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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

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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
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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