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Updated: Oct 10, 2025

Sequencing of mRNA from Whole Blood using Nanopore Sequencing
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Rapid absolute quantification of pathogens and ARGs by nanopore sequencing.

Yu Yang1, You Che1, Lei Liu1

  • 1Environmental Microbiome Engineering and Biotechnology Laboratory, Centre for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Hong Kong, China.

The Science of the Total Environment
|December 10, 2021
PubMed
Summary

A new rapid Nanopore sequencing method allows absolute quantification of pathogens and antibiotic resistance genes (ARGs) in wastewater. While treatment reduces most, some ARGs and pathogens persist or increase, highlighting the need for advanced microbial hazard management.

Keywords:
ARG removalInternal standardMinION sequencingPathogen removalWastewater treatment plantWastewater-based epidemiology

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Area of Science:

  • Environmental microbiology
  • Metagenomics
  • Wastewater treatment

Background:

  • Microbiome studies often use relative abundance data, limiting interpretation of microbial dynamics and cross-sample comparisons.
  • Absolute quantification of pathogens and antibiotic resistance genes (ARGs) is crucial for effective wastewater management and risk assessment.

Purpose of the Study:

  • To develop and validate a rapid method for absolute quantification and removal assessment of pathogens and ARGs in wastewater treatment plants (WWTPs).
  • To evaluate the efficacy of wastewater treatment in removing specific pathogens and ARGs.

Main Methods:

  • Coupling Nanopore metagenomic sequencing with cellular spike-in for rapid (1-hour) analysis.
  • Validating quantification results against established Illumina and culture-based methods using mock and real environmental samples.
  • Quantifying 46 predominant putative pathogenic species and 361 ARGs in three WWTP sample sets.

Main Results:

  • Nanopore sequencing demonstrated high consistency with Illumina and culture-based approaches for absolute quantification.
  • Wastewater treatment achieved high log removals for dominant pathogens (2.23 logs) and ARGs (1.98 logs).
  • Complete removal of all pathogens and ARGs was not achieved; some, like Mycobacterium spp. and 13 ARGs, showed low removal or increased abundance.

Conclusions:

  • The proposed rapid Nanopore sequencing method provides accurate absolute quantitation of microbial hazards in wastewater.
  • Wastewater treatment is effective but not complete in removing pathogens and ARGs, necessitating continuous monitoring.
  • This approach supports wastewater-based epidemiology, quantitative risk assessment, and improved microbial hazard management.