rchapman2000 / xgen-ont-pipeline

Assembles genomes from IDT's xGen amplicon sequencing data processed on Oxford Nanopore Sequencing Technology

Nanopore xGen Amplicon Assembly Pipeline

This pipeline automates the process of assembling genomes from IDT's xGen Amplicon Panels sequenced using Oxford Nanopore technology.

Technical Considerations

xGen Primer File

For primer clipping, this pipeline uses the tool Primerclip which was developed for IDT's xGen technology (formerly Swift Biosciences) specifically.

The input for Primerclip is different from the traditional BED file used for tools such as iVar. Instead, IDT creates a special "Masterfile" text file that contains information about each potential amplicon (amplicon coordinates, primer coordinates, primer sequences, etc). IDT will provided this file to those who have purchased one of their products.

This file is requires for the pipeline, and must be specified via the --primers option.

Medaka Model

Medaka requires information about the pore, sequencing device, and basecaller. This information is specified to the tool through a 'model', which is a string of text in the following format:

{pore}_{device}_{caller variant}_{caller version}

The pipeline requires a medaka model as input. To see a list of medaka models, use the command medaka tools list_models.

As well, it is important to note, the models will not contain individual versions of Guppy. Thus, you should choose the version most closest to and less than the version you used (Ex: using Guppy 6.00, enter R***_min_hac_g507)

Installation

To install this pipeline, enter the following commands:

# Clone the repository
git clone https://github.com/rchapman2000/xgen-ont-pipeline

# Create a conda environment using the provided environment.yml file
conda env create -f environment.yml

# Activate the conda environment
conda activate xGenONTAssembly

Updating the Pipeline

If you already have the pipeline installed, you can update it using the following commands:

# Navigate to your installation directory
cd xgen-ont-pipeline

# Use git to pull the latest update
git pull

# Activate the conda environment and use the environment.yml file to download updates
conda activate xGenONTAssembly
conda env update --file environment.yml --prune

Usage

To run the pipeline, use the following command:

# You must be either in the same directory as the main.nf file or reference the file location
nextflow run main.nf [options] --input INPUT_DIR --output OUTPUT_DIR --reference REFERENCE_FASTA --primers XGEN_MASTERFILE --model MEDAKA_MODEL

Optional Arguments

The pipeline also supports the following optional arguments:

Option	Type	Description
--minCov	int	The minimum depth of coverage required to report a site in the final consensus genome. Sites below this threshold will be masked [Default = 20]
--threads	int	The number of CPU threads that can be use to run pipeline tools in parallel

To view the list of options from the command line, use the following command:

nextflow run main.nf --help