Key concepts and tools

  • mix — interleaving two channels of the same type into one
  • container directive, -profile singularity — Singularity on the SCC
  • withLabel resource labels in nextflow.config — process_single, process_low, process_medium
  • withName per-process publishDir and ext.args in config
  • FastQC, Trimmomatic, MultiQC — standard short-read QC workflow
  • QC report interpretation: per-base quality, GC content, adapter content, read length distribution, duplication

This lab assembles a complete short-read QC pipeline — FastQC on raw reads, adapter trimming with Trimmomatic, FastQC on trimmed reads, and MultiQC to summarise everything in a single HTML report. All processes run in Singularity containers, which is how all subsequent projects are run on the SCC. The one new Nextflow concept is mix, used to merge the pre-trim and post-trim FastQC channels so MultiQC sees both sets of reports and can produce a before/after comparison.

Setup

module load miniconda
conda activate nextflow_latest
export NXF_SYNTAX_PARSER=v2

nextflow run main.nf -profile singularity,local    # local test
nextflow run main.nf -profile singularity,cluster  # submit to SCC

Pipeline

samplesheet.csv
    ↓ channel.fromList
samples_ch ──┬──→ FASTQC ──────────────────────────→ pre_qc_ch ──┐
             │                                                      ├─ mix → combined_qc → flatMap → collect → MULTIQC
             └──→ TRIMMOMATIC → trim_ch → FASTQC → post_qc_ch ───┘

mix is the join point: it interleaves pre_qc_ch and post_qc_ch into a single channel so all six FastQC zip files (3 samples × pre + post) reach MultiQC together.

Tasks

Task 1 — Read main.nf and the modules
Before writing anything, answer: what field does TRIMMOMATIC add that FASTQC doesn’t need? How many FASTQC tasks run for 3 samples? How does duck-typing allow FASTQC(trim_ch) to work when trim_ch carries a log field?

Task 2 — Fill in the mix call
main.nf has one ??? placeholder. Replace it with:

combined_qc = pre_qc_ch.mix(post_qc_ch)

Verify with -stub-run (expect 10 tasks: 6 FASTQC + 3 TRIMMOMATIC + 1 MULTIQC).

Task 3 — Run on the cluster
nextflow run main.nf -profile singularity,cluster. Open results/multiqc/multiqc_report.html and confirm both pre-trim and post-trim sections are present.

Task 4 — Resource labels
Trace how withLabel: process_low { cpus = 4 } in nextflow.config becomes -threads 4 in TRIMMOMATIC’s .command.sh. Change the value and re-run with -stub-run to confirm it propagates.

Task 5 — Interpreting the QC report
Working through five metrics: per-base quality, GC distribution, adapter content, read length distribution after trimming, duplication levels. Reference report: exercise_3/provided_results/full_report.html.

Updated: