HPV Genome Enrichment and Sequencing

The bait design, library preparation, HPV enrichment, and deep sequencing followed methods in original publication [17 (link)]. Briefly, the custom RNA bait (Agilent Technologies Inc., Santa Clara, CA) included 23, 941 probes (each 120 bases in length) complementary to one strand of the full-length genomes of 191 HPV genotypes/subtypes and 12 probes complementary to human haemoglobin subunit beta (HBB). Individual libraries with indexing for sample identification were prepared for each sample. Following indexing, equal amounts of 16 libraries were pooled for enrichment by overnight hybridization to the RNA custom bait and the captured fragments were amplified using 14 PCR cycles. The quality and quantity of HPV-enriched, pooled libraries were assessed by Bioanalyzer 2100 (Agilent Technologies, Inc.) and quantitative PCR using KAPA DNA library quantification kit (KAPA Biosystems, Wilmington, MA) on a LightCycler 480 (Roche Diagnostics, Indianapolis, IN). Each pooled library was paired-end sequenced on an Illumina HiSeq 2500 using TruSeq Rapid SBS Kit HS (200 cycle) (Illumina, San Diego, CA).