CFIA-NCFAD/nf-virontus

Oxford Nanopore viral sequence analysis pipeline.

Introduction

This pipeline performs read mapping and variant calling with Minimap2 and [Clair3]. A consensus sequence is generated from major variants and variants that would not cause potential frameshift mutations using Bcftools with masking of low coverage depth regions with N characters.

Optionally, amplicon primers can be trimmed with iVar if a BED file of primer coordinates is supplied.

If read mapping against the SARS-CoV-2 reference genome Wuhan-Hu-1 (MN908947.3), Pangolin lineage assignment will be done, as well as, Nextclade analysis.

Pipeline Overview

NOTE: This pipeline is still a work-in-progress. The following diagram shows the planned features and steps of the workflow:

flowchart LR
    classDef input fill:#fcba64,color:black
    classDef output fill:#b1fc9c,color:black
    subgraph legend["<b>Legend"]
        style legend fill:white,fill-opacity:0.5
        input([Input]):::input
        output([Output]):::output
        process[Process]
    end
    
    subgraph rqc["<b>fa:fa-dna Reads QA/QC</b>"]
        A([fa:fa-file Filtered Reads FASTQ]):::output
        FR["fa:fa-check Read QC & Filtering <br><small> fastp, nanoqc"]
        HR["fa:fa-cancel Dehosting <br><small> Kraken2 (optional)"]
        RR([fa:fa-file Raw Reads FASTQ]):::input --> FR 
        FR --> HR
        HR --> A
    end
    rqc --> rs
    subgraph rs[<b>fa:fa-crosshairs Reference Selection]
        RS["fa:fa-filter Ref Seq Selection <br><small> de novo assembly & BLAST, Mash, Kraken2"]
        frs_rs([fa:fa-file Reads FASTQ]):::input
        frs_rs --> RS
        R([fa:fa-file Ref Seqs FASTA]):::input --> RS
        RS --> T([fa:fa-file Top Ref Seq FASTA]):::output
    end

    rs --> rma
    rqc --> rma

    subgraph rma[<b>fa:fa-industry Reference Mapped Assembly]
        direction TB
        trs([fa:fa-file Top Ref FASTA]):::input
        fr([fa:fa-file FASTQ]):::input
        RM["fa:fa-bars-staggered Read Mapping <br><small> Minimap2"]
        PT["fa:fa-scissors Primer Trimming <br><small> iVar (optional)"]
        VC["fa:fa-code-compare Variant Calling <br><small> Clair3, Medaka"]
        VE["fa:fa-flask Variant Effect <br><small> SnpEff, SnpSift (optional)"]
        BAM([fa:fa-file BAM]):::output
        D["fa:fa-chart-area Coverage Stats <br><small> Mosdepth, Samtools"]
        CS["fa:fa-code-merge Make Consensus Sequence <br><small> Bcftools"]
        vcf([fa:fa-file VCF]):::output
        muts([fa:fa-table Amino Acid Mutations]):::output
        covbed([fa:fa-file Coverage BED]):::output
        fr --> RM
        trs --> RM
        trs --> VC
        RM --> BAM
        BAM --> PT
        PT --> BAM
        BAM --> D
        BAM --> VC
        vcf --> VE
        VE --> vcf
        VE --> muts
        VC --> vcf
        vcf --> CS
        trs --> CS
        D --> covbed
        covbed --> CS
        CS --> csf([fa:fa-file Consensus Sequence FASTA]):::output
    end
    rqc --> reporting
    rs --> reporting
    rma --> reporting
    subgraph reporting[<b>fa:fa-clipboard Reporting & Visualization]
        MQC[fa:fa-stethoscope MultiQC]
        MQCR([fa:fa-file MultiQC HTML Report]):::output
        CP[fa:fa-chart-area Seq Coverage Plots]
        png([fa:fa-image PNG]):::output
        pdf([fa:fa-file PDF]):::output
        MQC --> MQCR
        CP --> png & pdf
    end

Loading

Installation

You will need to install Nextflow in order to run the Virontus pipeline.

NB: Singularity or Docker is recommended for portable and reproducible execution of the pipeline with the -profile singularity or -profile docker command-line argument.

1) Install Nextflow

If you have Conda installed, you can install Nextflow with the following command:

conda install -c bioconda -c conda-forge nextflow

2) Install Docker and/or Singularity

Installing Docker and/or Singularity is optional but recommended for portability and reproducibility of results.

3) Install Virontus

Nextflow will automatically download the latest version of Virontus. You can show the Virontus help message with usage information with:

nextflow run CFIA-NCFAD/nf-virontus --help

Usage

Basic usage for mapping to SARS-CoV-2 reference genome MN908947.3 and ARTIC V3 protocol primers:

nextflow run CFIA-NCFAD/nf-virontus \
  --input samplesheet.csv \
  --genome MN908947.3 \
  --bed artic-ncov2019/primer_schemes/nCoV-2019/V3/nCoV-2019.bed

Can be simplified with:

nextflow run CFIA-NCFAD/nf-virontus \
  --input samplesheet.csv \
  --scov2 \
  --artic_v3
  # or `--freed` for Freed et al (2020) 1200bp amplicon method

Show usage information with

nextflow run CFIA-NCFAD/nf-virontus --help

NB: See the usage docs for more info.

Output

See the output docs for more info.

Credits

CFIA-NCFAD/nf-virontus was originally written by Peter Kruczkiewicz.

Bootstrapped with nf-core/tools nf-core create.

Thank you to the nf-core/tools team for a great tool for bootstrapping creation of a production ready Nextflow workflows.