RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing

Brinkmann, Annika; Uddin, Steven; Ulm, Sophie-Luisa; Pape, Katharina; Förster, Sophie; Enan, Khalid A; Nourlil, Jalal; Krause, Eva; Schaade, Lars; Michel, Janine; Nitsche, Andreas

doi:10.1371/journal.pone.0264855

Cited by 7 publications

(5 citation statements)

References 31 publications

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“…To maximise the scope of our method, we ensured our scheme could target an unprecedented 5 near-complete species' genomes from two coronaviral genera in the minimal 2 reaction format (21). To our knowledge, this is the broadest complete genome amplicon method presented to date and showcases the considerable potential of the method in sensitively targeting multiple viruses in whole, or part (51). We validated this by confirming clinically diagnosed coronavirus co-infections and identifying others missed by RT-qPCR multiplex assay panels, which can have poor discriminatory potential.…”

Section: Discussionmentioning

confidence: 99%

‘Vivaldi’: An amplicon-based whole genome sequencing method for the four seasonal human coronaviruses 229E, NL63, OC43 & HKU1, alongside SARS-CoV-2’

McClure,

Tsoleridis,

Holmes

et al. 2024

Preprint

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Prior to the emergence of SARS-CoV-2 in 2019, Alphacoronaviruses 229E and NL63 and Betacoronaviruses OC43 and HKU1 were already established endemic ‘common cold’ viral infections. Despite their collective contribution towards global respiratory morbidity and mortality and potential to inform the future trajectory of SARS-CoV-2 endemicity, they are infrequently sequenced. We therefore developed a 1200bp amplicon-based whole genome sequencing scheme targeting all four seasonal coronaviruses and SARS-CoV-2.The ‘Vivaldi’ method was applied retrospectively and prospectively using Oxford Nanopore Technology to approximately 400 seasonal coronavirus infections diagnosed in Nottingham, UK, from February 2016 to July 2023. We demonstrate that the amplicon multiplex strategy can be applied agnostically to determine complete genomes of five different species from two coronaviral genera. 304 unique seasonal coronavirus genomes of greater than 95% coverage were achieved: 64 for 229E, 85 for NL63, 128 for OC43 and 27 for HKU1. They collectively indicated a dynamic seasonal coronavirus genomic landscape, with co-circulation of multiple variants emerging and declining over the UK winter respiratory infection season, with further geographical distinction when compared to a global dataset. Prolonged infection with concomitant intra-host evolution was also observed for both Alpha-(NL63) and Betacoronaviruses (OC43).This data represents the largest single cohort of seasonal coronavirus genomes to date and also a novel amplicon scheme for their future global surveillance suitable for widespread and easy adoption in the post-SARS-CoV-2 era of viral genomics.

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Section: Discussionmentioning

confidence: 99%

‘Vivaldi’: An amplicon-based whole genome sequencing method for the four seasonal human coronaviruses 229E, NL63, OC43 & HKU1, alongside SARS-CoV-2’

McClure,

Tsoleridis,

Holmes

et al. 2024

Preprint

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“…The primers used in amplification were adopted from the RespiCov panel 15 . Briefly, 14 adenovirus primers were pooled into one tube and resuspended in nuclease free water to generate a 10μM working concentration.…”

Section: Methodsmentioning

confidence: 99%

“…HAdV genotyping is typically done by amplifying a region of the hexon gene, followed by Sanger sequencing 14 . Here we used adenovirus primers from the RespiCoV panel 15 , applying these primers to stool-derived nucleic acid extracts for the first time, and sequencing the amplicons on the Oxford Nanopore Technologies (ONT) platform.…”

Section: Introductionmentioning

confidence: 99%

Multi-species co-circulation of adenoviruses identified by next generation sequencing during an outbreak in coastal Kenya in 2023

Lambisia,

Mutunga,

Katama

et al. 2024

Preprint

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Background Although seven human adenovirus (HAdV) species are known to exist, only F (types 40 and 41) and G, are identified as diarrhoeal disease agents. The role of other HAdV species in diarrhoeal disease remains unclear and data of their prevalence is limited. We describe HAdV species and types in hospitalised children with diarrhoea in coastal Kenya. Methods 329 stool samples collected between June 2022 and August 2023 from children aged <13-years were screened for HAdV using quantitative polymerase chain reaction (qPCR). Positive HAdV cases were genotyped by adenovirus primers from the RespiCoV panel by amplification, next generation sequencing followed by phylogenetic analysis. Results 65 samples (20%) tested HadV positive from which five HAdV species were identified. Other than HAdV F, other species included A, B, C and D; these were detected as either mono-detections or coinfections. Six HAdV F identified by NGS had been missed by our q PCR typing method. This appeared to be as a result of a 133-nucleotide deletion in the long fiber protein which abrogated a primer and probe binding site. Based on VESIKARI scores grading of diarrhoeal disease severity, 93% of the HAdV cases presented with severe disease. One child with an HAdV F infection died. Conclusion Our study shows the enormous diversity and clinical characteristics of HAdV species in children with diarrhoea in coastal Kenya. These data offers an opportunity to improve current diagnostic assays, increase knowledge of HAdV in Africa for control of outbreaks in the future.

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“… 231 By combining nanopore sequencing with multiplex isothermal amplification, SARS‐CoV‐2 can be differentiated from a variety of viruses including influenza A virus (IAV), and human adenovirus. 232 By combining nanopore sequencing with RT‐PCR technology, the researchers were able to simultaneously screen 41 viruses including MERS‐CoV 233 while identifying SARS‐CoV‐2.…”

Section: Application Of Nanopore Sequencing Technology During the Cov...mentioning

confidence: 99%

Nanopore sequencing technology and its applications

Zheng

Zhou

Ding

et al. 2023

MedComm

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Since the development of Sanger sequencing in 1977, sequencing technology has played a pivotal role in molecular biology research by enabling the interpretation of biological genetic codes. Today, nanopore sequencing is one of the leading third‐generation sequencing technologies. With its long reads, portability, and low cost, nanopore sequencing is widely used in various scientific fields including epidemic prevention and control, disease diagnosis, and animal and plant breeding. Despite initial concerns about high error rates, continuous innovation in sequencing platforms and algorithm analysis technology has effectively addressed its accuracy. During the coronavirus disease (COVID‐19) pandemic, nanopore sequencing played a critical role in detecting the severe acute respiratory syndrome coronavirus‐2 virus genome and containing the pandemic. However, a lack of understanding of this technology may limit its popularization and application. Nanopore sequencing is poised to become the mainstream choice for preventing and controlling COVID‐19 and future epidemics while creating value in other fields such as oncology and botany. This work introduces the contributions of nanopore sequencing during the COVID‐19 pandemic to promote public understanding and its use in emerging outbreaks worldwide. We discuss its application in microbial detection, cancer genomes, and plant genomes and summarize strategies to improve its accuracy.

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RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing

Cited by 7 publications

References 31 publications

‘Vivaldi’: An amplicon-based whole genome sequencing method for the four seasonal human coronaviruses 229E, NL63, OC43 & HKU1, alongside SARS-CoV-2’

‘Vivaldi’: An amplicon-based whole genome sequencing method for the four seasonal human coronaviruses 229E, NL63, OC43 & HKU1, alongside SARS-CoV-2’

Multi-species co-circulation of adenoviruses identified by next generation sequencing during an outbreak in coastal Kenya in 2023

Nanopore sequencing technology and its applications

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