High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs

Mutation of the human genome ranges from single base-pair changes to whole-chromosome aneuploidy. Karyotyping, fluorescence in situ hybridization, and comparative genome hybridization are currently used to detect chromosome abnormalities of clinical significance. These methods, although powerful, suffer from limitations in speed, ease of use, and resolution, and they do not detect copy-neutral chromosomal aberrations--for example, uniparental disomy (UPD). We have developed a high-throughput approach for assessment of DNA copy-number changes, through use of high-density synthetic oligonucleotide arrays containing 116,204 single-nucleotide polymorphisms, spaced at an average distance of 23.6 kb across the genome. Using this approach, we analyzed samples that failed conventional karyotypic analysis, and we detected amplifications and deletions across a wide range of sizes (1.3-145.9 Mb), identified chromosomes containing anonymous chromatin, and used genotype data to determine the molecular origin of two cases of UPD. Furthermore, our data provided independent confirmation for a case that had been misinterpreted by karyotype analysis. The high resolution of our approach provides more-precise breakpoint mapping, which allows subtle phenotypic heterogeneity to be distinguished at a molecular level. The accurate genotype information provided on these arrays enables the identification of copy-neutral loss-of-heterozygosity events, and the minimal requirement of DNA (250 ng per array) allows rapid analysis of samples without the need for cell culture. This technology overcomes many limitations currently encountered in routine clinical diagnostic laboratories tasked with accurate and rapid diagnosis of chromosomal abnormalities.     

SNPs基因分型技术

介绍了SNP的概念、特点,集中讨论了各种技术的原理及优缺点,并对目前SNP在遗传图的绘制、疾病防治、药物设计及法医学等方面的应用及研究进展进行了综述。     

Dynamic model based algorithms for screening and genotyping over 100 K SNPs on oligonucleotide microarrays

MOTIVATION: A high density of single nucleotide polymorphism (SNP) coverage on the genome is desirable and often an essential requirement for population genetics studies. Region-specific or chromosome-specific linkage studies also benefit from the availability of as many high quality SNPs as possible. The availability of millions of SNPs from both Perlegen and the public domain and the development of an efficient microarray-based assay for genotyping SNPs has brought up some interesting analytical challenges. Effective methods for the selection of optimal subsets of SNPs spanning the genome and methods for accurately calling genotypes from probe hybridization patterns have enabled the development of a new microarray-based system for robustly genotyping over 100,000 SNPs per sample. RESULTS: We introduce a new dynamic model-based algorithm (DM) for screening over 3 million SNPs and genotyping over 100,000 SNPs. The model is based on four possible underlying states: Null, A, AB and B for each probe quartet. We calculate a probe-level log likelihood for each model and then select between the four competing models with an SNP-level statistical aggregation across multiple probe quartets to provide a high-quality genotype call along with a quality measure of the call. We assess performance with HapMap reference genotypes, informative Mendelian inheritance relationship in families, and consistency between DM and another genotype classification method. At a call rate of 95.91% the concordance with reference genotypes from the HapMap Project is 99.81% based on over 1.5 million genotypes, the Mendelian error rate is 0.018% based on 10 trios, and the consistency between DM and MPAM is 99.90% at a comparable rate of 97.18%. We also develop methods for SNP selection and optimal probe selection. AVAILABILITY: The DM algorithm is available in Affymetrix's Genotyping Tools software package and in Affymetrix's GDAS software package. See http://www.affymetrix.com for further information. 10 K and 100 K mapping array data are available on the Affymetrix website.     

Contrast normalization of oligonucleotide arrays.

Affymetrix high-density oligonucleotide array is a tool that has the capacity to simultaneously measure the abundance of thousands of mRNA sequences in biological samples. In order to allow direct array-to-array comparisons, normalization is a necessity. When deciding on an appropriate normalization procedure there are a couple questions that need to be addressed, e.g., on which level should the normalization be performed: On the level of feature intensities or on the level of expression indexes? Should all features/expression indexes be used or can we choose a subset of features likely to be unregulated? Another question is how to actually perform the normalization: normalize using the overall mean intensity or use a smooth normalization curve? Most of the currently used normalization methods are linear; e.g., the normalization method implemented in the Affymetrix software GeneChip is based on the overall mean intensity. However, along with alternative methods of summarizing feature intensities into an expression index, nonlinear methods have recently started to appear. For many of these alternative methods, the natural choice is to normalize on the level of feature intensities, either using all feature intensities or only perfect match intensities. In this report, a nonlinear normalization procedure aimed for normalizing feature intensities is proposed.     

Genotyping and annotation of Affymetrix SNP arrays

In this paper we develop a new method for genotyping Affymetrix single nucleotide polymorphism (SNP) array. The method is based on (i) using multiple arrays at the same time to determine the genotypes and (ii) a model that relates intensities of individual SNPs to each other. The latter point allows us to annotate SNPs that have poor performance, either because of poor experimental conditions or because for one of the alleles the probes do not behave in a dose–response manner. Generally, our method agrees well with a method developed by Affymetrix. When both methods make a call they agree in 99.25% (using standard settings) of the cases, using a sample of 113 Affymetrix 10k SNP arrays. In the majority of cases where the two methods disagree, our method makes a genotype call, whereas the method by Affymetrix makes a no call, i.e. the genotype of the SNP is not determined. By visualization it is indicated that our method is likely to be correct in majority of these cases. In addition, we demonstrate that our method produces more SNPs that are in concordance with Hardy–Weinberg equilibrium than the method by Affymetrix. Finally, we have validated our method on HapMap data and shown that the performance of our method is comparable to other methods.     

Sequencing of pools of nucleic acids on oligonucleotide arrays

In sequencing-by-hybridization methods, the nucleotide sequence of a nucleic acid is reconstructed by overlapping oligonucleotides capable of hybridizing with the nucleic acid. In their present form, the methods are hardly suitable for sequencing of long nucleic acid molecules because of the occurrence of non-unique overlaps between the oligonucleotides, and similarly to the conventional sequencing methods, it is necessary to obtain an individual molecule. In the method described here, most ambiguities in reconstruction of a sequence from the constituent oligonucleotides are eliminated by preparing on oligonucleotide arrays and separate surveying of the nucleic acid nested partials. This enables longer nucleic acids to be sequenced, and results in a high redundancy of the input data allowing most hybridization errors to be eliminated by algorithmic means. Furthermore, large pools of nucleic acid strands can be sequenced directly, without isolating individual strands.     

三色荧光技术在SNP检测中的应用

利用三色荧光标记的A、C、T双脱氧核苷酸单碱基延伸的方法结合编码寡核苷酸芯片技术检测单核苷酸多态性 (SNP)的基因型。以beta地中海贫血样本基因 (HBB基因 )突变作为模型的研究结果显示该方法能同时对多位点的SNP进行检测。     

Multiplex Genotyping of Cytokine Gene SNPs Using Fluorescence Bead Array

Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1α, IL-1R, IL-1RA, IL-1β, IL-2, IL-4, IL-4Rα, IL-6, IL-10, IL-12, TGF-β1, and TNF-α, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.     

Photolithographic synthesis of high-density oligonucleotide probe arrays

High-density DNA probe arrays provide a massively parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research and diagnostics. Light-directed combinatorial oligonucleotide synthesis has enabled the large-scale production of GeneChip probe arrays which contain several hundred of thousand oligonucleotide sequences on glass "chips" about one cm2 in size. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms ("genotyping"), and in a wide range of gene expression studies. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques adapted from the microelectronics industry. The present-generation methodology employs MeNPOC photo-activatable nucleoside monomers with proximity photolithography, and is currently capable of printing individual 10 microns 2 probe features at a density of 10(6) probes/cm2.     

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.     

Global assessment of cross-hybridization for oligonucleotide arrays.

Cross-hybridization is the tendency for chains of nucleic acids to bind to other chains of nucleic acids that have similar but not identical sequences. This has the potential to make the interpretation of microarray experiments difficult since intensity at a spot on the array does not simply depend on the quantity of target in the sample. We propose a method for evaluating the extent of cross-hybridization in oligonucleotide arrays for data arising from a typical microarray experiment. We show that the level of cross-hybridization can be quite substantial. We argue that this makes the interpretation of the difference calls provided by MAS 5.0 difficult.     

Selecting oligonucleotide probes for whole-genome tiling arrays with a cross-hybridization potential

For designing oligonucleotide tiling arrays popular, current methods still rely on simple criteria like Hamming distance or longest common factors, neglecting base stacking effects which strongly contribute to binding energies. Consequently, probes are often prone to cross-hybridization which reduces the signal-to-noise ratio and complicates downstream analysis. We propose the first computationally efficient method using hybridization energy to identify specific oligonucleotide probes. Our Cross-Hybridization Potential (CHP) is computed with a Nearest Neighbor Alignment, which efficiently estimates a lower bound for the Gibbs free energy of the duplex formed by two DNA sequences of bounded length. It is derived from our simplified reformulation of t-gap insertion-deletion-like metrics. The computations are accelerated by a filter using weighted ungapped q-grams to arrive at seeds. The computation of the CHP is implemented in our software OSProbes, available under the GPL, which computes sets of viable probe candidates. The user can choose a trade-off between running time and quality of probes selected. We obtain very favorable results in comparison with prior approaches with respect to specificity and sensitivity for cross-hybridization and genome coverage with high-specificity probes. The combination of OSProbes and our Tileomatic method, which computes optimal tiling paths from candidate sets, yields globally optimal tiling arrays, balancing probe distance, hybridization conditions, and uniqueness of hybridization.     

Long oligonucleotide arrays on nylon for large-scale gene expression analysis

To date, most studies of multigenic expression patterns by long DNA array have used DNA fragments as probes. These probes are usually obtained as PCR products, and this represents a time-consuming and error-prone approach, requiring strict quality control. The present study examines the use of 40- and 70-mer synthetic oligonucleotides as probes for DNA array analysis with radioactive labeled targets. Design, spotting onto nylon filters, and hybridization conditions were determined and optimized. In this approach, the sensitivity and the specificity of the hybridization appear comparable to the conventional long DNA probes assay, permitting the analysis of small samples of approximately 1 microg total RNA. The long oligonucleotide array thus provides a very convenient method for the analysis of gene expression patterns in biological specimens and in clinical research.     

Normalization of oligonucleotide arrays based on the least-variant set of genes

Background  

It is well known that the normalization step of microarray data makes a difference in the downstream analysis. All normalization methods rely on certain assumptions, so differences in results can be traced to different sensitivities to violation of the assumptions. Illustrating the lack of robustness, in a striking spike-in experiment all existing normalization methods fail because of an imbalance between up- and down-regulated genes. This means it is still important to develop a normalization method that is robust against violation of the standard assumptions     

Determining the influence of structure on hybridization using oligonucleotide arrays.

We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.     

Genotyping of eight polymorphic genes encoding drug-metabolizing enzymes and transporters using a customized oligonucleotide array

Polymorphisms in drug-metabolizing genes may lead to the production of dysfunctional proteins and consequently affect therapeutic efficacy and toxicity of drugs. Different frequencies of polymorphic alleles among the races have been postulated to account for the observed ethnic variations in drug responses. In the current study, we aimed to estimate the frequencies of 14 polymorphisms in eight genes (TPMT, NQO1, MTHFR, GSTP1, CYP1A1, CYP2D6, ABCB1, and SLC19A1) in the Singapore multiracial populations by screening 371 cord blood samples from healthy newborns. To improve genotyping efficacy, we designed an oligonucleotide array based on the principle of allele-specific primer extension (AsPEX) that was capable of detecting the 14 polymorphisms simultaneously. Cross-validation using conventional polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) assays demonstrated 99% concordant results. Measurements on the fluorescent intensity displayed clear distinctions among different genotypes. Statistical analyses showed significantly different allele distributions in several genes among the three races, namely Chinese, Malays, and Indians. Comparing the allelic frequencies in Chinese with previous studies in Caucasian populations, NQO1 609C>T and SLC19A1 80G>A were distinctly different, whereas close similarity was observed for MTHFR 677C>T. We have demonstrated an array-based methodology for rapid multiplex detection of genetic polymorphisms. The allelic frequencies reported in this study may have important therapeutic and prognostic implications in the clinical use of relevant drugs.