Streamlining whole genome sequencing for clinical diagnostics with ONT technology

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Whole-genome sequencing (WGS) offers significant potential for pathogen surveillance, outbreak response, and diagnostics.
• Clinical adoption of WGS is limited by high costs, technical complexity, and bioinformatics expertise requirements.

Purpose of the Study:

• To develop a cost-effective and accessible workflow for WGS-based pathogen analysis.
• To simplify WGS data analysis for routine clinical use, minimizing bioinformatics demands.

Main Methods:

• Mechanical shearing DNA extraction and multiplexed Oxford Nanopore Technologies (ONT) rapid barcoding.
• Automated de novo assembly using Flye, followed by polishing with Medaka and Homopolish.
• Analysis via the Pathogenwatch web platform for species identification, molecular typing, and antimicrobial resistance (AMR) prediction.

Main Results:

• The RapidONT workflow demonstrated high accuracy in multilocus sequence typing (MLST) and AMR identification for 90 bacterial strains.
• Successful genomic information generation for 48 isolates using a single ONT flow cell, significantly reducing costs.
• Identified limitations with Salmonella spp. and Neisseria gonorrhoeae, indicating areas for future refinement.

Conclusions:

• RapidONT provides a streamlined, cost-effective solution for WGS-based pathogen analysis.
• The workflow facilitates broader implementation of WGS in clinical diagnostics and public health surveillance.
• This approach reduces the need for extensive experimentation, accelerating the acquisition of crucial genomic data.