Medical Research Council, University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK.
Regional Virus Laboratory, Belfast Health and Social Care Trust, Belfast BT12 6BA, UK.
Viruses. 2023 May 26;15(6):1248. doi: 10.3390/v15061248.
Nanopore sequencing is becoming increasingly commonplace in clinical settings, particularly for diagnostic assessments and outbreak investigations, due to its portability, low cost, and ability to operate in near real-time. Although high sequencing error rates initially hampered the wider implementation of this technology, improvements have been made continually with each iteration of the sequencing hardware and base-calling software. Here, we assess the feasibility of using nanopore sequencing to determine the complete genomes of human cytomegalovirus (HCMV) in high-viral-load clinical samples without viral DNA enrichment, PCR amplification, or prior knowledge of the sequences. We utilised a hybrid bioinformatic approach that involved assembling the reads de novo, improving the consensus sequence by aligning reads to the best-matching genome from a collated set of published sequences, and polishing the improved consensus sequence. The final genomes from a urine sample and a lung sample, the former with an HCMV to human DNA load approximately 50 times greater than the latter, achieved 99.97 and 99.93% identity, respectively, to the benchmark genomes obtained independently by Illumina sequencing. Thus, we demonstrated that nanopore sequencing is capable of determining HCMV genomes directly from high-viral-load clinical samples with a high accuracy.
纳米孔测序由于其便携性、低成本和能够近乎实时运行,在临床环境中变得越来越普遍,特别是在诊断评估和疫情调查方面。尽管最初高测序错误率阻碍了这项技术的更广泛应用,但随着测序硬件和碱基调用软件的每次迭代,都在不断进行改进。在这里,我们评估了在没有病毒 DNA 富集、PCR 扩增或事先了解序列的情况下,使用纳米孔测序来确定高病毒载量临床样本中人巨细胞病毒 (HCMV) 完整基因组的可行性。我们采用了一种混合生物信息学方法,包括从头组装读取、通过将读取与从一组已发表序列中整理出的最佳匹配基因组进行对齐来改进共识序列,以及对改进后的共识序列进行润色。来自尿液样本和肺部样本的最终基因组,前者的 HCMV 与人 DNA 负载比后者大约高 50 倍,与通过 Illumina 测序独立获得的基准基因组分别达到了 99.97%和 99.93%的同一性。因此,我们证明了纳米孔测序能够直接从高病毒载量的临床样本中准确地确定 HCMV 基因组。