Assembly & AMR

The pipeline is currently under development!

Author

• Andreas Evenstad (https://github.com/aevenstad/)

Table of contents

• Introduction
• Requirements
  • Dependencies
  • Databases
• Quickstart
  • Download the pipeline
  • Get dependencies
  • Download databases
  • Arguments
  • Run the pipeline
  • Hybrid input
  • Long-read input
  • Short-read input

Introduction

This is a Nextflow pipeline written using the nf-core template, and is made for analyzing whole-genome sequencing data from bacterial isolates. Its main function is to asses antimicrobial resistance in the provided isolates and the main steps of the pipeline is: * Genome assembly: -hybridorlong` mode with hybracter (0.11.0) - Short read mode with shovill (1.1.0) (using spades assembler) * Multi locus sequence typing: - MLST (2.23.0) - rMLST (https://gist.github.com/kjolley/703fa2f00c3b2abeef9242fa193ea901#file-speciesapiupload-py) * Resistance analysis: - AMRFinderPlus (4.0.3) - Kleborate (3.1.2) (for Klebsiella) - PlasmidFinder (2.1.6) - LRE-Finder (1.0.0) (for enterobactales) * Annotation: - Bakta (1.10.4)

Requirements

Dependencies

This pipeline is developed with singularity for tool dependencies. Most of the containers used is accessible from public registries like quay and biocontainers but for the tools not available in the registries local builds are currently used.

Databases

In order for the pipeline to run these databases must be available on the system: * AMRFinderPlus database (release 2024-10-22.1 or later) * PlasmidFinder database * Bakta database (optionally for annotation)

Quickstart

Download the pipeline

Clone the repository: git clone https://github.com/aevenstad/assembly_amr.git

Get dependencies

You can optionally install dependencies before running the pipeline using the helper scripts in bin/.
It’s recommended to set the container directory using the Nextflow variable $NXF_SINGULARITY_CACHEDIR: ``` NXFSINGULARITYCACHEDIR=/path/to/containers/

to pull containers from public registries, run:

bash bin/pullcontainers.sh $NXFSINGULARITY_CACHEDIR

for dependencies without publicly available container images, use:

bash bin/buildcontainers.sh $NXFSINGULARITY_CACHEDIR `` This will build the required containers using custom definition files insingularity/`.

Download databases

AMRFinderPlus database: singularity exec <amrfinderplus_image> amrfinder_update -d <database_dir>

PlasmidFinder database: ```

go to preferred database directory

cd

clone the latest version of the database:

git clone https://bitbucket.org/genomicepidemiology/plasmidfinderdb.git cd plasmidfinderdb PLASMID_DB=$(pwd)

install the database:

singularity exec python3 INSTALL.py kma_index ```

Bakta database: cd <database_dir> singularity exec <bakta_image> bakta_db download --output ./ --type [light|full]

Arguments

-profile [string] Name of profile from `nextflow.conf` (Currently only <singularity> supported) --input [string] Path to input samplesheet --outdir [string] Path to output directory --assembly_type [string] Type of assembly to perform (accepted: hybrid, long, short) --amrfinder_db [string] Path to the AMRFinderPlus database --plasmidfinder_db [string] Path to the PlasmidFinder database --bakta [boolean] Run annotation with bakta [default: false] --bakta_db [string] Path to the bakta database

Run the pipeline

nextflow run /path/to/assembly_amr/main.nf \ -profile singularity \ --input samplesheet.csv \ --outdir <outdir> \ --assembly_type [hybrid|long|short] \ --amrfinder_db <amrfinder_db> \ --plasmidfinder_db <plasmidfinder_db>

Hybrid input

If you have both Nanopore and Illumina reads from the same isolate and want to run a hybrid assembly, input must be provided in a comma-separated file e.g. samplesheet.csv: sample,nanopore,illumina_R1,illumina_R2 isolate1,/path/to/nanopore/data/isolate1.fastq.gz,/path/to/illumina/data/isolate1_R1.fastq.gz,/path/to/illumina/data/isolate1_R2.fastq.gz isolate2,/path/to/nanopore/data/isolate2.fastq.gz,/path/to/illumina/data/isolate2_R1.fastq.gz,/path/to/illumina/data/isolate2_R2.fastq.gz

Long-read input

For long read only assembly with Nanopore reads: samplesheet.csv: sample,nanopore isolate1,/path/to/nanopore/data/isolate1.fastq.gz isolate2,/path/to/nanopore/data/isolate2.fastq.gz

Short-read input

For short read only assembly with Illumina reads: samplesheet.csv: sample,illumina_R1,illumina_R2 isolate1,/path/to/illumina/data/isolate1_R1.fastq.gz,/path/to/illumina/data/isolate1_R2.fastq.gz isolate2,/path/to/illumina/data/isolate2_R1.fastq.gz,/path/to/illumina/data/isolate2_R2.fastq.gz