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PubMed2006

A model-based approach to selection of tag SNPs.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) are the most common type of polymorphisms found in the human genome. Effective genetic association studies require the identification of sets of tag SNPs that capture as much haplotype information as possible. Tag SNP selection is analogous to the problem of data compression in information theory. According to Shannon's framework, the optimal tag set maximizes the entropy of the tag SNPs subject to constraints on the number of SNPs. This approach requires an appropriate probabilistic model. Compared to simple measures of Linkage Disequilibrium (LD), a good model of haplotype sequences can more accurately account for LD structure. It also provides a machinery for the prediction of tagged SNPs and thereby to assess the performances of tag sets through their ability to predict larger SNP sets. RESULTS: Here, we compute the description code-lengths of SNP data for an array of models and we develop tag SNP selection methods based on these models and the strategy of entropy maximization. Using data sets from the HapMap and ENCODE projects, we show that the hidden Markov model introduced by Li and Stephens outperforms the other models in several aspects: description code-length of SNP data, information content of tag sets, and prediction of tagged SNPs. This is the first use of this model in the context of tag SNP selection. CONCLUSION: Our study provides strong evidence that the tag sets selected by our best method, based on Li and Stephens model, outperform those chosen by several existing methods. The results also suggest that information content evaluated with a good model is more sensitive for assessing the quality of a tagging set than the correct prediction rate of tagged SNPs. Besides, we show that haplotype phase uncertainty has an almost negligible impact on the ability of good tag sets to predict tagged SNPs. This justifies the selection of tag SNPs on the basis of haplotype informativeness, although genotyping studies do not directly assess haplotypes. A software that implements our approach is available.

Databases, Genetic↗

PubMed1999

High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays.

A highly reliable and efficient technology has been developed for high-throughput DNA polymorphism screening and large-scale genotyping. Photolithographic synthesis has been used to generate miniaturized, high-density oligonucleotide arrays. Dedicated instrumentation and software have been developed for array hybridization, fluorescent detection, and data acquisition and analysis. Specific oligonucleotide probe arrays have been designed to rapidly screen human STSs, known genes and full-length cDNAs. This has led to the identification of several thousand biallelic single-nucleotide polymorphisms (SNPs). Meanwhile, a rapid and robust method has been developed for genotyping these SNPs using oligonucleotide arrays. Each allele of an SNP marker is represented on the array by a set of perfect match and mismatch probes. Prototype genotyping chips have been produced to detect 400, 600 and 3000 of these SNPs. Based on the preliminary results, using oligonucleotide arrays to genotype several thousand polymorphic loci simultaneously appears feasible.

Genetic Markers↗

PubMed2006

Dynamic variable selection in SNP genotype autocalling from APEX microarray data.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are DNA sequence variations, occurring when a single nucleotide--adenine (A), thymine (T), cytosine (C) or guanine (G)--is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX). This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart) is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias. RESULTS: Using a set of 32 Coriell DNA samples plus three negative PCR controls as a training data set, we have developed a fully-automated genotyping algorithm based on simple linear discriminant analysis (LDA) using dynamic variable selection. The algorithm combines separate analyses based on the multiple probe sets to give a final posterior probability for each candidate genotype. We have tested our algorithm on a completely independent data set of 270 DNA samples, with validated genotypes, from patients admitted to the intensive care unit (ICU) of St. Paul's Hospital (plus one negative PCR control sample). Our method achieves a concordance rate of 98.9% with a 99.6% call rate for a set of 96 SNPs. By adjusting the threshold value for the final posterior probability of the called genotype, the call rate reduces to 94.9% with a higher concordance rate of 99.6%. We also reversed the two independent data sets in their training and testing roles, achieving a concordance rate up to 99.8%. CONCLUSION: The strength of this APEX chemistry-based platform is its unique redundancy having multiple probes for a single SNP. Our model-based genotype calling algorithm captures the redundancy in the system considering all the underlying probe features of a particular SNP, automatically down-weighting any 'bad data' corresponding to image artifacts on the microarray slide or failure of a specific chemistry. In this regard, our method is able to automatically select the probes which work well and reduce the effect of other so-called bad performing probes in a sample-specific manner, for any number of SNPs.

Algorithms↗

PubMed2006

Rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms.

This report describes a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis is based on dynamic allele-specific hybridization (DASH). The DNA duplexes are conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. The method is based on random bead immobilization by microcontact printing. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Analyses that took more than 15 min could be performed in less that 1 min, enabling ultrarapid SNP analysis. In addition, an array version of the chip was implemented enabling the preparation of an array of bead arrays for high-throughput and rapid SNP genotyping.

Alleles↗

PubMed2000

High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates.

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.

Alleles↗

PubMed2002

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery.

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy

Alleles↗

PubMed1999

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Arabidopsis↗

PubMed2003

Effect of secondary structure on single nucleotide polymorphism detection with a porous microarray matrix; implications for probe selection.

Oligonucleotide arrays capable of detecting single nucleotide polymorphisms (SNPs) from amplified nucleic acid have many applications. The expected SNP is usually placed approximately in the center of the probe to ensure the maximum shift in Tm between complementary and SNP sequences. Unfortunately, different short probes (< 30 bases) selected using widely accepted criteria do not perform consistently in this type of assay. Here we present a systematic study on the effect of secondary structure on the ability of oligonucleotide probes to detect an SNP, using real-time array monitoring of a porous microarray substrate that incorporates a novel intra-array mixing system. These results demonstrate that, although positioning of an SNP in the middle of the probe is highly destabilizing, the effect of stable secondary structure on the signal obtained is so dramatic that such probes may be very insensitive. Therefore, if the SNP flanking sequence contains significant secondary structure, then more sensitive probes with good specificity may be obtained by positioning the mutation towards one end of the probe.

Artifacts↗

PubMed2006

Optimal two-stage genotyping designs for genome-wide association scans.

The much-anticipated fixed-array, genome-wide SNP genotyping technologies make large-scale genome-wide association scans now possible for large numbers of subjects. In this paper we reconsider the problem (Satagopan and Elston [2003] Genet Epidemiol 25:149-157) of optimizing a two-stage genotyping design to deal with important new issues that are relevant when studies are expanded from candidate gene size to a genome-wide scale. We investigate how the basic two-stage genotyping approach, in which all markers are genotyped in an initial group of subjects (stage I) and only the promising markers are genotyped in additional subjects (stage II), can be used to reduce genotyping cost in a genome-wide case-control association study even after allowing for much higher per genotype costs using specially designed assays in stage II, compared to the fixed array of SNPs used in stage I. In addition, we consider the problem of using measured SNPs to make (imperfect) prediction of unmeasured SNPs for association tests of all SNPs (measured or unmeasured) genome wide and the utility of expanding genotyping densities in stage II in the regions where significant associations were detected in stage I. Under a set of reasonable but conservative assumptions, we derive optimal two-stage design configurations (sample sizes and the thresholds of significance in both stages) with these optimal designs depending both on the total number of markers tested and upon the ratios of cost in stage II versus stage I. In addition we show how existing software for power and sample size calculations can be used for the purpose of designing two-stage studies, for a wide range of assumptions about the number of markers genotyped and the costs of genotyping in each stage of the study.

Case-Control Studies↗

PubMed2004

Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification.

Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.

Alleles↗

PubMed2006

Multiplexed SNP genotyping using nanobarcode particle technology.

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.

DNA↗

PubMed2005

[Application of oligonucleotide array in single nucleotide polymorphism typing of cytokines and cytokine receptors].

OBJECTIVE: To develop an oligonucleotide array for single nucleotide polymorphism (SNP) typing of cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-10, tumor growth factor (TGF)-betal, IL-4, and IL-6, and their receptors and evaluate its function by direct sequencing. METHODS: According to relevant literature, SNP database of NCBI and SNP500 Cancer database of NCI, SNP loci and sequences of cytokines of clinical importance, TNF-a, IL-10, TGF-bl, IL-4 and IL-6, and matched cytokine receptors were chosen and 59 synthesized oligonucleotide probes were immobilized on a glass support, then the primer and Cy5-dCTP were used in multi-PCR, thus the products were labeled with Cy5. The labeled PCR products were hybridized with the probes in the array, and the signals were scanned by Scanner and then analyzed by Image software. Peripheral blood samples were collected from 80 healthy donors and 50 patients with uremia to isolate lymphocytes and DNA so as to undergo typing by this array, and the results were validated with direct sequencing. RESULTS: All the samples with PCR products except for 10 from uremia patients had been genotyped by cytokine array successfully. No diversity was found in genotyping for three times. Incorrect locus was not found with direct sequencing. CONCLUSION: With high specificity, sensitivity, repetitiveness, and throughout, and easy to manipulate, oligonucleotide array technique is an ideal molecular method for SNP genotyping of cytokine and cytokine receptor.

Cytokines↗

PubMed2003

DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays.

Genotyping technologies need to be continually improved in terms of their flexibility, cost-efficiency, and throughput, to push forward genome variation analysis. To this end, we have leveraged the inherent simplicity of dynamic allele-specific hybridization (DASH) and coupled it to recent innovations of centrifugal arrays and iFRET. We have thereby created a new genotyping platform we term DASH-2, which we demonstrate and evaluate in this report. The system is highly flexible in many ways (any plate format, PCR multiplexing, serial and parallel array processing, spectral-multiplexing of hybridization probes), thus supporting a wide range of application scales and objectives. Precision is demonstrated to be in the range 99.8-100%, and assay costs are 0.05 USD or less per genotype assignment. DASH-2 thus provides a powerful new alternative for genotyping practice, which can be used without the need for expensive robotics support.

Alleles↗

PubMed2026

First genomic insights into the introgression of almond PPV-Marcus resistance into peach.

AIM: Sharka, caused by Plum pox virus (PPV), is one of the most damaging viral diseases of stone fruit crops, with peach among the most susceptible cultivated Prunus species. Almond is a promising source of resistance, but its genetic architecture and expression in a peach genetic background remain largely unknown. This study aimed to construct parental genetic linkage maps and identify genomic regions associated with PPV response in almond × peach interspecific populations. METHODS: Progenies derived from the almond cultivars 'Del Cid', 'Garrigues', and 'Mono' were evaluated by RT-PCR after graft inoculation with the PPV-Marcus (PPV-M) strain over consecutive infection cycles. Phenotypic data were summarized for each genotype using best linear unbiased estimates (BLUEs). High-density SNP almond and peach arrays were used to construct parental maps for 'Garrigues' and 'Mono' and perform quantitative trait locus (QTL) analysis. RESULTS: Phenotypic variation was observed among and within families. 'Del Cid'-derived progenies showed the greatest resistance, 'Garrigues'-derived progenies displayed intermediate responses, and 'Mono'-derived progenies showed greater susceptibility and variability. The parental maps covered 546.69 cM in 'Mono' and 521.72 cM in 'Garrigues', with average intervals of 0.61 and 1.26 cM per unique marker position, respectively, and showed strong collinearity with the reference genome. QTL associated with PPV-M response were detected on linkage groups (LG) 1 and 6 in 'Garrigues' and LG2 in 'Mono'. The main QTL in 'Garrigues' peaked near 22.39 Mb on LG1, whereas the 'Mono' QTL was located at 22.27-22.62 Mb on LG2; a weaker QTL was detected near 25.38 Mb on LG6 in 'Garrigues'. The results support a quantitative and genetic-background-dependent architecture of PPV resistance. CONCLUSION: This study provides the first evidence of genomic regions associated with PPV-Marcus response in almond × peach populations. The detected QTLs provide an initial basis to support the introgression of almond-derived resistance into peach breeding material.

Prunus↗

PubMed2000

A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays.

This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3'-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.

Alleles↗

PubMed2006

Genotyping pooled DNA using 100K SNP microarrays: a step towards genomewide association scans.

The identification of quantitative trait loci (QTLs) of small effect size that underlie complex traits poses a particular challenge for geneticists due to the large sample sizes and large numbers of genetic markers required for genomewide association scans. An efficient solution for screening purposes is to combine single nucleotide polymorphism (SNP) microarrays and DNA pooling (SNP-MaP), an approach that has been shown to be valid, reliable and accurate in deriving relative allele frequency estimates from pooled DNA for groups such as cases and controls for 10K SNP microarrays. However, in order to conduct a genomewide association study many more SNP markers are needed. To this end, we assessed the validity and reliability of the SNP-MaP method using Affymetrix GeneChip Mapping 100K Array set. Interpretable results emerged for 95% of the SNPs (nearly 110,000 SNPs). We found that SNP-MaP allele frequency estimates correlated 0.939 with allele frequencies for 97 605 SNPs that were genotyped individually in an independent population; the correlation was 0.971 for 26 SNPs that were genotyped individually for the 1028 individuals used to construct the DNA pools. We conclude that extending the SNP-MaP method to the Affymetrix GeneChip Mapping 100K Array set provides a useful screen of >100,000 SNP markers for QTL association scans.

DNA↗

PubMed2026

SNP genotyping in Pseudotsuga menziesii and Pinus radiata using targeted genotyping-by-sequencing (GBS): improved Bayesian SNP calling using a beta-binomial distribution and other optimized input parameters.

BACKGROUND: Single-nucleotide polymorphism markers (SNPs) have important applications in gene conservation, breeding, and fundamental genetics research. Our long-term goal is to develop routine approaches for SNP genotyping in forest trees. Ideally, these approaches would be inexpensive, able to accommodate a wide range of samples and SNPs, available through commercial providers, and produce high-quality SNP data. RESULTS: Using targeted genotyping-by-sequencing (GBS), we developed SNP assays for two highly heterozygous tree species, Douglas-fir (Pseudotsuga menziesii) and radiata pine (Pinus radiata). Using Douglas-fir haploid and diploid data, we optimized Bayesian SNP calling by testing four input parameters: (1) allele and genotype prior probabilities, (2) Rho, the beta-binomial dispersion parameter, (3) estimated read error (BayesReadError), and (4) the logPO cutoff used to filter low confidence SNP calls. logPO is the Bayesian posterior odds ratio for a called SNP. Compared to assuming a binomial distribution of read counts (Rho = 0), the beta-binomial distribution (Rho = 0.33) substantially reduced call error and heterozygote undercalling. Compared to the other Bayesian parameters, genotype priors had little effect on genotyping success. For Douglas-fir, we tested 5,360 SNP assays, and then studied the performance of the best 4,000. For radiata pine, we tested 6,000 SNP assays, and then studied the performance of the best 4,570. In Douglas-fir and radiata pine, our Bayesian approach resulted in median call rates of 95% to 98% for the top-ranked SNPs, with an estimated call error of 1.60% for known homozygous genotypes and 2.27% for known heterozygotes. In radiata pine, median and mean call rates were above 91% for GBS and SNP genotyping using an Axiom fixed genotyping array. Additionally, the median correspondence between the GBS and Axiom genotypes was about 98% overall (mean 96%). CONCLUSIONS: By optimizing Bayesian SNP calling, selecting the best 4-5 K SNPs, and excluding samples with low DNA amounts, we substantially reduced call error and heterozygote undercalling, resulting in SNP genotypes that were nearly identical to genotypes obtained using the Axiom array. Furthermore, genotyping performance should increase even further if our SNP rankings were used to develop less complex probe pools that target fewer SNPs.

Pinus↗

PubMed2005

DigiTag assay for multiplex single nucleotide polymorphism typing with high success rate.

As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost.

DNA↗