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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
Doença febril
Algoritmo de teste de grupo
Amplificação de deslocamento múltiplo
Detecção abrangente de patógenos
Febrile illness
Group testing algorithm
Multiple displacement amplification
Comprehensive pathogen detection
Autor
Afiliación
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan.
National Institute of Epidemiology and Health. Laboratory for Molecular Diagnostics. Hanoi, Vietnam.
Evercare Hospital Dhaka. Dhaka, Bangladesh.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivirus. Rio de Janeiro, RJ, Brasil.
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan. / Hokkaido University. International Collaboration Unit. International Institute for Zoonosis Control. Sapporo, Japan.
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan. / Hokkaido University. International Collaboration Unit. International Institute for Zoonosis Control. Sapporo, Japan.
University College Dublin. National Virus Reference Laboratory. Dublin, Ireland.
University College Dublin. School of Medicine and Medical Research. Dublin, Ireland / Global Virus Network. Baltimore, Maryland, USA.
National Institute of Epidemiology and Health. Laboratory for Molecular Diagnostics. Hanoi, Vietnam.
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan. / Hokkaido University. International Collaboration Unit. International Institute for Zoonosis Control. Sapporo, Japan.
National Institute of Epidemiology and Health. Laboratory for Molecular Diagnostics. Hanoi, Vietnam.
Evercare Hospital Dhaka. Dhaka, Bangladesh.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivirus. Rio de Janeiro, RJ, Brasil.
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan. / Hokkaido University. International Collaboration Unit. International Institute for Zoonosis Control. Sapporo, Japan.
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan. / Hokkaido University. International Collaboration Unit. International Institute for Zoonosis Control. Sapporo, Japan.
University College Dublin. National Virus Reference Laboratory. Dublin, Ireland.
University College Dublin. School of Medicine and Medical Research. Dublin, Ireland / Global Virus Network. Baltimore, Maryland, USA.
National Institute of Epidemiology and Health. Laboratory for Molecular Diagnostics. Hanoi, Vietnam.
Hokkaido University. International Institute for Zoonosis Control. Division of Collaboration and Education. Sapporo, Japan. / Hokkaido University. International Collaboration Unit. International Institute for Zoonosis Control. Sapporo, Japan.
Resumen en ingles
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.
Palabras clave en portugues
MetagenômicaDoença febril
Algoritmo de teste de grupo
Amplificação de deslocamento múltiplo
Detecção abrangente de patógenos
Palabras clave en ingles
MetagenomicFebrile illness
Group testing algorithm
Multiple displacement amplification
Comprehensive pathogen detection
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