Rapid viral sequencing for genomic surveillance of mpox using metagenomic or targeted approaches

Due to the ongoing outbreak, this workflow overview is only available online to allow for rapid updates. Please see the recent update to the analysis section below to ensure you are using the most up-to-date bioinformatics pipeline: artic-mpxv-nf.

Overview

Mpox is a viral zoonotic disease that can cause painful rashes, swollen lymph nodes, and fever as a result of infection with a virus called Orthopoxvirus monkeypox (MPXV)¹, with more severe symptoms often observed in those with comorbidities^2,3. It is a large (~200 kb), enveloped, linear, double-stranded DNA virus with ~200 genes, part of the Poxviridae family, and subclassified into two clades: clade I and II³. As well as zoonotic transmission, human-to-human transmission also occurs through close contact with an infected individual, including skin-to-skin contact, and via contaminated objects such as clothing³.

Mpox is endemic to West and Central African countries, but the world is currently experiencing the largest mpox outbreak, which is also affecting non-endemic countries^2,4. However, there is limited information about the circulating virus and no specific treatment for mpox^4,5. Rapid molecular assays are needed to detect MPXV and identify the clade and changes in viral characteristics to understand the epidemiology, prevent further spread, and develop specific treatments^4,5.

Most MPXV sequencing performed early in the current global outbreak used a metagenomic approach, but targeted sequencing has since been developed for MPXV whole-genome analysis⁶. With Oxford Nanopore Technologies, both metagenomic and targeted MPXV sequencing can be performed depending on your experimental needs.

For the generation of complete de novo MPXV reference genome assemblies, we recommend metagenomic sequencing. To investigate genomic epidemiology links, we recommend targeted MPXV sequencing of tiled PCR amplicons. Oxford Nanopore technology delivers fast and highly accurate sequencing reads of any length, and complex and repeat-rich regions can be sequenced in single nanopore reads, simplifying genome assembly. Furthermore, Oxford Nanopore sequencing is a cost-effective method with real-time data streaming to provide rapid, actionable results.

Here we provide a simple and rapid end-to-end workflow using either metagenomic or targeted sequencing and the EPI2ME analysis platform for the detection and characterisation of MPXV.

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Extraction: obtaining viral DNA

For metagenomic sequencing, we recommend extracting viral DNA from either nasopharyngeal or skin lesion swabs using Roche Diagnostics MagNA Pure Bacteria Lysis Buffer. In addition, we recommend preparing full-length cDNA from RNA in the sample using NEB LunaScript RT SuperMix Kit and Applied Biosystems Sequenase Version 2.0 DNA Polymerase⁷ in order to detect potential coinfections from RNA viruses.

For targeted MPXV sequencing, we recommend extracting viral DNA from skin lesion swabs using QIAGEN DNeasy Blood and Tissue Kit.

Find out more about the metagenomic sequencing extraction method in the legacy protocol

Library preparation: preparing DNA for sequencing

We recommend performing metagenomic sequencing to generate high-quality, complete de novo MPXV genome assemblies. To prepare your DNA for sequencing, we recommend using the Rapid PCR Barcoding Kit 24 V14 and following the standard protocol (panel a), which has been optimised for low quantities of starting input and can sequence MPXV clades I and II.

To perform rapid and cost-effective MPXV whole-genome sequencing for genomic epidemiological analysis, we recommend using the targeted sequencing approach where tiled PCR amplicons generate sufficient genome-wide coverage. To prepare your DNA for sequencing, we recommend using the 24- or 96-plex Rapid Barcoding Kits and the Midnight PCR tiling of SARS-CoV-2 virus protocol (panel b), replacing the midnight primers with the latest MPXV primer tiling scheme. Two popular tiling schemes have been developed by Welkers et al.⁸ and Chen, Chaguza, and Gagne et al.⁶.

Find out more about library preparation

Sequencing: generating MPXV sequencing reads

We recommend sequencing viral DNA libraries on MinION Flow Cells. These flow cells are compatible with portable MinION devices for rapid onsite sequencing. To scale up sequencing, up to five independent MinION Flow Cells can be run on GridION devices for large-scale pathogen surveillance.

The sequencing software MinKNOW provides a simple user interface to set up experiments easily. We recommend using the high-accuracy (HAC) basecalling model during sequencing for rapid results with real-time data streaming.

Find out more about nanopore sequencing devices

Analysis: assembling the viral genome

We recommend importing artic-mpxv-nf⁹ from ARTIC Network into the EPI2ME desktop application to ensure you are using the most up-to-date bioinformatics pipeline. EPI2ME is the data analysis platform, providing a user-friendly bioinformatics experience for all levels of expertise without the use of command line. This workflow generates a draft consensus assembly for further downstream analysis.

Learn more about importing the artic-mpxv-nf pipeline