Morrison Gretchen A, Fu Jianmin, Lee Grace C, Wiederhold Nathan P, Cañete-Gibas Connie F, Bunnik Evelien M, Wickes Brian L
Department of Microbiology, Immunology, and Molecular Genetics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
The University of Texas at Austin, College of Pharmacy, Austin, Texas, USA.
J Clin Microbiol. 2020 Nov 18;58(12). doi: 10.1128/JCM.01972-20.
Fungal infections are being caused by a broadening spectrum of fungi, yet in many cases, identification to the species level is required for proper antifungal selection. We investigated the fungal intergenic spacer (IGS) sequence in combination with nanopore sequencing for fungal identification. We sequenced isolates from two species complexes, and , which are the main pathogenic members of this genus, using the Oxford Nanopore Technologies MinION device and Sanger sequencing. There is enough variation within the two complexes to argue for further resolution into separate species, which we wanted to see if nanopore sequencing could detect. Using the R9.4.1 flow cell, IGS sequence identities averaged 99.57% compared to Sanger sequences of the same region. When the newer R10.3 flow cell was used, accuracy increased to 99.83% identity compared to the same Sanger sequences. Nanopore sequencing errors were predominantly in regions of homopolymers, with G homopolymers displaying the largest number of errors and C homopolymers displaying the least. Phylogenetic analysis of the nanopore- and Sanger-derived sequences resulted in indistinguishable trees. Comparison of average percent identities between the and species complexes resulted in only a 74 to 77% identity between the two complexes. Sequencing using the nanopore platform could be completed in less than an hour, and samples could be multiplexed in groups as large as 24 sequences in a single run. These results suggest that sequencing the IGS region using nanopore sequencing could be a potential new molecular diagnostic strategy.
真菌感染由种类日益增多的真菌引起，但在许多情况下，为了正确选择抗真菌药物，需要鉴定到种水平。我们研究了结合纳米孔测序的真菌基因间隔区（IGS）序列用于真菌鉴定。我们使用牛津纳米孔技术公司的MinION设备和桑格测序法对来自两个物种复合体（该属的主要致病成员）的分离株进行了测序。这两个复合体内部存在足够的差异，表明有进一步细分为不同物种的必要，我们想看看纳米孔测序能否检测到这些差异。使用R9.4.1流动槽，与同一区域的桑格序列相比，IGS序列同一性平均为99.57%。当使用更新的R10.3流动槽时，与相同的桑格序列相比，准确性提高到了99.83%的同一性。纳米孔测序错误主要出现在同聚物区域，其中G同聚物的错误数量最多，C同聚物的错误数量最少。对纳米孔测序和桑格测序所得序列进行系统发育分析，得到的树状图无法区分。比较两个物种复合体之间的平均同一性百分比，结果显示两个复合体之间的同一性仅为74%至77%。使用纳米孔平台进行测序可在不到一小时内完成，并且样本可以在单次运行中以多达24个序列的组进行多重分析。这些结果表明，使用纳米孔测序对IGS区域进行测序可能是一种潜在的新分子诊断策略。
