Viruses, Vol. 15, Pages 1248: Direct Nanopore Sequencing of Human Cytomegalovirus Genomes from High-Viral-Load Clinical Samples

Viruses, Vol. 15, Pages 1248: Direct Nanopore Sequencing of Human Cytomegalovirus Genomes from High-Viral-Load Clinical Samples

Viruses doi: 10.3390/v15061248

Authors: Kathy K. Li Betty Lau Nicolás M. Suárez Salvatore Camiolo Rory Gunson Andrew J. Davison Richard J. Orton

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.