Scalable long-read Nanopore HPV16 Amplicon-based Whole-Genome Sequencing

Abstract Human papillomavirus 16 (HPV16) drives precursor cervical lesions that often progress to cervical cancer (CC). Variation within the HPV16 genome has been associated with CC risk. Here, we developed an affordable and portable amplicon-based long-read whole genome sequencing (WGS) approach using Oxford Nanopore Technologies (ONT) to investigate HPV16 genetic diversity among women in sub-Saharan African countries. Applied to a control CaSki cell line and clinical samples (n = 12), our method generated complete HPV16 genomes at high coverage (median read coverage: 5,899–15,279×). Benchmarking our HPV16 controls showed high accuracy for two variant calling pipelines (Clair3 and PEPPER-Margin DeepVariant). Phylogenetic analysis identified all four previously defined HPV16 lineages (A–D) and their high-risk sublineages. All lineages exhibited strong concordance across de novo assembly, reference-based phylogenetics, and unsupervised clustering. Our pipeline effectively captured the full extent of genomic variation, including putative lineage-informative SNPs. This method offers a robust amplicon-based WGS and analysis pipeline for HPV16, making it well-suited for integration into surveillance, diagnostics, and epidemiological efforts in low-resource areas. Competing Interest Statement The authors have declared no competing interest. Funding Statement This study was supported by funding from the National Institutes of Health (NIH) (D43TW011317, United States of America; S. Cu-Uvin and O. Omenge) and the National Cancer Institute (NCI) (5U54CA190151-02 and 5U54CA254518-03, United States of America; Rachel A. Katzenellenbogen, D. Brown, O. Omenge, and P. Loehrer) and Providence/Boston CFAR (P30AI042853, United States of America; S. Cu-Uvin). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Written informed consent was obtained from all participants. Ethical approvals were obtained from the Institutional Research and Ethics Committee (IREC) of Moi Teaching and Referral Hospital (MTRH) and Moi University, Eldoret, Kenya (reference number IREC/371/2022), as well as from the Institutional Review Board (IRB) of Brown University, Providence, Rhode Island, USA (IRB-ID: 2023003553). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors