This is a Nextflow workflow to run analysis of ScaleBio Tagmentation Toolkit sequencing libraries. It processes data from sequencing reads to alignments, single-cell outputs (peak-count matrix, etc.), and QC reports. Optionally initial ATAC downstream analysis with ArchR can be run automatically.
- First install Nextflow
- Download this workflow to your machine
- Install dependencies
- Get the bead barcode sequence file
- 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
- Sequencing reads
- Path to the Illumina Sequencer RunFolder (bcl files)
- If you prefer to start from fastq files, generated outside (before) this workflow, see Fastq generation.
- Sample Table
- A .csv file listing all samples in the analysis with their library (PCR) index and (optional) tagmentation sample barcode sequences. See samples.csv.
- Reference Genome
- The workflow requires a reference genome, including a
bowtie2index for alignment and gene annotation; see Reference Genomes. - Pre-built reference genomes are currently available for human (grch38) and mouse (mm39).
- The workflow requires a reference genome, including a
The workflow requires information on the location and expected sequences for all cell-barcodes, see analysisParameters.md.
Before running any analysis the user needs to add a file with the bead barcode sequences from the underlying droplet system software. For ATAC this file is named 737K-cratac-v1.txt.gz and needs to be copied to ScaleTagToolkit/references before any analysis can be run.
The workflow produces alignments (.bam and fragments.bed), a cell-by-peak count-matrix (.mtx), QC reports and many other files. See Outputs for a full list.
A small test run, with all input data stored online, can be done with
nextflow run /PATH/TO/ScaleTagToolkit -profile PROFILE -params-file /PATH/TO/ScaleTagToolkit/docs/examples/runParams.yml --outDir output
See dependencies for the best PROFILE to use on your system.
Note that 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 the options to run nextflow on different systems (including HPC clusters and cloud compute).
Once output is generated, you may wish to rerun the QC filtering and HTML report generating scripts. This is particularly useful if you'd like to change the filter thresholds to include or exclude cells in the report. See Rerunning Scripts for details.
Analysis parameters (inputs, options, etc.) can be defined either in a runParams.yml file or directly on the nextflow command-line (e.g. --samples samples.csv). See analysisParameters for details on the options.
Additional parameters used by the QC filtering process (cellFilter) and automated ArchR Analysis process (archrAnalysis) are defined in qcAndArchR.yml. For
information on how best to modify see additionalInputParams.
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.
Different options to provide all required dependencies are described here. Follow one approach there and then run nextflow with the corresponding -profile.
Nextflow itself supports execution using AWS, Azure and Google Cloud.
In addition Nextflow tower offers another way to manage and execute nextflow workflows online.
See the Change log
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.