Optimising Long-Range PCR and Oxford Nanopore Sequencing for Whole-Genome Analysis of Human Cytomegalovirus

Abstract

Human Cytomegalovirus (HCMV) possesses the largest genome of all human herpesviruses, characterised by low complexity repeat regions and high Guanine (G) and Cytosine (C) content. This dissertation explores the development of a comprehensive method for HCMV whole-genome sequencing that uses long-range polymerase chain reaction (LR-PCR) combined with Oxford Nanopore Technologies (ONT) sequencing. The challenges in HCMV genomics include the limitations of current sequencing technologies in resolving complex genomic structures. This study designed 28 overlapping primer pairs spanning the HCMV genome, with amplicon lengths ranging from 5000bp and 9700bp. All primer pairs were successfully tested on the laboratory HCMV strain Merlin. Thereafter, multiplex PCR products were sequenced using the MinION platform; 66,789 reads were analysed, yielding 344.8 Mbases. The average quality score was 14.35, with a modal score of 15.15. Read lengths averaged 5,162 bases, with modal lengths of 9,702 bases, thus aligning with the 5kb- and 10kb-amplicon experimental design. Notably, 100% of reads were classified as 'workflow successful' by the EPI2ME analysis pipeline. Moreover, the method achieved 85.64% unique coverage of the HCMV genome, with a high percentage identity (99.91 ± 0.2619%) to the reference genome. These results demonstrate the effectiveness of this approach in generating high-quality, long-read sequencing data for the HCMV genome. The study considers the implications of this method for understanding HCMV genetic diversity, evolution and pathogenesis. It likewise discusses limitations and future directions, including the need for clinical sample validation.