IRIDA Viral Amplicon Consensus & Variants Pipeline Plugin

This project contains a pipeline implemented as a plugin for the IRIDA bioinformatics analysis system. This can be used to construct a consensus sequence for a viral genome that has been sequenced using a tiling amplicon strategy such as Primal Scheme.

Table of Contents

• IRIDA Viral Amplicon Consensus & Variants Pipeline Plugin
• Installation
  • Installing Galaxy Dependencies
  • Installing to IRIDA
  • Setting up your primer scheme file(s)
• Usage
  • Analysis Results
  • Metadata Table
• Building
  • Installing IRIDA to local Maven repository
  • Building the plugin
• Dependencies

Installation

Installing Galaxy Dependencies

In order to use this pipeline, you will also have to install the following Galaxy tools and data managers within your Galaxy instance. These can be found at:

Name	Tool Dependency Version	Owner	Metadata Revision	Galaxy Toolshed Link
trim_galore	0.6.3	bgruening	15 (2019-07-30)	trim_galore-15:084bbd8ba7b8
bwa	0.7.17	devteam	23 (2020-05-19)	bwa-23:3fe632431b68
samtools_view	1.9	iuc	7 (2020-01-21)	samtools_view-7:b01db2684fa5
suite_ivar	1.2.1	iuc	2 (2020-04-22)	suite_ivar-2:4b5c86ac057f
quast	5.0.2	iuc	7 (2019-07-24)	quast-7:59db8ea8c845

Installing to IRIDA

Please download the provided irida-plugin-viral-amplicon-consensus-variants-[version].jar from the [releases][] page and copy to your /etc/irida/plugins directory. Now you may start IRIDA and you should see the pipeline appear in your list of pipelines.

Note: This plugin requires you to be running IRIDA version >= 19.01. Please see the IRIDA documentation for more details.

Setting up your primer scheme file(s)

Primer schemes must be supplied to this pipeline via a Galaxy Tool Data Table called primer_scheme_bedfiles.

ARTIC primer scheme bedfiles are available from the artic-ncov2019 GitHub repo.

A Galaxy administrator will need to save the primer scheme bedfile(s) to an appropriate place in the Galaxy server that runs this pipeline, such as tool-data/primer_scheme_bedfiles.

Once the files are saved, make an entry in the primer_scheme_bedfiles.loc file that is installed with the ivar_trim tool. That file is located at tool-data/toolshed.g2.bx.psu.edu/repos/iuc/ivar_trim/cb903c9dc33d/primer_scheme_bedfiles.loc. Each entry in the primer_scheme_bedfiles.loc file should include three tab-delimited fields:

1. Value: A unique value, similar to a database primary key. Consider using a UUID.
2. Description: A human-readable label that is exposed to Galaxy and IRIDA end-users. eg: "ARTIC nCoV-2019 primers V3"
3. Path: The path to the primer scheme bedfile.

After editing the primer_scheme_bedfiles.loc file, confirm that the entries have been loaded using the Galaxy web interface. As an administrator, navigate to the 'Local Data' section of the Admin panel. Find 'primer_scheme_bedfiles' in the list of Tool Data Tables, and click on it. The entries should be displayed in a table:

If no entries are shown, a restart of the galaxy server process may be necessary. If the entries are still not shown, confirm that all lines in the primer_scheme_bedfiles.loc file are tab-delimited.

Usage

The plugin should now show up in the Analyses > Pipelines section of IRIDA.

Parameters

Note: As this pipeline is under active development, the list of exposed parameters and the default values of these parameters are subject to change.

Tool	Parameter	Description	Default Value
trim_galore	quality	Threshold for trimming low-quality ends from reads (phred scale)	20
trim_galore	min_length	Discard reads that became shorter than this length	30
ivar_trim	min_length	Minimum length of read to retain after trimming	30
ivar_trim	min_qual	Minimum quality threshold for sliding window to pass	20
ivar_trim	window_width	Sliding window width (bp)	4
ivar_trim	inc_primers	Include reads with no primers	true
ivar_consensus	min_depth	Minimum required depth to call a consensus base	10
ivar_consensus	min_freq	Minimum frequency that a base must match to be called as the consensus base at a position.	0.75
ivar_consensus	min_qual	Minimum quality score threshold to count base	20
ivar_consensus	gap	Use N instead of - for regions with less than minimum coverage	true
ivar_variants	min_freq	Minimum frequency threshold	0.25
ivar_variants	min_qual	Minimum quality score threshold to count base	20

Analysis Results

Analysis results include a consensus sequence in .fasta format, a table of variants in .tsv format.

In addition, quality-control results describing input read trimming and consensus sequence characteristics are created.

Metadata Table

This pipeline plugin does not currently write any metadata to the IRIDA 'Line List'.

Building

Building and packaging this code is accomplished using Apache Maven. However, you will first need to install IRIDA to your local Maven repository. The version of IRIDA you install will have to correspond to the version found in the irida.version.compiletime property in the pom.xml file of this project. Right now, this is IRIDA version 19.01.3.

Installing IRIDA to local Maven repository

To install IRIDA to your local Maven repository please do the following:

1. Clone the IRIDA project

git clone https://github.com/phac-nml/irida.git
cd irida

2. Checkout appropriate version of IRIDA

git checkout 19.01.3

3. Install IRIDA to local repository

mvn clean install -DskipTests

Building the plugin

Once you've installed IRIDA as a dependency, you can proceed to building this plugin. Please run the following commands:

cd irida-plugin-viral-amplicon-consensus-variants

mvn clean package

Once complete, you should end up with a file target/irida-plugin-viral-amplicon-consensus-variants-<version>.jar which can be installed as a plugin to IRIDA.

Dependencies

The following dependencies are required in order to make use of this plugin.

• IRIDA >= 0.23.0
• Java >= 1.8 and Maven (for building)