Next-generation fungal identification using target enrichment and Nanopore sequencing

Area of Science:

• Mycology
• Genomics
• Bioinformatics

Background:

• Accurate pathogen identification is crucial for effective disease management.
• Targeted sequencing offers higher resolution than single-locus methods for microbe identification.
• A novel tool was developed to focus high-throughput Nanopore sequencing on key fungal genes.

Purpose of the Study:

• To develop a broad-spectrum fungal identification tool using targeted Nanopore sequencing.
• To identify homologous regions across fungal species for probe design.
• To improve the efficiency and resolution of fungal molecular identification.

Main Methods:

• Extracted orthologs from 386 fungal reference genomes to design enrichment probes.
• Clustered DNA sequences and identified consensus sequences to create 26,000 probes targeting 114 genes.
• Applied targeted Nanopore sequencing to Ascomycota and Basidiomycota species.

Main Results:

• Enrichment efficiency ranged from 200 to 300 (mean target coverage/mean genome-wide coverage).
• Long-read enrichment increased coverage depth across targeted and flanking non-coding regions.
• Generated assemblies yielded well-resolved phylogenetic trees for accurate taxonomic assignment.

Conclusions:

• Targeted Nanopore sequencing is effective for fungal identification.
• The developed platform demonstrates utility for disease diagnostics and phylogenetic inference.
• This approach can be extended for identifying other phytopathogens.