STAR genome index
├── multiqc/                # MULTIQC report
├── pipeline_info/          # Pipeline reports and software versions
└── trimmed_reads/          # trimmed reads and their fastqc reports

File structure

bin/                        # Exectuable scripts used in the workflow (not used currently)
data/                       # Raw data (put your data here)
envs/                       # Conda environment definition files
modules/                    # Process definitions
results/                    # Results directory (is created)
work/                       # Working directory of nextflow (is created)
workflows/                  # RNA-seq workflow definition
main.nf                     # Entrypoint for the pipeline
nextflow.config             # (!!!) Configuration file
README.md