Fastq Barcode Demux

• A flexible tool for demultiplexing FASTQ files containing barcodes
• Splits FASTQ files into separate output files based on barcode sequence
• Supports both zstd and gzip compression formats
• Written in Rust using rust-bio and rayon

Usage

demux_barcodes \
    sample_R1.fastq.gz \
    sample_R2.fastq.gz \
    barcode_config.txt

Additional Options

• --output-path: Specify the directory where the output files will be written
• --nthreads: Set the number of threads to use
• --compress-algo: Choose the output compression algorithm (gz/gzip or zstd)
• --compress-level: Define the compression level (e.g., 0 means default; valid ranges: 0-9 for gzip, 0-22 for zstd).
• --verbose: Enable verbose output (0 or 1).

Installation

Rust must be installed:

• via official shell script (https://www.rust-lang.org/tools/install)
• or via conda conda install -c conda-forge rust

Compile executable:

cargo build --release

Executable demux_barcodes is located in targets folder and can be moved anywhere.

The barcode config file

• The barcode config file controls how barcodes are searched in each read pair
• There is a simple and advanced config format
• The configuration files can be tab or comma delimited

Simple configuration

Two columns, no column header: barcode_name and barcode_sequence. This will search for barcode sequences anywhere within the read pair, with a maximum edit distance of 1. If found, reads will be demultiplexed into files named based on barcode_name.

Don't use any weird symbols for barcode_name as it will be part of the output file path.

Advanced configuration

Five columns, no column header:

barcode_name, barcode_sequence, max_edit_distance, Read1_config, Read2_config (See next section)

Read Config: Barcode Direction / Match Position Configuration

For each read in the pair, this parameter is specified as a coupled value with two components separated by a slash. The first component is the Barcode Direction and the second is the Match Position.

Barcode Direction Allowed Values:

Forward barcode sequence only: `F`, `Fwd`, `Forward`
Reverse Complement barcode sequence only: `RC`, `RevComp`, `Reverse Complement`
Allow matches to either direction: `E`, `Either`

Match Position Allowed Values:

Match only at the start of the read: `S`, `Start`
Match only at the end of the read: `E`, `End`
Match anywhere in the read: `A`, `Any`, `Anywhere`

Usage Example

Specify as BarcodeDirection/MatchPosition. E.g., F/A for searching for the forward barcode sequence with a match allowed anywhere. Leave the value blank or use NA if not allowing any match for that read in the pair.