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trim galore

Demultiplexing

Trim Galore can demultiplex single-end FASTQ files by 3' inline barcode in a post-trim pass. Pass a barcode file via --demux. Demultiplexing is single-end only — combining --demux with --paired is rejected at startup.

How it works

Section titled “How it works”

After quality and adapter trimming, each read's 3' end is matched against the barcode list. Reads are written to per-barcode output files. Reads that do not match any barcode go to an "ambiguous" bucket.

Usage

Section titled “Usage”
Terminal window
trim_galore --demux barcodes.txt input.fq.gz

Per-barcode files are written alongside the trimmed FASTQ.

Notes

Section titled “Notes”
  • Demultiplexing runs after the regular trimming pipeline. Quality, adapter, and length filters all apply first.
  • Ambiguous reads are kept in a separate file, not discarded, so you can inspect what did not match.
  • For high-throughput inline-demux workflows, dedicated tools (e.g. Cutadapt's demux mode) can be more flexible. --demux here is a built-in convenience for standard 3' barcode designs in single-end data.