Sequence alignment

Raw paired-end reads were trimmed for adapters, HPV primers, quality (-q 20) and finally for minimum length (-m 50) using cutadapt (v1.10)⁵⁹. Trimmed reads were mapped to human (GRCh38/hg38) and HPV16, 18, 31, 33 and 45 reference genomes obtained from the PaVE database⁵⁵ using HISAT2 (v2.1.0)⁶⁰. Mapping statistics and sequencing coverage were calculated using the Pysam package⁶¹ with an in-house Python (v3.5.4) script. Downstream analysis was performed using an in-house R (v3.4.4) script. Results from both reactions of the same sample were combined and method performance was then evaluated based on the percentage of obtained reads mapped to the HPV reference genome, mean sequencing coverage and percentage of HPV reference genome coverage for each sample. Further analysis was performed when a sample had >20000 reads mapped to the target HPV reference genome. The target HPV genomes correspond to the HPV types for which the samples were reported positive by HPV genotyping.