Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis.
Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis.
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DOI：
10.7717/peerj.10778
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发表时间：
2021
期刊：
影响因子：
2.7
通讯作者：
West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT)
中科院分区：
文献类型：
--
作者：
Low L;Fuentes-Utrilla P;Hodson J;O'Neil JD;Rossiter AE;Begum G;Suleiman K;Murray PI;Wallace GR;Loman NJ;Rauz S;West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT)
微生物性角膜炎是全世界可预防性失明的主要原因。传统的采样和培养技术非常耗时，超过 40% 的病例培养结果呈阴性。纳米孔测序技术是便携式的，能够实时生成长测序读数。本研究的目的是评估直接从临床样本进行纳米孔测序诊断细菌性微生物角膜炎的潜力。使用来自定义的模拟微生物群落的全长 16S rRNA 扩增子序列，我们在三个不同的 16S rRNA 数据库（NCBI 16S RefSeq、RDP 和 SILVA）上评估和基准化了我们的生物信息学分析流程，以进行分类分配，聚类相似度为 97%、99% 和 100%。接下来，我们使用微生物角膜炎的离体猪模型优化了样品采集，以比较 12 种不同采集方法的 DNA 回收率：21 号针、PTFE 膜（4 毫米和 6 毫米）、Isohelix™ SK-2S、Sugi® Eyespear、棉签、人造丝、Dryswab™、Hydraflock®、白蛋白涂层、Purflock®、Purfoam 和聚酯拭子。作为一项概念验证研究，我们随后使用提供最高 DNA 回收率的采样技术以及优化的生物信息学流程，前瞻性地从疑似微生物性角膜炎患者身上采集样本。然后将所得纳米孔测序结果与标准微生物培养方法进行比较。我们发现，将比对过滤应用于纳米孔测序读数并以 100% 相似度比对到 NCBI 16S RefSeq 数据库，可以提供最准确的细菌分类群分配。不同采集方法之间的 DNA 浓度回收率存在显着差异 (p < 0.001)，其中 Sugi® Eyespear 拭子提供的 DNA 浓度平均等级最高。然后，将优化的收集方法和生物信息学流程直接应用于两名疑似微生物性角膜炎患者的样本，患者 A 的测序结果与培养结果一致，而培养结果阴性且既往使用过抗生素的患者 B 则显示 Nanopore 和 Illumina Miseq 测序结果一致。我们优化了收集方法，并展示了使用全长 16S 纳米孔测序鉴定细菌性微生物角膜炎的新颖工作流程。
Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.
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DOI：
10.1093/bioinformatics/bts565
发表时间：
2012-12-01
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Fu L;Niu B;Zhu Z;Wu S;Li W
通讯作者：
Li W
DOI：
10.1146/annurev-pathmechdis-012418-012751
发表时间：
2019-01-24
期刊：
Annual review of pathology
影响因子：
--
作者：
Gu W;Miller S;Chiu CY
通讯作者：
Chiu CY
影响因子：
5.8
作者：
Edgar, Robert C.
通讯作者：
Edgar, Robert C.
影响因子：
4.1
作者：
Ibrahim, Y. W.;Boase, D. L.;Cree, I. A.
通讯作者：
Cree, I. A.
影响因子：
4.2
作者：
Glassing A;Dowd SE;Galandiuk S;Davis B;Chiodini RJ
通讯作者：
Chiodini RJ