Development of an Amplicon-Based NGS Method for Dual Pol-Cap Genotyping of Norovirus GII

Sammanfattning

Noroviruses (NoVs) are the primary cause of acute non-bacterial gastroenteritis worldwide andare associated with hundreds of thousands of deaths annually. Based on the genomic major capsid and polymerase regions, NoVs can be phylogenetically classified into genogroups and P-groups. These groups are further divided into genotypes and P-types, respectively. To more accurately classify NoVs into their respective group, current typing methodologies would benefit from amplification of a single sequence covering both the capsid and the polymerase regions simultaneously. This is particularly important due to frequent occurrence of recombination in the ORF1/ORF2-junction between different variants. Therefore, the aim of this project was to develop an amplicon-based Next Generation Sequencing method for dual-typing of NoV genogroup II. Specifically, this project focused on the development of a polymerase chain reaction (PCR) method, in which primers were designed for amplification of the targeted region. A pipeline was implemented, consisting of data collection and curation, sequence alignment, primer design in varVAMP, evaluation using NoV Typing Tool and AssayBLAST. Using this approach, a degenerate primer pair generating a circa 839 base pair long amplicon, including primers, suitable for Illumina MiSeq i100 was identified. In total, 19 of the 24 genotypes and 28 of the 31 P-types represented in the analysis could produce amplicons, using the obtained primers. Although the genotype and P-type coverage were incomplete, the typing analysis demonstrated 100% accuracy for typing the capsid and 99.3% for typing the polymerase region. To further improve specificity, additional data is needed for future studies. A proposed workflow for in vitro implementation of the developed amplicon-based method was also presented. The workflow included a framework of relevant reaction parameters, as well as precautions and recommendations for conducting the PCR in which the targeted region spanning both the capsid and polymerase regions is amplified.