seismic demult

Purpose

seismic demult splits one or more multiplexed FASTQ files into per-sample FASTQ files by matching barcode sequences embedded in the reads. Use it when multiple samples were pooled before sequencing and share a single FASTQ file, distinguished only by their barcodes.

Requires seqkit (≥ 2.10.1).

Inputs

Multiplexed FASTQ files: Pass reads with --fastqz (-z, single-end), --fastqy (-y, paired interleaved), or --fastqx (-x, paired separate files). For FASTQ files that are already partially demultiplexed and need re-sorting, use --dmfastqz (-Z), --dmfastqy (-Y), or --dmfastqx (-X).
Reference FASTA: Positional argument. Required so SEISMIC-RNA knows which reference name corresponds to each barcode.

Outputs

All outputs go into {out}/{sample}/demult/. Reads that do not match any barcode go into an unmatched subdirectory.

Quick example

Demultiplex a single-end FASTQ with two barcodes defined on the command line:

seismic demult ref.fa -z reads.fastq.gz \
    --barcode sample1 ACGTACGT 5 \
    --barcode sample2 TTGGCCAA 5

Options

Define barcodes

--barcode NAME SEQ POS: Specify one barcode: its name (must match a reference in the FASTA), DNA sequence, and 1-based position in the read. Repeat for each sample.
--refs-meta FILE: Alternatively, read barcode definitions from a CSV file. See Metadata for References.
--barcode-start N / --barcode-end N: When all barcodes occupy the same region, set the 0-based start and end positions (half-open) of that region (default 0 for both).
--read-pos N: Expected 1-based position of the barcode in the read; defaults to --barcode-start when not set.

Matching tolerance

--mismatch-tolerance N: Allow up to N mismatches between a read and the expected barcode (default 0). Use caution above 2 — computation grows factorially with mismatches.
--index-tolerance N: Allow the barcode to appear up to N bases away from the expected position (default 0).
--allow-n/--no-allow-n: Count N bases as valid mismatches (default off). Requires --mismatch-tolerance ≥ 1; increases memory use.

Other

--phred-enc N: Phred score encoding of the FASTQ files (default 33).
--branch NAME (-b): Write outputs to {out}/{sample}/demult_{NAME}/. See Branches.

Performance

--num-cpus N — multiprocessing; see Parallelize Tasks. --force — overwrite existing outputs.

The auto-generated Command Line Reference lists every option with its current default.