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PubMed2004

Characteristics of mutations at the D5S818 locus studied with a tightly linked marker.

BACKGROUND: Mutations to STR alleles are not well understood in terms of the mechanism(s) underlying such mutations or their relative frequency among different alleles at the locus. STUDY DESIGN AND METHODS: A cytosine/thymosine (C/T) polymorphism was discovered 13 nucleotides upstream from the 5'-end of the tandem array of the D5S818 locus (-13SNP). The -13SNP coincidentally creates a restriction site polymorphism for the restriction endonuclease SnaBI that is tightly linked to the tandem array of D5S818. Forty D5S818 addition/deletion mutations in the tandem array were characterized with RFLP analysis with SnaBI. RESULTS: Among 40 mutations studied, 34 (approximately 85%) were of paternal origin, 1 ( approximately 3%) was of maternal origin, and 5 (approximately 13%) had an unclear lineage of origin. In 26 cases where the magnitude of the repeat change could be determined, 23 (88%) involved changes of a single repeat unit, whereas 3 (12%) involved changes of 2 or more repeats. The number of additions to the tandem array was roughly equal to the number of deletions from the tandem array. In 19 instances it was possible to identify the parental allele that underwent the mutation. Alleles 13 and 14 were prone to mutation, whereas Allele 11 was resistant. Thus, there is an unequal sensitivity to mutation among D5S818 alleles. CONCLUSIONS: The use of the linked -13SNP marker has revealed several features of mutations at the D5S818 locus: 1) Single repeat changes are the most commonly observed. 2) Mutations are more likely in the paternal lineage. 3) Not all alleles undergo mutation with equal frequency.

Adult↗

PubMed2005

SNiPer: improved SNP genotype calling for Affymetrix 10K GeneChip microarray data.

BACKGROUND: High throughput microarray-based single nucleotide polymorphism (SNP) genotyping has revolutionized the way genome-wide linkage scans and association analyses are performed. One of the key features of the array-based GeneChip Mapping 10K Array from Affymetrix is the automated SNP calling algorithm. The Affymetrix algorithm was trained on a database of ethnically diverse DNA samples to create SNP call zones that are used as static models to make genotype calls for experimental data. We describe here the implementation of clustering algorithms on large training datasets resulting in improved SNP call rates on the 10K GeneChip. RESULTS: A database of 948 individuals genotyped on the GeneChip Mapping 10K 2.0 Array was used to identify 822 SNPs that were called consistently less than 75% of the time. These SNPs represent on average 8.25% of the total SNPs on each chromosome with chromosome 19, the most gene-rich chromosome, containing the highest proportion of poor performers (18.7%). To remedy this, we created SNiPer, a new application which uses two clustering algorithms to yield increased call rates and equivalent concordance to Affymetrix called genotypes. We include a training set for these algorithms based on individual genotypes for 705 samples. SNiPer has the capability to be retrained for lab-specific training sets. SNiPer is freely available for download at http://www.tgen.org/neurogenomics/data. CONCLUSION: The correct calling of poor performing SNPs may prove to be key in future linkage studies performed on the 10K GeneChip. It would prove particularly invaluable for those diseases that map to chromosome 19, known to contain a high proportion of poorly performing SNPs. Our results illustrate that SNiPer can be used to increase call rates on the 10K GeneChip without sacrificing accuracy, thereby increasing the amount of valid data generated.

Algorithms↗

PubMed2001

[The Rsa I and Sma I polymorphisms in the von Willebrand factor gene discrimination by hybridization with oligonucleotide arrays].

OBJECTIVE: To develop a new assay for the Rsa I and Sma I polymorphisms (single nucleotide polymorphism, SNP) in the von Willebrand factor gene detection that utilizes oligonucleotide arrays (DNA chips) on glass supports. Expoloring the association of the RsaI and SmaI polymorphisms in the von Willebrand factor gene with thrombosis in Chinese. METHODS: Allele-specific oligonucleotides are covalently immobilized on glutaradehyde derivatized glass slides in arrays. Single strand PCR product of PCR-amplified genomic DNA is fluorescently labeled by assymetric PCR with fluorescently tagged dUTP and hybridized to the support-bound oligonucleotide array. The hybridization pattern is detected by fluorescence scenning. The effect of hybridization conditions were evaluated and optimized. The method was validated by the discrimination of blinded DNA samples identified by restriction enzymes. 50 patients with thrombosis were examined by this method. RESULTS: The genotypes scored by oligonucleotide arrays assay were in 100% agreement with restriction enzymes results. The genotype distributions GG, GA, AA and CC, CT, TT of RsaI and SmaI polymorphisms of patients or controls were 4.0%, 12.0%, 84.0% and 24.0%, 44.0%, 32.0% or 1.4%, 11.8%, 86.8% and 8.8%, 57.4%, 33.8%. The allele frequencies G, A and C, T were 10.0%, 90.0% and 46.0%, 54.0% or 7.4%, 92.6% and 37.5%, 62.5%. No statistical difference in genotype distribution and allele frequencies was observed between patients with thrombosis and the control groups (all P > 0.05). CONCLUSION: A rapid and accurate method for the RsaI and SmaI polymorphisms in the von Willebrand factor gene detection, or for the analysis of single nucleotide polymorphism (SNP) has been developed using oligonucleotide arrays on glass supports. No association was observed between the Rsa I and Sma I polymorphisms in the vWF gene and thrombotic disease.

Alleles↗

PubMed2006

Oligonucleotide-arrayed TFT photosensor applicable for DNA chip technology.

A thin film transistor (TFT) photosensor fabricated by semiconductor integrated circuit (IC) technology was applied to DNA chip technology. The surface of the TFT photosensor was coated with TiO2 using a vapor deposition technique for the fabrication of optical filters. The immobilization of thiolated oligonucleotide probes onto a TiO2-coated TFT photosensor using gamma-aminopropyltriethoxysilane (APTES) and N-(gamma-maleimidobutyloxy) sulfosuccinimide ester (GMBS) was optimized. The coverage value of immobilized oligonucleotides reached a plateau at 33.7 pmol/cm2, which was similar to a previous analysis using radioisotope-labeled oligonucleotides. The lowest detection limits were 0.05 pmol/cm2 for quantum dot and 2.1 pmol/cm2 for Alexa Fluor 350. Furthermore, single nucleotide polymorphism (SNP) detection was examined using the oligonucleotide-arrayed TFT photosensor. A SNP present in the aldehyde dehydrogenase 2 (ALDH2) gene was used as a target. The SNPs in ALDH2*1 and ALDH2*2 target DNA were detected successfully using the TFT photosensor. DNA hybridization in the presence of both ALDH2*1 and ALDH2*2 target DNA was observed using both ALDH2*1 and ALDH2*2 detection oligonucleotides-arrayed TFT photosensor. Use of the TFT photosensor will allow the development of a disposable photodetecting device for DNA chip systems.

Biosensing Techniques↗

PubMed2006

Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex changes and multiple forms of chromosomal instability in colorectal cancers.

Cancers with chromosomal instability (CIN) are held to be aneuploid/polyploid with multiple large-scale gains/deletions, but the processes underlying CIN are unclear and different types of CIN might exist. We investigated colorectal cancer cell lines using array-comparative genomic hybridization (CGH) for copy number changes and single-copy number polymorphism (SNP) microarrays for allelic loss (LOH). Many array-based CGH changes were not found by LOH because they did not cause true reduction-to-homozygosity. Conversely, many regions of SNP-LOH occurred in the absence of copy number change, comprising an average per cell line of 2 chromosomes with complete LOH; 1-2 terminal regions of LOH (mitotic recombination); and 1 interstitial region of LOH. SNP-LOH detected many novel changes, representing possible locations of uncharacterized tumor suppressor loci. Microsatellite unstable (MSI+) lines infrequently showed gains/deletions or whole-chromosome LOH, but their near-diploid karyotypes concealed mitotic recombination frequencies similar to those of MSI- lines. We analyzed p53 and chromosome 18q (SMAD4) in detail, including mutation screening. Almost all MSI- lines showed LOH and/or deletion of p53 and 18q; some near-triploid lines had acquired three independent changes at these loci. We found consistent results in primary colorectal cancers. Overall, the distributions of mitotic recombination and whole-chromosome LOH in the MSI- cell lines differed significantly from random, with some lines having much higher than expected levels of these changes. Moreover, lines with more LOH changes had significantly fewer copy number changes. These data suggest that CIN is not synonymous with copy number change and some cancers have a specific tendency to whole-chromosome deletion and regain or to mitotic recombination.

Cell Line, Tumor↗

PubMed2006

Long contiguous stretches of homozygosity in the human genome.

Single nucleotide polymorphisms (SNPs) are the most common sequence variation in the human genome; they have been successfully used in mapping disease genes and more recently in studying population genetics and cancer genetics. In a population-based association study using high-density oligonucleotide arrays for whole-genome SNP genotyping, we discovered that in the genomes of unrelated Han Chinese, 34 out of 515 (6.6%) individuals contained long contiguous stretches of homozygosity (LCSHs), ranging in the size from 2.94 to 26.27 Mbp (10.22+/-5.95 Mbp). Four out of four (100%) Taiwan aborigines also demonstrated this genetic characteristic. The number of LCSH regions increased markedly in the offspring of consanguineous marriages. LCSH was also detected in Caucasian samples (11/42; 26.2%) and African American samples (2/42; 4.76%). A total of 26 LCSH regions were recurrently detected among Han Chinese, Taiwan aborigines, and Caucasians. DNA copy number determination by hybridization intensity analysis and real-time quantitative PCR (qPCR) excluded deletion as the cause of LCSH. Our results suggest that LCSHs are common in the human genome of the outbred population and this genetic characteristic could have a significant impact on population genetics and disease gene studies.

Black or African American↗

PubMed2005

A genome-wide linkage analysis of alcoholism on microsatellite and single-nucleotide polymorphism data, using alcohol dependence phenotypes and electroencephalogram measures.

The Collaborative Study on the Genetics of Alcoholism (COGA) is a large-scale family study designed to identify genes that affect the risk for alcoholism and alcohol-related phenotypes. We performed genome-wide linkage analyses on the COGA data made available to participants in the Genetic Analysis Workshop 14 (GAW 14). The dataset comprised 1,350 participants from 143 families. The samples were analyzed on three technologies: microsatellites spaced at 10 cM, Affymetrix GeneChip Human Mapping 10 K Array (HMA10K) and Illumina SNP-based Linkage III Panel. We used ALDX1 and ALDX2, the COGA definitions of alcohol dependence, as well as electrophysiological measures TTTH1 and ECB21 to detect alcoholism susceptibility loci. Many chromosomal regions were found to be significant for each of the phenotypes at a p-value of 0.05. The most significant region for ALDX1 is on chromosome 7, with a maximum LOD score of 2.25 for Affymetrix SNPs, 1.97 for Illumina SNPs, and 1.72 for microsatellites. The same regions on chromosome 7 (96-106 cM) and 10 (149-176 cM) were found to be significant for both ALDX1 and ALDX2. A region on chromosome 7 (112-153 cM) and a region on chromosome 6 (169-185 cM) were identified as the most significant regions for TTTH1 and ECB21, respectively. We also performed linkage analysis on denser maps of markers by combining the SNPs datasets from Affymetrix and Illumina. Adding the microsatellite data to the combined SNP dataset improved the results only marginally. The results indicated that SNPs outperform microsatellites with the densest marker sets performing the best.

Alcoholism↗

PubMed2026

Genome-wide association study and KASP development for growth and leaf traits in Populus deltoides.

BACKGROUND: Populus deltoides is a valuable timber species of considerable importance in the study of forest genetic breeding. However, its genetic improvement continues to rely predominantly on conventional selection and hybridization strategies hampered by long breeding cycles and limited efficiency. RESULTS: A total of 209 P. deltoides accessions were genotyped using a 60K SNP (Single nucleotide polymorphism) liquid array. Following quality control, 46,031 high-quality SNPs were screened and analyzed alongside 15 phenotypic traits in a genome-wide association study (GWAS), which identified 219 SNPs significantly associated with the traits. After further screening and annotation, a final set of 57 target SNPs and 77 candidate genes was obtained. Using kompetitive allele-specific PCR (KASP) assays, we successfully developed 48 polymorphic KASP markers. Of these, 25 markers exhibited significant phenotypic differences (p < 0.05) across genotype groups. CONCLUSIONS: These 25 KASP markers can serve as reliable and practical tools for phenotype-assisted selection, providing efficient molecular resources for accelerating genetic improvement and marker-assisted breeding in poplar.

Populus↗

PubMed2003

A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings.

Detection of unknown single nucleotide polymorphism (SNP) relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos), the other includes oligos specific for known SNP within the same genomic region (variant-oligos). Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.

Journal Article↗

PubMed2005

A genome-wide scalable SNP genotyping assay using microarray technology.

Oligonucleotide probe arrays have enabled massively parallel analysis of gene expression levels from a single cDNA sample. Application of microarray technology to analyzing genomic DNA has been stymied by the sequence complexity of the entire human genome. A robust, single base-resolution direct genomic assay would extend the reach of microarray technology. We developed an array-based whole-genome genotyping assay that does not require PCR and enables effectively unlimited multiplexing. The assay achieves a high signal-to-noise ratio by combining specific hybridization of picomolar concentrations of whole genome-amplified DNA to arrayed probes with allele-specific primer extension and signal amplification. As proof of principle, we genotyped several hundred previously characterized SNPs. The conversion rate, call rate and accuracy were comparable to those of high-performance PCR-based genotyping assays.

Computational Biology↗

PubMed2006

MACGT: multi-dimensional automated clustering genotyping tool for analysis of microarray-based mini-sequencing data.

SUMMARY: Multi-dimensional Automated Clustering Genotyping Tool (MACGT) is a Java application that clusters complex multi-dimensional vector data derived from single nucleotide polymorphism (SNP) genotyping experiments using mini-sequencing based microarray chemistries such as arrayed primer extension (APEX). Spot intensity output files from microarray experiments across multiple samples are imported into MACGT. The datasets can include four channels of intensity data for each spot, replica spots for each SNP probe and multiple probe types (APEX and allele-specific APEX probes) on both DNA strands for each SNP. MACGT automatically clusters these multi-dimensionality datasets for each SNP across multiple samples. Incorporation of additional array datasets from known samples that have previously validated SNP genotype calls allows unknown samples to be automatically assigned a genotype based on the clustering, along with numerical measures of confidence for each genotype call. Calling accuracy by MACGT exceeds 98% when applied to genotyping data from APEX microarrays, and can be increased to >99.5% by applying thresholds to the confidence measures.

Algorithms↗

PubMed2006

CARAT: a novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays.

BACKGROUND: DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. RESULTS: We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100,000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. CONCLUSION: Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Algorithms↗

PubMed2005

Determination of affinity constants of locked nucleic acid (LNA) and DNA duplex formation using label free sensor technology.

Locked nucleic acid (LNA) is a nucleic acid analogue containing 2'-O,4'-C-methylene-beta-D-ribofuranosyl nucleotides, which have a bicyclic furanose unit locked in a RNA mimicking sugar conformation. Oligonucleotides containing LNA monomers show an enhanced thermal stability and robustness against nuclease mediated cleavage. Therefore special tailored LNA is a versatile tool for gene array analysis and single nucleotide polymorphism (SNP) analysis. The higher melting temperatures result from a higher affinity between the LNA and its complementary base. This was verified by the determination of the affinity constants of the duplex formation of 3 oligonucleotides: DNA, L-DNA, in which all thymidines are substituted by LNA, and a fully modified LNA, to their complementary DNA strand. Affinity constants were calculated to be 1.5 x 10(9), 4.0 x 10(9) and >10(12) L mol(-1). This was done using the label free and time resolved sensing technology reflectometric interference spectroscopy (RIfS), in an assay format similar to a titration called binding inhibition assay.

Chromatography, Affinity↗

PubMed2004

SNP Chart: an integrated platform for visualization and interpretation of microarray genotyping data.

UNLABELLED: SNP Chart is a Java application for the visualization and interpretation of microarray genotyping data primarily derived from arrayed primer extension-based chemistries. Spot intensity output files from microarray analysis tools are imported into SNP Chart, together with a multi-channel TIFF image of the original array experiment and a list of the actual single nucleotide polymorphisms (SNPs) being tested. Data from different and/or replicate probes that interrogate the same SNP, but that are scattered across the array grid, can be reassembled into a single chart format, specific for the SNP. This allows a quick and very effective 'visualization'/'quality control' of the data from multiple probes for the same SNP that can be easily interpreted and manually scored as a genotype. AVAILABILITY: http://www.snpchart.ca.

Computer Graphics↗

PubMed2003

Heteroduplex-based genotyping with microchip electrophoresis and dHPLC.

This work compares the methods of mutation detection via denaturing high-performance liquid chromatography (dHPLC) and a microchip-based heteroduplex analysis (HA) method. The mutations analyzed were 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 with, as additional examples, 188del11 and 5396 + 1G --> A in BRCA1. Our HA method is based upon the use of a replaceable, highly denaturing sieving matrix that has dynamic coating capabilities, rendering our method relatively insensitive to contamination. We have found significant advantages in the microchip analysis in terms of reagent consumption, ease of use, versatility, simplicity of the protocol, the lack of constraints upon sample preparation or content, and the lack of parameters that need be adjusted. Although HA methods have a lower sensitivity than that of dHPLC, the electropherograms of the present HA method appear to provide more information and may allow mutations within the same amplicon to be distinguished. Although the dHPLC method has a remarkably high sensitivity, with this sensitivity there come constraints that may prevent it, in its present form, from being used in some applications, particularly those involving higher levels of integration. The advantages of the present HA method, along with recent developments in microchip-based single-nucleotide polymorphism (SNP) detection and high-throughput arrays, suggest that microchip-based systems could provide compact and integrated platforms capable of large-scale genotyping or mutational screening.

BRCA1 Protein↗

PubMed2006

Genome-wide tagging SNPs with entropy-based Monte Carlo method.

The number of common single nucleotide polymorphisms (SNPs) in the human genome is estimated to be around 3-6 million. It is highly anticipated that the study of SNPs will help provide a means for elucidating the genetic component of complex diseases and variable drug responses. High-throughput technologies such as oligonucleotide arrays have produced enormous amount of SNP data, which creates great challenges in genome-wide disease linkage and association studies. In this paper, we present an adaptation of the cross entropy (CE) method and propose an iterative CE Monte Carlo (CEMC) algorithm for tagging SNP selection. This differs from most of SNP selection algorithms in the literature in that our method is independent of the notion of haplotype block. Thus, the method is applicable to whole genome SNP selection without prior knowledge of block boundaries. We applied this block-free algorithm to three large datasets (two simulated and one real) that are in the order of thousands of SNPs. The successful applications to these large scale datasets demonstrate that CEMC is computationally feasible for whole genome SNP selection. Furthermore, the results show that CEMC is significantly better than random selection, and it also outperformed another block-free selection algorithm for the dataset considered.

Algorithms↗

PubMed2006

Asthma families show transmission disequilibrium of gene variants in the vitamin D metabolism and signalling pathway.

The vitamin D prophylaxis of rickets in pregnant women and newborns may play a role in early allergic sensitization. We now asked if an already diseased population may have inherited genetic variants in the vitamin D turnover or signalling pathway. Serum levels of calcidiol (25-OH-D3) and calcitriol (1,25-(OH)2-D3) were retrospectively assessed in 872 participants of the German Asthma Family Study. 96 DNA single base variants in 13 different genes were genotyped with MALDI-TOF and a bead array system. At least one positive SNP with a TDT of p < 0.05 for asthma or total IgE and calcidiol or calcitriol was seen in IL10, GC, IL12B, CYP2R1, IL4R, and CYP24A1. Consistent strong genotypic association could not be observed. Haplotype association were found only for CYP24A1, the main calcidiol degrading enzyme, where a frequent 5-point-haplotype was associated with asthma (p = 0.00063), total IgE (p = 0.0014), calcidiol (p = 0.0043) and calcitriol (p = 0.0046). Genetic analysis of biological pathways seem to be a promising approach where this may be a first entry point into effects of a polygenic inherited vitamin D sensitivity that may affect also other metabolic, immunological and cancerous diseases.

Adolescent↗

PubMed2005

Silhouette scores for assessment of SNP genotype clusters.

BACKGROUND: High-throughput genotyping of single nucleotide polymorphisms (SNPs) generates large amounts of data. In many SNP genotyping assays, the genotype assignment is based on scatter plots of signals corresponding to the two SNP alleles. In a robust assay the three clusters that define the genotypes are well separated and the distances between the data points within a cluster are short. "Silhouettes" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a cluster. Thus "Silhouettes" can potentially be used as a quality measure for SNP genotyping results and for objective comparison of the performance of SNP assays at different circumstances. RESULTS: We created a program (ClusterA) for calculating "Silhouette scores", and applied it to assess the quality of SNP genotype clusters obtained by single nucleotide primer extension ("minisequencing") in the Tag-microarray format. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment has been arbitrary. In the present study we applied Silhouette scores to compare the performance of four DNA polymerases in our minisequencing system by analyzing 26 SNPs in both DNA polarities in 16 DNA samples. We found Silhouettes to provide a relevant measure for the quality of SNP assays at different reaction conditions, illustrated by the four DNA polymerases here. According to our result, the genotypes can be unequivocally assigned without manual inspection when the Silhouette score for a SNP assay is > 0.65. All four DNA polymerases performed satisfactorily in our Tag-array minisequencing system. CONCLUSION: "Silhouette scores" for assessing the quality of SNP genotyping clusters is convenient for evaluating the quality of SNP genotype assignment, and provides an objective, numeric measure for comparing the performance of SNP assays. The program we created for calculating Silhouette scores is freely available, and can be used for quality assessment of the results from all genotyping systems, where the genotypes are assigned by cluster analysis using scatter plots.

Alleles↗