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RNA Velocity Module Pipeline Specification

Pipeline Details

  • Name: RNA Velocity Module
  • Pipeline UUID: ec8fe071749a48cf953870c11ee25404
  • Version: 1.1.1
  • View Pipeline:

Overview

RNA Velocity Module pipeline is designed for analyzing RNA velocity in single-cell RNA-Seq data. It generates spliced and unspliced count matrices from cellranger output and uses it alongside Seurat-analyzed data to compute and visualize cellular differentiation trajectories, fate probabilities, and transition dynamics.

Key Use cases:

  • RNA Velocity Analysis: Compute RNA velocity to infer cell state transitions and differentiation trajectories
  • Fate Mapping: Identify terminal cell states and calculate fate probabilities for each cell
  • Trajectory Analysis: Visualize developmental paths using PAGA graphs and transition matrices
  • Condition Comparison: Compare velocity patterns and lineage trajectories across experimental conditions

Features

  • Integrated Workflow: Seamlessly merges Seurat h5ad objects with velocyto loom files based on matching barcodes
  • Multi-Sample Support: Processes multiple samples simultaneously while maintaining sample identity and metadata
  • Advanced Velocity Computation: Implements scVelo's dynamical, stochastic, and steady-state velocity models
  • CellRank Integration: Computes terminal states, fate probabilities, and lineage trajectories using CellRank
  • Condition-Based Analysis: Splits analysis by experimental conditions for comparative studies
  • Interactive Visualization: Web-based Shiny application for exploring velocity embeddings, PAGA graphs, and fate probabilities
  • Flexible Export: Download plots in multiple formats (PDF, SVG, PNG) for publication-ready figures
  • Batch Processing: Nextflow-based pipeline for automated, reproducible analysis at scale
  • Container Support: Uses specialized containers including quay.io/mustafapir/scvelo_shiny:1.0.2 and velocyto.py for reproducible environments

Input/Output Specification

Inputs

Required

BAM Files

  • Description: Aligned sequencing reads from single-cell RNA-seq experiments, typically from 10x Genomics Cell Ranger output
  • Format: .bam with corresponding .bai index files
  • Example File Path: /path/to/possorted_genome_bam.bam

GTF Annotation File

  • Description: Gene annotation file containing transcript and exon coordinates required for velocyto analysis
  • Format: .gtf
  • Example File Path: /path/to/annotation.gtf

Cell Barcodes

  • Description: Valid cell barcodes from filtered feature-barcode matrix
  • Format: .tsv.gz (compressed tab-separated values)
  • Example File Path: /path/to/filtered_feature_bc_matrix/barcodes.tsv.gz

h5ad Object

  • Description: Seurat-processed single-cell data in AnnData format containing cell embeddings and metadata
  • Format: .h5ad
  • Example File Path: /path/to/seurat_processed.h5ad

Optional Inputs

Mask GTF File

  • Description: GTF file containing repetitive elements and other regions to mask during velocyto analysis
  • Format: .gtf
  • Example File Path: /path/to/mask_repeats.gtf

Cell Ranger Output Directory

  • Description: Complete Cell Ranger output directory containing BAM files and feature-barcode matrices
  • Format: Directory structure
  • Example File Path: /path/to/cellranger_outs/

Outputs

Reported Outputs

  • Processed AnnData Object:
  • Description: Integrated h5ad file containing both gene expression data and velocity information
  • Format: .h5ad
  • Example File Path: /output/processed_adata.h5ad
  • Visualization App: Shiny Web Application (app9.py)

  • Location: Main output folder

  • Velocyto Loom File:

  • Description: Loom file containing spliced and unspliced count matrices generated by velocyto
  • Format: .loom
  • Example File Path: /output/sample_output.loom
  • Visualization App: scVelo, CellRank
  • Location: Velocyto output folder

Supporting Outputs

  • Interactive Visualization App:
  • Description: Web-based Shiny application for exploring velocity results across multiple analysis tabs
  • Format: Python application (app9.py)
  • Features: Velocity plots, PAGA trajectories, fate probabilities, condition comparisons

  • Location: Application folder

  • Preprocessed Input Files:

  • Description: Standardized input files prepared for velocyto analysis
  • Format: .bam, .bai, .tsv.gz
  • Example File Path: /intermediate/input.bam
  • Location: Preprocessing folder

Associated Processes

References & Additional Documentation

  • Related Papers:
  • Bergen, V., Lange, M., Peidli, S. et al. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol 38, 1408–1414 (2020).
  • Lange, M., Bergen, V., Klein, M. et al. CellRank for directed single-cell fate mapping. Nat Methods 19, 159–170 (2022).
  • scVelo Documentation: https://scvelo.readthedocs.io/
  • CellRank Documentation: https://cellrank.readthedocs.io/
  • Velocyto Documentation: http://velocyto.org/