Introduction

ViromeFlowX is a comprehensive Nextflow-based automated workflow for mining viral genomes from metagenomic sequencing Data. Understanding the link between the human gut virome and diseases has garnered significant interest in the research community. Extracting virus-related information from metagenomic sequencing data is crucial for unravelling virus composition, host interactions, and disease associations. However, current metagenomic analysis workflows for viral genomes vary in effectiveness, posing challenges for researchers seeking the most up-to-date tools. To address this, we present ViromeFlowX, a user-friendly Nextflow workflow that automates viral genome assembly, identification, classification, and annotation. This streamlined workflow integrates cutting-edge tools for processing raw sequencing data for taxonomic annotation and functional analysis. Application to a dataset of 200 metagenomic samples yielded high-quality viral genomes. ViromeFlowX enables efficient mining of viral genomic data, offering a valuable resource to investigate the gut virome’s role in virus-host interactions and virus-related diseases.

Pipeline summary

1. Quality Control ( trimmomatic bowtie2 )
2. Assembly ( metaspades )
3. Viral Taxonomic Classify ( Kraken2 )
4. Viral Contigs Identification ( VirFinder VirSorter2 CheckV Cdhit )
5. Gene Prediction & Functional Annotation ( Prodigal bedtools2 DIAMOND )
6. Viral Taxonomic Classify Assignment ( usearch Blast taxonkit CoverM )

Getting Start

Pre-requisites

To ensure the smoothest possible analysis using ViromeFlowX, we recommend taking the time to pre-build both the software components and the reference databases before you begin your analysis. This preparatory step will help guarantee a more efficient and hassle-free experience.

• Environment Setup: Most of the tools required by the pipeline can be conveniently installed using a Conda environment. Use the command below to create a new Conda environment based on the environment.yml configuration file.

bash conda env create -f environment.yml

Notes: The usearch tool is not supported for installation through conda. You need to manually download and install it. Please refer to the official documentation for installation instructions.

• Database Setup: Refer to the Database Installation and Configuration for downloading and configuring required databases.

Install and Usage

1. Clone the repository git clone https://github.com/01life/ViromeFlowX

2. Prepare a samplesheet samplesheet.csv with your input data that looks as follows csv id,reads1,read2 sample_2,/PATH/sample_L002_R1.fastq.gz,/PATH/sample_L002_R2.fastq.gz sample_3,/PATH/sample_L003_R1.fastq.gz,/PATH/sample_L003_R2.fastq.gz

3. Start running the pipeline nextflow run ViromeFlowX \ -profile <docker/singularity/conda/.../institute> \ --input samplesheet.csv \ --outdir <OUTDIR>

If you are new to Nextflow and nf-core, check the Nextflow installation guide. Ensure your setup passes the -profile test before processing real data.

1. Advance usage

The pipeline will run QC -> Metaspades(min_len=1k) -> Identify(VirFinder、VirSorter2、CheckV) -> Taxonomic Classify(Kraken2) -> Geneset -> Taxonomic Classify Assignment (demovir、pfam、protein、crAss、genome) -> Abundance. You can also use --help to see the parameters. For comprehensive tutorials and implementation guidelines, please refer to our Usage Documentation.

bash nextflow run /path/to/project/ViromeFlowX --help

1. Output information

To better understand the output files generated by ViromeFlowX and how to interpret them, refer to the Output Documentation.

Citation

If you found ViromeFlowX usefull in your research, please cite the publication: ViromeFlowX: a Comprehensive Nextflow-based Automated Workflow for Mining Viral Genomes from Metagenomic Sequencing Data.

Wang X, Ding Z, Yang Y, et al. ViromeFlowX: a Comprehensive Nextflow-based Automated Workflow for Mining Viral Genomes from Metagenomic Sequencing Data[J]. Microbial Genomics, 2024, 10(2): 001202.