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NanoMLST: A High-Throughput Bacterial Multi-Locus Sequence Typing Workflow Using Oxford Nanopore Next-Generation
Isabel García-Pérez1, Fernando Lázaro-Perona1, Diana Soledad Reyes-Zuñagua1
1Clinical Microbiology and Parasitology Department, Hospital Universitario La Paz, Instituto de Investigación Sanitaria del Hospital Universitario La Paz (IdiPAZ), Madrid, Spain.
NanoMLST provides a rapid, cost-effective, and less labor-intensive method for bacterial Multi-Locus Sequence Typing (MLST) using next-generation sequencing. This high-throughput workflow accurately identifies Sequence Types (STs) for clinically significant pathogens.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- Multi-Locus Sequence Typing (MLST) is crucial for bacterial isolate characterization.
- Traditional Sanger sequencing for MLST is time-consuming and labor-intensive.
- High-throughput methods are needed for large-scale pathogen surveillance.
Purpose of the Study:
- To develop and validate NanoMLST, a high-throughput MLST workflow.
- To enable rapid and efficient Sequence Type (ST) allocation for ESKAPE+E pathogens.
- To offer a cost-effective and less labor-intensive alternative to Sanger sequencing.
Main Methods:
- Multiplex PCR primer design for seven housekeeping genes.
- DNA extraction using heat lysis, mechanical lysis, and magnetic bead system.
- Oxford Nanopore Technologies (ONT) Next-Generation Sequencing on Flongle cells.
- Sequence Type (ST) determination using the Krocus program.
Main Results:
- Successfully obtained STs for 221 bacterial isolates.
- Average turnaround time of 12 hours for 24 isolates.
- 100% concordance with Sanger sequencing for representative isolates.
- Characterized K. pneumoniae and E. faecium as oligoclonal, others as polyclonal.
Conclusions:
- NanoMLST is a fast, affordable, and efficient alternative for large-scale MLST.
- The workflow is suitable for typing clinically important pathogens.
- Enables rapid surveillance and characterization of bacterial populations.
