Wurtzer Sebastien, Duvivier Mathilde, Accrombessi Heberte, Levert Morgane, Richard Elise, Moulin Laurent
Research & Development Department, Eau de Paris. DRDQE, FR-9400, France.
Paris Sorbonne Universite, CNRS, EPHE, UMR 7619 Metis, e-LTER Zone Atelier Seine, F-75005, Paris, France.
Biol Methods Protoc. 2024 Jul 27;9(1):bpae053. doi: 10.1093/biomethods/bpae053. eCollection 2024.
The development of high-throughput sequencing has greatly improved our knowledge of microbial diversity in aquatic environments and its evolution in highly diverse ecosystems. Relevant microbial diversity description based on high-throughput sequencing relies on the good quality of the nucleic acid recovered. Indeed, long genetic fragments are more informative for identifying mutation combinations that characterize variants or species in complex samples. This study describes a new analytical method based on digital Polymerase Chain Reaction (PCR) partitioning technology for assessing the fragmentation of nucleic acid and more specifically viral RNA. This method allows us to overcome limits associated with hydrolysis probe-based assay by focusing on the distance between different amplicons, and not, as usual, on the size of amplicons. RNA integrity can thus be determined as a new fragmentation index, the so-called Fragment size 50. The application of this method has provided information on issues that are inherent in environmental analyses, such as the storage impact of raw samples or extracted RNA, extraction methods, and the nature of the sample on the integrity of viral RNA. Finally, the estimation of fragment size by digital PCR (dPCR) showed a very strong similarity with the fragment size sequenced using Oxford Nanopore Technology. In addition to enabling objective improvements in analytical methods, this approach could become a systematic quality control prior to any long-read sequencing, avoiding insufficiently productive sequencing runs or biases in the representativeness of sequenced fragments.
高通量测序技术的发展极大地增进了我们对水生环境中微生物多样性及其在高度多样化生态系统中进化的了解。基于高通量测序的相关微生物多样性描述依赖于回收核酸的高质量。实际上，长基因片段对于识别复杂样本中表征变体或物种的突变组合更具信息性。本研究描述了一种基于数字聚合酶链反应（PCR）分区技术的新分析方法，用于评估核酸的片段化，更具体地说是病毒RNA的片段化。该方法使我们能够通过关注不同扩增子之间的距离，而不是像通常那样关注扩增子的大小，来克服基于水解探针检测的局限性。因此，RNA完整性可以确定为一种新的片段化指数，即所谓的片段大小50。该方法的应用提供了有关环境分析中固有问题的信息，例如原始样本或提取RNA的储存影响、提取方法以及样本性质对病毒RNA完整性的影响。最后，通过数字PCR（dPCR）估计的片段大小与使用牛津纳米孔技术测序的片段大小显示出非常强的相似性。除了能够客观地改进分析方法外，这种方法还可以成为任何长读长测序之前的系统质量控制，避免测序运行效率低下或测序片段代表性偏差。
