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Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA)

Patrick Reteng1, Linh Nguyen Thuy2, Mizanur Rahman3

  • 1Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan.

Msphere
|August 25, 2022
PubMed
Summary

This study introduces a novel PCR-free method, circular whole-transcriptome amplification (cWTA), for metagenomic next-generation sequencing (mNGS) pathogen detection. This approach simplifies library preparation and enhances high-throughput screening for infectious disease surveillance.

Keywords:
comprehensive pathogen detectionfebrile illnessgroup testing algorithmmetagenomicmultiple displacement amplification

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Area of Science:

  • Microbiology
  • Genomics
  • Infectious Diseases

Background:

  • Metagenomic next-generation sequencing (mNGS) is a powerful tool for hypothesis-free pathogen detection.
  • Current mNGS library construction is complex, costly, and time-consuming, limiting clinical applications.
  • A need exists for optimized, high-throughput methods for broad-range pathogen identification.

Purpose of the Study:

  • To develop and validate a simplified, PCR-free workflow for mNGS targeting RNA.
  • To assess the feasibility of a high-throughput detection system combining mNGS with a group testing algorithm.
  • To improve the diagnostic utility and scalability of mNGS for infectious disease surveillance.

Main Methods:

  • Developed circular whole-transcriptome amplification (cWTA), an isothermal, PCR-free amplification method.
  • Validated cWTA using clinical samples and nanopore sequencing.
  • Integrated cWTA with mNGS screening enhanced by a group testing algorithm (mEGA) for high-throughput analysis.

Main Results:

  • Detected dengue virus 2 and chikungunya virus in clinical samples from Bangladesh and Brazil, respectively.
  • Successfully identified dengue virus 2, hepatitis B virus, and parvovirus B19 in serum samples from Vietnam using cWTA and mEGA.
  • Demonstrated significant library size reduction and sample trackability with the mEGA approach.

Conclusions:

  • cWTA combined with nanopore sequencing enables hypothesis-free, on-site comprehensive pathogen diagnosis.
  • mEGA enhances sample throughput, making mNGS more practical for large-scale screening.
  • These optimized methods improve mNGS utility as a diagnostic tool and support infectious disease surveillance.