Array-based Methods Research Articles

Abstract C60: The integrative genomics viewer

Abstract Rapid improvements in next generation sequencing and array-based profiling methods have created a paradigm shift in how scientists approach the study of the cancer genome. Projects such as The Cancer Genome Atlas (TCGA) are harnessing these advances to probe the cancer genome at unprecedented resolution, generating comprehensively catalogs of the genetic alterations of hundreds of tumors. Visualization plays an important role in assimilating the large, muti-modal datasets produced by TCGA and similar projects. To be maximally effective tools must flexibly integrate genomic, functional, and phenotypic data from a variety of platforms, and efficiently handle large datasets. To address these critical challenges we developed the Integrative Genomics Viewer (IGV), a flexible, scalable, high-performance tool for the concurrent visualization of multiple large scale datasets. The IGV supports integrated views of any data type that can be mapped to genomic coordinates, including, but not limited to, next generation sequence data, copy number, gene expression, RNAi, ChIP-seq, RNA-seq, and genome annotations. Data can be annotated, filtered, grouped, and sorted according to clinical and phenotypic attributes. A particular strength of IGV is its ability to handle very large datasets, for example deep coverage sequence data from whole genome scans, or highresolution copy number data from thousands of samples, on standard desktop computers. IGV permits the user to move seamlessly across these datasets at any level of detail, and quickly zoom out to whole genome or drill down to base pair resolution. The ability to dynamically and flexibly integrate multiple data modalities and view them at any scale allows the user to elucidate complex biological relationships that would not otherwise be apparent. The IGV is freely available without restriction. It is currently under active development and use in The Cancer Genome Atlas, 1000 Genomes Project, and other large genomic projects. Citation Information: Cancer Res 2009;69(23 Suppl):C60.

High-throughput Biology in the Postgenomic Era

High-throughput Biology in the Postgenomic Era

Near-Field Effects on Array-Based Surface Wave Methods with Active Sources

Although the capabilities of engineering surface wave methods have improved in recent years due to several advances, a number of issues still require study to further improve the capabilities of modern surface wave methods. Near-field effects, which are one of these issues, have been studied for traditional surface wave methods with two receivers, and several criteria to mitigate the effects have been recommended. However, these criteria are not applicable to surface wave methods with multiple receivers. Moreover, the criteria are not quantitatively based and do not account for different types of soil profiles, which strongly influence near-field effects. A new study of near-field effects on surface wave methods with multiple receivers was conducted via numerical simulations, laboratory simulations, and field tests. Quantitatively based criteria for near-field effects in different soil profiles are presented using two normalized parameters developed in this study. There was excellent agreement between numerical and experimental results, and it was found that underestimation of measured Rayleigh phase velocities was the major symptom of near-field effects.

A Rational Approach to Genetic Testing for Sarcoma

Diagnosis of sarcoma increasingly relies on identifying genetic defects using modern molecular technologies. Each analytic method has unique advantages and specimen requirements that should be considered when allocating tissue for downstream testing. Karyotype on fresh tissue represents a genome-wide screen of gross chromosomal alterations, whereas fluorescence in situ hybridization and polymerase chain reaction detect specific defects that are characteristic of a given tumor type such as t(11;22) EWSR1-FLI1 in Ewing family tumors, t(X;18) SS18-SSX1 in synovial sarcoma, t(2;13) PAX3-FOXO1A in alveolar rhabdomyosarcoma, and MYCN gene amplification in neuroblastoma. Identifying a clonal genetic defect also provides a tumor marker that could help stage the extent of spread of the neoplasm or monitor the efficacy of therapy. In research laboratories, array-based methods identify genes and biochemical pathways contributing to tumor growth and maintenance, opening avenues for pharmacogenetic tests that predict which therapy is likely to overcome the biochemical defects with minimal toxicity. Array-based discoveries are also spurring validation of smaller test panels that rely on conventional technologies such as immunohistochemistry and reverse transcription polymerase chain reaction. The pathologist's expertise is critical in: (1) consulting with clinicians about specimen collection and handling; (2) preserving tissue for immediate testing and for any downstream testing that is indicated once morphology and immunophenotype are known; (3) performing tests that maximize outcome on the basis of the strengths and limitations of each assay in each available specimen type; and (4) conveying results to the rest of the healthcare team using proper gene nomenclature and interpreting the findings in a way that facilitates optimal clinical management.

Analysis of protein-protein interactions using array-based yeast two-hybrid screens.

The yeast two-hybrid system (Y2H) is a powerful tool to identify protein-protein interactions. Here we describe array-based two-hybrid methods that use defined libraries of open reading frames (ORFs) as opposed to random genomic or cDNA libraries. The array-based Y2H system is well suited for interactome studies of existing ORFeomes or subsets thereof, preferentially in a recombination-based cloning system. Array-based Y2H screens efficiently reduce false positives by using built-in controls, retesting, and evaluation of background activation. Hands-on time and the amount of used resources grow exponentially with the number of tested proteins; this is a disadvantage for large genome sizes. For large genomes, random library screen may be more efficient in terms of time and resources, but not as comprehensive as array screens, and they require an efficient sequencing facility. However, large array screens require some extent of automation although they can be carried out manually on smaller scales. Future-generation Y2H plasmid constructs including tightly regulated expression systems and features that facilitate biochemical characterization will provide more efficient and powerful tools to identify interacting proteins.

Bias analysis of MUSIC in the presence of mutual coupling

The effect of mutual coupling in antenna elements heavily deteriorates the performance of existing array-based bearing-finding methods. Such a bias of MUSIC in uniform and general linear arrays is analytically studied and an explicit expression of a relationship between this bias and the effect of mutual coupling together with the direction of the incident source is given. The biases of different arrays are also compared to provide a reference when choosing the right array under the considerable effect of mutual coupling.

Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code

AbstractEndoglin is an accessory receptor for TGF-β signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng −8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)–chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the −8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.

Evaluating the coupling efficiency of phosphorylated amino acids for SPOT synthesis

A high demand of interest concerning binding assays to study the consequences of posttranscriptional phosphorylation may be addressed by peptide array-based methods. A crucial factor for de novo chemical approaches to generate such arrays is the possibility to rationally permutate phosphorylation events along a huge number of sequences. The simple principle behind this advantage is the stepwise synthesis of peptides, which allows the incorporation of either phosphorylated or nonphosphorylated derivates at serine, threonine, and tyrosine positions. In spite of several reported applications of phosphopeptide arrays, there is, to our best knowledge, no reported analysis of the efficiency of the involved techniques. Here, we analyze different coupling conditions to introduce phosphoamino acids in standard SPOT synthesis. Our results clearly indicate that EEDQ is the preferable activator and can also be used in fully automated SPOT synthesis.

Microarray‐based DNA profiling to study genomic aberrations

High throughput microarrays were initially developed to analyse the expression of many RNA transcripts in parallel. The technology has since been adapted to a variety of applications, one of which is the analysis of the genome to study DNA dosage and sequence content. Advances in microarray fabrication and completion of large-scale genome sequencing projects have enabled the rapid development of affordable array-based methods for high-resolution genome-wide assessment of DNA alterations. This review will describe the evolution of microarray assays to study genomic aberrations and will highlight how they have enabled researchers to gain insight into the biology of human diseases and how they will benefit research in the future.

A study of near source effects in array-based (SPAC) microtremor surveys

Array-based methods for exploiting ambient seismic noise are receiving increasing attention in the literature, particularly for use in providing shear wave velocity profiles to assist with simulation of site-specific earthquake responses. Many of these microtremor methods—such as the spatial autocorrelation technique (SPAC)—operate under the fundamental assumption of plane wave propagation of surface waves. However, when there are significant microtremor sources in close proximity to an array, wave front curvature becomes significant, and the plane wave assumption becomes a tenuous one. This paper explains the use of a simplified geometry-based approach to examine the effect of source distance on SPAC spectra for hexagonal (six-station) and triangular (three-station) arrays. The results suggest that near source effects are generally minimal provided that most of the sources are located a distance equal to at least two array radii from the centre of the array, although this distance increases when attenuation is considered. Results of a field trial involving the deliberate use of near-sources in proximity to a hexagonal (six-station) array support the findings of the theoretical model. Near source effects generally result in the measured SPAC curve being offset to lower frequencies for wavenumbers less than the first secondary maximum of the SPAC curve (kr < 7.0; λ > 0.86R), resulting in underestimates of shear velocity values in the inverted layered earth model. For the field example, shear velocity was underestimated by up to approximately 17 per cent. Studies of near source effects in a number of theoretical source distributions for hexagonal arrays revealed that cyclic variation in the imaginary SPAC coefficients are indicative of near source effects, although field examples indicate this effect is less significant than the geometric modelling suggests. Modelling of linear microtremor source distributions (intended to represent traffic travelling along a road) resulted in less severe near source effects than full (360°) azimuthal source coverage—a result confirmed by field data. Model and field studies also indicated that in situations of a dominant, close microtremor source of limited azimuthal extent, use of a hexagonal (six-station) array significantly mitigates near source effects compared with a similarly aligned triangular (three-station) array.

A novel approach to determine cell formation, intracellular machine layout and cell layout in the CMS problem based on TOPSIS method

This paper deals with the cellular manufacturing system (CMS) that is based on group technology (GT) concepts. CMS is defined as identifying the similar parts that are processed on the same machines and then grouping them as a cell. The most proposed models for solving CMS problems are focused on cell formation and intracellular machine layout problem while cell layout is considered in few papers. In this paper we apply the multiple attribute decision making (MADM) concept and propose a two-stage method that leads to determine cell formation, intracellular machine layout and cell layout as three basic steps in the design of CMS. In this method, an initial solution is obtained from technique for order preference by similarity to the ideal solution (TOPSIS) and then this solution is improved. The results of the proposed method are compared with well-known approaches that are introduced in literature. These comparisons show that the proposed method offers good solutions for the CMS problem. The computational results are also reported. Scope and purpose In the previous array-based clustering methods, arrays are defined by binary numbers that are indicated as the set of machines that process each part. The main problem of these methods is that grouping parts and machines are made regardless of production volume, operational sequences, production cost, inventory and other production system's limitations. In this paper we consider the previous common problems of array-based clustering methods and apply the logical idea of TOPSIS method for solving the cellular manufacturing system problem in which arrays in the part-machine incidence matrix are defined by operational sequences. The TOPSIS is a multiple attribute decision making (MADM) technique in which the alternatives are ranked by their distances between positive and negative ideal solution.

Tissue microarray studies in bladder cancer

The introduction of tissue microarray (TMA) methodology 10 years ago has provided a valuable tool for high-throughput genomic and proteomic analyses. Using this method hundreds of minute tissue samples can be investigated on one microscopic glass slide. Several studies demonstrated that these small tissue areas are representative for the entire tumour block and can provide reliable information on the relation of molecular markers and clinical outcome of the patient. Types of TMA used in bladder cancer research include defined clinical case series, stage-specific series (e.g. pT1), stage progression series, TMAs containing all specimens from clinical trials for specific therapies, flat (pre)neoplastic lesions, cell culture pellets and mouse model TMAs. The TMA technique has frequently been used in bladder cancer research to evaluate immunohistochemical candidate markers for prognosis and to reveal the amplification frequency of candidate oncogenes in regions with copy number alterations detected by comparative genomic hybridization and array-based methods. In addition, multimarker expression studies of several specific biological functions (e.g. apoptosis or cell-cycle proteins) or signal transduction pathways have been performed. TMAs are also used for validation of array-based gene expression studies on the protein level. TMA technology represents a crucial technique for translating new information on molecular changes in bladder cancer into clinical practice.

RSC regulates nucleosome positioning at Pol II genes and density at Pol III genes

Nucleosomes can restrict the access of transcription factors to chromatin. RSC is a SWI/SNF-family chromatin-remodeling complex from yeast that repositions and ejects nucleosomes in vitro. Here, we examined these activities and their importance in vivo. We utilized array-based methods to examine nucleosome occupancy and positioning at more than 200 locations in the genome following the controlled destruction of the catalytic subunit of RSC, Sth1. Loss of RSC function caused pronounced and general reductions in new transcription from Pol I, II, and III genes. At Pol III genes, Sth1 loss conferred a general reduction in RNA Pol III occupancy and a gain in nucleosome density. Notably at the one Pol III gene examined, histone restoration was partly replication-dependent. In contrast, at Pol II promoters we observed primarily single nucleosome changes, including movement. Importantly, alterations near the transcription start site were more common at RSC-occupied promoters than at non-occupied promoters. Thus, RSC action affects both nucleosome density and positioning in vivo, but applies these remodeling modes differently at Pol II and Pol III genes.

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.

Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads

Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.

The Array Biosensor: Portable, Automated Systems

With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously. A key characteristic of this system is its two-dimensional configuration, which allows controls and standards to be analyzed in parallel with unknowns. Although the majority of our work has focused on instrument automation and immunoassay development, we have recently initiated efforts to utilize alternative recognition molecules, such as peptides and sugars, for detection of a wider variety of targets. The array biosensor has demonstrated utility for a variety of applications, including food safety, disease diagnosis, monitoring immune response, and homeland security, and is presently being transitioned to the commercial sector for manufacturing.

Computational analysis of rice (&lt;I&gt;Oryza sativa&lt;/I&gt; L.) panicle topology and ripening

The processes involved in rice (Oryza sativa L.) panicle ripening vary with time and topological grain position. Methods to describe the functioning and connectivity of the grains on a panicle could aid the analysis of these processes. Hence, we addressed the difficulty of encoding and representing panicle topology. Array-based decomposition and computational methods were developed to encode and analyse panicle topology and grain traits. The technique, applied to the analysis of dry matter accumulation, clearly represented the basipetal succession of asynchronous grain ripening on a panicle. These methods should be useful for the spatial and temporal analysis of a number of panicle processes and attributes, including molecular ones, involved with ripening.

Detection of small genomic imbalances using microarray-based multiplex amplifiable probe hybridization

Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200-600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype-phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.

Neue Möglichkeiten der pränatalen genetischen Diagnostik inklusive Präimplantationsdiagnostik

During recent years the progress with the most important practical impact in prenatal diagnosis has been the implementation of first trimester risk screening for common aneuploidies leading to a much improved identification of pregnancies at risk. Molecular methods for a rapid, cost-effective, but selective aneuploidy diagnosis such as interphase FISH or QF-PCR have been around for years, do have their specific indications, but will unlikely replace conventional cytogenetic tools in routine diagnosis. They apparently do also play a role as marketing instruments in the competition among cytogenetic laboratories. The most thrilling issue for all cytogeneticists in the years to come will be the introduction of array-based methods in the prenatal routine diagnosis of chromosomal abnormalities. Polar body diagnosis has been the only option available for preimplantation genetic diagnosis in german speaking countries. The overwhelming majority of all professionals involved and many families concerned share the hope that the legal situation will improve in these countries to allow the examination of early embryos in high risk situations.

In situ bisulfite modification of membrane-immobilized DNA for multiple methylation analysis

Increasing interest in DNA methylation has resulted in the recent development of array-based methods. However, these method require complex sample treatment processes, such as bisulfite modification in the liquid state of every sample. Here, we describe a microarray-based technology for interrogating DNA methylation status of multiple DNA samples at the same time, in which the DNA samples are first dotted on membranes and then treated with bisulfite directly. In this assay, plasmid pUC19 DNA is immobilized on nylon membranes and soaked in bisulfite reaction mixtures for 16 h to convert unmethylated Cs to Us, and methylated Cs remain unchanged. The probes for detecting the methylation patterns of CpG sites are hybridized with the converted DNA dotted on the membranes, and the signals are revealed by chemiluminescence of DIG-labeled probes. The experiments show that this method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This new method could be used as a convenient tool for detecting the methylation status of multiple genes for a large number of samples in the future.