背景:宏基因组学是检测未知和新型病原体的强大方法。基于Illumina短读测序的工作流程正在诊断实验室中建立。然而,高测序深度要求,较长的周转时间,有限的敏感性阻碍了更广泛的采用。我们调查了我们是否可以使用基于牛津纳米孔技术(ONT)的非靶向测序的方案克服这些限制,提供实时数据采集和分析,或者有针对性的小组方法,这允许对已知病原体进行选择性测序,并可以提高灵敏度。
方法:我们使用Illumina和ONT评估了现有的非靶向宏基因组工作流程对病毒的检测,以及使用Twist生物科学综合病毒研究小组(CVRP)的基于Illumina的富集方法,针对3153种病毒。我们测试了由人类DNA/RNA背景下的六病毒模拟群落的稀释系列组成的样本,设计类似于具有低微生物丰度和高宿主含量的临床标本。方案旨在保留宿主转录组,因为这可以帮助确认没有感染因子。我们进一步比较了常用分类分类器的性能。
结果:用TwistCVRP捕获比非靶向测序至少增加10-100倍的灵敏度,使其适用于低病毒载量的检测(60基因组拷贝每毫升(gc/ml)),但在诊断环境中可能需要其他方法来检测非靶向生物。虽然未靶向的ONT在高病毒载量(60,000gc/ml)下具有良好的敏感性,在较低的病毒载量(600-6000gc/ml),需要更长和更昂贵的测序运行才能达到与非靶向Illumina方案相当的灵敏度.非靶向ONT比非靶向Illumina测序提供更好的特异性。然而,在分类分类器之间应用鲁棒阈值标准化结果。宿主基因表达分析对于非靶向Illumina测序是最佳的,但对于CVRP和ONT都是可能的。
结论:宏基因组学有可能成为诊断的标准治疗方法,并且是发现新出现的病原体的有力工具。非靶向Illumina和ONT宏基因组学以及使用TwistCVRP捕获在灵敏度方面具有不同的优势,特异性,周转时间和成本,最佳方法将取决于临床情况。
BACKGROUND: Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity.
METHODS: We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers.
RESULTS: Capture with the Twist CVRP increased sensitivity by at least 10-100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600-6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT.
CONCLUSIONS: Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.