Abstract：
BACKGROUND:For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). CONTENT:This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. SUMMARY:In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.
journal_name
Clin Chemjournal_title
Clinical chemistryauthors
Voelkerding KV,Dames SA,Durtschi JDdoi
10.1373/clinchem.2008.112789subject
Has Abstractpub_date
2009-04-01 00:00:00pages
641-58issue
4eissn
0009-9147issn
1530-8561pii
clinchem.2008.112789journal_volume
55pub_type
杂志文章,评审abstract：:We examined the variability of lipid and lipoprotein concentrations in plasma from a population sample from the Jerusalem Lipid Research Clinic study. Coefficients of variation of about 8% for plasma cholesterol, 11% to 15% for low- and high-density-lipoprotein cholesterol, and about 30% for triglyceride were reported...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1985-07-01 00:00:00
abstract：:We describe specific EMIT homogeneous enzyme immunoassays for amitriptyline, nortriptyline, imipramine, and desipramine in patients' serum samples. Before analysis, an easily performed extraction step involving the use of 500 microL of sample and a 1-mL disposable column eliminates cross-reacting polar metabolites. Th...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1986-05-01 00:00:00
abstract：:We determined approximately 15,000 laboratory values in 236 individuals between the ages of 60 and 90 y, 22 individuals between 90 and 99 y, and 69 individuals greater than or equal to 100 y, and compared these with values in young adults. We tested 47 different analytes in the 60-90-y group and 93 analytes in the gre...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1992-06-01 00:00:00
abstract：:We detected the presence of creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) in serum by using an immunoinhibition technique to inactivate the creatine kinase isoenzyme of muscle origin (CK-MM). The GEMSAEC centrifugal analyzer is ideally suited for this procedure because it rapidly mixes reagents and has a throughpu...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1980-04-01 00:00:00
abstract：:The historical development of a charitable children's hospital and the evolution of its clinical laboratory are presented. With the appearance of practical quantitative blood chemistry tests in the period between the two World Wars, applications to pediatrics were hampered by the need for ultramicro procedures then un...
journal_title：Clinical chemistry
pub_type： 历史文章,杂志文章
doi：
更新日期：2000-07-01 00:00:00
abstract：BACKGROUND:Band 5 tartrate-resistant acid phosphatase (TrACP; EC 3. 1.3.2) consists of two isoenzymes, bands 5a and 5b, of which band 5b TrACP is considered to be derived from bone. However, no kinetic method for the specific measurement of band 5b TrACP in serum is available. Our aim was to develop a kinetic assay met...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：2000-04-01 00:00:00
abstract：:Values for insulin-mediated glucose disposal vary continuously throughout a population of apparently healthy individuals, with at least a sixfold variation between the most insulin sensitive and most insulin resistant of these individuals. The more insulin resistant a person, the more insulin must be secreted to preve...
journal_title：Clinical chemistry
pub_type： 杂志文章,评审
doi：10.1373/clinchem.2005.048611
更新日期：2005-06-01 00:00:00
abstract：BACKGROUND:Understanding the distribution of C-reactive protein (CRP) concentration among adult women in the US helps to establish the magnitude of women at increased risk for cardiovascular disease. METHODS:We examined the distribution and correlates of CRP, using data from 2205 women >or=20 years of age from the Nat...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2003.027359
更新日期：2004-03-01 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：BACKGROUND:The detection of monoclonal free light chains (FLCs) is an important diagnostic aid for a variety of monoclonal gammopathies and is especially important in light-chain diseases, such as light-chain myeloma, primary systemic amyloidosis, and light-chain-deposition disease. These diseases are more prevalent in...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：2002-09-01 00:00:00
abstract：BACKGROUND:In malignancy, gene mutations frequently occur in tumor suppressor genes such as p53 and are sporadically located. We describe a homogeneous method for amplification and mutation scanning, and apply the method to the p53 gene. METHODS:Using a series of overlapping fluorescein-labeled oligonucleotides comple...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：2002-08-01 00:00:00
abstract：BACKGROUND:The analytical and clinical performance of homogeneous LDL-cholesterol assays has been reported, but their reactions with subfractions of LDL and VLDL have not been described in detail. METHODS:We evaluated reaction selectivity of two homogeneous LDL-cholesterol assays, LDLk (Kyowa Medex) and LDLd (Daiichi ...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：2002-11-01 00:00:00
abstract：BACKGROUND:Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes tha...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2004.035907
更新日期：2004-10-01 00:00:00
abstract：BACKGROUND:Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and othe...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2019.301804
更新日期：2019-07-01 00:00:00
abstract：BACKGROUND:Gas6 is a vitamin K-dependent antiapoptotic protein that has been implicated in cardiovascular pathophysiology. We report the development and validation of an ELISA for Gas6, and the variation of plasma Gas6 with hormonal status in a study designed to evaluate the effect of oral contraception on plasma marke...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2007.089102
更新日期：2007-10-01 00:00:00
abstract：BACKGROUND:Genomic instability resulting in copy number variation is a hallmark of malignant transformation and may be identified through massive parallel sequencing. Tumor-specific cell free DNA (cfDNA) present in serum and plasma provides a real-time, easily accessible surrogate. METHODS:DNA was extracted from serum...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2014.226571
更新日期：2015-01-01 00:00:00
abstract：:Zinc protoporphyrin is the predominent fluorescent porphyrin accumulating in erythrocytes as a result of chronic lead absorption or iron-deficiency anemia. Although diagnostic concentrations or thresholds for it in erythrocytes have been inferred, normal adult averages have not been established. We quantitatively asse...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1980-09-01 00:00:00
abstract：:In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labe...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1985-07-01 00:00:00
abstract：BACKGROUND:There are 45 known genetic diseases that impair the lysosomal degradation of macromolecules. The loss of a single lysosomal hydrolase leads to the accumulation of its undegraded substrates in tissues and increases of related glycoconjugates in urine, some of which can be detected by screening of free oligosa...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2012.201053
更新日期：2013-09-01 00:00:00
abstract：:The molar absorptivity of unconjugated bilirubin in caffeine reagent is independent of the protein matrix. This finding, together with the simplicity of a dilution step for direct spectrophotometry, will improve the calibration methods of bilirubin and make them more nearly accurate. This is encouraging for the develo...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1986-07-01 00:00:00
abstract：:This relatively simple, rapid method for quantification of homovanillic acid (HVA) and vanillylmandelic acid (VMA) involves solvent extraction and capillary gas chromatography. With use of this extraction method, the overall analytical recovery from an aqueous solution is 92.5% for HVA and 79.2% for VMA. 3,4-Dihydroxy...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1983-05-01 00:00:00
abstract：BACKGROUND:We investigated agreement between self-reported prenatal alcohol exposure (PAE) and objective meconium alcohol markers to determine the optimal meconium marker and threshold for identifying PAE. METHODS:Meconium fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) were quantified...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2014.233718
更新日期：2015-03-01 00:00:00
abstract：:A modified pentamer formation assay (PFA) for quantification of tacrolimus and active metabolites after extraction from whole blood is described. The lower limit of detection was 2 microg/L. Intraassay precision (CV) was 5.7-13.7%, and the interassay CV was 6. 1-14.9%. Tacrolimus trough concentrations in 104 whole blo...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1998-12-01 00:00:00
abstract：BACKGROUND:The Dutch project "Calibration 2000" aims at harmonization of laboratory results via calibration by development of matrix-based secondary reference materials. We considered the selection, preparation, and characterization of 34 potential reference materials (PRMs). METHODS:Sixteen PRMs were prepared either ...
journal_title：Clinical chemistry
pub_type： 杂志文章,多中心研究
doi：
更新日期：2002-09-01 00:00:00
abstract：:Using a high-affinity solid-phase-bound antibody (Ka = 1.2 X 10(11) L/mol), we have standardized a two-step radioimmunoassay for free triiodothyronine (FT3) in serum, based on immunoextraction. The method was validated by comparison with an equilibrium-dialysis procedure (r = 0.96) involving RIA of T3 in the dialysate...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1987-03-01 00:00:00
abstract：:Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method ba...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1998-11-01 00:00:00
abstract：:In this automated system for assaying guanidino compounds, the compounds are resolved on an ion-exchange (Dowex 50) column, then reacted with phenanthrenequinone in alkaline solution. This mixture is then acidified and the resulting fluorescence is measured (lambda ex, 305 nm; lambda em, 395 nm). I applied the system ...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1984-02-01 00:00:00
abstract：BACKGROUND:Epidemiologists have observed that blood donation is associated with decreased risk of type 2 diabetes and cardiovascular disease. METHODS:We investigated the relationship between iron stores and insulin sensitivity, after controlling for known confounding factors, and compared insulin sensitivity between b...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2004.046847
更新日期：2005-07-01 00:00:00
abstract：:We studied the effects of some chelators on creatine kinase activity. Creatine kinase is competitively inhibited by endogenous polyvalent cations (e.g., calcium, Ki = 4.5 mmol/L); this can be reversed by adding chelators to the reagent, resulting in a mean increase in activity of 1.14-fold at 25 degrees C and 1.18-fol...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1979-03-01 00:00:00
abstract：:A dissociation-enhanced lanthanide fluoroimmunoassay of serum cortisol based on time-resolved fluorescence is described. The assay is a direct assay, where cortisol immobilized on the wall of a microtiter-strip well competes with cortisol in the sample for the europium-labeled polyclonal antibody. The amount of bound ...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1985-10-01 00:00:00
