Nanopore Basecalling Workflow

This Nextflow pipeline is designed to process Oxford Nanopore raw signal data (POD5 files) through basecalling and optional demultiplexing steps. It supports both simplex and duplex basecalling modes using Dorado.

Pipeline Description

Overview

The pipeline consists of a single workflow that processes Nanopore POD5 files through several phases:

1. A basecalling phase using Dorado in either simplex or duplex mode
2. An optional demultiplexing phase for barcoded samples
3. A final conversion phase to generate FASTQ files from BAM output

Pipeline Outputs

The workflow produces the following outputs:

1. raw/: Directory containing the final FASTQ files
2. unclassified/: Directory containing unclassified FASTQ files (only relevant for demultiplexing)

Using the Workflow

Installation & Setup

1. Install Nextflow (23.04.0+)
2. Install Docker
3. Set up AWS BATCH
4. Clone this repository

Running the Pipeline

Basic usage:

Create a new directory, name it after the delivery, copy in basecall.config as nextflow.config, and set the parameters. Params:

• duplex
  • Duplex basecalling or no? You can't combine duplex and demux
• demux
  • Demultiplex basecalling output?
• nanopore_run
  • Name of run/delivery
• kit
  • Name of ONT kit, needed for demux'ing

Additionally, add a barcodes.txt file to the directory, containing the barcodes to be demultiplexed, in the format:

01
02
12
...

Once that is done, you can switch into the directory and run

nextflow run .. -resume