Introduction

nfvibrio is a bioinformatics pipeline for the analysis Vibrio parahaemolyticus.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a highly portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies.

Pipeline summary

1. Read QC Filtering and Trimming (FASTP, TRIMMOMATIC)
2. Metagenomic read classification (optional, KRAKEN2)
3. Contamination detection (optional, CONFINDR)
4. Read QC Metrics (FastQC)
5. Assemble quality controlled reads (SHOVILL)
   1. Estimating genome size by counting unqiue kmer-mers (KMC)
   2. Correct sequencing errors in reads (Lighter)
   3. Merge paired-end reads (FLASH)
   4. Assemble with (SPAdes)/(SKESA)/(Megahit) with modified kmer range
   5. Correct minor assembly errors by mapping reads back to contigs (PILON)
6. Perform assembly QC (QUAST)
7. Annotate assembled contigs (PROKKA)
8. Pan-genome analysis (ROARY)
9. Virulence factor screening (ABRICATE)
   1. Custom heatmap plot
10. Find antimicrobial resistance genes and point mutations in assembled contigs (AMRFINDERPLUS)
11. Scan contig files against traditional PubMLST typing schemes (MLST)
12. Variant calling and consensus sequence generation (SNIPPY)
13. General QC report generation (MULTIQC)

graph TD
A[FASTQ Samplesheet]  --> AA(Input Check)
AA --> AAA(Trimmomatic \n- Adapter Removal)
AA --> AB(ConFindr \n- Contamination QC)
AAA --> AC(Fastp \n- Trim & Filter)
AC --> ACB(Kraken2 \n- Metagenomic Classifier)
AC --> ACA(FASTQC \n- QC Checks)
AC --> B(Shovill \n- De-novo Assembly)
B --> BA(Abricate \n- Virulence Factors)
BA --> BAA(Custom Heatmap Plot)
B --> BB(AMRFinderPlus \n- AMR Gene Detection)
B --> BC(MLST \n- Sequence Typing)
B --> BD(Quast \n- Assembly QC)
B --> BE(Prokka \n- Genome Annotation)
BE --> BEA(Roary \n- PanGenome Analysis)
B --> BF(Snippy \n- Variant Calls & Consensus)
AAA --> Z(MultiQC \n- QC Report)
AC --> Z
BF --> Z
BD --> Z
BE --> Z

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Quick Start

1. Install Nextflow (>=21.0.0)

2. Install any of Docker, Singularity (you can follow this tutorial), Podman, Shifter or Charliecloud for full pipeline reproducibility (you can use Conda both to install Nextflow itself and also to manage software within pipelines. Please only use it within pipelines as a last resort; see docs).

3. Given a directory of FASTQ files, use the extra/make_sample_sheet.sh script to automatically generate a corresponding samplesheet.

bash make_sample_sheet.sh [FASTQ_DIRECTORY] [SAMPLESHEET_FILENAME]

5. Download the pipeline and test it on a minimal dataset with a single command:

nextflow run nfvibrio/main.nf -profile <conda,singularity,YOURPROFILE>  --input sample_sheet.csv --outdir <OUTDIR> 

Note that some form of configuration will be needed so that Nextflow knows how to fetch the required software. This is usually done in the form of a config profile (YOURPROFILE in the example command above). You can chain multiple config profiles in a comma-separated string.

• The pipeline comes with config profiles called docker, singularity, podman, shifter, charliecloud and conda which instruct the pipeline to use the named tool for software management. For example, -profile test,docker.
• Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.
• If you are using singularity, please use the nf-core download command to download images first, before running the pipeline. Setting the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options enables you to store and re-use the images from a central location for future pipeline runs.
• If you are using conda, it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.

Documentation

Currently this README serves as the only documentation for this workflow. More detailed documentation is under development. If you have questions, open a issue or connect at [email protected].

Credits

The majority of the nfvibrio pipeline in its current state was developed by John Palmer for routine analysis of Vibrio parahaemolyticus at the BC Centre for Disease Control (BCCDC) and abroad. Special thanks to colleagues and supervisors who made this work possible at BCCDC (James Zlosnik, Linda Hoang, Tess MacIntyre). The original foundations of this pipeline were developed by Muhammad Zohaib Anwar under the supervision of William Hsiao at the Centre for Infectious Disease Genomics and One Health at Simon Fraser University.

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.