ONT Basecalling / Demux Pipeline

Nextflow pipeline to perform basecalling and (optional) demultiplexing of ONT data, collect QC metrics and generate a MultiQC report. Uses Dorado for basecalling and demultiplexing.

Requirements

• Nextflow (>= 22.04)
• Apptainer / Singularity
• Dorado (0.7.3 tested). It can be used via container, or installed locally from https://github.com/nanoporetech/dorado.

Quick Start

1. Clone this repository:

   git clone https://github.com/catg-umag/ont-basecalling-demultiplexing

2. Demultiplexing setup (optional):
   • If demultiplexing is needed, create a samples.csv file containing at least the barcode and sample columns.
   • Ensure the barcode column includes the barcode identifier (e.g., barcode01), and the sample column lists the sample name, which will be used in reports and as the FASTQ filename.
3. Configure parameters:
   • Copy the example parameters file:

      cp params.example.yml my_params.yml

   • Modify my_params.yml according to your needs. Ensure that the sample_data parameter points to your samples.csv file if you are demultiplexing.
4. Run the pipeline:

   nextflow run ont-basecalling-demultiplexing/ -profile apptainer -params-file my_params.yml

Pipeline Parameters

Parameter	Required	Default	Description
experiment_name	No	-	Name of the experiment, used for reports (title and filename).
data_dir	Yes	-	Path to the directory containing POD5 files.
sample_data	No	-	Path to the CSV file containing the sample data (if not provided, will not perform demultiplexing).
output_dir	No	results	Directory for saving results.
fastq_output	No	true	Generates FASTQ files if true; otherwise, generates UBAM files.
qscore_filter	No	10	Minimum QScore threshold for "pass" data, used in demultiplexing.
dorado_basecalling_model	No	sup	Model used for basecalling. Check Dorado help for available options.
dorado_basecalling_gpus	No	1	Number of GPUs to allocate for basecalling.
dorado_demux_kit	No	EXP-NBD196	Kit identifier used for demultiplexing.
dorado_demux_both_ends	No	false	Demultiplexes using barcodes on both ends (5' and 3') if true.
use_dorado_container	No	true	Uses Dorado via container if true; expects a local installation if false.
qc_tools	No	['fastqc', 'nanoq', 'toulligqc']	Specifies which QC tools to run. Options: 'nanoq', 'nanoplot', 'fastqc', 'toulligqc', 'pycoqc'.

Considerations

• The pipeline is compatible with SLURM clusters; use -profile slurm.
• GPU resources are required for basecalling. On SLURM, this pipeline will send jobs requesting GPUs with the --gres=gpu:X option.
• You can provide extra args to dorado basecalling and demultiplexing using ext.args.