Development of the TypeSeq Assay for Detection of 51 Human Papillomavirus Genotypes by Next-Generation Sequencing
Autor: | Mark Schiffman, Sarah Wagner, Rosemary E. Zuna, Michael Cullen, Nicolas Wentzensen, David Roberson, Joan L. Walker, Joseph Boland, Meredith Yeager, S. Terence Dunn, Lisa Mirabello |
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Jazyk: | angličtina |
Rok vydání: | 2019 |
Předmět: |
0301 basic medicine
Microbiology (medical) Hpv genotyping Genotype Genotyping Techniques Uterine Cervical Neoplasms Cervix Uteri Biology Sensitivity and Specificity DNA sequencing 03 medical and health sciences Roche Linear Array 0302 clinical medicine Virology Humans Human papillomavirus Genotyping Papillomaviridae Human papillomavirus 16 Human papillomavirus 18 Papillomavirus Infections High-Throughput Nucleotide Sequencing Sequence Analysis DNA Amplicon Viral Load Molecular biology 030104 developmental biology 030220 oncology & carcinogenesis DNA Viral Female Viral load Nucleic Acid Amplification Techniques |
Popis: | We have developed a new human papillomavirus (HPV) genotyping assay for detection of 51 HPV genotypes by next-generation sequencing (NGS). The TypeSeq assay consists of 3 PCR steps that equalize viral load and each type’s amplicon copies prior to genotyping by NGS, thereby maximizing multiple-type sensitivity with minimal sequencing reads. The analytical sensitivity of the TypeSeq assay is 10 copies per reaction for 49 of the 51 types, including 13 high-risk (HR) types. We tested 863 clinical cervical specimens previously evaluated with the Roche Linear Array HPV genotyping test (LA). TypeSeq achieved 94.4% positive agreement with LA for detection of any HR type. Positive agreement was 91.4% and 85.5% for HPV16 and HPV18, respectively. Low-risk (LR) types ranged from 40.0% positive agreement (HPV83) to 90.9% (HPV69). Our unique approach to HPV amplification achieved a multiple-type sensitivity comparable to that of LA, with 83.9% and 84.2% of specimens positive for multiple HPV types by TypeSeq or LA, respectively. A total of 48.2% of specimens showed perfect agreement for all 37 types common to both assays. The simplicity of our open-source TypeSeq assay allows for high-throughput yet scalable processing, with a single technician able to process up to 768 specimens within 3 days. By leveraging NGS sample multiplexing capabilities, the per-sample labor requirements are greatly reduced compared to those of traditional genotyping methods. These features and the broad spectrum of detectable types make TypeSeq highly suitable for a wide range of applications. |
Databáze: | OpenAIRE |
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