Next-generation fungal identification using target enrichment and Nanopore sequencing

Abstract

Background: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference.

Results: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification.

Conclusions: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

Keywords: Fungal identification; Oxford Nanopore Technologies; Probe-based target sequencing.

Fig. 1

Heatmaps provide depth of coverage of sequence surrounding the targeted genes in a 20-kb window. X-axes are the flanking genome regions of each targeted gene. Color keys represent the score of depth coverage per 20-kb genome regions computed by computeMatrix. Fc: Fusarium circinatum; Ss: Sclerotinia sclerotiorum; Ar: Athelia rolfsii; 0 = translation starting point; TG = targeted genes

Fig. 2

Taxonomic assignment based on majority rule consensus trees. The figures represent the phylogeny of (A)Fusarium species, (B)Sclerotinia species, and (C) species under Atheliales order. The values displayed on the branches indicate the proportion of trees analyzed that support the topology. The branch lengths are calculated based on the median of the branches across all individual trees