scholarly journals Development of the TypeSeq Assay for Detection of 51 Human Papillomavirus Genotypes by Next-Generation Sequencing

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Sarah Wagner ◽  
David Roberson ◽  
Joseph Boland ◽  
Meredith Yeager ◽  
Michael Cullen ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Next Generation Sequencing ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Perfect Agreement ◽  
Hpv Genotyping ◽  
Wide Range ◽  
Positive Agreement ◽  
Generation Sequencing ◽  
Multiple Type

ABSTRACTWe 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.

scholarly journals Evaluation of TypeSeq, a Novel High-Throughput, Low-Cost, Next-Generation Sequencing-Based Assay for Detection of 51 Human Papillomavirus Genotypes

2019 ◽  
Vol 220 (10) ◽  
pp. 1609-1619 ◽  
Author(s):  
Sarah Wagner ◽  
David Roberson ◽  
Joseph Boland ◽  
Aimée R Kreimer ◽  
Meredith Yeager ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Next Generation Sequencing ◽  
High Throughput ◽  
Vaccine Efficacy ◽  
Linear Array ◽  
Human Papillomaviruses ◽  
Next Generation ◽  
Hpv Genotyping ◽  
Positive Agreement ◽  
Generation Sequencing

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.

Multicenter validation study for the certification of a CFTR gene scanning method using next generation sequencing technology

2018 ◽  
Vol 56 (7) ◽  
pp. 1046-1053 ◽  
Author(s):  
Anne Bergougnoux ◽  
Valeria D’Argenio ◽  
Stefanie Sollfrank ◽  
Fanny Verneau ◽  
Antonella Telese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Genetic Analysis ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Sequencing Data ◽  
Scanning Method ◽  
Next Generation Sequencing Technology ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom’s CFTR MASTR Dx kit to obtain CE-IVD certification. Methods: A total of 164 samples, previously analyzed with well-established “reference” methods for the molecular diagnosis of the CFTR gene, were selected and re-sequenced using the Illumina MiSeq benchtop NGS platform. Sequencing data were analyzed using two different bioinformatic pipelines. Annotated variants were then compared to the previously obtained reference data. Results and conclusions: The analytical sensitivity, specificity and accuracy rates of the Multiplicom CFTR MASTR assay exceeded 99%. Because different types of CFTR mutations can be detected in a single workflow, the CFTR MASTR assay simplifies the overall process and is consequently well suited for routine diagnostics.

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

2015 ◽  
Vol 88 (5) ◽  
pp. 888-894 ◽  
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

scholarly journals Next-Generation Sequencing Identification and Characterization of MicroRNAs in Dwarfed Citrus Trees Infected With Citrus Dwarfing Viroid in High-Density Plantings

2021 ◽  
Vol 12 ◽  
Author(s):  
Tyler Dang ◽  
Irene Lavagi-Craddock ◽  
Sohrab Bodaghi ◽  
Georgios Vidalakis
Keyword(s):  
Next Generation Sequencing ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
High Density ◽  
Poncirus Trifoliata ◽  
Next Generation ◽  
Developmental Processes ◽  
Wide Range ◽  
Generation Sequencing ◽  
Citrus Trees

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

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

2015 ◽  
Vol 12 (1) ◽  
Author(s):  
María Guadalupe Flores-Miramontes ◽  
Luis Alberto Torres-Reyes ◽  
Liliana Alvarado-Ruíz ◽  
Salvador Angel Romero-Martínez ◽  
Verenice Ramírez-Rodríguez ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Next Generation Sequencing ◽  
Linear Array ◽  
Next Generation ◽  
Western Mexico ◽  
Generation Sequencing

scholarly journals 355. A Novel Likelihood-Based Model to Estimate SARS-CoV-2 Viral Titer from Next-Generation Sequencing Data

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S281-S282
Author(s):  
Heather L Wells ◽  
Joseph Barrows ◽  
Mara Couto-Rodriguez ◽  
Xavier O Jirau Serrano ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Board Member ◽  
Clinical Specimen ◽  
Next Generation Sequencing Data ◽  
Viral Titer ◽  
Next Generation ◽  
Genome Coverage ◽  
Ct Value ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background The quantitative level of pathogens present in a host is a major driver of infectious disease (ID) state and outcome. However, the majority of ID diagnostics are qualitative. Next-generation sequencing (NGS) is an emerging ID diagnostics and research tool to provide insights, including tracking transmission, evolution, and identifying novel strains. Methods We built a novel likelihood-based computational method to leverage pathogen-specific genome-wide NGS data to detect SARS-CoV-2, profile genetic variants, and furthermore quantify levels of these pathogens. We used de-identified clinical specimens tested for SARS-CoV-2 using RT-PCR, SARS-CoV-2 NGS Assay (hybrid capture, Twist Bioscience), or ARTIC (amplicon-based) platform, and COVID-DX software. A training (n=87) and validation (n=22) set was selected to establish the strength of our quantification model. We fit non-uniform probabilistic error profiles to a deterministic sigmoidal equation that more realistically represents observed data and used likelihood maximized over several different read depths to improve accuracy over a wide range of values of viral load. Given the proportion of the genome covered at varying depths for a single sample as input data, our model estimated the Ct of that sample as the value that produces the maximum likelihood of generating the observed genome coverage data. Results The model fit on 87 SARS-CoV-2 NGS Assay training samples produced a good fit to the 22 validation samples, with a coefficient of correlation (r2) of ~0.8. The accuracy of the model was high (mean absolute % error of ~10%, meaning our model is able to predict the Ct value of each sample within a margin of ±10% on average). Because of the nature of the commonly used ARTIC protocol, we found that all quantitative signals in this data were lost during PCR amplification and the model is not applicable for quantification of samples captured this way. The ability to model quantification is a major advantage of the SARS-CoV-2 NGS assay protocol. The likelihood-based model to estimate SARS-CoV-2 viral titer Left Observed genome coverage (y-axis) plotted against Ct value (x-axis). The best-fitting logistic curve is demonstrated with a red line with shaded areas above and below representing the fitted error profile. RIGHT: Model-estimated Ct values (y-axis) compared to laboratory Ct values (x-axis) with grey bars representing estimated confidence intervals. The 1:1 diagonal is shown as a dotted line. Conclusion To our knowledge, this is the first model to incorporate sequence data mapped across the genome of a pathogen to quantify the level of that pathogen in a clinical specimen. This has implications in ID diagnostics, research, and metagenomics. Disclosures Heather L. Wells, MPH, Biotia, Inc. (Consultant) Joseph Barrows, MS, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Marilyne Debieu, PhD, Biotia (Employee) Karen Wessel, PhD, Labor Zotz/Klimas (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)

scholarly journals Development and Validation of a Next-Generation Sequencing Panel for Syndromic and Nonsyndromic Hearing Loss

2020 ◽  
Vol 5 (3) ◽  
pp. 467-479 ◽  
Author(s):  
Malinda Butz ◽  
Amber McDonald ◽  
Patrick A Lundquist ◽  
Melanie Meyer ◽  
Sean Harrington ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Copy Number Variants ◽  
Analytical Sensitivity ◽  
Nonsyndromic Hearing Loss ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Small Indels ◽  
Syndromic Hearing Loss ◽  
Generation Sequencing

Abstract Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Mutation enrichment next-generation sequencing for quantitative detection of KRAS mutations in urine cell-free DNA from patients with advanced colorectal and other cancers.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 602-602
Author(s):  
Filip Janku ◽  
Afsaneh Barzi ◽  
Andrea Sartore-Bianchi ◽  
Takeo Fujii ◽  
Andrea Cassingena ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Treatment Failure ◽  
Tumor Tissue ◽  
Quantitative Detection ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Invasive Approach ◽  
Time To Treatment ◽  
Time To Treatment Failure ◽  
Generation Sequencing

602 Background: Molecular testing of cell-free (cf) DNA from urine is a completely non-invasive approach for detection of actionable mutations in cancer. Methods: A quantitative mutation enrichment next-generation sequencing (NGS) urine cell-free (cf) DNA KRASG12/G13mutation test was developed and results compared to clinical testing of archival tumor tissue and research testing of plasma cfDNA from patients with advanced colorectal (n=56, 79%) and other advanced cancers (n=15, 21%). Results: The analytical sensitivity of the KRASG12/G13 cfDNA test was 0.002%-0.006% mutant copies in wild-type background. In 71 patients, the agreement between urine cfDNA and tumor was 73% (sensitivity 63%; specificity 96%); the agreement increased to 89% for patients with recommended 90-110mL of urine. In 33 patients with available plasma samples, the agreement with tumor was 94% (sensitivity 92%; specificity 100%). In patients treated with systemic therapy there was lower number of KRASG12/G13 copies in urine and plasma cfDNA on therapy compared to baseline and progression ( P<0.003). Decrease in urine and plasma cfDNA KRASG12/G13 copies on therapy compared to no change/increase was associated with longer median time to treatment failure ( P<0.05). Conclusions: Mutation enrichment NGS detection of KRASG12/G13 mutations in urine cfDNA has good concordance with archival tumor tissue. Changes in urine cfDNA correspond with time to treatment failure.

scholarly journals Zombies in TCGA

2015 ◽  
Vol 89 (8) ◽  
pp. 4044-4046 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document