1. Complete HPV genomes from cervical samples using next generation sequencing in Luxembourg
A. Latsuzbaia¹, J. Tapp¹, C. Ragimbeau¹, M. Fischer, S. Weyers², M. Arbyn³, J. Mossong¹
1. Laboratoire National de Santé, Luxembourg; 2. Scientific Institute of Public Health, Belgium.
3. Ghent University Hospital, Belgium
Background & Objectives Next generation sequencing using rolling circle amplification (NGS) is promising approach for detecting a wide spectrum of human papillomavirus (HPV) in clinical samples.
Previously NGS was conducted in HIV-HPV co-infected women. We performed a pilot study for detecting and assembling complete HPV genomes in cervical samples from a cohort of young women attending cervical screening with access to HPV vaccination in Luxembourg, a low HIV-prevalence setting. Methods
DNA extracts of 81 cervical swabs from women (mean age 23 y.) positive for HPV by Anyplex II HPV 28 (Seegene) were enriched by rolling circle amplification and sequenced on Illumina Miseq. Metagenomic classification was performed using the webtool Taxonomer.com and reads were mapped to 182 HPV types of the PAVE reference collection using BBMap (Geneious) and de novo assembled using Velvet. Complete assembled genomes were aligned with genomes published in Genbank using MEGA7 (Maximum likelihood, Kimura-2 parameter).
Results
Overall, on average 1% of reads were classified as papillomavirus, 7% as bacteria, 85% as human.
Anyplex detected a total of 206 genotypes in 81 samples. NGS detected 186 genotypes in 71 samples spanning 36 of the 51 known mucosal HPV types. HPV types 42, 53, 58, 51, and 66 were most frequently detected by Anyplex (Fig. 1). Genotypes only detected by NGS included types 90 (N=9), 67 (N=6), 30 (N=5) and 62 (N=5).
106 HPV genotypes detected by NGS were concordant with Anyplex II HPV28 (Fig. 1).
Detection of concordant types was strongly associated with semi-quantitative viral load of Anyplex: 86%, 45% and 4% of HPV types with high, intermediate and low viral loads, respectively, were detected by NGS.
67 complete HPV genomes were recovered with a mean coverage of 270x (range: 16x-2100x). Two novel lineages of HPV90 and HPV66 (Fig. 2) and two novel sublineages of HPV67 and HPV73 (not shown) were found.
Fig2. Phylogenetic trees showing study samples and reference strains of variant lineages and sublineages of HPV66 and HPV90. Blue circles indicate study samples representing putatively novel lineages. Yellow circles indicate study samples belonging to pre-existing lineages
Conclusion
NGS-RCA is a powerful method for HPV detection and recovering complete genomes from cervical samples. Concordance between Anyplex and NGS is high on samples with high viral load. NGS frequently detects genotypes not covered by Anyplex II HPV28.
After eight years of the vaccination programme in Luxembourg, vaccine-related types 6, 11, 16 & 18 are less frequently detected than other types suggesting replacement.
Fig.1 HPV type-specific detections by Anyplex and NGS
This study received CORE programme funding by
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