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Xinjing Wang, Wadih M Zein, Leera D'Souza, Chimere Roberson, Keith Wetherby, Hong He, Angela Villarta, Amy Turriff, Kory R Johnson, Yang C Fann
BACKGROUND: Inherited Retinal dystrophy (IRD) is a broad group of inherited retinal disorders with heterogeneous genotypes and phenotypes. Next generation sequencing (NGS) methods have been broadly applied for analyzing patients with IRD. Here we report a novel approach to enrich the target gene panel by microdroplet PCR. METHODS: This assay involved a primer library which targeted 3071 amplicons from 2078 exons comprised of 184 genes involved in retinal function and/or retinal development...
August 24, 2017: BMC Ophthalmology
P Chandrani, K Prabhash, R Prasad, V Sethunath, M Ranjan, P Iyer, J Aich, H Dhamne, D N Iyer, P Upadhyay, B Mohanty, P Chandna, R Kumar, A Joshi, V Noronha, V Patil, A Ramaswamy, A Karpe, R Thorat, P Chaudhari, A Ingle, A Choughule, A Dutt
Background: Lung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin. Materials and methods: Forty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry...
March 1, 2017: Annals of Oncology: Official Journal of the European Society for Medical Oncology
Gerwyn M Jones, Eloise Busby, Jeremy A Garson, Paul R Grant, Eleni Nastouli, Alison S Devonshire, Alexandra S Whale
Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR...
December 2016: Biomolecular Detection and Quantification
Anniina Färkkilä, Melissa K McConechy, Winnie Yang, Aline Talhouk, Ying Ng, Amy Lum, Ryan D Morin, Kevin Bushell, Annika Riska, Jessica N McAlpine, C Blake Gilks, Leila Unkila-Kallio, Mikko Anttonen, David G Huntsman
Adult granulosa cell tumors (AGCTs) of the ovary are molecularly characterized by the pathognomonic FOXL2 402C>G (C134W) mutation. To improve diagnostics and follow-up, we developed a specific digital droplet PCR (ddPCR) assay to detect the FOXL2 mutation in the circulating tumor DNA (ctDNA) of AGCT patients. Optimization of the ddPCR assay was performed using a TaqMan primer/probe with the RainDance RainDrop digital PCR system. The ddPCR assay was performed on circulating cell-free DNA extracted from 120 serial plasma samples collected prospectively from 35 AGCT patients...
January 2017: Journal of Molecular Diagnostics: JMD
R T Hayden, Z Gu, S S Sam, Y Sun, L Tang, S Pounds, A M Caliendo
A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV). Both commercial quantitative viral standards and 16 patient samples (n = 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results. Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results...
October 2016: Journal of Clinical Microbiology
Julien Philippe, Mehdi Derhourhi, Emmanuelle Durand, Emmanuel Vaillant, Aurélie Dechaume, Iandry Rabearivelo, Véronique Dhennin, Martine Vaxillaire, Franck De Graeve, Olivier Sand, Philippe Froguel, Amélie Bonnefond
Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex)...
2015: PloS One
Funda Orkunoglu-Suer, Arthur F Harralson, David Frankfurter, Paul Gindoff, Travis J O'Brien
BACKGROUND: One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility...
2015: BMC Genomics
Alicia A Tone, Melissa K McConechy, Winnie Yang, Jiarui Ding, Stephen Yip, Esther Kong, Kwong-Kwok Wong, David M Gershenson, Helen Mackay, Sohrab Shah, Blake Gilks, Anna V Tinker, Blaise Clarke, Jessica N McAlpine, David Huntsman
BACKGROUND: Ovarian low-grade serous carcinoma (LGSC) has fewer mutations than ovarian high-grade serous carcinoma (HGSC) and a less aggressive clinical course. However, an overwhelming majority of LGSC patients do not respond to conventional chemotherapy resulting in a poor long-term prognosis comparable to women diagnosed with HGSC. KRAS and BRAF mutations are common in LGSC, leading to clinical trials targeting the MAPK pathway. We assessed the stability of targetable somatic mutations over space and/or time in LGSC, with a view to inform stratified treatment strategies and clinical trial design...
December 18, 2014: BMC Cancer
Donavan T Cheng, Janice Cheng, Talia N Mitchell, Aijazuddin Syed, Ahmet Zehir, Nana Yaa T Mensah, Alifya Oultache, Khedoudja Nafa, Ross L Levine, Maria E Arcila, Michael F Berger, Cyrus V Hedvat
Amplicon-based methods for targeted resequencing of cancer genes have gained traction in the clinic as a strategy for molecular diagnostic testing. An 847-amplicon panel was designed with the RainDance DeepSeq system, covering most exons of 28 genes relevant to acute myeloid leukemia and myeloproliferative neoplasms. We developed a paired-sample analysis pipeline for variant calling and sought to assess its sensitivity and specificity relative to a set of samples with previously identified mutations. Thirty samples with known mutations in JAK2, NPM1, DNMT3A, MPL, IDH1, IDH2, CEBPA, and FLT3, were profiled and sequenced to high depth...
September 2014: Journal of Molecular Diagnostics: JMD
Hongsheng Gui, Jessie Yunjuan Bao, Clara Sze-Man Tang, Man-Ting So, Diem-Ngoc Ngo, Anh-Quynh Tran, Duc-Hau Bui, Duy-Hien Pham, Thanh-Liem Nguyen, Amy Tong, Si Lok, Pak-Chung Sham, Paul Kwong-Hang Tam, Stacey S Cherny, Maria-Mercè Garcia-Barcelo
To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform...
September 2014: Annals of Human Genetics
Sadia Saeed, Amélie Bonnefond, Jaida Manzoor, Julien Philippe, Emmanuelle Durand, Mohsin Arshad, Olivier Sand, Taeed A Butt, Mario Falchi, Muhammad Arslan, Philippe Froguel
OBJECTIVE: Mutations in leptin receptor gene (LEPR) result in early onset extreme adiposity. However, their prevalence in different populations is not known. Indeed, LEPR screening by gold standard Sanger sequencing has been limited by its large size and the cost. One-step PCR-based targeted enrichment could be an option for rapid and cost effective molecular diagnosis of monogenic forms of obesity. METHODS: The study is based on 39 unrelated severely obese Pakistani children, previously shown to be negative for leptin (LEP) and melanocortin 4 receptor (MC4R) gene mutations, from an initial cohort of 62 probands...
April 2014: Obesity
Amélie Bonnefond, Julien Philippe, Emmanuelle Durand, Jean Muller, Sadia Saeed, Muhammad Arslan, Rosa Martínez, Franck De Graeve, Véronique Dhennin, Iandry Rabearivelo, Michel Polak, Hélène Cavé, Luis Castaño, Martine Vaxillaire, Jean-Louis Mandel, Olivier Sand, Philippe Froguel
OBJECTIVE: Accurate etiological diagnosis of monogenic forms of diabetes and obesity is useful as it can lead to marked improvements in patient care and genetic counseling. Currently, molecular diagnosis based on Sanger sequencing is restricted to only a few genes, as this technology is expensive, time-consuming, and labor-intensive. High-throughput next-generation sequencing (NGS) provides an opportunity to develop innovative cost-efficient methods for sensitive diabetes and obesity multigene screening...
February 2014: Diabetes Care
Shale Dames, Lan-Szu Chou, Ye Xiao, Tyler Wayman, Jennifer Stocks, Marc Singleton, Karen Eilbeck, Rong Mao
Sanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes...
July 2013: Journal of Molecular Diagnostics: JMD
C Alexander Valencia, Arunkanth Ankala, Devin Rhodenizer, Shruti Bhide, Martin Robert Littlejohn, Lisa Mari Keong, Anne Rutkowski, Susan Sparks, Carsten Bonnemann, Madhuri Hegde
The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications...
2013: PloS One
Whitney L Wooderchak-Donahue, Brendan O'Fallon, Larissa V Furtado, Jacob D Durtschi, Parker Plant, Perry G Ridge, Alan F Rope, Angela T Yetman, Pinar Bayrak-Toydemir
BACKGROUND: Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques. METHODS: The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy...
2012: BMC Medical Genomics
Melanie A Jones, Shruti Bhide, Ephrem Chin, Bobby G Ng, Devin Rhodenizer, Victor W Zhang, Jessica J Sun, Alice Tanner, Hudson H Freeze, Madhuri R Hegde
PURPOSE: Congenital disorders of glycosylation are a heterogeneous group of disorders caused by deficient glycosylation, primarily affecting the N-linked pathway. It is estimated that more than 40% of congenital disorders of glycosylation patients lack a confirmatory molecular diagnosis. The purpose of this study was to improve molecular diagnosis for congenital disorders of glycosylation by developing and validating a next generation sequencing panel for comprehensive mutation detection in 24 genes known to cause congenital disorders of glycosylation...
November 2011: Genetics in Medicine: Official Journal of the American College of Medical Genetics
Dale J Hedges, Toumy Guettouche, Shan Yang, Guney Bademci, Ashley Diaz, Ashley Andersen, William F Hulme, Sara Linker, Arpit Mehta, Yvonne J K Edwards, Gary W Beecham, Eden R Martin, Margaret A Pericak-Vance, Stephan Zuchner, Jeffery M Vance, John R Gilbert
Despite the ever-increasing throughput and steadily decreasing cost of next generation sequencing (NGS), whole genome sequencing of humans is still not a viable option for the majority of genetics laboratories. This is particularly true in the case of complex disease studies, where large sample sets are often required to achieve adequate statistical power. To fully leverage the potential of NGS technology on large sample sets, several methods have been developed to selectively enrich for regions of interest...
2011: PloS One
Kajari Mondal, Amol Carl Shetty, Viren Patel, David J Cutler, Michael E Zwick
We used a RainDance Technologies (RDT) expanded content library to enrich the human X chromosome exome (2.5 Mb) from 26 male samples followed by Illumina sequencing. Our multiplex primer library covered 98.05% of the human X chromosome exome in a single tube with 11,845 different PCR amplicons. Illumina sequencing of 24 male samples showed coverage for 97% of the targeted sequences. Sequence from 2 HapMap samples confirmed missing data rates of 2-3% at sites successfully typed by the HapMap project, with an accuracy of at least ~99...
October 2011: Genomics
Katharina Hopp, Christina M Heyer, Cynthia J Hommerding, Susan A Henke, Jamie L Sundsbak, Shail Patel, Priyanka Patel, Mark B Consugar, Peter G Czarnecki, Troy J Gliem, Vicente E Torres, Sandro Rossetti, Peter C Harris
Meckel syndrome (MKS) is an embryonic lethal, autosomal recessive disorder characterized by polycystic kidney disease, central nervous system defects, polydactyly and liver fibrosis. This disorder is thought to be associated with defects in primary cilia; therefore, it is classed as a ciliopathy. To date, six genes have been commonly associated with MKS (MKS1, TMEM67, TMEM216, CEP290, CC2D2A and RPGRIP1L). However, mutation screening of these genes revealed two mutated alleles in only just over half of our MKS cohort (46 families), suggesting an even greater level of genetic heterogeneity...
July 1, 2011: Human Molecular Genetics
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