Development of an amplicon-based sequencing approach in response to the global emergence of mpox

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 inf...

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Published in:	PLoS biology Vol. 21; no. 6; p. e3002151
Main Authors:	Chen, Nicholas F G, Chaguza, Chrispin, Gagne, Luc, Doucette, Matthew, Smole, Sandra, Buzby, Erika, Hall, Joshua, Ash, Stephanie, Harrington, Rachel, Cofsky, Seana, Clancy, Selina, Kapsak, Curtis J, Sevinsky, Joel, Libuit, Kevin, Park, Daniel J, Hemarajata, Peera, Garrigues, Jacob M, Green, Nicole M, Sierra-Patev, Sean, Carpenter-Azevedo, Kristin, Huard, Richard C, Pearson, Claire, Incekara, Kutluhan, Nishimura, Christina, Huang, Jian Ping, Gagnon, Emily, Reever, Ethan, Razeq, Jafar, Muyombwe, Anthony, Borges, Vítor, Ferreira, Rita, Sobral, Daniel, Duarte, Silvia, Santos, Daniela, Vieira, Luís, Gomes, João Paulo, Aquino, Carly, Savino, Isabella M, Felton, Karinda, Bajwa, Moneeb, Hayward, Nyjil, Miller, Holly, Naumann, Allison, Allman, Ria, Greer, Neel, Fall, Amary, Mostafa, Heba H, McHugh, Martin P, Maloney, Daniel M, Dewar, Rebecca, Kenicer, Juliet, Parker, Abby, Mathers, Katharine, Wild, Jonathan, Cotton, Seb, Templeton, Kate E, Churchwell, George, Lee, Philip A, Pedrosa, Maria, McGruder, Brenna, Schmedes, Sarah, Plumb, Matthew R, Wang, Xiong, Barcellos, Regina Bones, Godinho, Fernanda M S, Salvato, Richard Steiner, Ceniseros, Aimee, Breban, Mallery I, Grubaugh, Nathan D, Gallagher, Glen R, Vogels, Chantal B F
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 13-06-2023 Public Library of Science (PLoS)
Subjects:	Bioinformatics Biology and Life Sciences Causes of Control Coronaviruses COVID-19 COVID-19 - epidemiology Deoxyribonucleic acid Disease transmission DNA DNA sequencing DNA viruses Epidemics Gene sequencing Genetic aspects Genomes Genomics Health surveillance Human monkeypox Humans Identification and classification Laboratories Medicine and Health Sciences Metagenomics Methods Monkeypox Mpox (monkeypox) Nucleotide sequence Nucleotide sequencing Outbreaks Pandemics Pathogens Polymerase chain reaction Public health Research and Analysis Methods SARS-CoV-2 - genetics Severe acute respiratory syndrome coronavirus 2 Surveillance Vector-borne diseases Viral diseases Virus research Viruses Whole genome sequencing Zika Virus Zika Virus Infection United States United States > US Massachusetts Connecticut
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Summary:	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. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 NFGC, CC, and LG share first authorship on this work. GRG and CBFV are co-senior authors on this work. 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.
ISSN:	1545-7885 1544-9173 1545-7885
DOI:	10.1371/journal.pbio.3002151