DNA Pooling: a tool for large-scale association studies
TL;DR: Recent developments in quantitative genotyping assays and in the design and analysis of pooling studies are discussed.
Abstract: DNA pooling is a practical way to reduce the cost of large-scale association studies to identify susceptibility loci for common diseases. Pooling allows allele frequencies in groups of individuals to be measured using far fewer PCR reactions and genotyping assays than are used when genotyping individuals. Here, we discuss recent developments in quantitative genotyping assays and in the design and analysis of pooling studies. Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and inter-loci interactions, and that allow the analysis of haplotypes.
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TL;DR: Genome-wide association studies will soon become possible, and could open new frontiers in the understanding and treatment of disease, however, the execution and analysis of such studies will require great care.
Abstract: Genetic factors strongly affect susceptibility to common diseases and also influence disease-related quantitative traits. Identifying the relevant genes has been difficult, in part because each causal gene only makes a small contribution to overall heritability. Genetic association studies offer a potentially powerful approach for mapping causal genes with modest effects, but are limited because only a small number of genes can be studied at a time. Genome-wide association studies will soon become possible, and could open new frontiers in our understanding and treatment of disease. However, the execution and analysis of such studies will require great care.
2,912 citations
Wellcome Trust Centre for Human Genetics1, University of Oxford2, University of Michigan3, Fred Hutchinson Cancer Research Center4, Duke University5, University of Ottawa6, Tufts University7, Foundation for Research & Technology – Hellas8, Broad Institute9, Harvard University10, Boston Children's Hospital11
TL;DR: This Review highlights the knowledge gained, defines areas of emerging consensus, and describes the challenges that remain as researchers seek to obtain more complete descriptions of the susceptibility architecture of biomedical traits of interest and to translate the information gathered into improvements in clinical management.
Abstract: The past year has witnessed substantial advances in understanding the genetic basis of many common phenotypes of biomedical importance. These advances have been the result of systematic, well-powered, genome-wide surveys exploring the relationships between common sequence variation and disease predisposition. This approach has revealed over 50 disease-susceptibility loci and has provided insights into the allelic architecture of multifactorial traits. At the same time, much has been learned about the successful prosecution of association studies on such a scale. This Review highlights the knowledge gained, defines areas of emerging consensus, and describes the challenges that remain as researchers seek to obtain more complete descriptions of the susceptibility architecture of biomedical traits of interest and to translate the information gathered into improvements in clinical management.
2,908 citations
TL;DR: Although ascertainment bias is a problem for some applications, SNPs can often generate equivalent statistical power whilst providing broader genome coverage and higher quality data than can either microsatellites or mtDNA, suggesting that SNPs could become an efficient and cost-effective genetic tool.
Abstract: Over the past two decades, new molecular genetic techniques have had substantial impacts on the fields of ecology, evolution and conservation. However, our current toolbox of genetic methodologies remains inadequate for answering many questions and there are significant technological and analytical limitations. We review the possible uses of single nucleotide polymorphisms (SNPs) as novel genetic markers for common questions in population genetics. Furthermore, we evaluate the potential of SNPs relative to frequently used genetic markers, such as microsatellite loci and mitochondrial DNA (mtDNA) sequences, and we discuss statistical power, analytical approaches, and technological improvements and limitations. Although ascertainment bias is a problem for some applications, SNPs can often generate equivalent statistical power whilst providing broader genome coverage and higher quality data than can either microsatellites or mtDNA, suggesting that SNPs could become an efficient and cost-effective genetic tool.
961 citations
TL;DR: Analytical methods that explicitly look for statistical interactions between loci are shown to be computationally feasible, even for studies of hundreds of thousands of loci, and to be more powerful than traditional analyses under a range of models for interlocus interactions.
Abstract: After nearly 10 years of intense academic and commercial research effort, large genome-wide association studies for common complex diseases are now imminent. Although these conditions involve a complex relationship between genotype and phenotype, including interactions between unlinked loci1, the prevailing strategies for analysis of such studies focus on the locus-by-locus paradigm. Here we consider analytical methods that explicitly look for statistical interactions between loci. We show first that they are computationally feasible, even for studies of hundreds of thousands of loci, and second that even with a conservative correction for multiple testing, they can be more powerful than traditional analyses under a range of models for interlocus interactions. We also show that plausible variations across populations in allele frequencies among interacting loci can markedly affect the power to detect their marginal effects, which may account in part for the well-known difficulties in replicating association results. These results suggest that searching for interactions among genetic loci can be fruitfully incorporated into analysis strategies for genome-wide association studies.
939 citations
TL;DR: Fundamental issues remain to be resolved, particularly regarding complex traits, before marker-assisted selection realizes its full potential in public sector breeding programs, including the development of high throughput precision phenotyping systems for QTL mapping, improved understanding of genotype by environment interaction and epistasis, and development of publicly available computational tools tailored to the needs of molecular breeding programs.
Abstract: The volume of publications on the development and to a lesser extent the application of molecular markers in plant breeding has increased dramatically during the last decade. However, most of the publications result from investments from donors with a strategic science quality or biotech advocacy mandate leading to insufficient emphasis on applied value in plant breeding. Converting promising publications into practical applications requires the resolution of many logistical and genetical constraints that are rarely addressed in journal publications. This results in a high proportion of published markers failing at one or more of the translation steps from research arena to application domain. The rate of success is likely to increase due to developments in gene-based marker development, more efficient quantitative trait locus (QTL) mapping procedures, and lower cost genotyping systems. However, some fundamental issues remain to be resolved, particularly regarding complex traits, before marker-assisted selection realizes its full potential in public sector breeding programs. These include the development of high throughput precision phenotyping systems for QTL mapping, improved understanding of genotype by environment interaction and epistasis, and development of publicly available computational tools tailored to the needs of molecular breeding programs.
809 citations
Cites background from "DNA Pooling: a tool for large-scale..."
...In human research, it is possible to carry out genome-wide association mapping by using an integrated technology package including selective genotyping, pooled DNA analysis, and microarray-based SNP genotyping with 100,000 markers (Sham et al., 2002; Meaburn et al., 2006; Yang et al., 2006)....
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References
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Book•
01 Dec 1969TL;DR: The concepts of power analysis are discussed in this paper, where Chi-square Tests for Goodness of Fit and Contingency Tables, t-Test for Means, and Sign Test are used.
Abstract: Contents: Prefaces. The Concepts of Power Analysis. The t-Test for Means. The Significance of a Product Moment rs (subscript s). Differences Between Correlation Coefficients. The Test That a Proportion is .50 and the Sign Test. Differences Between Proportions. Chi-Square Tests for Goodness of Fit and Contingency Tables. The Analysis of Variance and Covariance. Multiple Regression and Correlation Analysis. Set Correlation and Multivariate Methods. Some Issues in Power Analysis. Computational Procedures.
115,069 citations
TL;DR: Bulk segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome and will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.
Abstract: We developed bulked segregant analysis as a method for rapidly identifying markers linked to any specific gene or genomic region. Two bulked DNA samples are generated from a segregating population from a single cross. Each pool, or bulk, contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. The two bulks can be made for any genomic region and from any segregating population. The bulks are screened for differences using restriction fragment length polymorphism probes or random amplified polymorphic DNA primers. We have used bulked segregant analysis to identify three random amplified polymorphic DNA markers in lettuce linked to a gene for resistance to downy mildew. We showed that markers can be reliably identified in a 25-centimorgan window on either side of the targeted locus. Bulked segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome. Genetic walking will be possible by multiple rounds of bulked segregation analysis; each new pair of bulks will differ at a locus identified in the previous round of analysis. This approach will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.
4,492 citations
"DNA Pooling: a tool for large-scale..." refers methods in this paper
...Subsequently, it has been used for linkage studies in plant...
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Journal Article•
TL;DR: The statistical basis for this "transmission test for linkage disequilibrium" (transmission/disequilibrium test] is described and the relationship of this test to tests of cosegregation that are based on the proportion of haplotypes or genes identical by descent in affected sibs is shown.
Abstract: A population association has consistently been observed between insulin-dependent diabetes mellitus (IDDM) and the "class 1" alleles of the region of tandem-repeat DNA (5' flanking polymorphism [5'FP]) adjacent to the insulin gene on chromosome 11p. This finding suggests that the insulin gene region contains a gene or genes contributing to IDDM susceptibility. However, several studies that have sought to show linkage with IDDM by testing for cosegregation in affected sib pairs have failed to find evidence for linkage. As means for identifying genes for complex diseases, both the association and the affected-sib-pairs approaches have limitations. It is well known that population association between a disease and a genetic marker can arise as an artifact of population structure, even in the absence of linkage. On the other hand, linkage studies with modest numbers of affected sib pairs may fail to detect linkage, especially if there is linkage heterogeneity. We consider an alternative method to test for linkage with a genetic marker when population association has been found. Using data from families with at least one affected child, we evaluate the transmission of the associated marker allele from a heterozygous parent to an affected offspring. This approach has been used by several investigators, but the statistical properties of the method as a test for linkage have not been investigated. In the present paper we describe the statistical basis for this "transmission test for linkage disequilibrium" (transmission/disequilibrium test [TDT]). We then show the relationship of this test to tests of cosegregation that are based on the proportion of haplotypes or genes identical by descent in affected sibs. The TDT provides strong evidence for linkage between the 5'FP and susceptibility to IDDM. The conclusions from this analysis apply in general to the study of disease associations, where genetic markers are usually closely linked to candidate genes. When a disease is found to be associated with such a marker, the TDT may detect linkage even when haplotype-sharing tests do not.
3,791 citations
TL;DR: The performance of the genomic control method is quite good for plausible effects of liability genes, which bodes well for future genetic analyses of complex disorders.
Abstract: A dense set of single nucleotide polymorphisms (SNP) covering the genome and an efficient method to assess SNP genotypes are expected to be available in the near future. An outstanding question is how to use these technologies efficiently to identify genes affecting liability to complex disorders. To achieve this goal, we propose a statistical method that has several optimal properties: It can be used with case control data and yet, like family-based designs, controls for population heterogeneity; it is insensitive to the usual violations of model assumptions, such as cases failing to be strictly independent; and, by using Bayesian outlier methods, it circumvents the need for Bonferroni correction for multiple tests, leading to better performance in many settings while still constraining risk for false positives. The performance of our genomic control method is quite good for plausible effects of liability genes, which bodes well for future genetic analyses of complex disorders.
3,130 citations
"DNA Pooling: a tool for large-scale..." refers methods in this paper
...This factor is then used to adjust association test statistic...
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TL;DR: The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification.
Abstract: The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
3,050 citations
"DNA Pooling: a tool for large-scale..." refers background in this paper
...Higher hybridization specificity can usually be achieved in liquid phase (by using allele-specific PCR, or exonuclease assays, such as TAQMAN™ ), but as each allele is distinguished by oligonucleotides of slightly different sequence, there is the potential for alleles to be differentially amplifie...
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