Phylo-Plex: a phylogenetically informed, low-cost amplicon sequencing platform for deployable high-resolution genomic...

Area of Science:

• Genomic epidemiology
• Molecular diagnostics
• Public health microbiology

Background:

• Genomic pathogen surveillance is crucial but often limited by cost and accessibility in low-resource settings.
• Existing sub-genomic typing methods often compromise resolution for cost-effectiveness.
• Effective tools are needed for high-resolution pathogen lineage tracking in diverse global health contexts.

Purpose of the Study:

• To develop and evaluate Phylo-Plex, a novel, cost-effective method for high-resolution genomic surveillance of pathogens.
• To maximize phylogenetic information while minimizing the number of genomic regions analyzed.
• To enable pathogen lineage tracking in resource-limited environments.

Main Methods:

• Developed Phylo-Plex, a multiplex PCR sequencing scheme targeting information-rich genomic regions.
• Applied the scheme to Treponema pallidum and Neisseria gonorrhoeae, focusing on different genome variation extremes.
• Validated the Treponema pallidum scheme using laboratory and field settings with MinION sequencing of 72 clinical samples.

Main Results:

• The Phylo-Plex scheme for T. pallidum, using 59 amplicons, achieved high discrimination of sublineages, comparable to whole-genome sequencing.
• Demonstrated a qPCR detection limit of ≤Ct 32 for the T. pallidum scheme.
• Variant calls from MinION amplicon sequencing showed high correlation with Illumina whole-genome sequencing data.
• Successfully deployed the method in a low-resource laboratory in Zimbabwe at a cost of <£300/24 samples (£12.47/sample).

Conclusions:

• Phylo-Plex provides a high-resolution, low-cost solution for genomic surveillance of priority pathogens.
• The method is effective in both laboratory and field settings, including low-resource environments.
• Phylo-Plex enables scalable tracking of pathogenic lineages, enhancing global public health capabilities.