Rapid identification, capsular typing and molecular characterization of Streptococcus pneumoniae by using whole genome nanopore sequencing.
Rapid identification, capsular typing and molecular characterization of Streptococcus pneumoniae by using whole genome nanopore sequencing.
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DOI：
10.1186/s12866-020-02032-x
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发表时间：
2020-11-13
期刊：
影响因子：
4.2
通讯作者：
Muñoz-Almagro C
中科院分区：
文献类型：
--
作者：
Garcia-Garcia S;Perez-Arguello A;Henares D;Timoneda N;Muñoz-Almagro C
全基因组测序已成为病原体鉴定和分子表征的有用工具。MinION（牛津纳米孔）是实时第三代测序仪，其便携性，可负担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测序仪在儿童医院分离的肺炎链球菌的病原体鉴定和分子特征。采用标准方法（Quellung反应、多重PCR和Sanger-MLST）对32株肺炎链球菌侵袭性分离株进行全基因组测序。采用ZymoBIOMICS DNA Microprep试剂盒提取DNA。分别用Qubit和Nanodrop对DNA的定量和纯度进行评估。使用快速条形码试剂盒进行文库制备。实时工作流EPI2ME平台“What 's it in my pot”用于物种鉴定。用Albacore软件将Fast5序列转化为FASTQ。读取用CANU软件进行组装。使用病原体观察、基因组流行病学和pubmlst在线工具进行荚膜分型和/或全基因组- mlst分析。采用“我锅里有什么”的方法，实现了肺炎链球菌的快速鉴定。所有32个分离株血清组水平和24个血清型水平的荚膜分型都被病原体观察正确地分配。31株分离株的基因组流行病学和pubmlst全基因组- mlst结果与Sanger-MLST双位点变异克隆复合体结果一致。MinION测序仪为流行病学目的提供了一种快速、经济、有前途的方法，但其测序精度的提高将使其在临床微生物实验室的应用更具吸引力。在线版本包含补充材料，可在10.1186/s12866-020-02032-x上获得。
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. The online version contains supplementary material available at 10.1186/s12866-020-02032-x.
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影响因子：
2.7
作者：
Kapatai G;Sheppard CL;Al-Shahib A;Litt DJ;Underwood AP;Harrison TG;Fry NK
通讯作者：
Fry NK
影响因子：
3.7
作者：
Mauffrey F;Fournier É;Demczuk W;Martin I;Mulvey M;Martineau C;Lévesque S;Bekal S;Domingo MC;Doualla-Bell F;Longtin J;Lefebvre B
通讯作者：
Lefebvre B
开放式细菌种群基因组学：BIGSDB软件，pubmlst.org网站及其应用。
影响因子：
--
作者：
Jolley KA;Bray JE;Maiden MCJ
通讯作者：
Maiden MCJ
影响因子：
5.2
作者：
Ardanuy, Carmen;Rolo, Dora;Linares, Josefina
通讯作者：
Linares, Josefina
影响因子：
2.4
作者：
Pareek, Chandra Shekhar;Smoczynski, Rafal;Tretyn, Andrzej
通讯作者：
Tretyn, Andrzej