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Custom amplicon

Allison B Chambliss, Molly Resnick, Athena K Petrides, William A Clarke, Mark A Marzinke
BACKGROUND: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. METHODS: Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes...
October 12, 2016: Clinical Chemistry and Laboratory Medicine: CCLM
Robert Makowsky, Pema Lhaki, Howard W Wiener, Madhav P Bhatta, Michael Cullen, Derek C Johnson, Rodney T Perry, Mingma Lama, Joseph F Boland, Meredith Yeager, Sarita Ghimire, Thomas R Broker, Sadeep Shrestha
OBJECTIVES/BACKGROUND: Sequence variants in HPV16 confer differences in oncogenic potential; however, to date there have not been any HPV sequence studies performed in Nepal. The objective of this study was to characterize HPV16 viral genome sequences from Nepal compared to a reference sequence in order to determine their lineages. Additionally, we sought to determine if five High-grade Squamous Intraepithelial Lesion (HSIL) subjects were genetically distinct from the non-HSIL subjects...
October 7, 2016: Infection, Genetics and Evolution
Dario Kringel, Jörn Lötsch
BACKGROUND: The opioid system is involved in the control of pain, reward, addictive behaviors and vegetative effects. Opioids exert their pharmacological actions through the agonistic binding at opioid receptors and variation in the coding genes has been found to modulate opioid receptor expression or signaling. However, a limited selection of functional opioid receptor variants is perceived as insufficient in providing a genetic diagnosis of clinical phenotypes and therefore, unrestricted access to opioid receptor genetics is required...
October 8, 2016: Clinica Chimica Acta; International Journal of Clinical Chemistry
Frank R Wendt, David H Warshauer, Xiangpei Zeng, Jennifer D Churchill, Nicole M M Novroski, Bing Song, Jonathan L King, Bobby L LaRue, Bruce Budowle
Short tandem repeat (STR) loci are the traditional markers used for kinship, missing persons, and direct comparison human identity testing. These markers hold considerable value due to their highly polymorphic nature, amplicon size, and ability to be multiplexed. However, many STRs are still too large for use in analysis of highly degraded DNA. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), may be better suited for analyzing compromised samples, and their allele size differences are amenable to analysis by capillary electrophoresis...
September 20, 2016: Forensic Science International. Genetics
Alan J Fox, Matthew C Hiemenz, David B Lieberman, Shrey Sukhadia, Barnett Li, Joseph Grubb, Patrick Candrea, Karthik Ganapathy, Jianhua Zhao, David Roth, Evan Alley, Alison Loren, Jennifer J D Morrissette
As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer...
September 20, 2016: Journal of Visualized Experiments: JoVE
Hanna Hartikainen, David Bass, Andrew G Briscoe, Hazel Knipe, Andy J Green, Beth Okamura
Amplicon sequencing on a High Throughput Sequencing (HTS) platform (custom barcoding) was used to detect and characterise myxosporean communities in environmental DNA (eDNA) samples from marine and freshwater environments and in faeces of animals that may serve as hosts or whose prey may host myxosporean infections. A diversity of myxozoans in filtered water samples and in faeces of piscivores (otters and great cormorants) was detected, demonstrating the suitability of lineage-specific amplicons for characterising otherwise difficult to sample parasite communities...
September 10, 2016: International Journal for Parasitology
Jordan O Cox, Teresa Sikes DeCarmen, Yiwen Ouyang, Briony Strachan, Hillary Sloane, Cathey Connon, Kemper Gibson, Kimberly Jackson, James P Landers, Tracey Dawson Cruz
This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme-based assay for DNA preparation which allows for faster processing times and reduced sample handling. Infrared-mediated PCR (IR-PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 minutes while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment...
September 12, 2016: Electrophoresis
Michelangelo Aloisio, Danilo Licastro, Luciana Caenazzo, Valentina Torboli, Angela D'Eustacchio, Giovanni Maria Severini, Emmanouil Athanasakis
At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification...
October 2016: Molecular Medicine Reports
Saeam Shin, In Sik Hwang, Seung-Tae Lee, Jong Rak Choi
The recent advances in the next-generation sequencing (NGS) technology have enabled fast, accurate, and cost-effective genetic testing. Here, we evaluated the performance of a targeted NGS panel for BRCA1/2 sequencing and confirmed its applicability in routine clinical diagnostics. We tested samples from 88 patients using the TruSeq custom panel (Illumina Inc, USA) and a MiSeq sequencer (Illumina) and compared the results to the outcomes of conventional Sanger sequencing. All 1015 sequence variations identified by Sanger sequencing were detected by NGS, except for one missense variant that might have been missed due to a rare mutation on a primer-binding site...
August 2016: Breast Cancer Research and Treatment
Michael J Kluk, R Coleman Lindsley, Jon C Aster, Neal I Lindeman, David Szeto, Dimity Hall, Frank C Kuo
Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total...
July 2016: Journal of Molecular Diagnostics: JMD
Yongjie Zhang, Ida Skaar, Michael Sulyok, Xingzhong Liu, Mingyong Rao, John W Taylor
Pu-erh is a tea produced in Yunnan, China by microbial fermentation of fresh Camellia sinensis leaves by two processes, the traditional raw fermentation and the faster, ripened fermentation. We characterized fungal and bacterial communities in leaves and both Pu-erhs by high-throughput, rDNA-amplicon sequencing and we characterized the profile of bioactive extrolite mycotoxins in Pu-erh teas by quantitative liquid chromatography-tandem mass spectrometry. We identified 390 fungal and 629 bacterial OTUs from leaves and both Pu-erhs...
2016: PloS One
Cathy K Wang, Ana Aleksic, Michael S Xu, Ric M Procyshyn, Colin J Ross, Fidel Vila-Rodriguez, Alfredo Ramos-Miguel, Ryan Yan, William G Honer, Alasdair M Barr
AIMS: Catechol-O-methyltransferase (COMT) is an enzyme involved in the degradation of catecholamine neurotransmitters. Due to its role in neurotransmitter flux, multiple COMT variants have been associated with the development of psychiatric disorders. Notably, select single-nucleotide polymorphisms (SNPs) of the COMT gene have been implicated in schizophrenia risk, severity, and treatment response. In recognition of the value of a streamlined genotyping method for COMT SNP detection, this study was designed to develop a simple and economical tetra-primer amplification refractory mutation system (T-ARMS) assay for the concurrent detection of eight COMT SNPs: rs4680, rs737865, rs165599, rs2075507, rs4633, rs4818, rs6269, and rs165774...
August 2016: Genetic Testing and Molecular Biomarkers
Jérôme Morinière, Bruno Cancian de Araujo, Athena Wai Lam, Axel Hausmann, Michael Balke, Stefan Schmidt, Lars Hendrich, Dieter Doczkal, Berthold Fartmann, Samuel Arvidsson, Gerhard Haszprunar
The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection...
2016: PloS One
Guy Froyen, An Broekmans, Femke Hillen, Karin Pat, Ruth Achten, Jeroen Mebis, Jean-Luc Rummens, Johan Willemse, Brigitte Maes
The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively...
2016: PloS One
Stephan Bartels, Elisa Schipper, Britta Hasemeier, Hans Kreipe, Ulrich Lehmann
Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions.To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192)...
May 24, 2016: Oncotarget
Ram Vinay Pandey, Walter Pulverer, Pulverer Walter, Rainer Kallmeyer, Gabriel Beikircher, Stephan Pabinger, Albert Kriegner, Andreas Weinhäusel
BACKGROUND: Methylation-sensitive restriction enzymes-polymerase chain reaction (MSRE-PCR) has been used in epigenetic research to identify genome-wide and gene-specific DNA methylation. Currently, epigenome-wide discovery studies provide many candidate regions for which the MSREqPCR approach can be very effective to confirm the findings. MSREqPCR provides high multiplexing capabilities also when starting with limited amount of DNA-like cfDNA to validate many targets in a time- and cost-effective manner...
2016: Clinical Epigenetics
Takao Togawa, Tokio Sugiura, Koichi Ito, Takeshi Endo, Kohei Aoyama, Kei Ohashi, Yutaka Negishi, Toyoichiro Kudo, Reiko Ito, Atsuo Kikuchi, Natsuko Arai-Ichinoi, Shigeo Kure, Shinji Saitoh
OBJECTIVES: To ascertain a molecular genetic diagnosis for subjects with neonatal/infantile intrahepatic cholestasis (NIIC) by the use of next-generation sequencing (NGS) and to perform a genotype-phenotype correlation. STUDY DESIGN: We recruited Japanese subjects with NIIC who had no definitive molecular genetic diagnosis. We developed a diagnostic custom panel of 18 genes, and the amplicon library was sequenced via NGS. We then compared clinical data between the molecular genetically confirmed subjects with NIIC...
April 2016: Journal of Pediatrics
Erzsébet Csernák, János Molnár, Gábor E Tusnády, Erika Tóth
The implementation of targeted therapies revolutionized oncology. As the number of new oncogenic driver mutations, which provide molecular targets for prediction of effective and selective therapies, is increasing, the implementation of fast and reliable methods by molecular pathology labs is very important. Here we report our results with TruSeq Custom Amplicon assay performed on formalin-fixed and paraffin-embedded material. The oligo capture probes targeted the hotspot regions of 10 well-known oncogenes linked to clinical diagnosis and treatment of lung and colorectal adenocarcinomas, melanomas, and gastrointestinal stromal tumors...
January 22, 2016: Applied Immunohistochemistry & Molecular Morphology: AIMM
R Scott Cornman, Clint R V Otto, Deborah Iwanowicz, Jeffery S Pettis
Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota apiaries across multiple years. We used the Illumina MiSeq platform to generate sequence scaffolds from non-overlapping 300-bp paired-end sequencing reads of the ribosomal internal transcribed spacers (ITS). Full-length sequence scaffolds represented ~530 bp of ITS sequence after adapter trimming, drawn from the 5' of ITS1 and the 3' of ITS2, while skipping the uninformative 5...
2015: PloS One
Michael Cullen, Joseph F Boland, Mark Schiffman, Xijun Zhang, Nicolas Wentzensen, Qi Yang, Zigui Chen, Kai Yu, Jason Mitchell, David Roberson, Sara Bass, Laurie Burdette, Moara Machado, Sarangan Ravichandran, Brian Luke, Mitchell J Machiela, Mark Andersen, Matt Osentoski, Michael Laptewicz, Sholom Wacholder, Ashlie Feldman, Tina Raine-Bennett, Thomas Lorey, Philip E Castle, Meredith Yeager, Robert D Burk, Lisa Mirabello
For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method...
December 1, 2015: Papillomavirus Research
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