Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA.
Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL, 32608, USA.
BMC Genomics. 2023 Oct 2;24(1):581. doi: 10.1186/s12864-023-09691-w.
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.
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.
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.
疾病管理需要快速准确的病原体鉴定。与测序整个基因组相比，靶向测序可用于将测序资源引导至微生物鉴定感兴趣的基因，并减轻单基因分子鉴定提供的低分辨率。这项工作描述了一种广谱真菌鉴定工具，用于将高通量纳米孔测序集中在常用于疾病诊断和系统发育推断的基因上。
从六个门的 386 个真菌参考基因组中提取靶向基因的直系同源物，以鉴定用于富集的同源区域。为了在不降低系统发育能力的情况下降低生产探针的成本，首先对 DNA 序列进行聚类，然后在每个聚类中确定共识序列，以产生针对 114 个基因的 26000 个探针。为了测试我们探针的功效，我们将该技术应用于三个代表子囊菌和担子菌的物种。以平均目标覆盖率与平均全基因组覆盖率的比率来量化的富集效率范围为 200 到 300。此外，长读段的富集增加了目标基因和非编码侧翼序列的覆盖深度。从富集样本生成的组装提供了用于分类学分配和分子鉴定的分辨率良好的系统发育树。
我们的工作提供了支持靶向纳米孔测序用于真菌鉴定的效用的数据，并提供了一个可能扩展到其他植物病原体的平台。
