Abstract
Laboratory Automation and High-Throughput Chemistry
A Nanochannel-Based Online Universal Logic Ion Sensing Platform
In this work, a novel ion-sensing platform is constructed in a microfluidic chip based on a very easy nano-fabrication technique, with which the nanoscale channel generated along the junction of the PDMS and metal strip can serve as a salt bridge for electrochemical measurements. More important, Chen et al. propose a flexible and universal ion-sensing strategy based on Boolean logic, which can rapidly report the concentration of analyte by the approach method. First, the performance of the nanochannel-based salt bridge is characterized, and the results show that the nanoscale salt bridge behaves comparably to the traditional ones. To illustrate the promising applications of this design, an IrOx electrode is employed to construct the online pH sensing device as an example, and a wide linearity range (pH 2–12) is obtained with a high sensitivity of 74.15 mV per pH unit. Owing to the use of the logic-sensing strategy, the authors achieve rapid identification of the sample pH online and demonstrate the broad potential of the system in designing sensing devices with extremely high integration, automation, and throughput. (Chen et al., Nanoscale,
Online Simultaneous and Rapid Separation of Anions and Cations from a Single Sample Using Dual-Capillary Sequential Injection-Capillary Electrophoresis
A novel capillary electrophoresis (CE) approach has been developed for the simultaneous rapid separation and identification of common environmental inorganic anions and cations from a single sample injection. The method uses a sequential injection–capillary electrophoresis instrument (SI-CE) with capacitively coupled contactless conductivity detection (C(4)D) constructed in house from commercial off-the-shelf components. Oppositely charged analytes from a single sample plug are simultaneously injected electrokinetically onto two separate capillaries for independent separation and detection. Injection is automated and may occur from a syringe or be directly coupled to an external source in a continuous manner. Software control enables high sample throughput (17 runs per hour for the target analyte set), and the inclusion of an isolation valve allows the separation capillaries to be flushed, increasing throughput by removing slow migrating species as well as improving repeatability.
Various environmental and industrial samples (subjected only to filtering) are analyzed in the laboratory with a 3-min analysis time, which allows the separation of 23 inorganic and small organic anions and cations. Finally, the system is applied to an extended automated analysis of Hobart Southern Water tap water for a period of 48 h. The overall repeatability of the migration times of a 14-analyte standard sample is less than 0.74% under laboratory conditions. Limits of detection range from 5 to 61 µg/L–1. The combination of automation, high confidence of peak identification, and low limits of detection make this a useful system for the simultaneous identification of a range of common inorganic anions and cations for discrete or continuous monitoring applications. (Gaudry et al., Anal. Chim. Acta,
Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses
Dispensing and dilution processes may profoundly influence estimates of biological activity of compounds. Published data show that Ephrin-type B receptor 4 IC50 values obtained via tip-based serial dilution and dispensing versus acoustic dispensing with direct dilution differ by orders of magnitude with no correlation or ranking of data sets. Ekins et al. generate computational 3D pharmacophores based on data derived by both acoustic and tip-based transfer. The computed pharmacophores differ significantly depending on dispensing and dilution methods.
The acoustic dispensing–derived pharmacophore correctly identifies active compounds in a subsequent test set where the tip-based method fails. Data from acoustic dispensing generate a pharmacophore containing two hydrophobic features, one hydrogen bond donor and one hydrogen bond acceptor. This is consistent with X-ray crystallography studies of ligand-protein interactions and automatically generated pharmacophores derived from these structural data. In contrast, the tip-based data suggest a pharmacophore with two hydrogen bond acceptors, one hydrogen bond donor, and no hydrophobic features. This pharmacophore is inconsistent with the X-ray crystallographic studies and automatically generated pharmacophores. In short, traditional dispensing processes are another important source of error in high-throughput screening that affects computational and statistical analyses. These findings have far-reaching implications in biological research. (Ekins et al., Plos One,
Detailed Study of Precipitation of a Poorly Water Soluble Test Compound Using Methodologies as in Activity and Solubility Screening: Mixing and Automation Effects
Storage of pharmaceutical discovery compounds dissolved in DMSO is commonplace within the industry. Often, the DMSO stock solution is added to an aqueous system (e.g., in bioassay or kinetic solubility testing). Because most test compounds are hydrophobic, precipitation could occur. Little is known about the factors affecting this precipitation process at the low (µM) concentrations used in screening analyses.
Here, a poorly water-soluble test compound (tolnaftate) is used to compare manual and automated pipetting, and the effect of mixing variables on precipitation is explored. The amount of drug present in the supernatant after precipitation and centrifugation of the samples is quantified. Unusual results are obtained in three different laboratories. Results of experiments performed initially are statistically significantly higher than those performed after a few days in the same laboratory. No significant differences are found between automated and manual pipetting, including in variability. Vortex mixing is found to give significantly lower supernatant amounts compared with milder mixing types. The mixing employed affects the particle growth of the precipitate. These findings are of relevance to discovery stage bioassay and kinetic solubility analyses. (Gillespie et al., Comb. Chem. High Throughput Screen.,
Automation in Haemostasis
The initially small and relatively simple, standalone automation instruments have been replaced by more complex systems that allow for multitasking. Integration of automated coagulation testing into total laboratory automation has become possible in recent years. Automation has many strengths and opportunities if weaknesses and threats are respected. On the positive side, standardization, reduction of errors, reduction of cost, and increase of throughput are clearly beneficial. Dependence on manufacturers, high initiation cost, and somewhat expensive maintenance are less favorable factors. The modern laboratory and especially today’s laboratory technicians and academic personnel in the laboratory do not add value for the doctor and his or her patients by spending a lot of time behind the machines. In the future, the laboratory needs to contribute at the bedside, suggesting laboratory testing and providing support and interpretation of the obtained results. The human factor will continue to play an important role in testing in hemostasis yet under different circumstances. (Huber et al., Hamostaseologie, in press)
An Automatic High-Throughput Single Nucleotide Polymorphism Genotyping Approach Based on Universal Tagged Arrays and Magnetic Nanoparticles
Recent developments in highly parallel genome-wide studies are transforming the association of human health and diseases. For studies where multiple single-nucleotide polymorphism (SNP) loci from a large amount of samples need to be investigated to obtain a result with a high degree of confidence, Li et al. describe a novel, cost-effective, and automated method for high-throughput SNPs genotyping based on universal tagged array and magnetic separation. By using two kinds of functionalized magnetic nanoparticles, the entire operation procedure, including genome DNA extraction and SNP genotyping, can be automatically performed by a JANUS automated workstation (PerkinElmer, Waltham, MA).
Four different SNP loci from 80 samples were scored using only one pair of universal dual-color probes, and the phase of numerous SNPs could be automatically assessed simultaneously. The results demonstrate that the expected scores and good discrimination were obtained between the two alleles from these four SNP loci. By taking advantage of high parallel readout and intrinsically scalable properties of microarray, the automated magnetic separation handling technology is highly adaptable for multiplexing sample preparation and automated SNP analysis. It also avoids the complex procedure, including purification and concentration. The new strategy is high throughput, simple, flexible, cost-effective, and very suitable for large-scale genotyping. (Li et al., J. Biomed Nanotechnol.,
Membrane Protein Structure Determination: The Next Generation
The field of membrane protein structural biology has grown significantly since its first landmark in 1985 with the first 3D atomic resolution structure of a membrane protein. Nearly 26 years later, the crystal structure of the β2 adrenergic receptor in complex with G protein has contributed to another landmark in the field, leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane proteins structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration, and third-generation synchrotrons, has enhanced membrane protein structure determination rates. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible.
In this review, Moraes et al. draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition, the authors highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a special issue titled “Structural and Biophysical Characterization of Membrane Protein-Ligand Binding.” (Moraes et al., Biochim. Biophys. Acta, in press)
Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR
A microneutralization assay using an enzyme-linked immunosorbent assay (ELISA)–based end-point assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. Teferedegne et al. use a quantitative reverse transcription PCR-based end-point assessment (qPCR-MN) to improve upon technical limitations associated with ELISA-MN.
For qPCR-MN, infected MDCK-London cells in 96-well cell culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrate that the qRT-PCR signal detects at 6 h postinfection, reflecting an amplification of at least 100-fold over input. Using ferret antisera, the authors establish the feasibility of measuring virus neutralization at 6 h postinfection, a duration likely confined to a single virus replication cycle.
The neutralization titer for qPCR-MN is defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal. This end point is in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN is robust with respect to assay duration (6 vs. 12 h). In addition, qPCR-MN appears to be compliant with the percentage law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500–12,000 TCID50). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and interlaboratory variability while affording suitability for automation and high-throughput uses. Finally, the qRT-PCR–based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses. (Teferedegne et al., Plos One,
Microfluidic Chip Technology and Micro Reactor Technology
Nanobiotechnology Advanced Antifouling Surfaces for the Continuous Electrochemical Monitoring of Glucose in Whole Blood Using a Lab-on-a-Chip
Picher et al. have developed a lab-on-a-chip containing embedded amperometric sensors in four microreactors that can be addressed individually and that are coated with crystalline surface protein monolayers to provide a continuous, stable, reliable, and accurate detection of blood glucose. It is envisioned that the microfluidic device will be used in a feedback loop mechanism to assess natural variations in blood glucose levels during hemodialysis to allow the individual adjustment of glucose.
Reliable and accurate detection of blood glucose is accomplished by simultaneously performing (a) blood glucose measurements, (b) autocalibration routines, (c) mediator-interferences detection, and (d) background subtractions. The electrochemical detection of blood glucose variations in the absence of electrode fouling events is performed by integrating crystalline surface layer proteins (S-layer) that function as an efficient antifouling coating, a highly oriented immobilization matrix for biomolecules, and an effective molecular sieve with pore sizes of 4 to 5 nm. The authors demonstrate that the S-layer protein SbpA (from Lysinibacillus sphaericus CCM 2177) readily forms monomolecular lattice structures at the various microchip surfaces (e.g., glass, PDMS, platinum, and gold) within 60 min, eliminating unspecific adsorption events in the presence of human serum albumin, human plasma, and freshly drawn blood samples. The highly isoporous SbpA coating allows undisturbed diffusion of the mediator between the electrode surface, thus enabling bioelectrochemical measurements of glucose concentrations between 500 µM and 50 mM (calibration slope DI/Dc of 8.7 nA mM–1). Final proof-of-concept implementing the four-microfluidic microreactor design is demonstrated using freshly drawn blood. Accurate and drift-free assessment of blood glucose concentrations (6. 4 mM) is accomplished over 130 min at 37 °C using immobilized enzyme glucose oxidase by calculating the difference between autocalibration (10 mM glc) and background measurements. This novel combination of biologically derived nanostructured surfaces with microchip technology constitutes a powerful new tool for multiplexed analysis of complex samples. (Picher et al., LabChip,
Microfluidic Devices for High-Throughput Proteome Analyses
In this review, Chao et al. focus on recent developments and strategies to enable and integrate proteomic workflows into microfluidic devices. Over the past decades, microfabricated bioanalytical platforms have gained enormous interest due to their potential to revolutionize biological analytics. Their popularity is based on several key properties, such as high flexibility of design, low sample consumption, rapid analysis time, and minimization of manual handling steps, which are of interest for proteomics analyses. An ideal totally integrated chip-based microfluidic device could allow rapid automated workflows starting from cell cultivation and ending with mass spectrometry–based proteome analysis. By reducing or eliminating sample handling and transfer steps and increasing the throughput of analyses, these workflows could dramatically improve the reliability, reproducibility, and throughput of proteomic investigations. While these complete devices do not exist for routine use yet, many improvements have been made in the translation of proteomic sample handling and separation steps into microfluidic formats. (Chao and Hansmeier, Proteomics,
High-Throughput Analytics
Automated Disposable Small Scale Reactor for High-Throughput Bioprocess Development: A Proof of Concept Study
The acceleration of bioprocess development for biologics and vaccines can be enabled by automated high-throughput technologies. This alleviates the significant resource burden from the multifactorial statistical experimentation required for controlling product quality attributes of complex biologics. Recent technology advances have improved clone evaluation and screening but have struggled to combine the scale-down criteria required for both high cell density cell culture and microbial processes, with sufficient automation and disposable technologies to accelerate process development.
This article describes the proof-of-concept evaluations of an automated disposable small-scale reactor for high-throughput upstream process development. Characterization studies establish the small-scale stirred tank disposable 250-mL reactor as similar to those of laboratory and pilot scale. The reactor generates equivalent process performance for industrial biologics processes for therapeutic protein and monoclonal antibody production using Chinese hamster ovary (CHO) cell culture, Pichia pastoris, and Escherichia coli. This includes similar growth, cell viability, product titer, and product quality. The technology is shown to be robust across multiple runs and meets the requirements for running high cell density processes (>400 g/L wet cell weight) with exponential feeds and sophisticated event-triggered processes. Combining this reactor into an automated array of reactors could ultimately be part of a high-throughput process development strategy. This combines upstream, small-scale purification with rapid analytics, which could dramatically shorten timelines and costs of developing biological processes. (Bareither et al., Biotechnol. Bioeng., in press)
High-Throughput Solution-Based Measurement of Antibody-Antigen Affinity and Epitope Binning
Advances in human antibody discovery have allowed for the selection of hundreds of high-affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high-throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface-related artifacts can limit the accuracy of high-affinity measurements.
When comparing BLI and solution equilibrium-based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) are observed. KinExA direct association (ka) rate constant measurements suggest that this is mainly caused by inaccurate ka measurements associated with BLI-related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, Estep et al. demonstrate a high-throughput 96-well plate format assay, using a Meso Scale Discovery (Rockville, MD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods. (Estep et al., MAbs,
Identifying Analytics for High-Throughput Bioprocess Development Studies
In recent years, high-throughput screening (HTS) studies have been increasingly employed as integral elements of bioprocess development activities. These studies are often limited by an analytical bottleneck; they generate multiple samples for analysis, and the available analytical methods cannot always cope with the added analytical burden. A potential solution to this challenge is offered by the deployment of appropriate analytics. This article outlines features of analytical methods that affect their fit to high-throughput (HT) applications. These are discussed for a range of analytics frequently used in bioprocess development studies of monoclonal antibodies. The authors also outline how these features need to be considered to classify analytical methods in terms of their particular application in high-throughput scenarios. (Konstantinidis et al., Biotechnol. Bioeng.,
Nutritional Lipidomics: Molecular Metabolism, Analytics, and Diagnostics
The field of lipidomics is providing nutritional science with a more comprehensive view of lipid intermediates. Lipidomics research takes advantage of the increase in accuracy and sensitivity of mass detection of mass spectrometry with new bioinformatics toolsets to characterize the structures and abundances of complex lipids. Yet, translating lipidomics to practice via nutritional interventions is still in its infancy. No single instrumentation platform is able to solve the varying analytical challenges of the different molecular lipid species. Biochemical pathways of lipid metabolism remain incomplete, and the tools to map lipid compositional data to pathways are still being assembled. Biology itself is dauntingly complex, and simply separating biological structures remains a key challenge to lipidomics. Nonetheless, the strategy of combining tandem analytical methods to perform the sensitive, high-throughput, quantitative, and comprehensive analysis of lipid metabolites of very large numbers of molecules is poised to drive the field forward rapidly. Among the next steps for nutrition to understand the changes in structures, compositions, and function of lipid biomolecules in response to diet is to describe their distribution within discrete functional compartments lipoproteins. In addition, lipidomics must tackle the task of assigning the functions of lipids as signaling molecules, nutrient sensors, and intermediates of metabolic pathways. (Smilowitz et al., Mol. Nutr. Food Res.
Automation Systems
Minimized Cell Usage for Stem Cell–Derived and Primary Cells on an Automated Patch Clamp System
Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high-quality, high-throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, because cells are not targeted individually but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell–derived cardiomyocytes or neurons.
Becker et al. establish application protocols for CHO cells, IPS cell-derived neurons (iCell Neurons; Cellular Dynamics International, Madison, WI), cardiomyocytes (Cor.4U; Axiogenesis, Cologne, Germany), and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion’s Patchliner (Nanion, Munich, Germany). Use of a 5-µL cell suspension per well for densities between 55,000 and 400,000 cells/mL depending on cell type results in good cell capture.
The authors’ new cell application procedure achieves a >80% success rates while using as little as 300 to 2000 cells per well depending on cell type. The authors demonstrate that this protocol works for standard cell lines, as well as for stem cell–derived neurons and cardiomyocytes, and for primary pancreatic islet cells. Recordings for these cell types are presented and demonstrate that high-data quality are not compromised by altered cell application. This new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared with other standard automated patch clamp systems, the work reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability. (Becker, J. Pharmacol. Toxicol. Methods,
Cell to Whole-Plant Phenotyping: The Best Is Yet to Come
Imaging and image processing have revolutionized plant phenotyping and are now a major tool for phenotypic trait measurement. Dhondt et al. review plant phenotyping systems by examining three important characteristics: throughput, dimensionality, and resolution. First, whole-plant phenotyping systems are highlighted together with advances in automation that enable significant throughput increases. Organ- and cellular-level phenotyping and its tools, often operating at a lower throughput, are then discussed as a means to obtain high-dimensional phenotypic data at elevated spatial and temporal resolution. The significance of recent developments in sensor technologies that give access to plant morphology and physiology-related traits is shown. Overall, attention is focused on spatial and temporal resolution because these are crucial aspects of imaging procedures in plant phenotyping systems. (Dhondt et al., Trends Plant Sci.,
Primary Hepatocyte Cultures for Pharmaco-Toxicological Studies: At the Busy Crossroad of Various Anti-Dedifferentiation Strategies
Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with potential to become new successful drugs. However, before patients can benefit from newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavorable drug candidates. Cost-effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, have stimulated the shift of the safety studies to the earliest stages of drug discovery and development.
In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, matching the requirements of HTS approaches while satisfying ethical considerations. Cultures of primary hepatocytes are already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment (as recently witnessed in the cosmetics industry). Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In this review, both classical and innovative methodologies to stabilize the in vivo–like hepatocyte phenotype in a culture of primary hepatocytes are presented and discussed. (Fraczek et al., Arch Toxicol.,
Establishing a High-Throughput and Automated Cancer Cell Proliferation Panel for Oncology Lead Optimization
Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification and optimization, and determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency.
Lei et al. describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines are selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance are developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) is applied to analyze the data and generate data reports. Using tool compounds, Lei et al. show that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, the authors demonstrate that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, drive cellular structure-activity relationships, and profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization. (Lei et al., J. Biomol. Screen., in press)
Genomics and Proteomics: How Long Do We Need to Reach Clinical Results?
Discovery of the ideal biomarker for clinical care remains a major challenge. Recent progress in genomic and proteomic technologies has allowed the identification of thousands of potential markers, although the benefits of these findings in clinical routine use are not completely evident yet.
Major genomics and proteomics approaches are outlined in this report and their clinical applications are described. Future developments in clinical nephrology are discussed. Genomics and proteomics technologies, used to measure gene expression at the transcript and at the protein levels, provide complementary information, which paves the way for systems biology. The fields of genomics and proteomics continue to develop rapidly, and it is evident that there is great potential for their ability to predict diseases and outcomes. However, several tasks must be accomplished to convert all these “-omics” approaches into clinical practice. Collaboration between clinicians, scientists, and health care funding organizations together with specific guideline development and high-throughput analytical automation is identified as crucial for achieving the potential of these technologies. (Matafora, Blood Purif.,
Recent Advances in Microchip Electrophoresis for Amino Acid Analysis
With the maturation of microfluidic technologies, microchip electrophoresis has been widely employed for amino acid analysis owing to its advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. In this article, Ou et al. review recent progress in amino acid analysis using microchip electrophoresis from 2007 to 2012. Innovations in microchip materials, surface modification, sample introduction, microchip electrophoresis, and detection methods are documented, as well as nascent applications of amino acid analysis in single-cell analysis, microdialysis sampling, food analysis, and extraterrestrial exploration. Without a doubt, more applications of microchip electrophoresis in amino acid analysis may be expected soon. (Ou et al., Anal Bioanal Chem, in press)
Advances in Next-Gen Sequencing
Short Read (Next-Generation) Sequencing: A Tutorial with Cardiomyopathy Diagnostics as an Exemplar
Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel, or “next generation” [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the past year is exome sequencing. Whole-exome sequencing covers all protein coding genes in the genome (~1.1% of genome), and an exome test for a single patient generates about 6 gigabases (109 bp) of DNA sequence data.
A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study shows that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients finds that ~27% of patients show mutations of the titin gene, the most complex gene in the genome (363 exons). The authors use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. (Punetha and Hoffman, Circ. Cardiovasc. Genet.,