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Strainer

This module evaluates index mixing across flowcell lanes. It scans the undetermined FASTQ files produced by a demultiplexing run and identifies reads whose index sequences match (within one mismatch) a sample that was assigned to a different lane. Such cross-lane contamination can occur when sample plates are accidentally loaded into the wrong lane position.

Usage

The minimal command is biomate strainer --input-path <FLOWCELL_DIR>. The module expects to find the undetermined FASTQ files and a SampleSheet.csv inside the input directory.

Options

  • --input-path: Path to the input flowcell directory. The directory must contain at least one file matching **/Undetermined*R1*.fastq.gz. This argument is required.
  • --output-path: Path to the output directory where the results CSV will be written (default: current directory). The directory will be created automatically if it does not exist.
  • --sample-sheet: Path to an explicit Sample Sheet file. If not provided, the module looks for SampleSheet.csv inside --input-path.
  • --threads: Number of parallel worker processes used to read the undetermined FASTQ files. Default is 1 (single process). Increasing this value speeds up the extraction step on multi-core machines.

Sample Sheet Format

The sample sheet must contain a [Data] or [BCLConvert_Data] section with at least the following columns:

Column Description
Lane Lane number
Sample_Project Project name
index Index 1 sequence
index2 Index 2 sequence (optional — single-index samples may omit this column)

Lane values are normalised to the LXXX format (e.g. lane 1 becomes L001) so that they can be matched against the lane extracted from each undetermined filename.

Matching Logic

For each sample in the sample sheet the module searches through the top-1000 most-frequent indexes from every other lane's undetermined file. An undetermined read is flagged as unexpected if:

  • Its combined index string contains a sequence that matches index within one substitution (s≤1), and
  • If index2 is present, the combined index string also contains a sequence matching index2 within one substitution.

Reads from the same lane as the sample are never flagged.

Output

A single CSV file named <flowcell_name>_unexpected_indexes.csv is written to --output-path. Each row describes one unexpected index observation:

Column Description
Lane Lane in which the unexpected index was found
Index The combined index string from the undetermined read
Count Number of times this index was observed
Sample_Project Project to which the matching sample belongs
Lane_Project Lane to which the matching sample was assigned
index1 Index 1 of the matching sample
index2 Index 2 of the matching sample (empty for single-index samples)

If no unexpected indexes are found the file is still written but will contain only the header row.

Example

biomate strainer \
    --input-path /data/flowcells/20260310_LM43899_0385_A12GGASZR5 \
    --output-path ./strainer_results \
    --threads 4

The above command processes all undetermined FASTQ files in the flowcell directory using 4 parallel workers and writes the results to:

strainer_results/
└── 20260310_LM43899_0385_A12GGASZR5_unexpected_indexes.csv

Notes

  • Only the top 1000 most-common undetermined indexes per lane are considered. Extremely rare cross-lane contamination events below this threshold will not be reported.
  • Indexes containing four or more consecutive G bases (GGGG) are excluded from the analysis, as these are characteristic of sequencing errors on Illumina instruments rather than genuine index sequences.
  • The module performs fuzzy matching with at most one substitution per index. Two substitutions or any insertion/deletion will not be matched.