Phased amplicon multiplex sequencing for cost-effective detection of high-risk human papillomavirus from cervical samples

Access to accurate cost-effective technologies for typing high-risk human papillomaviruses (hrHPV) is critical to expand cervical cancer screening and inform vaccination strategies. Compared with clinical-standard quantitative polymerase chain reaction (qPCR) assays, HPV genotyping by next-generation sequencing (NGS) provides greater flexibility, scalability, and genotype specificity. We have developed a method for HPV genotyping, HPV Phased Amplicon Multiplex Sequencing (PhAM-Seq), that uses combinatorial barcoding of amplicons with short, variable-length inline sequences to enable higher throughput and lower per-sample costs than conventional amplicon sequencing approaches. We evaluated HPV PhAM-Seq using degenerate and type-specific primers targeting the L1 and E6-E7 gene loci in a blinded cohort of 170 cervical samples previously typed by the Seegene Anyplex II HPV28 Detection qPCR assay. Across eight common hrHPV types (HPV16, 18, 31, 33, 35, 45, 52, and 58), HPV PhAM-Seq demonstrated >80% overall agreement with qPCR using degenerate L1-targeting primers, with the highest sensitivity for HPV16, 31, 33, and 58. Sensitivity for HPV35, 45, and 52 improved to 85% or greater with type-specific primers targeting genes E6/E7. Parallel processing and sequencing enables a single technician to assay hundreds of samples per week at a reagent cost of around $10 USD per sample, with laboratory automation and sequencing on higher-output platforms enabling further scaling and cost reduction to a scale amenable to population-level surveillance. We include a detailed SOP; tools for primer design, sequencing library construction, and sample tracking; and all scripts needed for data analysis to ensure HPV PhAM-Seq can be readily implemented for scalable, cost-effective hrHPV genotyping or extended to other similar applications.