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Hokkaido University Collection of Scholarly and Academic Papers >
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Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification
| Title: | Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification |
| Authors: | Reteng, Patrick Browse this author | | Nguyen Thuy, Linh Browse this author | | Rahman, Mizanur Browse this author | | de Filippis, Ana Maria Bispo Browse this author | | Hayashida, Kyoko Browse this author | | Sugi, Tatsuki Browse this author | | Gonzalez, Gabriel Browse this author | | Hall, William W. Browse this author | | Nguyen Thi, Lan Anh Browse this author | | Yamagishi, Junya Browse this author →KAKEN DB |
| Keywords: | metagenomic | | febrile illness | | group testing algorithm | | multiple displacement amplification | | comprehensive pathogen detection |
| Issue Date: | 25-Aug-2022 |
| Publisher: | American Society for Microbiology |
| Journal Title: | mSphere |
| Volume: | 7 |
| Issue: | 5 |
| Start Page: | e00332-22 |
| Publisher 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 wide-range 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. |
| Type: | article |
| URI: | http://hdl.handle.net/2115/86852 |
| Appears in Collections: | 人獣共通感染症国際共同研究所 (International Institute for Zoonosis Control) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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