Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.
Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.
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DOI：
10.1038/s41598-018-31873-w
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发表时间：
2018-09-13
影响因子：
4.6
通讯作者：
Rossen JW
中科院分区：
文献类型：
--
作者：
Couto N;Schuele L;Raangs EC;Machado MP;Mendes CI;Jesus TF;Chlebowicz M;Rosema S;Ramirez M;Carriço JA;Autenrieth IB;Friedrich AW;Peter S;Rossen JW
高通量测序已被提议作为直接从患者样本进行诊断和分子分型的一站式解决方案，允许及时和适当地实施治疗、感染预防和控制措施。然而，目前尚不清楚各种可用方法如何影响最终结果。我们使用三种不同的方法将鸟枪宏基因组学应用于不同类型的患者样本，以在DNA提取之前耗尽人类DNA。用Illumina化学法制备文库并测序。使用基因组学临床微生物学实验室可能提供的方法分析数据。将微生物鉴定结果与标准的基于培养的微生物方法进行比较。平均而言，75%的读数对应于人类DNA，这是分析结果的主要决定因素。没有一个试剂盒明显具有上级优势，这表明宿主和微生物DNA之间的初始比率或其他样品特征是微生物读数比例的主要决定因素。通过培养鉴定的大多数病原体也通过宏基因组学鉴定，但在分类学分类工具之间存在实质性差异。在两种情况下，大量的人类读段导致细菌DNA的测序深度不足以进行鉴定。在三个样本中，我们可以推断可能的多位点序列类型的最丰富的物种。用于分类学分类和抗菌药物耐药性鉴定的工具和数据库对结果产生了关键影响，建议如果宏基因组学常规用于临床微生物学，则需要努力将分析方法标准化。
High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.
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DOI：
10.1093/jac/dkx217
发表时间：
2017-10-01
期刊：
The Journal of antimicrobial chemotherapy
影响因子：
--
作者：
Zankari E;Allesøe R;Joensen KG;Cavaco LM;Lund O;Aarestrup FM
通讯作者：
Aarestrup FM
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
14.9
作者：
Zolfo M;Tett A;Jousson O;Donati C;Segata N
通讯作者：
Segata N
影响因子：
3
作者：
Peabody MA;Van Rossum T;Lo R;Brinkman FS
通讯作者：
Brinkman FS
影响因子：
4.9
作者：
Willmann, Matthias;El-Hadidi, Mohamed;Peter, Silke
通讯作者：
Peter, Silke