Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

Autor:	Chen NFG; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA., Chaguza C; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA., Gagne L; Massachusetts Department of Public Health, Boston, MA, USA., Doucette M; Massachusetts Department of Public Health, Boston, MA, USA., Smole S; Massachusetts Department of Public Health, Boston, MA, USA., Buzby E; Massachusetts Department of Public Health, Boston, MA, USA., Hall J; Massachusetts Department of Public Health, Boston, MA, USA., Ash S; Massachusetts Department of Public Health, Boston, MA, USA., Harrington R; Massachusetts Department of Public Health, Boston, MA, USA., Cofsky S; Massachusetts Department of Public Health, Boston, MA, USA., Clancy S; Massachusetts Department of Public Health, Boston, MA, USA., Kapsak CJ; Theiagen Genomics, Highlands Ranch, CO, USA., Sevinsky J; Theiagen Genomics, Highlands Ranch, CO, USA., Libuit K; Theiagen Genomics, Highlands Ranch, CO, USA., Park DJ; Broad Institute, Cambridge, MA, USA., Hemarajata P; Los Angeles County Public Health Laboratories, Downey, CA, USA., Garrigues JM; Los Angeles County Public Health Laboratories, Downey, CA, USA., Green NM; Los Angeles County Public Health Laboratories, Downey, CA, USA., Sierra-Patev S; Rhode Island Department of Health, Rhode Island State Health Laboratory, Providence, RI, USA., Carpenter-Azevedo K; Rhode Island Department of Health, Rhode Island State Health Laboratory, Providence, RI, USA., Huard RC; Rhode Island Department of Health, Rhode Island State Health Laboratory, Providence, RI, USA., Pearson C; Connecticut Department of Public Health, Rocky Hill, CT, USA., Incekara K; Connecticut Department of Public Health, Rocky Hill, CT, USA., Nishimura C; Connecticut Department of Public Health, Rocky Hill, CT, USA., Huang JP; Connecticut Department of Public Health, Rocky Hill, CT, USA., Gagnon E; Connecticut Department of Public Health, Rocky Hill, CT, USA., Reever E; Connecticut Department of Public Health, Rocky Hill, CT, USA., Razeq J; Connecticut Department of Public Health, Rocky Hill, CT, USA., Muyombwe A; Connecticut Department of Public Health, Rocky Hill, CT, USA., Borges V; Genomics and Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal., Ferreira R; Genomics and Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal., Sobral D; Genomics and Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal., Duarte S; Technology and Innovation Unit, Department of Human Genetics, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal., Santos D; Technology and Innovation Unit, Department of Human Genetics, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal., Vieira L; Technology and Innovation Unit, Department of Human Genetics, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal., Gomes JP; Genomics and Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal.; Faculty of Veterinary Medicine, Lusófona University, Lisbon, Portugal., Aquino C; Delaware Public Health Laboratory, Smyrna, DE, USA., Savino IM; Delaware Public Health Laboratory, Smyrna, DE, USA., Felton K; Delaware Public Health Laboratory, Smyrna, DE, USA., Bajwa M; Delaware Public Health Laboratory, Smyrna, DE, USA., Hayward N; Delaware Public Health Laboratory, Smyrna, DE, USA., Miller H; Delaware Public Health Laboratory, Smyrna, DE, USA., Naumann A; Delaware Public Health Laboratory, Smyrna, DE, USA., Allman R; Delaware Public Health Laboratory, Smyrna, DE, USA., Greer N; Delaware Public Health Laboratory, Smyrna, DE, USA., Fall A; Johns Hopkins School of Medicine, Baltimore, MD, USA., Mostafa HH; Johns Hopkins School of Medicine, Baltimore, MD, USA., McHugh MP; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK.; School of Medicine, University of St Andrews, St Andrews, UK., Maloney DM; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK.; Institute of Ecology and Evolution, University of Edinburgh, Edinburgh, UK., Dewar R; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Kenicer J; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Parker A; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Mathers K; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Wild J; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Cotton S; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Templeton KE; Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, UK., Churchwell G; Florida Department of Health, Bureau of Public Health Laboratories, Jacksonville, FL, USA., Lee PA; Florida Department of Health, Bureau of Public Health Laboratories, Jacksonville, FL, USA., Pedrosa M; Florida Department of Health, Bureau of Public Health Laboratories, Jacksonville, FL, USA., McGruder B; Florida Department of Health, Bureau of Public Health Laboratories, Jacksonville, FL, USA., Schmedes S; Florida Department of Health, Bureau of Public Health Laboratories, Jacksonville, FL, USA., Plumb MR; Minnesota Department of Health, Public Health Laboratory, St. Paul, MN, USA., Wang X; Minnesota Department of Health, Public Health Laboratory, St. Paul, MN, USA., Barcellos RB; Centro Estadual de Vigilância em Saúde, Secretaria Estadual da Saúde do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil., Godinho FMS; Centro Estadual de Vigilância em Saúde, Secretaria Estadual da Saúde do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil., Salvato RS; Centro Estadual de Vigilância em Saúde, Secretaria Estadual da Saúde do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil., Ceniseros A; Idaho Bureau of Laboratories, Boise, ID, USA., Breban MI; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA., Grubaugh ND; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA.; Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, USA., Gallagher GR; Massachusetts Department of Public Health, Boston, MA, USA.; Rhode Island Department of Health, Rhode Island State Health Laboratory, Providence, RI, USA., Vogels CBF; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA.
Jazyk:	angličtina
Zdroj:	MedRxiv : the preprint server for health sciences [medRxiv] 2023 Jan 13. Date of Electronic Publication: 2023 Jan 13.
DOI:	10.1101/2022.10.14.22280783
Abstrakt:	The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has 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 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 samples that tested presumptive 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 (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 cycle threshold 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.
Databáze:	MEDLINE
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