RNA-seq Nextflow Pipeline with Docker

Minimal end-to-end RNA-seq pipeline: FASTQ -> FastQC -> cutadapt -> STAR -> sorted BAM -> featureCounts -> MultiQC → DESeq2

This repository includes a small test setup so you can run an end-to-end check locally.

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Requirements

• Nextflow (tested with 25.10.3)
• Docker
• Git

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Quick start (test run)

From the repo root:

# (Optional) build the local containers (only needed if you use local Docker builds)
docker build -t rnaseq-tools:0.1.0 containers/rnaseq-tools
docker build -t rnaseq-r:0.1.0 containers/rnaseq-r

# run the test
nextflow run main.nf -profile docker \
  --samplesheet tests/samplesheet.test.csv \
  --genome_fasta tests/ref/genome.fasta \
  --genome_gtf tests/ref/genes.gtf \
  --design tests/design.test.tsv \
  --outdir results_test \
  -with-trace  "trace-$(date +%Y%m%d-%H%M%S).txt" \
  -with-report "report-$(date +%Y%m%d-%H%M%S).html" \
  -with-timeline "timeline-$(date +%Y%m%d-%H%M%S).html" \
  -resume

Note on report/timeline filenames

If you reuse the same report.html / timeline.html, Nextflow will refuse to overwrite them.
Using timestamped names (as above) avoids that.

Proof it runs (Docker profile)

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Inputs

Sample sheet (--samplesheet)

CSV with columns:

• sample - unique sample ID
• fastq_1 - path to R1 FASTQ.gz
• fastq_2 - path to R2 FASTQ.gz

Example: tests/samplesheet.test.csv

Design matrix (--design)

TSV with columns:

• sample
• condition (e.g. WT / KO)

Example: tests/design.test.tsv

Reference

• --genome_fasta - reference genome FASTA
• --genome_gtf - annotation GTF

Test reference in: tests/ref/

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Outputs

Output folder: --outdir (example: results_test/)

Key outputs:

• counts.tsv - gene counts (featureCounts)
• qc/multiqc_report.html - aggregated QC report
• deseq2_results.tsv - DESeq2 differential expression results
• deseq2_ma_plot.png - MA plot
• deseq2_pca_plot.png - PCA plot

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Repository structure

• main.nf - Nextflow pipeline (DSL2)
• nextflow.config - pipeline configuration
• bin/deseq2.R - DESeq2 script
• containers/ - container build context(s)
• tests/ - test samplesheet/design/reference and helper scripts

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Notes / limitations

• The test reference and test FASTQs are intentionally small; they are for validating the workflow wiring, not biological interpretation.
• For real datasets, update references, resources (cpus/memory), and output locations accordingly.