A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome...

Area of Science:

• Clinical Microbiology
• Genomics
• Molecular Diagnostics

Background:

• Whole-genome sequencing (WGS) adoption is increasing in local clinical microbiology labs.
• Low-volume labs face challenges with existing WGS protocols designed for high throughput.
• A need exists for efficient, adaptable WGS methods for diverse bacterial targets.

Purpose of the Study:

• To develop and validate a universal protocol for simultaneous DNA extraction and WGS.
• To enable mixed-species sequencing runs suitable for variable laboratory demand.
• To support rapid turnaround times and low-volume processing in clinical settings.

Main Methods:

• A universal protocol combining DNA extraction and library preparation for WGS was developed.
• The QIAamp mini DNA kit was optimized with specific reagent combinations (lysozyme plus lysostaphin) for broad bacterial lysis.
• Testing involved 20 clinically relevant bacterial species, with DNA extraction times assessed (24-72 hours).

Main Results:

• Lysozyme plus lysostaphin ensured sufficient DNA yield across all 20 tested bacterial species.
• DNA extraction was successful for 15 species within 24 hours, with others requiring 48-72 hours.
• The protocol demonstrated 100% reproducibility and enabled accurate species identification, outbreak detection, and antimicrobial resistance gene identification.

Conclusions:

• A single, universal protocol facilitates simultaneous DNA extraction and WGS for multiple bacterial species.
• This approach is highly suitable for low-volume clinical microbiology laboratories requiring rapid and reproducible results.
• The protocol supports key diagnostic applications including species identification, outbreak surveillance, and antimicrobial resistance profiling.