scrnaseq-cellranger-count

Skill: scRNA-seq Cell Ranger Count

Use When

• User has raw FASTQ files from a droplet-based single-cell protocol (10x Chromium, Parse Biosciences, etc.) and needs a feature-barcode count matrix.
• User wants to run Cell Ranger count, STARsolo, or alevin-fry to produce per-cell gene expression matrices.
• User needs to assess sequencing saturation or cell calling statistics before downstream analysis.
• User is generating input for the scrnaseq-quality-control skill.

Inputs

• Required:
  • Raw FASTQ files (Cell Ranger naming: {sample}_S{n}_L00{lane}_R{read}_001.fastq.gz)
  • Reference transcriptome (Cell Ranger reference package, STAR genome index, or salmon index)
• Optional:
  • Aligner choice: cellranger, starsolo, or alevin-fry (default: cellranger)
  • 10x chemistry: auto, threeprime, fiveprime, SC3Pv2, SC3Pv3, SC3Pv3.1, SC3Pv4 (default: auto)
  • Number of threads (default: 8)
  • Expected number of cells (leave unset to use automatic knee-point detection)
  • Output directory (default: ./cellranger_output)
  • Cell barcode whitelist (required for STARsolo; bundled in Cell Ranger reference)

Workflow

1. Validate FASTQ naming convention. Cell Ranger requires {sample}_S{n}_L00{lane}_R{read}_001.fastq.gz; rename files if needed. STARsolo accepts generic paired FASTQ names.
2. If Cell Ranger: run cellranger count with --id, --transcriptome, --fastqs, --sample, and --chemistry flags.
3. If STARsolo: run STAR with --soloType CB_UMI_Simple, providing --soloCBwhitelist, --soloCBstart/End, --soloUMIstart/End for the appropriate chemistry.
4. If alevin-fry: run simpleaf quant with the pre-built index and chemistry string; use --resolution cr-like for Cell Ranger-compatible output.
5. Report cell calling statistics: estimated cells, mean reads per cell, median genes per cell, sequencing saturation, fraction reads in cells.
6. Verify output: confirm existence of filtered and raw (unfiltered) feature-barcode matrices in MEX format.

Output Contract

• Filtered feature-barcode matrix directory (filtered_feature_bc_matrix/): matrix.mtx.gz, barcodes.tsv.gz, features.tsv.gz
• Raw (unfiltered) feature-barcode matrix directory (raw_feature_bc_matrix/)
• Web summary HTML (Cell Ranger: web_summary.html) or equivalent STARsolo/alevin-fry summary
• Per-barcode metrics CSV (metrics_summary.csv for Cell Ranger)
• BAM file and index (Cell Ranger only: possorted_genome_bam.bam, .bai)

Limits

• Cell Ranger requires a commercial license (free for academic) and approximately 10–30 GB disk per sample for intermediate files.
• STARsolo requires a STAR genome index and the appropriate cell barcode whitelist (bundled in Cell Ranger references or available from 10x Genomics).
• alevin-fry is fastest and lowest-memory but requires salmon index built with simpleaf index.
• Cell calling at the knee-point may under-call rare cell types; consider running EmptyDrops (via DropletUtils in R) on the raw matrix for improved sensitivity.
• Ambient RNA contamination is not removed at this step; handle in the scrnaseq-quality-control skill.
• Reference genome and transcriptome annotation must match (same genome build and GTF version).
• Common failure cases:
  • FASTQ naming mismatch causing Cell Ranger to find no reads for the sample.
  • Chemistry mismatch (auto detection fails on unusual protocols); specify explicitly.
  • Insufficient disk space for BAM file (~10x raw FASTQ size).
  • Reference built with a different Cell Ranger version causing incompatibility.