Ngs Bcl To Fastq

BCL To FASTQ

Use this skill when the input is an Illumina BCL run folder or the user asks to demultiplex a sequencing run. This is a deep demultiplexing and run-validation skill, not only a command wrapper.

Essential Inputs

Confirm:

• run folder path with RunInfo.xml
• sample sheet path and format
• output directory
• instrument/run metadata from RunInfo.xml and RunParameters.xml
• lane handling: split by lane or combine lanes
• index mismatch tolerance
• index read structure and dual-index orientation
• UMI layout, if any
• whether adapter trimming/masking should happen during conversion
• whether undetermined reads and demultiplexing metrics should be reviewed before downstream analysis

Public Tool Boundary

Prefer bcl-convert if it is already installed. It is free for local use but proprietary and RPM-distributed by Illumina, so do not auto-download without explicit user approval.

Legacy bcl2fastq may exist in older environments. Use it only when BCL Convert is unavailable or the run requires legacy compatibility.

Preflight

python plugins/ngs-analysis/scripts/ngs_preflight.py --pipeline bcl_to_fastq --emit-install-plan

Also check run-folder structure:

test -f /path/to/run/RunInfo.xml
test -f /path/to/SampleSheet.csv
find /path/to/run -maxdepth 4 -type d -name BaseCalls

Local Execution Package

Use the plugin-owned runner when the user provides a local run folder and sample sheet:

python plugins/ngs-analysis/scripts/run_bcl_to_fastq.py \
  --run-folder /path/to/run \
  --sample-sheet /path/to/SampleSheet.csv \
  --output-directory /path/to/fastq_out

Add --execute only when conversion is requested. The runner validates RunInfo.xml, optional RunParameters.xml, the BaseCalls directory, sample-sheet rows, duplicate lane/index combinations, and index length compatibility. With --execute, it uses installed bcl-convert, then legacy bcl2fastq if available; if neither exists, it records the blocker instead of downloading proprietary software.

Validation Checklist

Before conversion, validate:

• RunInfo.xml exists and its read structure matches the expected sequencing design.
• SampleSheet.csv exists, is the intended version, and has no duplicate sample/index combinations within each lane.
• Index sequence lengths match the index reads and any trimming/masking requested by the sample sheet.
• Dual-index orientation is explicit for the instrument and library prep; do not infer i5 orientation from filenames.
• UMI bases are assigned to the intended read or index read and carried through to FASTQ headers or output metadata as needed.
• Lane-splitting, sample-name normalization, and output directory behavior are agreed before running.
• Disk space is sufficient for output FASTQs, reports, and temporary files.

Kickoff Pattern

First produce a preflight plan with paths and sample sheet validation. Then run conversion only after the user confirms:

bcl-convert \
  --bcl-input-directory /path/to/run \
  --output-directory /path/to/fastq_out \
  --sample-sheet /path/to/SampleSheet.csv

Metrics Review

After conversion, inspect and report:

• total clusters, clusters passing filter, and yield by lane
• percent assigned by sample and percent undetermined by lane
• top undetermined index sequences when available
• per-sample FASTQ counts and read-pair consistency
• unexpected index hopping, barcode collision, or sample-sheet mismatch signals

Record software version, command, sample sheet checksum, run-folder path, output path, and conversion metrics. Do not start downstream analysis until severe demultiplexing anomalies are surfaced.