Development of an amplicon-based sequencing approach in response to the global emergence of mpox
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. 
Published: June 2023

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.