Targeted tiled amplicon based protocol for sequencing the Hemagglutinin (HA) gene segment of seasonal influenza A and influenza B virus from wastewater at high depth of coverage

Wastewater based epidemiology has emerged as a compelling tool to monitor the spread and evolution of pathogens of public health concern. Next generation sequencing (NGS) of pathogens detected in wastewater enables sequence characterization which is essential for monitoring the changing genomic landscape of pathogens. Influenza virus, with its potential to cause epidemics and pandemics, poses a serious risk to human health. Genomic surveillance of the virus is essential for safeguarding public health by monitoring the virus’s evolution, and developing preventive vaccines and therapeutics. As such, methods for successfully sequencing influenza virus at a consistent high depth of coverage across the genome can aid in this endeavor. Here, we present a novel targeted tiled amplicon based sequencing protocol that uses short tiled amplicons (<250 bp in length) to successfully capture the Hemagglutinin (HA) gene segment of seasonal influenza A subtypes (H1 and H3) and Influenza B at high depth of coverage. We observed near consistent coverage across the HA gene segment for wastewater samples that had influenza viral target dPCR detections of at least 10 ³ copies/L. We were able to successfully detect low frequency single nucleotide variants (SNVs) at high depth of coverage demonstrating the utility of the data to characterize the diversity of circulating influenza A and B viruses at the community level. Our approach is flexible and future directions include expanding this approach to sequence additional influenza virus HA subtypes and gene segments.