Figure 1.

Overview of HPV-seq and dual-strand hybrid capture. A, HPV-seq conducted on plasma cfDNA is designed to provide quantitative and qualitative information about ctDNA in patients with HPV-associated cancers. In addition to being highly sensitive and quantitative, HPV-seq can report on ctDNA fragment size and HPV genotype. Each full-length viral genome (episome or linearized genome) is expected to yield approximately 50 distinct cfDNA fragments. B, HPV-seq is conducted using hybrid-capture sequencing with single-stranded [sense (+) and/or antisense (−)] biotinylated baits tiled across the HPV genome: (i) single-strand viral genome hybrid capture, (ii) sequential dual-strand hybrid capture, and (iii) simultaneous dual-strand hybrid capture. C, Compared with single-strand hybrid capture (i), dual-strand hybrid capture using either a sequential (ii) or simultaneous (iii) approach recovers more HPV molecules. Results were normalized to the degree of HPV sequence enrichment with a single round of capture using single-stranded baits (left-most bar). Subjecting the unbound library to another round of hybrid capture using baits targeting the same strand (second bar from left) did not improve HPV sequence enrichment. The degree of HPV DNA enrichment in postcapture libraries was determined using HPV-16 E6 and E7 dPCR assays. N = 4 per condition. Error bars represent SD. Asterisk indicates statistical significance (P < 0.05) in comparison with single-strand capture conditions.