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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
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Targeting RNA with Next- and Third-Generation Sequencing Improves Pathogen Identification in Clinical Samples.

Na Zhao1, Jiabao Cao1,2, Jiayue Xu1

  • 1CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|October 23, 2021
PubMed
Summary

RNA-targeted sequencing, including meta-transcriptomics using next-generation sequencing (mtNGS) and third-generation sequencing (mtTGS) with Oxford Nanopore Technology (ONT), rapidly identifies microbial pathogens in clinical samples. This approach improves diagnostic accuracy and speed for infections.

Keywords:
Oxford Nanopore Technologydirect RNA sequencingmetagenomemetatranscriptome

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Area of Science:

  • Microbiology
  • Genomics
  • Infectious Diseases

Background:

  • Accurate microbial pathogen identification is crucial for effective infection treatment.
  • Traditional culture-based methods are slow and often supplemented by metagenomic next-generation sequencing (mNGS).
  • Reducing host sequences and improving speed are key challenges in clinical metagenomics.

Purpose of the Study:

  • To establish RNA/cDNA-targeted sequencing (meta-transcriptomics using NGS - mtNGS) to enhance microbial read ratios in clinical samples.
  • To integrate Oxford Nanopore Technology (ONT) platforms (meta-transcriptomics using third-generation sequencing - mtTGS) for accelerated sequencing.
  • To evaluate the diagnostic potential of these advanced sequencing techniques for pathogen detection.

Main Methods:

  • Development and application of mtNGS to reduce host sequences and increase microbial reads.
  • Integration of ONT platforms for mtTGS, leveraging longer reads and faster sequencing.
  • Utilizing multiple strategies for bacterial identification and discovery of fungi, viruses, and antibiotic resistance genes.
  • Exploration of direct-RNA sequencing and targeted ONT sequencing.

Main Results:

  • mtNGS significantly improved the ratio of microbial reads, facilitating pathogen identification.
  • mtNGS successfully identified bacteria, fungi, viruses, and antibiotic resistance genes, aligning with clinical findings.
  • mtTGS, utilizing longer ONT reads, further enhanced pathogen identification accuracy and accelerated clinical diagnosis.
  • Initial ONT direct-RNA and targeted sequencing showed promise but require further optimization.

Conclusions:

  • RNA-targeted sequencing, particularly mtNGS, is a powerful tool for identifying diverse microbial pathogens in clinical samples.
  • Combining mtNGS with ONT's mtTGS offers significant advantages in speed and accuracy for clinical diagnostics.
  • ONT platforms hold substantial potential for advancing rapid clinical pathogen identification, warranting further development.