bcl2fastq

bcl2fastq is a command‑line application developed by Illumina for converting raw binary BCL (Base Call) files generated by Illumina sequencing instruments into the FASTQ format widely used for downstream bioinformatics analysis. The software reads the compressed BCL data from a sequencing run directory, assembles the bases and quality scores for each read, and writes separate FASTQ files for the forward and reverse read(s) as well as the index reads. It also optionally generates a sample sheet summary and can demultiplex samples based on index sequences, producing separate FASTQ files per sample. bcl2fastq is typically executed on the instrument server or a connected workstation during the run’s post‑processing step.

The program first appeared in the Illumina Pipeline Software (Version 1.x) and was later incorporated into the Illumina BaseSpace Sequence Hub as part of the Illumina Genome Analyzer (IGA) workflow. Since 2018 the original bcl2fastq was succeeded by bcl2fastq v2, compatible with newer instruments such as NovaSeq and NovaSeq6000. On Windows it can be run via the standalone Windows installer, while on Linux/macOS it is bundled with a set of 64‑bit binaries and supports multi‑core processing to accelerate conversion.

bcl2fastq is distributed under Illumina’s proprietary license, requiring a valid instrument license for use. The software is free for users who have a licensed Illumina sequencer but is not available as open source. The tool has been widely adopted in genomics laboratories for its speed, reliability, and integration with Illumina’s standard sequencing data output.