| Author | Reteng, Patrick | |
| Author | Thuy, Linh Nguyen | |
| Author | Rahman, Mizanur | |
| Author | Filippis, Ana Maria Bispo de | |
| Author | Hayashida, Kyoko | |
| Author | Sugi, Tatsuki | |
| Author | Gonzalez, Gabriel | |
| Author | Hall, William W. | |
| Author | Thi, Lan Anh Nguyen | |
| Author | Yamagishi, Junya | |
| Access date | 2023-01-06T13:20:25Z | |
| Available date | 2023-01-06T13:20:25Z | |
| Document date | 2022 | |
| Citation | RETENG, Patrick et al. Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification. mSphere, v. 7, n. 5, p. 1-10, Aug. 2022. | en_US |
| ISSN | 2379-5042 | en_US |
| URI | https://www.arca.fiocruz.br/handle/icict/56346 | |
| Language | eng | en_US |
| Publisher | American Society for Microbiology | en_US |
| Rights | open access | |
| Subject in Portuguese | Metagenômica | en_US |
| Subject in Portuguese | Doença febril | en_US |
| Subject in Portuguese | Algoritmo de teste de grupo | en_US |
| Subject in Portuguese | Amplificação de deslocamento múltiplo | en_US |
| Subject in Portuguese | Detecção abrangente de patógenos | en_US |
| Title | Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification | en_US |
| Type | Article | |
| DOI | 10.1128/msphere.00332-22 | |
| Abstract | Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from
Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a widerange of pathogens by detecting the presence of their nucleic acids in the sample.
However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance. | en_US |
| Affilliation | Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. | en_US |
| Affilliation | Laboratory for Molecular Diagnostics, National Institute of Epidemiology and Health, Hanoi, Vietnam. | en_US |
| Affilliation | Evercare Hospital Dhaka, Dhaka, Bangladesh. | en_US |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brasil. | en_US |
| Affilliation | Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan / International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. | en_US |
| Affilliation | Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan / International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. | en_US |
| Affilliation | National Virus Reference Laboratory, University College Dublin, Dublin, Ireland. | en_US |
| Affilliation | School of Medicine and Medical Research, University College Dublin, Dublin, Ireland / Global Virus Network, Baltimore, Maryland, USA. | en_US |
| Affilliation | Laboratory for Molecular Diagnostics, National Institute of Epidemiology and Health, Hanoi, Vietnam. | en_US |
| Affilliation | Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan / International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. | en_US |
| Subject | Metagenomic | en_US |
| Subject | Febrile illness | en_US |
| Subject | Group testing algorithm | en_US |
| Subject | Multiple displacement amplification | en_US |
| Subject | Comprehensive pathogen detection | en_US |
| xmlui.metadata.dc.subject.ods | 03 Saúde e Bem-Estar | |
