Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Abstract

Background: Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.

Methods: Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.

Results: Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively.

Conclusions: Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Figure 1

Schematic representation of the pipeline designed for the analysis of the Illumina reads and detection of HPV genotypes present.

Figure 2

Pie chart summary of mapping of Illumina reads from specimen HH015 to human and HPV reference sequences using CLC Genomics Workbench 4.6.1 (Global alignment, mismatch cost 2, limit 5). Numbers on the pie chart on the right indicate HPV genotypes. The Genbank accession numbers for the sequences used as references in the mappings are given in Table 2.

Figure 3

Representative agarose gel analyses following PCR amplification of a type-specific region within the E6/E7 genes of HPV types 30 (A), 74 (B), 86 (C) and 90 (D) from clinical specimens. A negative control with water (Negative) and a positive control using DNA from HH015 (Positive) were included.