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Bio-Rad Laboratories, Inc. Omnition Single-Cell Analysis Software

⚠️ Beta Release Notice

This version of Omnition is a beta release. If you want general information on Omnition and the latest stable release v1.1 which supports 3' RNA-Seq and ATAC-Seq please see below: Introduction to Omnition

Please note:

  • Stability: This release may contain bugs and is not fully feature-complete, especially for the newly added Cytometry and CITE-Seq (Cytometry + RNA) assay support.
  • Recommended for Testing Only: If you need a stable production pipeline, switch to the main branch of this repository and use that version instead.
  • Feedback Welcome: This is an open beta. Please report any issues, share feedback, and suggest features—we’ll prioritize improvements based on your input.

✅ Changes Affecting All Modalities

  • Mixed Species YAML Format
    When supplying YAMLs for mixed-species experiments, the two sets of references (FASTA and GTF files) are now specified on a single line, separated by a space.
    See the example-parameters directory for updated formatting examples.

  • Updated Output Directory Structure
    With multi-omics support, the sample_files directory now includes an additional folder layer under each sample ID, organized by modality (e.g., RNA, Cytometry, ATAC).

🆕 New Modalities: Cytometry & CITE-seq

  • Run Modes Added:

    • Cytometry
    • CITE-Seq (Cytometry + RNA)

  • Supported Inputs:
    Any ADT or HTO with polyA capture can be analyzed alone or combined with the Bio-Rad RNA kit (protocols coming soon).

  • Configuration:

    • Specify cytometry in the YAML file.
    • See cytometry-full.yaml and cite-seq-full.yaml in example-parameters.
    • Provide a reference file (e.g., cytometry-adt-file.csv in example-parameters).

🔍 Current Beta Limitations

  • Reporting:
    No HTML report for Cytometry or CITE-seq yet. Instead, key quality metrics are output to report/metric_summary. 3' RNA-Seq reports are still generated when analysing CITE-Seq (no cytometry data included)

  • Outputs Available:

    • Assay-specific h5ad files
    • Pan-assay h5mu files
    • Count matrices
    • MultiQC report
    • Seurat object

If there are additional outputs you need, please let us know—we’ll prioritize adding them.

💬 Feedback & Issues

Your input is critical! Please share:

  • Bugs or errors you encounter
  • Feature requests
  • Suggestions for improving usability

Thank you for helping us make Omnition better!

Introduction to Omnition

Omnition is a pipeline designed to process data generated with Bio-Rad's ddSEQ™ Single-Cell 3’ RNA-Seq Kit or ddSEQ™ SureCell ATAC-Seq Library Prep Kit and the dsciATAC protocol. It performs debarcoding, alignment, bead merging, cell calling, and feature counting. Output is an HTML report and files for downstream biological analysis. 

This page is intended to be a quick start. For the complete Omnition user guides, please see: 

Omnition Single-cell 3’ RNA-Seq 

Omnition Single-cell ATAC-Seq

Table of Contents
  1. Introduction to Omnition
  2. Installation
  3. Getting started

Installation

Omnition Analysis Software utilizes the Nextflow framework to connect individual processes, and runs the processes in virtual environments called containers using the Docker or Singularity container programs. Once Omnition is installed on a system meeting minimum requirements, Nextflow integration with GitHub and Docker will prepare workflows without any additional configuration.

System requirements

  • Omnition is designed to run on a local Linux server, high performance computing (HPC) cluster, or cloud virtual machine, and has been tested on the 64-bit CentOS 7 and 8, Amazon Linux 2, and Ubuntu 18.04.6, 20.04 LTS, 21.04, and 21.10 Linux operating systems.

    NOTE: Although they might be functional, Bio-Rad does not support additional Linux variants or other versions of the specified operating systems.

  • Internet connection

    NOTE: For installation without direct internet access, please see user manual.

Hardware requirements

Requirement ATAC seq analysis Combinatorial ATAC seq analysis RNA seq analysis Recommended for >=12x samples
CPU 16 16 16 64
RAM 64 GB 128 GB 64 GB 512 GB
IOPS* 3,000 3,000 3,000 16,000
I/O throughput* 125 mbps 125 mbps 125 mbps 1,000 mbps
EBS volume type* gp3 gp3 gp3 gp3

*AWS specific cloud computing specifications

Software requirements

  • Nextflow (v22.04.0 to v23.10.1) or Nextflow with Conda

    NOTE: Specify the Nextflow version in the Conda installation by using the following command: conda install –c bioconda nextflow=<version>

  • Only one of the following container programs are needed: Either Docker (>=20.10.7) or Singularity (>=3.6.4)

    NOTE: If using Docker, your USER must be added to the docker root user group before executing the pipeline. On shared systems, such as HPC clusters, this may not be possible due to security risks and the pipeline should be executed using the Singularity profile (default) instead. The user must verify with their system administrator that Docker or Singularity is available before using Omnition.

Download Omnition

To download and run Omnition, use nextflow to retrieve the latest Omnition version from GitHub.

nextflow pull BioRadOpenSource/omnition

Verify installation

Omnition includes small demonstration datasets to verify that the environment has been properly built and all software dependencies are in place. To verify the success of the installation for each analysis type, run the Nextflow command for the container system that is installed on your computer (Singularity or Docker).

To verify each analysis workflow is installed correctly, run the following commands with either Docker or Singularity.

NOTE: Working files (and output files, unless otherwise specified) will be generated in the same directory the pipeline was run from on the command line.

mkdir /home/ubuntu/demo_data
cd /home/ubuntu/demo_data

NOTE: Specified file paths are for example purposes

Docker:

# Verify single and mixed species 3’ RNA workflows
nextflow run BioRadOpenSource/omnition -profile demo_rna_single,docker --core.outputDir /home/ubuntu/demo_data/rna
nextflow run BioRadOpenSource/omnition -profile demo_rna_mixed_options,docker --core.outputDir /home/ubuntu/demo_data/rna_mixed

# Verify ATAC-seq and combinatorial ATAC-seq workflows
nextflow run BioRadOpenSource/omnition -profile demo_atac,docker --core.outputDir /home/ubuntu/demo_data/atac
nextflow run BioRadOpenSource/omnition -profile demo_catac,docker --core.outputDir /home/ubuntu/demo_data/catac

Singularity:

# Verify single and mixed species 3’ RNA workflows
nextflow run BioRadOpenSource/omnition -profile demo_rna_single,standard --core.outputDir /home/ubuntu/demo_data/rna
nextflow run BioRadOpenSource/omnition -profile demo_rna_mixed_options,standard --core.outputDir /home/ubuntu/demo_data/rna_mixed

# Verify ATAC-seq and combinatorial ATAC-seq workflows
nextflow run BioRadOpenSource/omnition -profile demo_atac,standard --core.outputDir /home/ubuntu/demo_data/atac
nextflow run BioRadOpenSource/omnition -profile demo_catac,standard --core.outputDir /home/ubuntu/demo_data/catac

NOTE: Please note the use of - and -- in the execution commands. Arguments with a single - in front are Nextflow arguments and those with -- are user-defined parameters. You may also use the Nextflow -r flag to specify a git tag (i.e. release), branch, or hash to execute the pipeline at that point in the git history.

NOTE: When launching a nextflow run there will be a randomly generated [adjective_names] that appear in the terminal. These names are from a list prepared by Nextflow. This is an inherent feature of nextflow, not something added by Bio-Rad.

When the run finishes, you should see a message that it has completed without any failed tasks.

Getting started

Before running a workflow, Omnition requires the following files to run:

  • Genome FASTA and GTF files from ENSEMBL.
  • Genome reference sequences must be formatted as FASTA files.
  • Annotations must be formatted as GTF files.
  • Sequence names in the FASTA and GTF files must match.
  • Directory with input FASTQs

NOTE: Input reference files can be compressed as gzip (.gz) files

References

Omnition is compatible with references from ENSEMBL. The ENSEMBL Human/GRCh38 and Mouse/GRCm39 references are supported by Omnition. Other species from ENSEMBL are not supported and references produced by other sources (NCBI, GENCODE, etc.) are not compatible.

Human and mouse references can be obtained with the following commands.

Human genome

mkdir ~/references/human
cd ~/references/human

NOTE: Specified file paths are for example purposes

curl -o Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz \
http://ftp.ensembl.org/pub/release-106/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

curl -o Homo_sapiens.GRCh38.106.gtf.gz \
http://ftp.ensembl.org/pub/release-106/gtf/homo_sapiens/Homo_sapiens.GRCh38.106.gtf.gz

Mouse Genome

mkdir ~/references/mouse
cd ~/references/mouse

curl -o Mus_musculus.GRCm39.dna.primary_assembly.fa.gz \
http://ftp.ensembl.org/pub/release-106/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.primary_assembly.fa.gz

curl -o Mus_musculus.GRCm39.106.gtf.gz \
http://ftp.ensembl.org/pub/release-106/gtf/mus_musculus/Mus_musculus.GRCm39.106.gtf.gz

Running Omnition

YAML configuration

The pipeline requires a YAML-formatted file with input/output paths and assay-specific parameters when not running the test data. A detailed description of the file contents, available parameters, and how to format them can be found in the user manual. Example YAML configs can be found under the example-yamls folder of this repo.

Run RNA pipeline

With Singularity:

nextflow run BioRadOpenSource/omnition -params-file <path to rna_example.yaml>

With Docker:

nextflow run BioRadOpenSource/omnition -params-file <path to rna_example.yaml> -profile docker

Run ATAC pipeline

With Singularity:

nextflow run BioRadOpenSource/omnition -params-file <path to atac_example.yaml>

With Docker:

nextflow run BioRadOpenSource/omnition -params-file <path to atac_example.yaml> -profile docker

Outputs

After completion, reports can be found in the results/report/ subdirectory and intermediate files can be found in the results/Sample_Files/ subdirectory. Additionally, a Nextflow cache directory (.nextflow/) and working directory (work/) will be created in the directory where Nextflow was executed from. This allows interrupted/failed analyses to resume from their point of failure. If you would like to continue a run, add -resume to the execution commands below. You can delete the cache and working directories to save space after completion though this will inhibit the -resume feature.


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