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 Cryptococcus species complexes, C. gattii and C. neoformans, 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 C. gattii and C. neoformans 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.

中文翻译：

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真菌间基因间隔子（IGS）序列的纳米孔测序可作为一种潜在的快速诊断分析方法。

真菌感染是由范围越来越广的真菌引起的，但是在许多情况下，为了正确选择抗真菌剂，需要鉴定到物种水平。我们调查了真菌基因间隔区（IGS）序列与纳米孔测序相结合的真菌鉴定。我们测序的菌株由两种隐球菌种复合物，隐球菌和隐球菌使用牛津纳米孔技术MinION装置和Sanger测序，是该属的主要致病成员。两种复合物中存在足够的差异，可以争辩进一步分离成单独的物种，我们希望了解纳米孔测序是否可以检测到。与相同区域的Sanger序列相比，使用R9.4.1流通池时，IGS序列同一性平均为99.57％。当使用更新的R10.3流通池时，与相同的Sanger序列相比，准确性提高到99.83％。纳米孔测序错误主要发生在均聚物区域，G均聚物显示最多错误，C均聚物显示最少。系统发育分析的纳米孔和桑格派生的序列导致无法区分树木。隐球菌和隐球菌物种络合物导致只有两种复合物之间的74至77％的同一性。使用纳米孔平台的测序可以在不到一个小时的时间内完成，并且样品可以在一次运行中以多达24个序列的组形式进行多路复用。这些结果表明，使用纳米孔测序对IGS区域进行测序可能是潜在的新分子诊断策略。