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Cell Ranger Multi Pipeline Specification

Pipeline Details

  • Name: Cell Ranger Multi Pipeline
  • Pipeline UUID: 681a4cccc74e41fe9d43a1ddf38fa77a
  • Version: 4.7.1
  • View Pipeline:

Overview

Cell Ranger Multi Pipeline is designed for processing Chromium single-cell RNA-seq output to align reads, generate feature-barcode matrices and perform clustering and gene expression analysis. It automates data preprocessing, quality control, and downstream analysis to ensure reliable and reproducible results using Cell Ranger v9.0.1.

Key Use cases:

  • 3' Gene Expression + Cell Multiplexing: Analysis of multiplexed single-cell samples with cell multiplexing oligos (CMOs) and optional antibody/CRISPR guide capture.
  • Flex Gene Expression Analysis: Processing of 10x Flex gene expression libraries with optional antibody or CRISPR guide capture.
  • 5' Gene Expression + V(D)J Profiling: Combined gene expression and immune profiling analysis for T-cell and B-cell receptor sequencing.
  • Multimodal Single-Cell Analysis: Integrated analysis combining gene expression, protein detection (antibody capture), and immune profiling in a single workflow.

Features

  • Support for Multiple Library Types: Handles Gene Expression, Antibody Capture, CRISPR Guide Capture, Multiplexing Capture, VDJ, VDJ-T, VDJ-T-GD, VDJ-B, Antigen Capture, and Custom libraries.
  • BCL Conversion Integration: Optional BCLConvert v4.3 integration for direct demultiplexing from Illumina sequencer output.
  • Modular Design: All tools are integrated as modules allowing selective enabling/disabling of components including FastQC, MultiQC, scRNA-seq analysis, RNA velocity, pySCENIC, and Slingshot modules.
  • Cloud Integration: Supports reading input data directly from S3 or GCP with automatic handling.
  • Advanced Analysis Modules: Includes optional RNA velocity analysis, gene regulatory network inference with pySCENIC, and trajectory analysis with Slingshot.
  • Automated Cell Type Annotation: Built-in cell type annotation using sc-type tool with tissue-specific reference datasets.
  • Scalability: Adaptable to various compute environments and data sizes with customizable cell detection and filtering parameters.

Input/Output Specification

Inputs

Required

Run BCL-Convert

  • Description: Choose whether to demultiplex BCL files to FASTQ format using BCLConvert v4.3.
  • Format: DROPDOWN
  • Options: "yes", "no"
  • Default: "no"

Genome Build

  • Description: Reference genome selection for alignment and quantification.
  • Format: DROPDOWN
  • Options: human_hg38_gencode_v32_cellranger_v6, human_hg38_cellranger_GRCh38-2024-A, mouse_mm10_gencode_vm23_cellranger_v6, mouse_GRCm39_cellranger_GRCm39-2024-A, human-mouse_cellranger_GRCh38-GRCm39-2024-A, zebrafish_GRCz11plus_ensembl, d_melanogaster_BDGP6_32_ensembl_105_cellranger_v6, d_melanogaster_flybase_r6_45_cellranger_v6, custom

libraries

  • Description: Table defining FASTQ files and their corresponding feature types for Cell Ranger Multi analysis.
  • Format: TABLE
  • Required Columns: fastq_id, group (optional), feature_types
  • Supported Feature Types: Gene Expression, Antibody Capture, CRISPR Guide Capture, Multiplexing Capture, VDJ, VDJ-T, VDJ-T-GD, VDJ-B, Antigen Capture, Custom

Optional Inputs

reads

  • Description: Cloud/local dataset containing input FASTQ.GZ files. Required when Run BCL-Convert is set to "no".
  • Format: FASTQ.GZ

Sample Separation

  • Description: Table for demultiplexing multiplexed samples using CMO IDs, hashtag IDs, OCM barcode IDs, or probe barcode IDs.
  • Format: TABLE
  • Required Columns: sample_id, group, cmo_ids (for CMO), hashtag_ids (for antibody hashing), ocm_barcode_ids (for OCM), probe_barcode_ids (for FLEX), description (optional)

feature_reference

  • Description: Feature barcode reference CSV file for antibody capture or CRISPR guide capture libraries.
  • Format: CSV
  • Required Columns: id, name, read, pattern, sequence, feature_type

cmo_set

  • Description: Cell Multiplexing Oligos (CMO) reference file. Optional - Cell Ranger uses built-in CMO set by default.
  • Format: CSV
  • Required Columns: id, name, read, pattern, sequence, feature_type

VDJ_reference

  • Description: V(D)J reference for immune profiling analysis. Required for VDJ-related feature types.
  • Format: Folder or tar.gz archive

Metadata

  • Description: Tab-separated sample metadata file for downstream analysis and visualization.
  • Format: TABLE
  • Required Columns: Sample ID, Condition, Replicate

Outputs

Reported Outputs

  • geneexpressionmatrix.csv:
  • Description: Normalized gene expression matrix from Cell Ranger Multi analysis
  • Format: CSV
  • Location: results/geneexpressionmatrix.csv

  • Visualization App: DE Browser, UCSC Genome Browser

  • summaryreport.html:

  • Description: Comprehensive summary report of Cell Ranger Multi run with QC metrics and analysis results
  • Format: HTML

  • Location: results/report.html

  • alignedreads.bam:

  • Description: Aligned sequencing reads in BAM format from Cell Ranger Multi
  • Format: BAM
  • Location: results/alignments/alignedreads.bam
  • Visualization App: IGV, UCSC Genome Browser

Supporting Outputs

  • fastqcreport.html:
  • Description: FastQC quality control report for raw sequencing data (if enabled)
  • Format: HTML

  • Location: results/qc/fastqcreport.html

  • scvelo_out.h5ad:

  • Description: H5AD file containing RNA velocity analysis results for trajectory inference
  • Format: h5ad
  • Location: scVelo_out/scvelo_out.h5ad

  • Visualization App: scVelo Shiny App

  • sce_fitgam.rds:

  • Description: Seurat object with Slingshot trajectory analysis results
  • Format: rds
  • Location: fitgam_rds/sce_fitgam.rds

  • Visualization App: Slingshot Shiny App

  • scenic_integrated.loom:

  • Description: Loom file with integrated pySCENIC gene regulatory network analysis and AUCell scores
  • Format: loom

  • Location: pySCENIC_loom/scenic_integrated.loom

  • pyscenic_out.zip:

  • Description: Compressed archive containing pySCENIC pipeline results including adjacencies, regulons, and AUCell matrices
  • Format: zip
  • Location: pySCENIC_out/pyscenic_out.zip

Associated Processes

References & Additional Documentation

  • Related Papers/links:
  • https://www.10xgenomics.com/support/software/cell-ranger/latest
  • https://www.10xgenomics.com/support/software/cell-ranger/downloads
  • https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/multi
  • BCLConvert v4.3 User Guide: https://help.dragen.illumina.com/product-guides/dragen-v4.3/bcl-conversion
  • Pipeline Repository: https://github.com/10XGenomics/cellranger