Accurate human papillomavirus genotyping by 454 pyrosequencing

Abstract

Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization

“…HPV typing by NGS is different in that it is not dependent on probes for detection that may cause cross-hybridizations. At least three previous HPV-genotyping assays using NGS have found NGS to be both sensitive and accurate: Arroyo et al and Militello et al both used the PGMY set of primers for the amplification of a 450 bp fragment [18,19], while Yi et al [43] used a series of unique primers for the amplification of a 150 bp fragment of the L1 gene, which is similar in length to the fragments produced by the MGP primers used here.…”

“…Confirmation of analysis results; sensitivity and specificity calculations. All single identifications made with just one of the two methods were confirmed with a third method using either real time PCR with HPV type-specific primers (HPV6, 16, 18, 31, 33, 35, 39, 42, 43, 45, 51, 52, 56, 58, 66, 68, 90) and SYBR ® Select Master Mix (Thermo Fischer Scientific) or by sequencing of a 450 bp PGMY amplicon [18,19] on a MiSeq (Illumina) platform (HPV30, 32, 40, 53, 54, 62, 67, 73, 74, 82, 87, 89, 91 and 114). Type-specific HPV primers were designed using Primer3 [33] for HPV types: 68 (forward: , reverse: , T A = 54.2°C) and 90 (forward: , reverse: , T A = 51.9°C).…”

“…The MGP primers are based the GP5+/6+ primers [15–17] and were further developed by the WHO HPV LabNet Global Reference Laboratory [13]. Similar studies reported at the time of planning this study, typically combined the HPV consensus primers MY09/11,PGMY or a modified PGMY, which amplify a 450 nt fragment, in combination with 454 sequencing technology [18–20]. A recent study utilized the Ion Torrent platform in combination with the HPV broad-spectrum general primers (BSGP5+/6+) on formalin-fixed, paraffin-embedded cancer specimens, showing high sensitivity and specificity [21].…”

Moderne Diagnostik sexuell übertragbarer Infektionen

“…Bei Vorliegen von mehr als einem HPV-Typ ist die direkte Sequenzierung der PCR-Produkte ("bulk sequencing") infolge Sequenzüberlagerungen oft nicht auswertbar. Neue Sequenziermethoden (NGS) ermöglichen auch bei Mischinfektionen die korrekte Identifizierung der zugrunde liegenden HPV-Typen [18]. Mit dem GS FLX-System (454 Life Sciences) waren durch AmpliconSequenzierung HPV-Typen zuverlässig nachweisbar, deren Anteil an der Gesamtpopulation nur 1 % betrug.…”

“…Mit dem GS FLX-System (454 Life Sciences) waren durch AmpliconSequenzierung HPV-Typen zuverlässig nachweisbar, deren Anteil an der Gesamtpopulation nur 1 % betrug. NGSVerfahren sind darüber hinaus potenziell in der Lage, neue Varianten und Subtypen zu identifizieren [18].…”