Development of an amplicon-based sequencing approach in response to the global emergence of mpox
Nicholas F. G. Chen, Chrispin Chaguza, Luc Gagne, Matthew Doucette, Sandra Smole, Erika Buzby, Joshua Hall, Stephanie Ash, Rachel Harrington, Seana Cofsky, Selina Clancy, Curtis J. Kapsak, Joel Sevinsky, Kevin Libuit, Daniel J. Park, Peera Hemarajata, Jacob M. Garrigues, Nicole M. Green, Sean Sierra-Patev, Kristin Carpenter-Azevedo, Richard C. Huard, Claire Pearson, Kutluhan Incekara, Christina Nishimura, Jian Ping Huang, Emily Gagnon, Ethan Reever, Jafar Razeq, Anthony Muyombwe, Vítor Borges, Rita Ferreira, Daniel Sobral, Silvia Duarte, Daniela Santos, Luís Vieira, João Paulo Gomes, Carly Aquino, Isabella M. Savino, Karinda Felton, Moneeb Bajwa, Nyjil Hayward, Holly Miller, Allison Naumann, Ria Allman, Neel Greer, Amary Fall, Heba H. Mostafa, Martin P. McHugh, Daniel M. Maloney, Rebecca Dewar, Kenicer, Abby Parker, Katharine Mathers, Jonathan Wild, Seb Cotton, Kate E. Templeton, George Churchwell, Philip A. Lee, Maria Pedrosa, Brenna McGruder, Sarah Schmedes, Matthew R. Plumb, Xiong Wang,Regina Bones Barcellos, Fernanda M. S. Godinho, Richard Steiner Salvato, Aimee Ceniseros, Mallery I. Breban, Nathan D. Grubaugh, Glen R. Gallagher, Chantal B. F. Vogels
Abstract
The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches.
Introduction
The integration of pathogen whole-genome sequencing with public health surveillance provides a powerful tool to inform outbreak control [1,2]. While the feasibility of real-time genomic surveillance was demonstrated during the 2013 to 2016 Ebola outbreak [3], the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has launched a revolution in viral genomics [4]. To date, more than 14 million SARS-CoV-2 genomes have been sequenced and shared publicly [5], furthering our understanding of viral transmission and evolution. The rapid advancement in pathogen genomics in public health laboratories was facilitated in the United States through significant investments by the Centers of Disease Control and Prevention’s Office of Advanced Molecular Detection. These programs situated sequencing equipment in state and local public health laboratories and provided practical training in laboratory and bioinformatics approaches to allow for the rapid adoption of new sequencing methods.
Material and methods
Ethics statement
As part of this study, we sequenced remnant clinical specimens that tested presumptive positive for monkeypox virus. Ethical oversight for each institution is indicated in Table 1. All data were de-identified prior to sharing and sample codes as included in the manuscript are not known outside the research groups.
Primer scheme design
The human monkeypox primer scheme (v1) was designed with PrimalScheme [15] using a pre-outbreak clade IIb reference genome (GenBank accession: MT903345) belonging to the A.1 lineage, following the newly proposed monkeypox virus naming system [8]. The primer scheme comprises a total of 163 primer pairs with an amplicon length ranging between 1,597 and 2,497 bp (average length of 1,977 bp; S1 Table).
Results
In May 2022, a growing cluster of mpox cases in humans was reported outside its endemic region [6,10]. Difficulties in obtaining sufficient coverage with metagenomic sequencing approaches led us to develop a primer scheme for use with amplicon-based sequencing approaches. Given that many of the early B.1 outbreak clade genomes had low coverage, we used the closely related pre-outbreak A.1 clade genome (GenBank accession: MT903345) as a reference for the primer scheme. The primer scheme, designed using PrimalScheme [15], consists of 163 primer pairs with an amplicon length ranging between 1,597 and 2,497 bp (average length of 1,977 bp; S1 Table). For the initial validation, we sequenced 10 clinical specimens with a range of PCR Ct values with both amplicon-based and metagenomic sequencing approaches at the Massachusetts State Public Health Laboratory (MASPHL). Clinical specimens ranged in Ct value from 15.0 (highest DNA concentration) to 34.6 (lowest DNA concentration).
Discussion
We developed an amplicon-based sequencing approach for human monkeypox virus to provide a more sensitive, lower cost, and higher throughput alternative to metagenomic sequencing. We used PrimalScheme to develop primers for human monkeypox virus based on a pre-outbreak A.1 lineage genome (GenBank accession: MT903345) and tested the primer scheme with clinical specimens in 2 independent laboratories. After initial validation, we shared primer pool aliquots with 10 additional public health laboratories, who successfully implemented the scheme in their existing amplicon-based sequencing workflows. Our findings showed greater breadth and depth of genome coverage when using the amplicon-based sequencing approach as compared to metagenomics, particularly for specimens with lower DNA concentrations. We identified Ct value and number of sequencing reads as 2 factors that influence percent genome coverage. Based on our findings, we made the following recommendations for amplicon-based sequencing of human monkeypox virus:
Acknowledgments
We thank Cornelius Roemer for help with the logistic function analysis.
Citation: Chen NFG, Chaguza C, Gagne L, Doucette M, Smole S, Buzby E, et al. (2023) Development of an amplicon-based sequencing approach in response to the global emergence of mpox. PLoS Biol 21(6): e3002151. https://doi.org/10.1371/journal.pbio.3002151
Academic Editor: Bill Sugden, University of Wisconsin-Madison, UNITED STATES
Received: January 12, 2023; Accepted: May 5, 2023; Published: June 13, 2023
Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Genomic data are available on NCBI Sequence Read Archive (SRA), GenBank, and GISAID (see accession numbers in S3 Table). All other data are included in the manuscript and the Supporting Information.
Funding: This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: NDG is a consultant for Tempus Labs and the National Basketball Association for work related to COVID-19. All other authors have declared that no competing interests exist.
Abbreviations: CDPH, Connecticut Department of Public Health; CEVS, Centro Estadual de Vigilância em Saúde; COVID-19, Coronavirus Disease 2019; Ct, cycle threshold; DPHL, Delaware Public Health Lab; FDA, US Food and Drug Administration; FDH, Florida Department of Health; IBL, Idaho Bureau of Laboratories; INSA, National Institute of Health Dr. Ricardo Jorge; JHMI, Johns Hopkins Medical Institutions; LACPHL, Los Angeles County Public Health Lab; LMIC, low- or middle-income country; MASPHL, Massachusetts State Public Health Laboratory; MDH, Minnesota Department of Health; NHS Lothian, National Health Service Lothian; RIDOH RISHL, Rhode Island Department of Health/Rhode Island State Health Laboratory; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; YSPH, Yale School of Public Health.
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002151#abstract0
