HPVPool-Seq: a genotype-guided pooling strategy for cost-effective next-generation sequencing detection of HPV...

Integration of high-risk human papillomavirus (hrHPV) DNA is a critical event in carcinogenesis and a promising biomarker for risk stratification. However, the high cost of next-generation sequencing (NGS) limits its widespread clinical adoption. We developed HPVPool-Seq, an innovative pooling strategy that leverages the inherent diversity of HPV genotypes as natural barcodes, enabling cost-effective, scalable integration detection. Samples were pooled based on qPCR-derived HPV genotypes and viral loads prior to targeted NGS and bioinformatic decoding. A web-based automation tool was implemented to streamline pooling and decoding workflows. In a proof-of-concept study of 175 clinical specimens, HPVPool-Seq achieved 77.1% exact genotype concordance and 97.1% combined sensitivity compared with qPCR. Cost simulations demonstrated a 60% reduction in per-sample sequencing expenses. Self-correcting capability through targeted retesting further enhanced reliability. HPVPool-Seq offers a novel, traceable, and economically viable solution for high-throughput HPV integration profiling, balancing cost, scalability, and clinical precision. This strategy sets a new framework for molecular screening in HPV-associated cancers.

Accurate detection of high-risk HPV integration is critical for identifying individuals at true risk of progression to malignancy. However, the high cost of next-generation sequencing (NGS) has limited its widespread clinical application. Here, we propose HPVPool-Seq, a novel pooling-based sequencing strategy that uses HPV genotypes as intrinsic barcodes to guide sample pooling without compromising detection sensitivity. This method dramatically reduces sequencing costs while maintaining genotype-level traceability and offers a built-in mechanism for selective retesting of discordant cases. By addressing both technical and economic barriers, our approach provides a scalable, clinically applicable solution for HPV integration profiling in large cohorts, with important implications for precision screening, triage, and epidemiological surveillance.