High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Buetow, Kenneth H.; Edmonson, Michael N.; MacDonald, Richard; Clifford, Robert; Yip, Ping; Kelley, Jenny M.; Little, Damon P.; Strausberg, Robert L.; Koester, H.; Cantor, Charles R.; Braun, Andreas

doi:10.1073/pnas.98.2.581

Cited by 403 publications

(135 citation statements)

References 12 publications

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“…Fifteen SNPs from dbSNP were confirmed to be polymorphic in a pool of 94 Centre d'Etude du Polymorphisme Humaine DNA samples as described (21). The relative locations of these SNPs are shown in Fig.…”

Section: Preparation Of Single-stranded Template For Massextend (Seque-mentioning

confidence: 99%

Association testing by DNA pooling: An effective initial screen

Bansal

Boom²,

Kammerer³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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146

116

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With an ever-increasing resource of validated single-nucleotide polymorphisms (SNPs), the limiting factors in genome-wide association analysis have become genotyping capacity and the availability of DNA. We provide a proof of concept of the use of pooled DNA as a means of efficiently screening SNPs and prioritizing them for further study. This approach reduces the final number of SNPs that undergo full, sample-by-sample genotyping as well as the quantity of DNA used overall. We have examined 15 SNPs in the cholesteryl ester transfer protein (CETP) gene, a gene previously demonstrated to be associated with serum high-density lipoprotein cholesterol levels. The SNPs were amplified in two pools of DNA derived from groups of individuals with extremely high and extremely low serum highdensity lipoprotein cholesterol levels, respectively. P values <0.05 were obtained for 14 SNPs, supporting the described association. Genotyping of the individual samples showed that the average margin of error in frequency estimate was Ϸ4% when pools were used. These findings clearly demonstrate the potential of pooling techniques and their associated technologies as an initial screen in the search for genetic associations.A ssociation testing may be regarded as a comparison of allele frequency between cases and controls, in which a statistically significant difference is used to implicate a locus in a disease etiology. The methodology relies on the investigator testing either the causal variant itself or a marker in linkage disequilibrium (LD) with the causal variant. It is now well known that the distance over which LD extends is highly variable, not only between populations (1-3) but also across genomic regions (4, 5). A comprehensive genome scan by association analysis is therefore likely to require tens or hundreds of thousands of markers (6, 7).To date, we have performed validation experiments on Ϸ200,000 putative single-nucleotide polymorphisms (SNPs) from the public domain, and the confirmation rate is Ϸ60% in a Caucasian sample (unpublished results). The theoretical advantages of analyzing these SNPs by an association rather than by a linkage approach are clear (8). The reality, however, is hindered by the expense of generating the necessarily large number of genotypes. The use of pooled DNA has been offered as an alternative in the case of qualitative traits (9-11), but practical extensions to quantitative traits have not been presented thus far.To evaluate the applicability of pooled samples, SNPs in the cholesteryl ester transfer protein (CETP) gene were tested for association by using a pair of pools containing DNA from individuals with low and high serum high-density lipoprotein cholesterol (HDL-C) levels, respectively. CETP is known to play an essential role in reverse cholesterol metabolism. It is believed to mediate the exchange of cholesteryl ester in HDL-C for triglyceride in low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL) (12)(13)(14).

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Section: Preparation Of Single-stranded Template For Massextend (Seque-mentioning

confidence: 99%

Association testing by DNA pooling: An effective initial screen

Bansal

Boom²,

Kammerer³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

146

116

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“…haplotype tags), which could be useful in linkage disequilibrium studies. Expressed sequence tags (ESTs) are partial single-pass 400-600 bp sequences generated from cDNA libraries that provide an opportunity to detect these single-nucleotide differences among sequences derived from a same gene (Buetow et al, 2001). Since cDNA libraries are generated from a wide range of cancerous and normal tissues, some variations in genes might be directly related to the cancer phenotype.…”

Section: Introductionmentioning

confidence: 99%

In silico whole-genome scanning of cancer-associated nonsynonymous SNPs and molecular characterization of a dynein light chain tumour variant

et al. 2005

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Last decade has led to the accumulation of large amounts of data on cancer genetics, opening an unprecedented access to the mapping of cancer genes in the human genome. Single-nucleotide polymorphisms (SNPs), the most common form of DNA variation in humans, emerge as an invaluable tool for cancer association studies. These genotypic markers can be used to assay how alleles of candidate genes correlate with the malignant phenotype, and may provide new clues into the genetic modifications that characterize cancer onset. In this cancer-oriented study, we detail an SNP mining strategy based on the analysis of expressed sequence tags among publicly available databases. Our whole-genome approach provides a comprehensive and unbiased description of nonsynonymous SNPs (nsSNPs) in tumoral versus normal tissues. To gain further insights into the possible relationships between genetic variation and altered phenotype, locations of a subset of nsSNPs were mapped onto protein domains known to be critical for protein function. Computational methods were also used to predict the potential impact of these cancer-associated nsSNPs on protein structure and function. We illustrate our approach through the detailed biochemical and structural characterization of a previously unknown cancer-associated mutation (G79C) affecting the 8 kDa dynein light chain (DNCL1).

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“…This similarity of assay conditions permits extensive use of robotics, which limits human error. Mass spectrometry data collection is fast and automated, based on the size of extended products.The precision of mass spectrometry has been evaluated in a limited number of studies (19)(20)(21). Ross and coworkers (19) tested the quantitative range and detection limits of the technique and were able to quantitate allele frequencies as low as 0.05.…”

mentioning

confidence: 99%

“…The precision of mass spectrometry has been evaluated in a limited number of studies (19)(20)(21). Ross and coworkers (19) tested the quantitative range and detection limits of the technique and were able to quantitate allele frequencies as low as 0.05.…”

mentioning

confidence: 99%

High-throughput screening for evidence of association by using mass spectrometry genotyping on DNA pools

Mohlke

Erdos

Scott

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

120

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To facilitate positional cloning of complex trait susceptibility loci, we are investigating methods to reduce the effort required to identify trait-associated alleles. We examined primer extension analysis by matrix-assisted laser desorption͞ionization time-offlight mass spectrometry to screen single-nucleotide polymorphisms (SNPs) for association by using DNA pools. We tested whether this method can accurately estimate allele frequency differences between pools while maintaining the high-throughput nature of assay design, sample handling, and scoring. We follow up interesting allele frequency differences in pools by genotyping individuals. We tested DNA pools of 182, 228, and 499 individuals using 16 SNPs with minor allele frequencies 0.026 -0.486 and allele frequency differences 0.001-0.108 that we had genotyped previously on individuals and 381 SNPs that we had not. Precision, as measured by the average standard deviation among 16 semidependent replicates, was 0.021 ؎ 0.011 for the 16 SNPs and 0.018 ؎ 0.008 for the 291͞381 SNPs used in further analysis. For the 16 SNPs, the average absolute error in predicting allele frequency differences between pools was 0.009; the largest errors were 0.031, 0.028, and 0.027. We determined that compensating for unequal peak heights in heterozygotes improved precision of allele frequency estimates but had only a very minor effect on accuracy of allele frequency differences between pools. Based on these data and assuming pools of 500 individuals, we conclude that at significance level 0.05 we would have 95% (82%) power to detect population allele frequency differences of 0.07 for control allele frequencies of 0.10 (0.50).A ssociation studies provide a powerful approach to identify the DNA variants underlying complex traits (1). Currently, association studies can be especially useful for narrowing a complex trait candidate interval identified by linkage analysis (2, 3), although improved genotyping technology and a map of single-nucleotide polymorphisms (SNPs) identifying the common haplotypes in the human genome may enable association studies of loci spanning the entire genome. A rate-limiting step for association studies is to obtain the large number of genotypes needed. Currently, a linkage region expected to contain a complex trait locus typically spans 10-20 Mb, and even with a priori knowledge of the linkage disequilibrium between DNA variants, thousands of densely spaced SNPs with a range of allele frequencies may need to be screened (4). In addition, sample sizes of hundreds or even thousands of individuals may be required to have sufficient power to detect loci with modest effect.A reliable screening method to identify SNPs associated with disease without genotyping all individuals would be efficient and economical. Screening SNPs by typing a limited number of DNA pools representing cases and controls in principle requires vastly fewer genotypes for each SNP, reducing labor and reagent costs. Genotyping cost becomes essentially independent of sample size, allowing lar...

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High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cited by 403 publications

References 12 publications

Association testing by DNA pooling: An effective initial screen

Association testing by DNA pooling: An effective initial screen

In silico whole-genome scanning of cancer-associated nonsynonymous SNPs and molecular characterization of a dynein light chain tumour variant

High-throughput screening for evidence of association by using mass spectrometry genotyping on DNA pools

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