fqdemux Nextflow Pipeline

Introduction

This pipeline demultiplexes FASTQ files based on inline barcodes using fqtk demux. It takes multiplexed FASTQ files, along with definitions for barcodes, UMIs and sample names, and produces demultiplexed FASTQ files and a samplesheet compatible with the nf-core/rnaseq pipeline.

Usage

nextflow run <path/to/pipeline>/main.nf \
  --barcodes_samplesheet <path/to/barcodes_samplesheet.tsv> \
  --readstructure_samplesheet <path/to/readstructure_samplesheet.tsv> \
  --outdir <output_directory> \
  -profile apptainer  # e.g., local, docker, singularity

Replace placeholders like <path/to/pipeline>, <path/to/barcodes_samplesheet.tsv>, etc., with your actual paths.

Input Parameters

• --barcodes_samplesheet (Required): Path to a tab-separated values (TSV) file defining the barcodes and their corresponding sample names. Example format:

  sample_id	barcode
  A01	AATGCGGCTTGACC
  A02	ATGGTAATCCAAGG
  A03	ACTGTGCAGCTGAG
  A04	AACCGCCGAGAAGG

• --readstructure_samplesheet (Required): Path to a tab-separated values (TSV) file defining the input FASTQ files and their associated read structures. The pipeline expects columns:

  • filename: Full path to the FASTQ file (e.g., /path/to/your/data/example_R1.fastq.gz).
  • read_structure: The read structure string compatible with fqtk (e.g., 10B141T for 10bp barcode followed by 141bp transcript). Example format:

  filename	read_structure
  /path/to/BRBseq_1.fq.gz	14B14M122T
  /path/to/BRBseq_2.fq.gz	150T

  See the fqtk documentation for more details on the read structure string format.

• --outdir: Path to the directory where output files will be saved. Defaults to ./results.

• --strandedness (Optional, default: 'auto') - set the strandedness column for the samplesheet.csv output ('auto', 'forward' or 'reverse').

Output

The pipeline will create the specified --outdir and place the following files inside:

1. Demultiplexed FASTQ files: FASTQ files split by sample based on the provided barcodes.
2. Samplesheet: A samplesheet.csv CSV file compatible with nf-core/rnaseq.