Abstract:
BACKGROUND:Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS:We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform. RESULTS:The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS:With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.
journal_name
Clin Chemjournal_title
Clinical chemistryauthors
Abou Tayoun AN,Tunkey CD,Pugh TJ,Ross T,Shah M,Lee CC,Harkins TT,Wells WA,Tafe LJ,Amos CI,Tsongalis GJdoi
10.1373/clinchem.2013.206466subject
Has Abstractpub_date
2013-10-01 00:00:00pages
1481-8issue
10eissn
0009-9147issn
1530-8561pii
clinchem.2013.206466journal_volume
59pub_type
杂志文章abstract:BACKGROUND:An association has been described between mortality in children with meningococcal disease and functional polymorphisms in the interleukin-1 (IL1) cluster. We undertook a multicenter study to evaluate associations of these polymorphisms in a Central European population. PATIENTS AND METHODS:The study involv...
journal_title:Clinical chemistry
pub_type: 杂志文章,多中心研究
doi:10.1373/clinchem.2005.058537
更新日期:2006-03-01 00:00:00
abstract::We describe a time-resolved immunofluorometric assay (IFMA) for corticotropin in unextracted human plasma, based on the use of two monoclonal antibodies: europium-labeled antibody 1A12 and antibody 2A3 coated onto microtiter wells. We compared the results of this assay with those of an immunoradiometric assay (IRMA) p...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1987-10-01 00:00:00
abstract:BACKGROUND:Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virus-specific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding color-coded particles. METHODS:We used layer-by-layer technology ...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:10.1373/clinchem.2005.065789
更新日期:2006-08-01 00:00:00
abstract::Establishing associations between environmental agents and disease presents challenges to both epidemiologists and toxicologists, particularly in cases of complex gene-environment interactions and when there is a long latency between exposure and disease. Biologic markers, physiological signals that reflect exposure, ...
journal_title:Clinical chemistry
pub_type: 杂志文章,评审
doi:
更新日期:1994-07-01 00:00:00
abstract:BACKGROUND:Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. A methodology using immunocapture purification with liqui...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:10.1373/clinchem.2009.134643
更新日期:2010-02-01 00:00:00
abstract::We measured changes in concentrations of magnesium, zinc, calcium, potassium, cholesterol [total and high-density lipoproteins (HDL)], total creatine kinase (CK), and CK isoenzyme-MB in plasma (PI) and/or erythrocytes (Erc) from apparently healthy subjects and from patients with either pre-infarction syndrome (PIS) or...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1988-10-01 00:00:00
abstract::We review recently developed reference methods based on mass fragmentography (specific ion monitoring) with use of isotope-labeled internal standard, for determination of cholesterol, triglycerides, urea, glucose, cortisol, progesterone, and testosterone. With an optimal ratio between standard and material to be deter...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1976-11-01 00:00:00
abstract::In this manual fluorometric method, blood samples are used that have been impregnated on the filter paper, a convenient collection technique that is widely used to screen newborns for phenylketonuria. The modified procedure, based on the method of McCaman and Robins [J. Lab Clin. Med. 59, 885 (1962)], includes elution...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1979-03-01 00:00:00
abstract::Mesenteric vascular occlusion and intestinal obstruction are difficult-to-diagnose medical emergencies. We evaluated a large panel of biochemical markers as diagnostic and prognostic indicators in a rat model of intestinal infarction and partial, complete, and strangulated intestinal obstruction. After intestinal infa...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1988-02-01 00:00:00
abstract::We evaluated a newly developed solid-phase immunoassay (EIA) of terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.3) and compared it with the enzymatic assay of TdT involving DNA polymerase. We assessed the precision, performance characteristics, and clinical efficacy of the EIA procedure, using 249 specimens of pe...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1987-02-01 00:00:00
abstract:BACKGROUND:Variability among assays used to measure intact parathyroid hormone (iPTH) is of particular concern because of the routine use of iPTH assay results to guide management of osteodystrophy and calcium metabolism in patients with end-stage renal disease (ESRD). The aim of this study was to determine the extent ...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:10.1373/clinchem.2006.071589
更新日期:2006-09-01 00:00:00
abstract::In the diagnosis of chronic (as opposed to acute) liver diseases, combinations of indicators are needed to improve specificity. Alanine aminopeptidase (AAP; microsomal aminopeptidase, EC 3.4.11.2) activity in serum reportedly is a very sensitive indicator of intrahepatic cholestasis and biliary obstruction; it is also...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1987-03-01 00:00:00
abstract::Lactate dehydrogenase (LD) isoenzymes 1 and 2 in human serum were separated on a column of diethylaminoethyl-Sephadex. Samples layered on mini-columns were eluted with buffered sodium chloride (100, 150, and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and thei...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1978-03-01 00:00:00
abstract::The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical metho...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1987-10-01 00:00:00
abstract::Using Syva "EMIT" reagents and the Cobas Bio centrifugal analyzer, we have developed cost-effective assays for therapeutic drug monitoring of carbamazepine, phenobarbital, and phenytoin. With these optimized methods one can assay up to 2470 samples with a single 100-test EMIT kit with good precision and accuracy. Betw...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1989-08-01 00:00:00
abstract::Gene probes can now be used to detect a variety of mutations that produce single-gene disorders. In present clinical practice, restriction endonuclease analysis is used for the prenatal diagnosis of sickle cell anemia, alpha-thalassemia, and beta-thalassemia. Direct detection of the mutation is possible in alpha-thala...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1985-09-01 00:00:00
abstract::Serum specimens from patients on primidone therapy were assayed by two currently available procedures: a commercially available enzyme immunoassay (EMIT) and a published gas-chromatographic procedure. Both procedures were done with commercially available materials and equipment. Results by the two procedures agreed we...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1976-06-01 00:00:00
abstract:BACKGROUND:Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. METHODS:Parvovirus DNA target sequences and competitor sequences were coamplified and direc...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1999-09-01 00:00:00
abstract::We report a simple, enzymatic, end point method for determining nitrate in serum and urine with use of nitrate reductase from Aspergillus sp. (EC 1.6.6.2). The decrease in absorbance at 340 nm as a result of the oxidation of beta-NADPH is measured. We used FAD as a supplementary electron carrier and added an internal ...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1995-06-01 00:00:00
abstract::Antiserum performance in a nephelometric system can be characterized by parameters derived from measuring reaction rates. The characterization process is derived from a series of dose-response curves (elicited nephelometric response vs antigen concentration) generated from various dilutions of the antiserum being test...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1981-11-01 00:00:00
abstract:BACKGROUND:Uremic syndrome is the consequence of the retention of solutes usually cleared by the healthy kidneys. p-Cresol can be considered a prototypic protein-bound uremic toxin. It is conceivable, analogous with drugs, that the non-protein-bound fraction of p-cresol exerts toxicity. This aspect had never been evalu...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:10.1373/49.3.470
更新日期:2003-03-01 00:00:00
abstract::We developed a solid-phase two-site immunoenzymometric assay (IEMA) of the estrogen-induced 52-kDa cathepsin D (EC 3.4.23.5) and its processed forms (48-kDa and 34-kDa proteins) in cytosols of breast cancer tissues, using two monoclonal antibodies directed against two different epitopes of these antigens. The first an...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1989-01-01 00:00:00
abstract::Creatine kinase (CK) isoenzymes MM, MB, and BB are located primarily in the cell cytosol, and increased CKMB in plasma is the hallmark of myocardial infarction. However, whether CK is released with reversible ischemic injury remains controversial. Here, we assessed plasma CK activity--cytosolic and mitochondrial CK--i...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1997-03-01 00:00:00
abstract:BACKGROUND:Whether low plasma 25-hydroxyvitamin D concentrations cause osteoporotic fractures is unclear. We tested the hypothesis that low plasma 25-hydroxyvitamin D concentrations are associated with increased risk of osteoporotic fractures using a Mendelian randomization analysis. METHODS:We genotyped 116 335 rando...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:10.1093/clinchem/hvaa049
更新日期:2020-05-01 00:00:00
abstract::Quantification of ethanol by a radiative energy attenuation (REA) technique was evaluated and compared with results by gas-liquid chromatography (GLC) and by the Du Pont aca. Within-assay CVs were less than 5.5%. Between-assay CVs ranged from 1.9% to 6.0% for serum and blood controls at concentrations of 0.5, 1.0, and...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1984-11-01 00:00:00
abstract::In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine dinucleotide-labeled TBG react sequentially with antiserum. Then apoglucose oxidase is added to combine with the free fraction and generate glucose oxidase activit...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1986-05-01 00:00:00
abstract::At the clinical level, we have seen the emergence of a subspecialty of affective disorders within psychiatry. Mood clinics, depression units, and affective disorders centers are appearing in many academic and clinical settings, where clinical skill and knowledge can be concentrated and new research furthered. By using...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1988-05-01 00:00:00
abstract:BACKGROUND:The hepatocyte-derived acute-phase reactant C-reactive protein (CRP) has been the subject of intense research over the last 2 decades for its possible role in the pathogenesis of cardiovascular diseases. This research has spawned interest in the use of the blood concentration of CRP for predicting a first co...
journal_title:Clinical chemistry
pub_type: 杂志文章,评审
doi:10.1373/clinchem.2008.115923
更新日期:2009-02-01 00:00:00
abstract::Serum amyloid A (SAA), a sensitive acute-phase protein, is the precursor of AA fibrils in reactive amyloidosis. However, SAA is poorly immunogenic, and development and standardization of immunoassays of this protein have been difficult. We established an automated polyclonal/monoclonal microparticle capture enzyme imm...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1994-07-01 00:00:00
abstract::We determined the LD-1/LD-2 isoenzyme ratio in hemolysates of erythrocytes by electrophoresis on cellulose acetate and on agarose. A ratio exceeding 1.0 was found with the former but not the latter. Results were similar for in vitro models of hemolytic disorders. Using cellulose acetate electrophoresis, we determined ...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1981-08-01 00:00:00