nf-core pipeline · bases2fastq, bcl2fastq, demultiplexing
nf-core/demultiplex
Nextflow pipeline for demultiplexing raw data from various sequencing platforms into FASTQ files.
What nf-core/demultiplex is
nf-core/demultiplex is a bioinformatics pipeline designed to demultiplex raw sequencing data generated across different next-generation sequencing instruments. It was originally written by Chelsea Sawyer from The Bioinformatics & Biostatistics Group at The Francis Crick Institute in London, and later re-written in Nextflow DSL2 with primary maintenance by Matthias De Smet from Ghent University and Edmund Miller from Element Biosciences.
The primary challenge the pipeline addresses is processing raw sequencer outputs—such as base call files or multiplexed FASTQ files—into sample-specific FASTQ reads across distinct commercial technologies. Demultiplexing is the essential first computational step for pooled sequencing libraries before any downstream biological interpretation can take place.
Built within the nf-core community framework, the pipeline leverages Nextflow DSL2 and per-process containerisation via Docker or Singularity. This architecture ensures high reproducibility and portable deployment across diverse computing environments.
What the pipeline does
The pipeline takes an input samplesheet, optionally validating Illumina v2 samplesheets with samshee. Depending on the input sequencer data and configuration, it directs execution conditionally to one of several demultiplexing tools: bcl-convert, bcl2fastq, bases2fastq, sgdemux, fqtk, mkfastq, or mgikit.
Following demultiplexing, bcl2fastq runs can optionally undergo quality evaluation using checkqc. Downstream processes execute adapter and quality trimming via fastp, raw read quality assessment using Falco, and MD5 (128-bit) checksum generation for every resulting FASTQ file. Finally, an aggregate MultiQC report compiles results from across the entire pipeline execution.
Key facts
- Pipeline engine
- Nextflow DSL2
- Supported platforms
- Illumina, Element Biosciences, Singular Genomics, 10x Genomics, MGI
- Demultiplexing tools
- bcl-convert, bcl2fastq, bases2fastq, sgdemux, fqtk, mkfastq, mgikit
- Quality control tools
- samshee, checkqc, fastp, Falco, MultiQC
- Primary maintainers
- Matthias De Smet and Edmund Miller
- Original author
- Chelsea Sawyer (The Francis Crick Institute)
- Latest release
- 1.7.1 (2026-04-17)
What you can use it for
- Illumina BCL Conversion
- Converts raw Illumina BCL files into separate FASTQ files per sample using either bcl2fastq or bcl-convert.
- Element Biosciences Data Processing
- Processes raw bases files from Element Biosciences instruments into demultiplexed FASTQs via bases2fastq.
- Single-Cell Sequencing Demultiplexing
- Demultiplexes raw Illumina BCL files from 10x Genomics single-cell libraries using 10x Genomics mkfastq.
- Singular Genomics Demultiplexing
- Demultiplexes bgzipped FASTQ files produced by Singular Genomics sequencing systems using sgdemux.
- MGI and Custom Read Structure Demultiplexing
- Splits FASTQ files generated by MGI sequencers via mgikit, or processes custom user-supplied read structures using fqtk.
Run it here
The research agent runs nf-core/demultiplex with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
Strengths and limits
Good at
- Supports a wide variety of sequencing platforms including Illumina, Element Biosciences, Singular Genomics, 10x Genomics, and MGI.
- Integrated quality assurance workflow combining sample sheet validation, read trimming, MD5 checksum verification, and MultiQC reporting.
- Modular architecture built on Nextflow DSL2 using single-process containers to simplify updates and ensure reproducibility.
Watch out for
- The post-demultiplexing checkqc step is restricted exclusively to outputs from bcl2fastq.
- Samplesheet validation using samshee is limited to Illumina v2 samplesheets.
- Pipeline configuration files cannot be used to pass pipeline parameters, which must be specified via CLI or parameter files.
Questions
Can I run nf-core/demultiplex without my own cluster?
The research agent runs nf-core/demultiplex with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
What licence is nf-core/demultiplex under?
MIT, like every nf-core pipeline.
Which tools handle standard Illumina BCL files in the pipeline?
Raw Illumina BCL files can be demultiplexed into FASTQ format using either bcl2fastq or bcl-convert.
Can user-defined read structures be demultiplexed?
Yes, FASTQ files with custom user-supplied read structures can be demultiplexed using the fqtk toolkit.
How does the pipeline verify file integrity after demultiplexing?
The pipeline generates a 128-bit MD5 checksum for every output FASTQ file via md5sum.
How to cite it
10.5281/zenodo.7153103
Written from nf-core/demultiplex's own README. Statements citing a figure that does not appear there are removed before publication. Licence and running details come from our registry.