Petty Tom J, Cordey Samuel, Padioleau Ismael, Docquier Mylène, Turin Lara, Preynat-Seauve Olivier, Zdobnov Evgeny M, Kaiser Laurent
Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Swiss Institute of Bioinformatics, Geneva, Switzerland
Division of Infectious Diseases, Laboratory of Virology and Division of Laboratory Medicine, University Hospitals of Geneva, Geneva, Switzerland Department of Medicine, University of Geneva Medical School, Geneva, Switzerland.
J Clin Microbiol. 2014 Sep;52(9):3351-61. doi: 10.1128/JCM.01389-14. Epub 2014 Jul 9.
High-throughput sequencing (HTS) provides the means to analyze clinical specimens in unprecedented molecular detail. While this technology has been successfully applied to virus discovery and other related areas of research, HTS methodology has yet to be exploited for use in a clinical setting for routine diagnostics. Here, a bioinformatics pipeline (ezVIR) was designed to process HTS data from any of the standard platforms and to evaluate the entire spectrum of known human viruses at once, providing results that are easy to interpret and customizable. The pipeline works by identifying the most likely viruses present in the specimen given the sequencing data. Additionally, ezVIR can generate optional reports for strain typing, can create genome coverage histograms, and can perform cross-contamination analysis for specimens prepared in series. In this pilot study, the pipeline was challenged using HTS data from 20 clinical specimens representative of those most often collected and analyzed in daily practice. The specimens (5 cerebrospinal fluid, 7 bronchoalveolar lavage fluid, 5 plasma, 2 serum, and 1 nasopharyngeal aspirate) were originally found to be positive for a diverse range of DNA or RNA viruses by routine molecular diagnostics. The ezVIR pipeline correctly identified 14 of 14 specimens containing viruses with genomes of <40,000 bp, and 4 of 6 specimens positive for large-genome viruses. Although further validation is needed to evaluate sensitivity and to define detection cutoffs, results obtained in this pilot study indicate that the overall detection success rate, coupled with the ease of interpreting the analysis reports, makes it worth considering using HTS for clinical diagnostics.
高通量测序（HTS）提供了以前所未有的分子细节分析临床标本的方法。虽然这项技术已成功应用于病毒发现及其他相关研究领域，但HTS方法尚未用于临床常规诊断。在此，设计了一种生物信息学流程（ezVIR），用于处理来自任何标准平台的HTS数据，并一次性评估已知人类病毒的全谱，提供易于解读且可定制的结果。该流程通过根据测序数据识别标本中最可能存在的病毒来工作。此外，ezVIR可以生成用于毒株分型的可选报告，创建基因组覆盖直方图，并对系列制备的标本进行交叉污染分析。在这项初步研究中，使用来自20份临床标本的HTS数据对该流程进行了验证，这些标本代表了日常实践中最常采集和分析的标本类型。这些标本（5份脑脊液、7份支气管肺泡灌洗液、5份血浆、2份血清和1份鼻咽抽吸物）最初通过常规分子诊断发现对多种DNA或RNA病毒呈阳性。ezVIR流程正确识别了14份含有基因组小于40,000 bp病毒的标本中的14份，以及6份对大基因组病毒呈阳性的标本中的4份。尽管需要进一步验证以评估敏感性并确定检测阈值，但这项初步研究获得的结果表明，总体检测成功率以及分析报告易于解读，使得考虑将HTS用于临床诊断是值得的。
