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...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-06-01
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| Series: | PLoS Biology |
| Online Access: | https://doi.org/10.1371/journal.pbio.3002151 |
| _version_ | 1797377804656967680 |
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| author | 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 Juliet 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 |
| author_facet | 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 Juliet 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 |
| author_sort | Nicholas F G Chen |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T19:58:43Z |
| format | Article |
| id | doaj.art-f6624948d6784e6da2c1bd198855dab0 |
| institution | Directory Open Access Journal |
| issn | 1544-9173 1545-7885 |
| language | English |
| last_indexed | 2024-03-08T19:58:43Z |
| publishDate | 2023-06-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Biology |
| spelling | doaj.art-f6624948d6784e6da2c1bd198855dab02023-12-24T05:31:09ZengPublic Library of Science (PLoS)PLoS Biology1544-91731545-78852023-06-01216e300215110.1371/journal.pbio.3002151Development of an amplicon-based sequencing approach in response to the global emergence of mpox.Nicholas F G ChenChrispin ChaguzaLuc GagneMatthew DoucetteSandra SmoleErika BuzbyJoshua HallStephanie AshRachel HarringtonSeana CofskySelina ClancyCurtis J KapsakJoel SevinskyKevin LibuitDaniel J ParkPeera HemarajataJacob M GarriguesNicole M GreenSean Sierra-PatevKristin Carpenter-AzevedoRichard C HuardClaire PearsonKutluhan IncekaraChristina NishimuraJian Ping HuangEmily GagnonEthan ReeverJafar RazeqAnthony MuyombweVítor BorgesRita FerreiraDaniel SobralSilvia DuarteDaniela SantosLuís VieiraJoão Paulo GomesCarly AquinoIsabella M SavinoKarinda FeltonMoneeb BajwaNyjil HaywardHolly MillerAllison NaumannRia AllmanNeel GreerAmary FallHeba H MostafaMartin P McHughDaniel M MaloneyRebecca DewarJuliet KenicerAbby ParkerKatharine MathersJonathan WildSeb CottonKate E TempletonGeorge ChurchwellPhilip A LeeMaria PedrosaBrenna McGruderSarah SchmedesMatthew R PlumbXiong WangRegina Bones BarcellosFernanda M S GodinhoRichard Steiner SalvatoAimee CeniserosMallery I BrebanNathan D GrubaughGlen R GallagherChantal B F VogelsThe 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.https://doi.org/10.1371/journal.pbio.3002151 |
| spellingShingle | 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 Juliet 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 Development of an amplicon-based sequencing approach in response to the global emergence of mpox. PLoS Biology |
| title | Development of an amplicon-based sequencing approach in response to the global emergence of mpox. |
| title_full | Development of an amplicon-based sequencing approach in response to the global emergence of mpox. |
| title_fullStr | Development of an amplicon-based sequencing approach in response to the global emergence of mpox. |
| title_full_unstemmed | Development of an amplicon-based sequencing approach in response to the global emergence of mpox. |
| title_short | Development of an amplicon-based sequencing approach in response to the global emergence of mpox. |
| title_sort | development of an amplicon based sequencing approach in response to the global emergence of mpox |
| url | https://doi.org/10.1371/journal.pbio.3002151 |
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