ScaleBio Seq Suite: RNA Workflow

This is a Nextflow workflow to run analysis of ScaleBio Single Cell RNA Sequencing libraries. It processes data from sequencing reads to alignments, single-cell outputs (gene-expression matrix, etc.), and QC reports.

The QuantumScale RNA assay is supported with version 2.0 and later.

Getting started

• First install Nextflow (version 23.10 or later)
• Download this workflow to your machine
• Setup dependencies
• Launch the small pipeline test run
• Download / configure a reference genome for your samples
• Create a samples.csv table for your samples
• Create runParams.yml, specifying inputs and analysis options for your run
• Launch the workflow for your run

Requirements

• Linux system with GLIBC >= 2.17 (such as CentOS 7 or later)
• Java 11 or later
• 64GB of RAM and 12 CPU cores

Required Inputs

• Sequencing reads (one of the following)
  • An Illumina sequencer RunFolder (BCL files)
  • A directory with FASTQ files including index reads; see Fastq generation.
  • A directory containing unaligned cram files from Ultima Genomics sequencing
• Sample table
  • A .csv file listing all samples for this analysis run, optionally split by RT barcode. See samples.csv.
• Reference genome
  • The workflow requires a reference genome, including a STAR index for alignment, and gene annotation. See Reference Genomes
• Kit version / Library structure definition
  • If using something other than Quantum RNA, see Analysis Parameters

Outputs

The workflow produces per-sample and per-library ScaleBio demultiplexed reads (fastqs), alignments (bam), QC reports (html), a cell-by-gene count-matrix (mtx) and more; See Outputs for a full list.

ScalePlex

To analyze your ScaleRNA sequencing data with ScalePlex labeling and assignment, see ScalePlex

Workflow Execution

Workflow test

A small test run, with all input data stored online, can be run with the following command:

nextflow run /PATH/TO/ScaleRna -profile PROFILE -params-file /PATH/TO/ScaleRna/docs/examples/runParams.yml --outDir output

-profile docker is the preferred option if the system supports Docker containers; See Dependency Management for alternatives.

With this command, nextflow will automatically download the input data from the internet (AWS S3), so please ensure that the compute nodes have internet access and storage space. Alternatively you can manually download the data first (using AWS CLI):

aws s3 sync s3://scale.pub/testData/rna/202506_quantumV1/fastq fastq --no-sign-request
aws s3 sync s3://scale.pub/testData/rna/GRCh38_chr20_genome GRCh38_chr20_genome --no-sign-request

and then run with

nextflow run /PATH/TO/ScaleRna/ -profile PROFILE -params-file /PATH/TO/ScaleRna/docs/examples/runParams.yml --genome GRCh38_chr20_genome/genome.json --fastqDir fastq --outDir /PATH/TO/OUTPUT_DIR

Note that this test run is merely a quick and easy way to verify that the pipeline executes properly and does not represent a complete or realistic dataset.

Nextflow command line (CLI)

nextflow options are given with a single - (e.g. -profile), while workflow parameters (e.g. --outDir) are given with a double dash --. See the Nextflow command line documentation for further details.

A typical command to run the workflow would be:

nextflow run /PATH/TO/ScaleRna/ -profile docker --samples samples.csv --genome /PATH/TO/GRCh38/grch38.json --runFolder /PATH/TO/230830_A00525_1087_BHLFF3DSX7/ --outDir output

Configuration

Specifying Analysis Parameters

Analysis parameters (inputs, options, etc.) can be defined either in a runParams.yml file or directly on the nextflow command-line. See Analysis Parameters for details on the options.

Config File

In addition to the analysis parameters, a user-specific nextflow configuration file can be used for system settings (compute and storage resources, resource limits, storage paths, etc.):

-c path/to/user.config

See Nextflow configuration for the way different configuration files, parameter files and the command-line interact.

Dependency Management

The Nextflow workflow can automatically use pre-built docker containers with all dependencies included. Activating the included -profile docker enables the required Nextflow settings. For details and alternatives see Dependencies.

Running in the cloud

Nextflow itself supports execution on AWS, Azure, and Google Cloud.

In addition Seqera Platform offers another simple way to manage and execute nextflow workflows in Amazon AWS.

Versions and Updates

See the change log

License

By purchasing product(s) and downloading the software product(s) of ScaleBio, You accept all of the terms of the License Agreement. If You do not agree to these terms and conditions, You may not use or download any of the software product(s) of ScaleBio.