Human papillomavirus genotyping by Linear Array and Next-Generation Sequencing in cervical samples from Western Mexico

Abstract

BackgroundThe Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS.MethodsTwo hundred thirty three cervical samples from women Without cervical lesions (WCL, n = 48), with Cervical intraepithelial neoplasia grade 1 (CIN I, n = 98), or with Cervical cancer (CC, n = 87) were recruited, DNA was extracted, and HPV positivity was determined by PCR amplification using PGMY09/11 primers. All HPV- positive samples were genotyped individually by LA. Additionally, pools of amplicons from the PGMY-PCR products were sequenced using 454 NGS technology. Results obtained by NGS were compared with those of LA for each group of samples.ResultsWe identified 35 HPV genotypes, among which 30 were identified by both technologies; in addition, the HPV genotypes 32, 44, 74, 102 and 114 were detected by NGS. These latter genotypes, to our knowledge, have not been previously reported in Mexican population. Furthermore, we found that LA did not detect, in some diagnosis groups, certain HPV genotypes included in the test, such as 6, 11, 16, 26, 35, 51, 58, 68, 73, and 89, which indicates possible variations at the species level.ConclusionsThere are HPV genotypes in Mexican population that cannot be detected by LA, which is, at present, the most complete commercial genotyping test. More studies are necessary to determine the impact of HPV-44, 74, 102 and 114 on the risk of developing CC. A greater number of samples must be analyzed by NGS for the most accurate determination of Mexican HPV variants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0391-4) contains supplementary material, which is available to authorized users.

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

“…On the other hand, even with only 15 epidemiologic samples, eWGS detected 7 HPV types (HPV30, -43, -68a, -87, -90, -91, and -114) not targeted by LA. The ability of this eWGS method to detect HPV types not targeted by current assays indicates it could be useful in evaluation of HPV-negative cervical lesions ( 29 ) and in epidemiologic studies of HPV in geographic regions that may have uncommon types in circulation ( 30 ).…”

Detection of Alpha, Beta, Gamma, and Unclassified Human Papillomaviruses in Cervical Cancer Samples From Mexican Women

“…First, the DNA of each sample was amplified by two independent conventional PCRs with PGMY09/11 primers on one side and FAP59/64 primers on the other side (Forslund et al, 1999;Gravitt et al, 2000). Then, a second PCR reaction was performed using PGMY09/11 on one side and FAP6085/64 primer pairs on the other side, coupled to a universal tail sequence as previously described (Flores-Miramontes et al, 2015). PCR products were quantified using the Qubit dsDNA HS (Cat.…”

“…For research purposes, sensitive, and specific technologies that cover a broad range of HPV genotypes have been developed (Nilyanimit et al, 2018). More precisely, the use of PGMY and FAP primers couple with Next-Generation Sequencing (NGS) strategies detect α-, β-, and γ -PVs simultaneously in different tissues have helped to broaden the knowledge in this field (Flores-Miramontes et al, 2015;Sias et al, 2019). The aim of this work was to study the HPV genotypes present in cervical samples from women diagnosed specifically with CC using NGS with primer pairs (both PGMY11/09 and FAP) that allow amplification not only of α-PVs, but also of multiple β-and γ -PVs.…”

Effects of 60�kDa prolactin and estradiol on metabolism and cell survival in cervical cancer: Co‑expression of their hormonal receptors during cancer progression

“…open reading frames were amplified from genomic DNA obtained from the biopsies of patients infected with HPV 16 or HPV 18 in a previous study (33). Polymerase chain reaction (PCR) was performed using the Expand High Fidelity PCR system kit (cat.…”