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ngs-bcl-to-fastq

Validates Illumina BCL run folders and converts them to demultiplexed FASTQ files.

5.3k|765|Updated Mar 4, 2026
Search Tags:#sequencing#ngs#fastq#demultiplexing#illumina#bcl-convert#sample sheet
One-click install
npx skills add https://github.com/openai/plugins --skill ngs-bcl-to-fastq
Or copy as Structured Prompt for Agent▼
Please help me install this Agent Skill.
Skill: ngs-bcl-to-fastq
Source: https://github.com/openai/plugins/tree/main/plugins/ngs-analysis/skills/ngs-bcl-to-fastq
Command: npx skills add https://github.com/openai/plugins --skill ngs-bcl-to-fastq

SYSTEM DOCUMENTATION & REQUIREMENTS

What problem does it solve?

Demultiplexing Illumina sequencing runs requires careful validation of run folders, sample sheets, index orientation, and UMI layout before conversion, and mistakes silently corrupt downstream analysis. This Skill guides the full BCL-to-FASTQ workflow with preflight checks, controlled execution, and demultiplexing metrics review.

Core Features & Use Cases

  • Run and Sample Sheet Validation: Checks RunInfo.xml, RunParameters.xml, BaseCalls directories, duplicate lane/index combinations, and index length compatibility before conversion.
  • Controlled Conversion Execution: Uses installed bcl-convert or legacy bcl2fastq without auto-downloading proprietary software, recording blockers when tools are missing.
  • Demultiplexing Metrics Review: Reports clusters passing filter, percent assigned per sample, undetermined reads, and index hopping signals before downstream analysis begins.
  • Use Case: A core facility receives a NovaSeq run folder and sample sheet; the Skill validates dual-index orientation and UMI layout, runs bcl-convert, and flags a lane with high undetermined reads before FASTQs are released.

Quick Start

Inspect this Illumina run folder and sample sheet, validate the demultiplexing parameters, and prepare a conversion plan with bcl-convert.

Frequently Asked Questions about ngs-bcl-to-fastq

High-intent search queries and answers about installing and using this skill.

FAQPage Schema
How do I convert Illumina BCL files to FASTQ?▼

BCL to FASTQ conversion uses bcl-convert with the run folder, sample sheet, and output directory as inputs. Validate RunInfo.xml and SampleSheet.csv first, then run bcl-convert with --bcl-input-directory, --sample-sheet, and --output-directory flags.

bcl-convert vs bcl2fastq which should I use?▼

bcl-convert is the preferred modern tool and is free for local use but proprietary and RPM-distributed by Illumina. Legacy bcl2fastq should only be used when bcl-convert is unavailable or the run requires legacy compatibility.

What should I validate in a sample sheet before demultiplexing?▼

Check for duplicate sample and index combinations within each lane, confirm index sequence lengths match the index reads, and verify dual-index orientation explicitly for the instrument. Never infer i5 orientation from filenames.

Can bcl-convert be downloaded automatically during conversion?▼

No, bcl-convert is proprietary software distributed by Illumina as RPM packages and should not be auto-downloaded without explicit user approval. If neither bcl-convert nor bcl2fastq is installed, the blocker is recorded instead.

Why does demultiplexing produce high undetermined reads?▼

High undetermined percentages typically indicate index hopping, barcode collisions, sample sheet mismatches, or incorrect index orientation. Review top undetermined index sequences and per-lane metrics before starting downstream analysis.

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