King Jacqueline, Harder Timm, Beer Martin, Pohlmann Anne
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493, Greifswald, Insel Riems, Germany.
BMC Infect Dis. 2020 Sep 3;20(1):648. doi: 10.1186/s12879-020-05367-y.
Due to the frequent reassortment and zoonotic potential of influenza A viruses, rapid gain of sequence information is crucial. Alongside established next-generation sequencing protocols, the MinION sequencing device (Oxford Nanopore Technologies) has become a serious competitor for routine whole-genome sequencing. Here, we established a novel, rapid and high-throughput MinION multiplexing workflow based on a universal RT-PCR.
Twelve representative influenza A virus samples of multiple subtypes were universally amplified in a one-step RT-PCR and subsequently sequenced on the MinION instrument in conjunction with a barcoding library preparation kit from the rapid family and the MinIT performing live base-calling. The identical PCR products were sequenced on an IonTorrent platform and, after final consensus assembly, all data was compared for validation. To prove the practicability of the MinION-MinIT method in human and veterinary diagnostics, we sequenced recent and historical influenza strains for further benchmarking.
The MinION-MinIT combination generated over two million reads for twelve samples in a six-hour sequencing run, from which a total of 72% classified as quality screened, trimmed and mapped influenza reads to produce full genome sequences. Identities between the datasets of > 99.9% were achieved, with 100% coverage of all segments alongside a sufficient confidence and 4492fold mean depth. From RNA extraction to finished sequences, only 14 h were required.
Overall, we developed and validated a novel and rapid multiplex workflow for influenza A virus sequencing. This protocol suits both clinical and academic settings, aiding in real time diagnostics and passive surveillance.
由于甲型流感病毒频繁重配且具有人畜共患病潜力，快速获取序列信息至关重要。除了既定的下一代测序方案外，MinION测序设备（牛津纳米孔技术公司）已成为常规全基因组测序的有力竞争对手。在此，我们基于通用逆转录聚合酶链反应（RT-PCR）建立了一种新型、快速且高通量的MinION多重测序工作流程。
12个多种亚型的代表性甲型流感病毒样本通过一步法RT-PCR进行通用扩增，随后在MinION仪器上进行测序，并结合快速家族的条形码文库制备试剂盒以及进行实时碱基识别的MinIT。相同的PCR产物在IonTorrent平台上进行测序，最终经过一致性组装后，对所有数据进行比较以验证。为证明MinION-MinIT方法在人类和兽医诊断中的实用性，我们对近期和历史流感毒株进行测序以作进一步基准测试。
在6小时的测序运行中，MinION-MinIT组合为12个样本产生了超过200万个读数，其中共有72%被分类为经过质量筛选、修剪和映射的流感读数，从而产生全基因组序列。数据集之间的同一性达到>99.9%，所有片段的覆盖率为100%，同时具有足够的可信度和4492倍的平均深度。从RNA提取到完成序列仅需14小时。
总体而言，我们开发并验证了一种用于甲型流感病毒测序的新型快速多重工作流程。该方案适用于临床和学术环境，有助于实时诊断和被动监测。
