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ngs-bulk-rnaseq-counts-qc

Run or plan bulk RNA-seq FASTQ-to-count processing with sample-sheet, strandedness, genome annotation, alignment or pseudoalignment, MultiQC, and count-matrix QC checks.

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  • SKILL.md3.9 KB
  • agents/openai.yaml291 B

SKILL.md(原文)

インストールする前に、エージェントに与えられる指示の中身を確認できます。

Bulk RNA-seq Counts QC

Use this skill for bulk RNA-seq read processing, quantification, and count-matrix generation. If the user already has a count matrix and wants contrasts or statistics, use ngs-bulk-rnaseq-differential-expression.

Essential Inputs

Confirm:

  • FASTQ or aligned-read inputs and paired-end/single-end status
  • organism, genome build, FASTA, GTF, and gene ID convention
  • strandedness or permission to infer strandedness
  • sample sheet with biological condition, replicate, batch, and library metadata
  • desired quantification: gene counts, transcript estimates, or both
  • alignment strategy: STAR/Salmon, Salmon-only, featureCounts from BAMs, or existing lab protocol

Route

Prefer nf-core/rnaseq for standard processing when a stable container or HPC runtime is available. Use the local_light Snakemake/Salmon path for small local/devbox feasibility runs when Docker, registry egress, or Nextflow process containers are the blocker.

The plugin-owned local runner is:

python plugins/ngs-analysis/scripts/run_bulk_rnaseq_counts_qc.py \
  --sample-sheet samplesheet.csv \
  --fastq-root path/to/fastqs \
  --transcriptome-fasta reference/transcriptome.fasta \
  --genome-fasta reference/genome.fa \
  --annotation-gtf reference/genes.gtf \
  --execute

Omit --execute for validation plus Snakemake workflow validation only. Use --no-dry-run only when the user wants input validation and run-envelope preparation without workflow graph validation.

The runner emits a run-local resources/ readiness bundle with resource_plan.json, resource_manifest.tsv, resource_env.sh, and resource_readiness.md. Resource checks are advisory by default for custom or reduced references; add --genome-build, --bundle-root <bundle>=<path>, and --require-resource-plan when a registered genome bundle must be complete before the run is considered ready.

Preflight command:

python plugins/ngs-analysis/scripts/ngs_preflight.py --pipeline bulk_rnaseq_counts_qc --emit-install-plan
python plugins/ngs-analysis/scripts/ngs_preflight.py --profile local_light --emit-install-plan

Decision Points

  • If strandedness is unknown, infer it before final counting; do not lock in a design based on library guesses.
  • If strandedness is provided, carry it into the quantification command and flag any disagreement between the configured library type and Salmon's inferred format.
  • Keep genome FASTA, GTF, transcriptome, and aligner indexes from the same build/release.
  • Inspect per-sample reads, mapping rate, rRNA/mitochondrial fraction when available, duplication, insert size, gene-body bias, and assignment rate.
  • Preserve raw counts separately from normalized expression.
  • Carry sample metadata forward exactly; downstream DE depends on this table.

Outputs

Produce:

  • sample sheet and command/profile
  • reference manifest with genome and GTF release
  • MultiQC or equivalent processing summary
  • Salmon quant.sf outputs, TPM/NumReads/effective-length matrices, and carried-forward sample metadata
  • Gene-level expected-count and TPM matrices derived from transcript-level Salmon outputs, plus a tx2gene provenance table
  • Compact QC verdict JSON covering mapping rate, duplication, library-type agreement, and outlier samples
  • Browser-safe MultiQC helper HTML pages and a localhost launch hint for reliable in-app review
  • Run-local reference readiness artifacts under resources/, including the resource plan, manifest, environment exports, and Markdown readiness summary
  • issues that block differential expression, such as missing replicates, mislabeled groups, or severe batch/library failures
  • standard run envelope: run_manifest.json, config.json, validation/, logs/, versions/, artifact_index.json, and summary.md

レビュー

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