A Modified Next-Generation Sequencing Methodology for Norovirus Genotyping

Adam Bissonnette, Marshfield Clinic Research Institute

Alex Linz, PhD; Marshfield Clinic Research Institute Sherri Guzinski; Marshfield Clinic Research Institute Thao Le; Marshfield Clinic Research Institute Josh Petrie, PhD, MPH; Marshfield Clinic Research Institute Huong Nguyen, PhD, MPH; Marshfield Clinic Research Institute Jennifer K. Meece, PhD; Marshfield Clinic Research Institute Adam Bissonnette, MS; Marshfield Clinic Research Institute

Advances in diagnostics and detection

Norovirus gastroenteritis (NGE) places significant burden on the US healthcare system annually. Development of interventions including vaccines have been challenged by genomic diversity of norovirus (NoV) and scarcity of genomic data from NGE cases. Improving next-generation sequencing (NGS) genotyping methods to characterize local NGE cases could inform vaccine design. We converted a Sanger sequencing single amplicon NoV genotyping protocol into a multiplexed Illumina-based methodology capable of genotyping ≥180 clinical samples per run. We performed PCR spanning the polymerase and capsid region, then fragment into a 330 bp final product size. These fragments are sequenced using a MiSeq v2 2x250bp reagent kit, and the resulting reads are filtered, clustered, and assembled using VSEARCH and MEGAHIT. The consensus sequence for each sample is classified using the CDC Human Calicivirus Typing Tool. Any unclassified sequences were compared to the NIH Core Nucleotide Database using BLASTn. Contamination from gut microbiota was minimal and easily distinguished from NoV sequences. An initial method validation study correctly identified 40 previously genotyped clinical samples with 100% accuracy evenly distributed between Group I and Group II NoV. Following this initial validation, we attempted to sequence 78 unique clinical fecal specimens (Group I, n=3; Group II, n=75) and achieved Calicivirus Typing results for 86% of the samples (n=67). Failed samples were a nearly equal mix of low viral load (Ct > 25) and poor PCR amplification. By combining multiple sample types onto a single flow cell (e.g, influenza, SARS-CoV-2, NoV) we demonstrate an agile approach to increasing NoV genotyping capability.