Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel
Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel
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DOI：
10.1128/jcm.03060-15
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发表时间：
2016-04-01
影响因子：
9.4
通讯作者：
Schlaberg, Robert
中科院分区：
文献类型：
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作者：
Graf, Erin H.;Simmon, Keith E.;Schlaberg, Robert
目前的传染病分子检测主要是病原体特异性的，需要根据患者的症状选择检测方法。对于由大量病毒、细菌或真菌病原体引起的许多综合征，如呼吸道感染，这需要大量的测试，并且产量有限。相比之下，下一代基于测序的宏基因组学可用于任何预期或意外病原体的无偏检测。然而，其诊断实施的障碍包括分析性能的不完全理解和序列数据分析的复杂性。我们比较了使用基于RNA测序（RNA-seq）的宏基因组学方法和Taxplatin（一种超快速、交互式、基于网络的宏基因组学数据分析工具）与FDA批准的呼吸道病毒面板（RVP; GenMark eSensor）对已知呼吸道病毒阳性（n = 42）和非阳性（n = 67）儿科鼻咽拭子的检测。非靶向宏基因组学检测到86%的已知呼吸道病毒感染，额外的PCR检测仅证实了2个（33%）不一致样本的RVP结果。在RVP样本中，非靶向宏基因组学与RVP有很好的一致性（93%）。此外，非靶向宏基因组学检测到另外12种病毒，这些病毒要么不是RVP靶向的，要么是由于基因组序列高度不同而错过的。非靶向宏基因组学的归一化病毒读段计数与通过定量PCR测定的病毒负荷相关，并显示出高的运行内和运行间重现性。在86%的RNA-seq阳性样本中生成了部分或全长病毒基因组序列，从而可以评估抗病毒耐药性、毒株水平分型和系统发育相关性。总体而言，非靶向宏基因组学与敏感的RVP具有高度一致性，检测到RVP不靶向的病毒，并产生流行病学和临床有价值的序列信息。
Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient's symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n = 42) and unselected (n = 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.