Genotyping of high-risk anal human papillomavirus (HPV): ion torrent-next generation sequencing vs. linear array

AbstractBackgroundHuman papillomaviruses (HPV) cause over 500 000 cervical cancers each year, most of which occur in low-resource settings. Human papillomavirus genotyping is important to study natural history and vaccine efficacy. We evaluated TypeSeq, a novel, next-generation, sequencing-based assay that detects 51 HPV genotypes, in 2 large international epidemiologic studies.MethodsTypeSeq was evaluated in 2804 cervical specimens from the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED) and in 2357 specimens from the Costa Rica Vaccine Trial (CVT). Positive agreement and risks of precancer for individual genotypes were calculated for TypeSeq in comparison to Linear Array (SUCCEED). In CVT, positive agreement and vaccine efficacy were calculated for TypeSeq and SPF10-LiPA.ResultsWe observed high overall and positive agreement for most genotypes between TypeSeq and Linear Array in SUCCEED and SPF10-LiPA in CVT. There was no significant difference in risk of precancer between TypeSeq and Linear Array in SUCCEED or in estimates of vaccine efficacy between TypeSeq and SPF10-LiPA in CVT.ConclusionsThe agreement of TypeSeq with Linear Array and SPF10-LiPA, 2 well established standards for HPV genotyping, demonstrates its high accuracy. TypeSeq provides high-throughput, affordable HPV genotyping for world-wide studies of cervical precancer risk and of HPV vaccine efficacy.

Download Full-text

Characterization and Diversity of 243 Complete Human Papillomavirus Genomes in Cervical Swabs Using Next Generation Sequencing

Viruses ◽

10.3390/v12121437 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1437

Author(s):

Ardashel Latsuzbaia ◽

Anke Wienecke-Baldacchino ◽

Jessica Tapp ◽

Marc Arbyn ◽

Irma Karabegović ◽

...

Keyword(s):

Human Papillomavirus ◽

Next Generation Sequencing ◽

High Risk ◽

Pairwise Distance ◽

Next Generation ◽

Hpv Genotypes ◽

Complete Genomes ◽

Hpv Types ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

In recent years, next generation sequencing (NGS) technology has been widely used for the discovery of novel human papillomavirus (HPV) genotypes, variant characterization and genotyping. Here, we compared the analytical performance of NGS with a commercial PCR-based assay (Anyplex II HPV28) in cervical samples of 744 women. Overall, HPV positivity was 50.2% by the Anyplex and 45.5% by the NGS. With the NGS, we detected 25 genotypes covered by Anyplex and 41 additional genotypes. Agreement between the two methods for HPV positivity was 80.8% (kappa = 0.616) and 84.8% (kappa = 0.652) for 28 HPV genotypes and 14 high-risk genotypes, respectively. We recovered and characterized 243 complete HPV genomes from 153 samples spanning 40 different genotypes. According to phylogenetic analysis and pairwise distance, we identified novel lineages and sublineages of four high-risk and 16 low-risk genotypes. In total, 17 novel lineages and 14 novel sublineages were proposed, including novel lineages of HPV45, HPV52, HPV66 and a novel sublineage of HPV59. Our study provides important genomic insights on HPV types and lineages, where few complete genomes were publicly available.

Download Full-text

Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

Journal of International Medical Research ◽

10.1177/0300060520967778 ◽

2020 ◽

Vol 48 (12) ◽

pp. 030006052096777

Author(s):

Peisong Chen ◽

Xuegao Yu ◽

Hao Huang ◽

Wentao Zeng ◽

Xiaohong He ◽

...

Keyword(s):

Next Generation Sequencing ◽

Ion Torrent ◽

Next Generation ◽

Large Deletions ◽

Different Types ◽

Variant Detection ◽

Polymerase Chain ◽

Next Generation Sequencing Ngs ◽

Accuracy And Repeatability ◽

Generation Sequencing

Introduction To evaluate a next-generation sequencing (NGS) workflow in the screening and diagnosis of thalassemia. Methods In this prospective study, blood samples were obtained from people undergoing genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain reaction (PCR)-amplified and sequenced using the Ion Torrent system and results compared with traditional genetic analyses. Results Of the 359 subjects, 148 (41%) were confirmed to have thalassemia. Variant detection identified 35 different types including the most common. Identification of the mutational sites by NGS were consistent with those identified by Sanger sequencing and Gap-PCR. The sensitivity and specificities of the Ion Torrent NGS were 100%. In a separate test of 16 samples, results were consistent when repeated ten times. Conclusion Our NGS workflow based on the Ion Torrent sequencer was successful in the detection of large deletions and non-deletional defects in thalassemia with high accuracy and repeatability.

Download Full-text

Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing

Journal of Medical Virology ◽

10.1002/jmv.24413 ◽

2015 ◽

Vol 88 (5) ◽

pp. 888-894 ◽

Cited By ~ 14

Author(s):

Allex Jardim da Fonseca ◽

Renata Silva Galvão ◽

Angelica Espinosa Miranda ◽

Luiz Carlos de Lima Ferreira ◽

Zigui Chen

Keyword(s):

Human Papillomavirus ◽

Next Generation Sequencing ◽

Multiplex Pcr ◽

Nested Pcr ◽

Dna Detection ◽

Detection Methods ◽

Next Generation ◽

Papillomavirus Dna ◽

Generation Sequencing

Download Full-text

HPV genotypes detected by linear array and next-generation sequencing in anal samples from HIV positive men who have sex with men in Mexico

Archives of Virology ◽

10.1007/s00705-017-3697-2 ◽

2018 ◽

Vol 163 (4) ◽

pp. 925-935 ◽

Cited By ~ 3

Author(s):

Luz A. González-Hernández ◽

María G. Flores-Miramontes ◽

Adriana Aguilar-Lemarroy ◽

Krissya S. Quintanilla-Peña ◽

Fabiola L. Martin-Amaya-Barajas ◽

...

Keyword(s):

Next Generation Sequencing ◽

Linear Array ◽

Hiv Positive ◽

Next Generation ◽

Hpv Genotypes ◽

Sex With Men ◽

Generation Sequencing ◽

Hiv Positive Men

Download Full-text

Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer

PeerJ ◽

10.7717/peerj.6661 ◽

2019 ◽

Vol 7 ◽

pp. e6661 ◽

Cited By ~ 7

Author(s):

Arianna Nicolussi ◽

Francesca Belardinilli ◽

Yasaman Mahdavian ◽

Valeria Colicchia ◽

Sonia D’Inzeo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sanger Sequencing ◽

Germline Mutations ◽

Ion Torrent ◽

Next Generation ◽

Training Set ◽

Brca1 And Brca2 ◽

Ion Torrent Pgm ◽

Validation Set ◽

Generation Sequencing

Background Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. Methods We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). Results By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. Discussion Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

Download Full-text

Identification of Human Papillomavirus Infection in Cancer Tissue by Targeted Next-generation Sequencing

Applied Immunohistochemistry & Molecular Morphology ◽

10.1097/pai.0000000000000215 ◽

2016 ◽

Vol 24 (7) ◽

pp. 490-495 ◽

Cited By ~ 6

Author(s):

Nathan D. Montgomery ◽

Joel S. Parker ◽

David A. Eberhard ◽

Nirali M. Patel ◽

Karen E. Weck ◽

...

Keyword(s):

Human Papillomavirus ◽

Next Generation Sequencing ◽

Cancer Tissue ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Human Papillomavirus Infection ◽

Papillomavirus Infection ◽

Generation Sequencing

Download Full-text

A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2016-0194-cp ◽

2016 ◽

Vol 140 (10) ◽

pp. 1085-1091 ◽

Cited By ~ 21

Author(s):

Eric J. Duncavage ◽

Haley J. Abel ◽

Jason D. Merker ◽

John B. Bodner ◽

Qin Zhao ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

In Silico ◽

Absolute Difference ◽

Ion Torrent ◽

Next Generation ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Clinical Laboratories ◽

Generation Sequencing

Context.—Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. Objective.—To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

Download Full-text

Zombies in TCGA

Journal of Virology ◽

10.1128/jvi.00170-15 ◽

2015 ◽

Vol 89 (8) ◽

pp. 4044-4046 ◽

Cited By ~ 1

Author(s):

Daniel DiMaio

Keyword(s):

Human Papillomavirus ◽

Next Generation Sequencing ◽

Hela Cells ◽

Somatic Mutations ◽

Hpv Infection ◽

Next Generation ◽

Human Cancers ◽

Human Papillomavirus 18 ◽

Tumor Types ◽

Generation Sequencing

Next-generation sequencing results obtained to detect somatic mutations in human cancers can also be searched for viruses that contribute to cancer. Recently, human papillomavirus 18 RNA was detected in tumor types not typically associated with HPV infection. Analyses reported in this issue ofJournal of Virologydemonstrate that the apparent presence of HPV18 RNA in these atypical tumors is due in at least some cases to contamination of samples with HeLa cells, which harbor HPV18.

Download Full-text