Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment

We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library prepara...

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Bibliographic Details
Published in:Journal of clinical microbiology 2017-03, Vol.55 (3), p.811-823
Main Authors: Li, Tengguo, Unger, Elizabeth R, Batra, Dhwani, Sheth, Mili, Steinau, Martin, Jasinski, Jean, Jones, Jennifer, Rajeevan, Mangalathu S
Format: Article
Language:English
Subjects:
BASIC BIOLOGICAL SCIENCES
broad-spectrum assay
Genome, Viral
Genomics
Genotype
Genotyping Techniques - methods
HPV typing
Humans
Microbiology
Papillomaviridae - classification
Papillomaviridae - genetics
Papillomaviridae - isolation & purification
Papillomavirus Infections - diagnosis
Papillomavirus Infections - virology
Sequence Analysis, DNA
target enrichment
Virology
whole-genome sequencing
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Summary:We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology.
ISSN:0095-1137
1098-660X
DOI:10.1128/JCM.02132-16