Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children’s Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform “What’s it in my pot” was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile. Rapid identification of Streptococcus pneumoniae was achieved by “What’s in my pot”. Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates. MinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.

中文翻译：

---

利用全基因组纳米孔测序对肺炎链球菌进行快速鉴定、荚膜分型和分子表征

全基因组测序已成为病原体识别和分子表征的有用工具。MinION（牛津纳米孔）是一款实时第三代测序仪，其便携性、经济性和数据生成速度使其成为全基因组测序的有吸引力的设备。本研究的目的是评估 MinION 测序仪对儿童医院分离出的肺炎链球菌的病原体鉴定和分子特征。对 32 种肺炎链球菌侵袭性分离株进行了全基因组测序，之前通过标准方法（Quellung 反应、多重 PCR 和 Sanger-MLST）进行了表征。使用 ZymoBIOMICS DNA Microprep 试剂盒提取 DNA。DNA 的定量和纯度分别通过 Qubit 和 Nanodrop 进行评估。使用快速条形码试剂盒进行文库制备。实时工作流程 EPI2ME 平台“What's it in my pot”用于物种识别。Fast5序列通过Albacore软件转化为FASTQ。使用 CANU 软件组装读数。PathogenWatch、基因组流行病学和 pubmlst 在线工具用于荚膜分型和/或全基因组 MLST 分析。通过“What's in my pot”实现了肺炎链球菌的快速鉴定。使用 PathogenWatch 在血清组水平上的所有 32 个分离株和血清型水平上的 24 个分离株中正确分配了荚膜分型。基因组流行病学和pubmlst获得的全基因组-MLST结果与Sanger-MLST在31个分离株中获得的双位点变异克隆复合物一致。MinION 测序仪提供了一种快速、经济高效且有前途的途径，通过用于流行病学目的的小型设备进行全基因组测序，但提高其测序准确性将使其在临床微生物学实验室中使用更具吸引力。