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1.  Accuracy of genome-wide imputation of untyped markers and impacts on statistical power for association studies 
BMC Genetics  2009;10:27.
Background
Although high-throughput genotyping arrays have made whole-genome association studies (WGAS) feasible, only a small proportion of SNPs in the human genome are actually surveyed in such studies. In addition, various SNP arrays assay different sets of SNPs, which leads to challenges in comparing results and merging data for meta-analyses. Genome-wide imputation of untyped markers allows us to address these issues in a direct fashion.
Methods
384 Caucasian American liver donors were genotyped using Illumina 650Y (Ilmn650Y) arrays, from which we also derived genotypes from the Ilmn317K array. On these data, we compared two imputation methods: MACH and BEAGLE. We imputed 2.5 million HapMap Release22 SNPs, and conducted GWAS on ~40,000 liver mRNA expression traits (eQTL analysis). In addition, 200 Caucasian American and 200 African American subjects were genotyped using the Affymetrix 500 K array plus a custom 164 K fill-in chip. We then imputed the HapMap SNPs and quantified the accuracy by randomly masking observed SNPs.
Results
MACH and BEAGLE perform similarly with respect to imputation accuracy. The Ilmn650Y results in excellent imputation performance, and it outperforms Affx500K or Ilmn317K sets. For Caucasian Americans, 90% of the HapMap SNPs were imputed at 98% accuracy. As expected, imputation of poorly tagged SNPs (untyped SNPs in weak LD with typed markers) was not as successful. It was more challenging to impute genotypes in the African American population, given (1) shorter LD blocks and (2) admixture with Caucasian populations in this population. To address issue (2), we pooled HapMap CEU and YRI data as an imputation reference set, which greatly improved overall performance. The approximate 40,000 phenotypes scored in these populations provide a path to determine empirically how the power to detect associations is affected by the imputation procedures. That is, at a fixed false discovery rate, the number of cis-eQTL discoveries detected by various methods can be interpreted as their relative statistical power in the GWAS. In this study, we find that imputation offer modest additional power (by 4%) on top of either Ilmn317K or Ilmn650Y, much less than the power gain from Ilmn317K to Ilmn650Y (13%).
Conclusion
Current algorithms can accurately impute genotypes for untyped markers, which enables researchers to pool data between studies conducted using different SNP sets. While genotyping itself results in a small error rate (e.g. 0.5%), imputing genotypes is surprisingly accurate. We found that dense marker sets (e.g. Ilmn650Y) outperform sparser ones (e.g. Ilmn317K) in terms of imputation yield and accuracy. We also noticed it was harder to impute genotypes for African American samples, partially due to population admixture, although using a pooled reference boosts performance. Interestingly, GWAS carried out using imputed genotypes only slightly increased power on top of assayed SNPs. The reason is likely due to adding more markers via imputation only results in modest gain in genetic coverage, but worsens the multiple testing penalties. Furthermore, cis-eQTL mapping using dense SNP set derived from imputation achieves great resolution, and locate associate peak closer to causal variants than conventional approach.
doi:10.1186/1471-2156-10-27
PMCID: PMC2709633  PMID: 19531258
2.  Simultaneous Analysis of All SNPs in Genome-Wide and Re-Sequencing Association Studies 
PLoS Genetics  2008;4(7):e1000130.
Testing one SNP at a time does not fully realise the potential of genome-wide association studies to identify multiple causal variants, which is a plausible scenario for many complex diseases. We show that simultaneous analysis of the entire set of SNPs from a genome-wide study to identify the subset that best predicts disease outcome is now feasible, thanks to developments in stochastic search methods. We used a Bayesian-inspired penalised maximum likelihood approach in which every SNP can be considered for additive, dominant, and recessive contributions to disease risk. Posterior mode estimates were obtained for regression coefficients that were each assigned a prior with a sharp mode at zero. A non-zero coefficient estimate was interpreted as corresponding to a significant SNP. We investigated two prior distributions and show that the normal-exponential-gamma prior leads to improved SNP selection in comparison with single-SNP tests. We also derived an explicit approximation for type-I error that avoids the need to use permutation procedures. As well as genome-wide analyses, our method is well-suited to fine mapping with very dense SNP sets obtained from re-sequencing and/or imputation. It can accommodate quantitative as well as case-control phenotypes, covariate adjustment, and can be extended to search for interactions. Here, we demonstrate the power and empirical type-I error of our approach using simulated case-control data sets of up to 500 K SNPs, a real genome-wide data set of 300 K SNPs, and a sequence-based dataset, each of which can be analysed in a few hours on a desktop workstation.
Author Summary
Tests of association with disease status are normally conducted one SNP at a time, ignoring the effects of all other genotyped SNPs. We developed a computationally efficient method to simultaneously analyse all SNPs, either in a genome-wide association (GWA) study, or a fine-mapping study based on re-sequencing and/or imputation. The method selects a subset of SNPs that best predicts disease status, while controlling the type-I error of the selected SNPs. This brings many advantages over standard single-SNP approaches, because the signal from a particular SNP can be more clearly assessed when other SNPs associated with disease status are already included in the model. Thus, in comparison with single-SNP analyses, power is increased and the false positive rate is reduced because of reduced residual variation. Localisation is also greatly improved. We demonstrate these advantages over the widely used single-SNP Armitage Trend Test using GWA simulation studies, a real GWA dataset, and a sequence-based fine-mapping simulation study.
doi:10.1371/journal.pgen.1000130
PMCID: PMC2464715  PMID: 18654633
3.  Comprehensive evaluation of imputation performance in African Americans 
Journal of human genetics  2012;57(7):411-421.
Imputation of genome-wide single-nucleotide polymorphism (SNP) arrays to a larger known reference panel of SNPs has become a standard and an essential part of genome-wide association studies. However, little is known about the behavior of imputation in African Americans with respect to the different imputation algorithms, the reference population(s) and the reference SNP panels used. Genome-wide SNP data (Affymetrix 6.0) from 3207 African American samples in the Atherosclerosis Risk in Communities Study (ARIC) was used to systematically evaluate imputation quality and yield. Imputation was performed with the imputation algorithms MACH, IMPUTE and BEAGLE using several combinations of three reference panels of HapMap III (ASW, YRI and CEU) and 1000 Genomes Project (pilot 1 YRI June 2010 release, EUR and AFR August 2010 and June 2011 releases) panels with SNP data on chromosomes 18, 20 and 22. About 10% of the directly genotyped SNPs from each chromosome were masked, and SNPs common between the reference panels were used for evaluating the imputation quality using two statistical metrics—concordance accuracy and Cohen’s kappa (κ) coefficient. The dependencies of these metrics on the minor allele frequencies (MAF) and specific genotype categories (minor allele homozygotes, heterozygotes and major allele homozygotes) were thoroughly investigated to determine the best panel and method for imputation in African Americans. In addition, the power to detect imputed SNPs associated with simulated phenotypes was studied using the mean genotype of each masked SNP in the imputed data. Our results indicate that the genotype concordances after stratification into each genotype category and Cohen’s κ coefficient are considerably better equipped to differentiate imputation performance compared with the traditionally used total concordance statistic, and both statistics improved with increasing MAF irrespective of the imputation method. We also find that both MACH and IMPUTE performed equally well and consistently better than BEAGLE irrespective of the reference panel used. Of the various combinations of reference panels, for both HapMap III and 1000 Genomes Project reference panels, the multi-ethnic panels had better imputation accuracy than those containing only single ethnic samples. The most recent 1000 Genomes Project release June 2011 had substantially higher number of imputed SNPs than HapMap III and performed as well or better than the best combined HapMap III reference panels and previous releases of the 1000 Genomes Project.
doi:10.1038/jhg.2012.43
PMCID: PMC3477509  PMID: 22648186
concordance; GWAS; Hapmap; imputation; imputation accuracy; kappa; 1000 genomes
4.  Re-Ranking Sequencing Variants in the Post-GWAS Era for Accurate Causal Variant Identification 
PLoS Genetics  2013;9(8):e1003609.
Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website.
Author Summary
As next-generation sequencing (NGS) costs continue to fall and genome-wide association study (GWAS) platform coverage improves, the human genetics community is positioned to identify potentially causal variants. However, current NGS or imputation-based studies of either the whole genome or regions previously identified by GWAS have not yet been very successful in identifying causal variants. A major hurdle is the development of methods to distinguish disease-causing variants from their highly-correlated proxies within an associated region. We show that various common factors, such as differential sequencing or imputation accuracy rates and linkage disequilibrium patterns, with or without GWAS-informed region selection, can substantially decrease the probability of identifying the correct causal SNP, often by more than half. We then describe a novel and easy-to-implement re-ranking procedure that can double the probability that the causal SNP is top-ranked in many settings. Application to the NCI Breast and Prostate Cancer (BPC3) Cohort Consortium aggressive prostate cancer data identified new top SNPs within two associated loci previously established via GWAS, as well as several additional possible causal SNPs that had been previously overlooked.
doi:10.1371/journal.pgen.1003609
PMCID: PMC3738448  PMID: 23950724
5.  One Thousand Genomes Imputation in the National Cancer Institute Breast and Prostate Cancer Cohort Consortium Aggressive Prostate Cancer Genome-wide Association Study 
The Prostate  2012;73(7):677-689.
BACKGROUND
Genotype imputation substantially increases available markers for analysis in genome-wide association studies (GWAS) by leveraging linkage disequilibrium from a reference panel. We sought to (i) investigate the performance of imputation from the August 2010 release of the 1000 Genomes Project (1000GP) in an existing GWAS of prostate cancer, (ii) look for novel associations with prostate cancer risk, (iii) fine-map known prostate cancer susceptibility regions using an approximate Bayesian framework and stepwise regression, and (iv) compare power and efficiency of imputation and de novo sequencing.
METHODS
We used 2,782 aggressive prostate cancer cases and 4,458 controls from the NCI Breast and Prostate Cancer Cohort Consortium aggressive prostate cancer GWAS to infer 5.8 million well-imputed autosomal single nucleotide polymorphisms.
RESULTS
Imputation quality, as measured by correlation between imputed and true allele counts, was higher among common variants than rare variants. We found no novel prostate cancer associations among a subset of 1.2 million well-imputed low-frequency variants. At a genome-wide sequencing cost of $2,500, imputation from SNP arrays is a more powerful strategy than sequencing for detecting disease associations of SNPs with minor allele frequencies above 1%.
CONCLUSIONS
1000GP imputation provided dense coverage of previously-identified prostate cancer susceptibility regions, highlighting its potential as an inexpensive first-pass approach to fine-mapping in regions such as 5p15 and 8q24. Our study shows 1000GP imputation can accurately identify low-frequency variants and stresses the importance of large sample size when studying these variants.
doi:10.1002/pros.22608
PMCID: PMC3962143  PMID: 23255287
rare variants; association; fine mapping
6.  Practical Issues in Imputation-Based Association Mapping 
PLoS Genetics  2008;4(12):e1000279.
Imputation-based association methods provide a powerful framework for testing untyped variants for association with phenotypes and for combining results from multiple studies that use different genotyping platforms. Here, we consider several issues that arise when applying these methods in practice, including: (i) factors affecting imputation accuracy, including choice of reference panel; (ii) the effects of imputation accuracy on power to detect associations; (iii) the relative merits of Bayesian and frequentist approaches to testing imputed genotypes for association with phenotype; and (iv) how to quickly and accurately compute Bayes factors for testing imputed SNPs. We find that imputation-based methods can be robust to imputation accuracy and can improve power to detect associations, even when average imputation accuracy is poor. We explain how ranking SNPs for association by a standard likelihood ratio test gives the same results as a Bayesian procedure that uses an unnatural prior assumption—specifically, that difficult-to-impute SNPs tend to have larger effects—and assess the power gained from using a Bayesian approach that does not make this assumption. Within the Bayesian framework, we find that good approximations to a full analysis can be achieved by simply replacing unknown genotypes with a point estimate—their posterior mean. This approximation considerably reduces computational expense compared with published sampling-based approaches, and the methods we present are practical on a genome-wide scale with very modest computational resources (e.g., a single desktop computer). The approximation also facilitates combining information across studies, using only summary data for each SNP. Methods discussed here are implemented in the software package BIMBAM, which is available from http://stephenslab.uchicago.edu/software.html.
Author Summary
Genotype imputation is becoming a popular approach to comparing and combining results of multiple association studies that used different SNP genotyping platforms. The basic idea is to exploit the fact that, due to correlation among untyped and typed SNPs, genotypes of untyped SNPs in each study can be inferred (“imputed”) from the genotypes at typed SNPs, often with high accuracy. In this paper, we consider several issues that arise when applying these methods in practice, including factors affecting imputation accuracy, the importance of taking account of imputation uncertainty when testing for association between imputed SNPs and phenotype, how imputation accuracy affects power, and how to combine results across studies when only single-SNP summary data can be shared among research groups.
doi:10.1371/journal.pgen.1000279
PMCID: PMC2585794  PMID: 19057666
7.  A Flexible and Accurate Genotype Imputation Method for the Next Generation of Genome-Wide Association Studies 
PLoS Genetics  2009;5(6):e1000529.
Genotype imputation methods are now being widely used in the analysis of genome-wide association studies. Most imputation analyses to date have used the HapMap as a reference dataset, but new reference panels (such as controls genotyped on multiple SNP chips and densely typed samples from the 1,000 Genomes Project) will soon allow a broader range of SNPs to be imputed with higher accuracy, thereby increasing power. We describe a genotype imputation method (IMPUTE version 2) that is designed to address the challenges presented by these new datasets. The main innovation of our approach is a flexible modelling framework that increases accuracy and combines information across multiple reference panels while remaining computationally feasible. We find that IMPUTE v2 attains higher accuracy than other methods when the HapMap provides the sole reference panel, but that the size of the panel constrains the improvements that can be made. We also find that imputation accuracy can be greatly enhanced by expanding the reference panel to contain thousands of chromosomes and that IMPUTE v2 outperforms other methods in this setting at both rare and common SNPs, with overall error rates that are 15%–20% lower than those of the closest competing method. One particularly challenging aspect of next-generation association studies is to integrate information across multiple reference panels genotyped on different sets of SNPs; we show that our approach to this problem has practical advantages over other suggested solutions.
Author Summary
Large association studies have proven to be effective tools for identifying parts of the genome that influence disease risk and other heritable traits. So-called “genotype imputation” methods form a cornerstone of modern association studies: by extrapolating genetic correlations from a densely characterized reference panel to a sparsely typed study sample, such methods can estimate unobserved genotypes with high accuracy, thereby increasing the chances of finding true associations. To date, most genome-wide imputation analyses have used reference data from the International HapMap Project. While this strategy has been successful, association studies in the near future will also have access to additional reference information, such as control sets genotyped on multiple SNP chips and dense genome-wide haplotypes from the 1,000 Genomes Project. These new reference panels should improve the quality and scope of imputation, but they also present new methodological challenges. We describe a genotype imputation method, IMPUTE version 2, that is designed to address these challenges in next-generation association studies. We show that our method can use a reference panel containing thousands of chromosomes to attain higher accuracy than is possible with the HapMap alone, and that our approach is more accurate than competing methods on both current and next-generation datasets. We also highlight the modeling issues that arise in imputation datasets.
doi:10.1371/journal.pgen.1000529
PMCID: PMC2689936  PMID: 19543373
8.  Designing Genome-Wide Association Studies: Sample Size, Power, Imputation, and the Choice of Genotyping Chip 
PLoS Genetics  2009;5(5):e1000477.
Genome-wide association studies are revolutionizing the search for the genes underlying human complex diseases. The main decisions to be made at the design stage of these studies are the choice of the commercial genotyping chip to be used and the numbers of case and control samples to be genotyped. The most common method of comparing different chips is using a measure of coverage, but this fails to properly account for the effects of sample size, the genetic model of the disease, and linkage disequilibrium between SNPs. In this paper, we argue that the statistical power to detect a causative variant should be the major criterion in study design. Because of the complicated pattern of linkage disequilibrium (LD) in the human genome, power cannot be calculated analytically and must instead be assessed by simulation. We describe in detail a method of simulating case-control samples at a set of linked SNPs that replicates the patterns of LD in human populations, and we used it to assess power for a comprehensive set of available genotyping chips. Our results allow us to compare the performance of the chips to detect variants with different effect sizes and allele frequencies, look at how power changes with sample size in different populations or when using multi-marker tags and genotype imputation approaches, and how performance compares to a hypothetical chip that contains every SNP in HapMap. A main conclusion of this study is that marked differences in genome coverage may not translate into appreciable differences in power and that, when taking budgetary considerations into account, the most powerful design may not always correspond to the chip with the highest coverage. We also show that genotype imputation can be used to boost the power of many chips up to the level obtained from a hypothetical “complete” chip containing all the SNPs in HapMap. Our results have been encapsulated into an R software package that allows users to design future association studies and our methods provide a framework with which new chip sets can be evaluated.
Author Summary
Genome-wide association studies are a powerful and now widely-used method for finding genetic variants that increase the risk of developing particular diseases. These studies are complex and must be planned carefully in order to maximize the probability of finding novel associations. The main design choices to be made relate to sample sizes and choice of commercially available genotyping chip and are often constrained by cost, which can currently be as much as several million dollars. No comprehensive comparisons of chips based on their power for different sample sizes or for fixed study cost are currently available. We describe in detail a method for simulating large genome-wide association samples that accounts for the complex correlations between SNPs due to LD, and we used this method to assess the power of current genotyping chips. Our results highlight the differences between the chips under a range of plausible scenarios, and we demonstrate how our results can be used to design a study with a budget constraint. We also show how genotype imputation can be used to boost the power of each chip and that this method decreases the differences between the chips. Our simulation method and software for comparing power are being made available so that future association studies can be designed in a principled fashion.
doi:10.1371/journal.pgen.1000477
PMCID: PMC2688469  PMID: 19492015
9.  Application of imputation methods to the analysis of rheumatoid arthritis data in genome-wide association studies 
BMC Proceedings  2009;3(Suppl 7):S24.
Most genetic association studies only genotype a small proportion of cataloged single-nucleotide polymorphisms (SNPs) in regions of interest. With the catalogs of high-density SNP data available (e.g., HapMap) to researchers today, it has become possible to impute genotypes at untyped SNPs. This in turn allows us to test those untyped SNPs, the motivation being to increase power in association studies. Several imputation methods and corresponding software packages have been developed for this purpose. The objective of our study is to apply three widely used imputation methods and corresponding software packages to a data from a genome-wide association study of rheumatoid arthritis from the North American Rheumatoid Arthritis Consortium in Genetic Analysis Workshop 16, to compare the performances of the three methods, to evaluate their strengths and weaknesses, and to identify additional susceptibility loci underlying rheumatoid arthritis. The software packages used in this paper included a program for Bayesian imputation-based association mapping (BIMBAM), a program for imputing unobserved genotypes in case-control association studies (IMPUTE), and a program for testing untyped alleles (TUNA). We found some untyped SNP that showed significant association with rheumatoid arthritis. Among them, a few of these were not located near any typed SNP that was found to be significant and thus may be worth further investigation.
PMCID: PMC2795921  PMID: 20018014
10.  Assessment of genotype imputation methods 
BMC Proceedings  2009;3(Suppl 7):S5.
Several methods have been proposed to impute genotypes at untyped markers using observed genotypes and genetic data from a reference panel. We used the Genetic Analysis Workshop 16 rheumatoid arthritis case-control dataset to compare the performance of four of these imputation methods: IMPUTE, MACH, PLINK, and fastPHASE. We compared the methods' imputation error rates and performance of association tests using the imputed data, in the context of imputing completely untyped markers as well as imputing missing genotypes to combine two datasets genotyped at different sets of markers. As expected, all methods performed better for single-nucleotide polymorphisms (SNPs) in high linkage disequilibrium with genotyped SNPs. However, MACH and IMPUTE generated lower imputation error rates than fastPHASE and PLINK. Association tests based on allele "dosage" from MACH and tests based on the posterior probabilities from IMPUTE provided results closest to those based on complete data. However, in both situations, none of the imputation-based tests provide the same level of evidence of association as the complete data at SNPs strongly associated with disease.
PMCID: PMC2795949  PMID: 20018042
11.  Imputation reliability on DNA biallelic markers for drug metabolism studies 
BMC Bioinformatics  2012;13(Suppl 14):S7.
Background
Imputation is a statistical process used to predict genotypes of loci not directly assayed in a sample of individuals. Our goal is to measure the performance of imputation in predicting the genotype of the best known gene polymorphisms involved in drug metabolism using a common SNP array genotyping platform generally exploited in genome wide association studies.
Methods
Thirty-nine (39) individuals were genotyped with both Affymetrix Genome Wide Human SNP 6.0 (AFFY) and Affymetrix DMET Plus (DMET) platforms. AFFY and DMET contain nearly 900000 and 1931 markers respectively. We used a 1000 Genomes Pilot + HapMap 3 reference panel. Imputation was performed using the computer program Impute, version 2. SNPs contained in DMET, but not imputed, were analysed studying markers around their chromosome regions. The efficacy of the imputation was measured evaluating the number of successfully imputed SNPs (SSNPs).
Results
The imputation predicted the genotypes of 654 SNPs not present in the AFFY array, but contained in the DMET array. Approximately 1000 SNPs were not annotated in the reference panel and therefore they could not be directly imputed. After testing three different imputed genotype calling threshold (IGCT), we observed that imputation performs at its best for IGCT value equal to 50%, with rate of SSNPs (MAF > 0.05) equal to 85%.
Conclusions
Most of the genes involved in drug metabolism can be imputed with high efficacy using standard genome-wide genotyping platforms and imputing procedures.
doi:10.1186/1471-2105-13-S14-S7
PMCID: PMC3439717  PMID: 23095502
12.  Enhanced Statistical Tests for GWAS in Admixed Populations: Assessment using African Americans from CARe and a Breast Cancer Consortium 
PLoS Genetics  2011;7(4):e1001371.
While genome-wide association studies (GWAS) have primarily examined populations of European ancestry, more recent studies often involve additional populations, including admixed populations such as African Americans and Latinos. In admixed populations, linkage disequilibrium (LD) exists both at a fine scale in ancestral populations and at a coarse scale (admixture-LD) due to chromosomal segments of distinct ancestry. Disease association statistics in admixed populations have previously considered SNP association (LD mapping) or admixture association (mapping by admixture-LD), but not both. Here, we introduce a new statistical framework for combining SNP and admixture association in case-control studies, as well as methods for local ancestry-aware imputation. We illustrate the gain in statistical power achieved by these methods by analyzing data of 6,209 unrelated African Americans from the CARe project genotyped on the Affymetrix 6.0 chip, in conjunction with both simulated and real phenotypes, as well as by analyzing the FGFR2 locus using breast cancer GWAS data from 5,761 African-American women. We show that, at typed SNPs, our method yields an 8% increase in statistical power for finding disease risk loci compared to the power achieved by standard methods in case-control studies. At imputed SNPs, we observe an 11% increase in statistical power for mapping disease loci when our local ancestry-aware imputation framework and the new scoring statistic are jointly employed. Finally, we show that our method increases statistical power in regions harboring the causal SNP in the case when the causal SNP is untyped and cannot be imputed. Our methods and our publicly available software are broadly applicable to GWAS in admixed populations.
Author Summary
This paper presents improved methodologies for the analysis of genome-wide association studies in admixed populations, which are populations that came about by the mixing of two or more distant continental populations over a few hundred years (e.g., African Americans or Latinos). Studies of admixed populations offer the promise of capturing additional genetic diversity compared to studies over homogeneous populations such as Europeans. In admixed populations, correlation between genetic variants exists both at a fine scale in the ancestral populations and at a coarse scale due to chromosomal segments of distinct ancestry. Disease association statistics in admixed populations have previously considered either one or the other type of correlation, but not both. In this work we develop novel statistical methods that account for both types of genetic correlation, and we show that the combined approach attains greater statistical power than that achieved by applying either approach separately. We provide analysis of simulated and real data from major studies performed in African-American men and women to show the improvement obtained by our methods over the standard methods for analyzing association studies in admixed populations.
doi:10.1371/journal.pgen.1001371
PMCID: PMC3080860  PMID: 21541012
13.  Gene-based interaction analysis by incorporating external linkage disequilibrium information 
Gene–gene interactions have an important role in complex human diseases. Detection of gene–gene interactions has long been a challenge due to their complexity. The standard method aiming at detecting SNP–SNP interactions may be inadequate as it does not model linkage disequilibrium (LD) among SNPs in each gene and may lose power due to a large number of comparisons. To improve power, we propose a principal component (PC)-based framework for gene-based interaction analysis. We analytically derive the optimal weight for both quantitative and binary traits based on pairwise LD information. We then use PCs to summarize the information in each gene and test for interactions between the PCs. We further extend this gene-based interaction analysis procedure to allow the use of imputation dosage scores obtained from a popular imputation software package, MACH, which incorporates multilocus LD information. To evaluate the performance of the gene-based interaction tests, we conducted extensive simulations under various settings. We demonstrate that gene-based interaction tests are more powerful than SNP-based tests when more than two variants interact with each other; moreover, tests that incorporate external LD information are generally more powerful than those that use genotyped markers only. We also apply the proposed gene-based interaction tests to a candidate gene study on high-density lipoprotein. As our method operates at the gene level, it can be applied to a genome-wide association setting and used as a screening tool to detect gene–gene interactions.
doi:10.1038/ejhg.2010.164
PMCID: PMC3025792  PMID: 20924406
gene–gene interaction; linkage disequilibrium; imputation
14.  Comparative analysis of methods for detecting interacting loci 
BMC Genomics  2011;12:344.
Background
Interactions among genetic loci are believed to play an important role in disease risk. While many methods have been proposed for detecting such interactions, their relative performance remains largely unclear, mainly because different data sources, detection performance criteria, and experimental protocols were used in the papers introducing these methods and in subsequent studies. Moreover, there have been very few studies strictly focused on comparison of existing methods. Given the importance of detecting gene-gene and gene-environment interactions, a rigorous, comprehensive comparison of performance and limitations of available interaction detection methods is warranted.
Results
We report a comparison of eight representative methods, of which seven were specifically designed to detect interactions among single nucleotide polymorphisms (SNPs), with the last a popular main-effect testing method used as a baseline for performance evaluation. The selected methods, multifactor dimensionality reduction (MDR), full interaction model (FIM), information gain (IG), Bayesian epistasis association mapping (BEAM), SNP harvester (SH), maximum entropy conditional probability modeling (MECPM), logistic regression with an interaction term (LRIT), and logistic regression (LR) were compared on a large number of simulated data sets, each, consistent with complex disease models, embedding multiple sets of interacting SNPs, under different interaction models. The assessment criteria included several relevant detection power measures, family-wise type I error rate, and computational complexity. There are several important results from this study. First, while some SNPs in interactions with strong effects are successfully detected, most of the methods miss many interacting SNPs at an acceptable rate of false positives. In this study, the best-performing method was MECPM. Second, the statistical significance assessment criteria, used by some of the methods to control the type I error rate, are quite conservative, thereby limiting their power and making it difficult to fairly compare them. Third, as expected, power varies for different models and as a function of penetrance, minor allele frequency, linkage disequilibrium and marginal effects. Fourth, the analytical relationships between power and these factors are derived, aiding in the interpretation of the study results. Fifth, for these methods the magnitude of the main effect influences the power of the tests. Sixth, most methods can detect some ground-truth SNPs but have modest power to detect the whole set of interacting SNPs.
Conclusion
This comparison study provides new insights into the strengths and limitations of current methods for detecting interacting loci. This study, along with freely available simulation tools we provide, should help support development of improved methods. The simulation tools are available at: http://code.google.com/p/simulation-tool-bmc-ms9169818735220977/downloads/list.
doi:10.1186/1471-2164-12-344
PMCID: PMC3161015  PMID: 21729295
15.  Genome Wide Association Studies Using a New Nonparametric Model Reveal the Genetic Architecture of 17 Agronomic Traits in an Enlarged Maize Association Panel 
PLoS Genetics  2014;10(9):e1004573.
Association mapping is a powerful approach for dissecting the genetic architecture of complex quantitative traits using high-density SNP markers in maize. Here, we expanded our association panel size from 368 to 513 inbred lines with 0.5 million high quality SNPs using a two-step data-imputation method which combines identity by descent (IBD) based projection and k-nearest neighbor (KNN) algorithm. Genome-wide association studies (GWAS) were carried out for 17 agronomic traits with a panel of 513 inbred lines applying both mixed linear model (MLM) and a new method, the Anderson-Darling (A-D) test. Ten loci for five traits were identified using the MLM method at the Bonferroni-corrected threshold −log10 (P) >5.74 (α = 1). Many loci ranging from one to 34 loci (107 loci for plant height) were identified for 17 traits using the A-D test at the Bonferroni-corrected threshold −log10 (P) >7.05 (α = 0.05) using 556809 SNPs. Many known loci and new candidate loci were only observed by the A-D test, a few of which were also detected in independent linkage analysis. This study indicates that combining IBD based projection and KNN algorithm is an efficient imputation method for inferring large missing genotype segments. In addition, we showed that the A-D test is a useful complement for GWAS analysis of complex quantitative traits. Especially for traits with abnormal phenotype distribution, controlled by moderate effect loci or rare variations, the A-D test balances false positives and statistical power. The candidate SNPs and associated genes also provide a rich resource for maize genetics and breeding.
Author Summary
Genotype imputation has been used widely in the analysis of genome-wide association studies (GWAS) to boost power and fine-map associations. We developed a two-step data imputation method to meet the challenge of large proportion missing genotypes. GWAS have uncovered an extensive genetic architecture of complex quantitative traits using high-density SNP markers in maize in the past few years. Here, GWAS were carried out for 17 agronomic traits with a panel of 513 inbred lines applying both mixed linear model and a new method, the Anderson-Darling (A-D) test. We intend to show that the A-D test is a complement to current GWAS methods, especially for complex quantitative traits controlled by moderate effect loci or rare variations and with abnormal phenotype distribution. In addition, the traits associated QTL identified here provide a rich resource for maize genetics and breeding.
doi:10.1371/journal.pgen.1004573
PMCID: PMC4161304  PMID: 25211220
16.  Quick, “Imputation-free” meta-analysis with proxy-SNPs 
BMC Bioinformatics  2012;13:231.
Background
Meta-analysis (MA) is widely used to pool genome-wide association studies (GWASes) in order to a) increase the power to detect strong or weak genotype effects or b) as a result verification method. As a consequence of differing SNP panels among genotyping chips, imputation is the method of choice within GWAS consortia to avoid losing too many SNPs in a MA. YAMAS (Yet Another Meta Analysis Software), however, enables cross-GWAS conclusions prior to finished and polished imputation runs, which eventually are time-consuming.
Results
Here we present a fast method to avoid forfeiting SNPs present in only a subset of studies, without relying on imputation. This is accomplished by using reference linkage disequilibrium data from 1,000 Genomes/HapMap projects to find proxy-SNPs together with in-phase alleles for SNPs missing in at least one study. MA is conducted by combining association effect estimates of a SNP and those of its proxy-SNPs. Our algorithm is implemented in the MA software YAMAS. Association results from GWAS analysis applications can be used as input files for MA, tremendously speeding up MA compared to the conventional imputation approach. We show that our proxy algorithm is well-powered and yields valuable ad hoc results, possibly providing an incentive for follow-up studies. We propose our method as a quick screening step prior to imputation-based MA, as well as an additional main approach for studies without available reference data matching the ethnicities of study participants. As a proof of principle, we analyzed six dbGaP Type II Diabetes GWAS and found that the proxy algorithm clearly outperforms naïve MA on the p-value level: for 17 out of 23 we observe an improvement on the p-value level by a factor of more than two, and a maximum improvement by a factor of 2127.
Conclusions
YAMAS is an efficient and fast meta-analysis program which offers various methods, including conventional MA as well as inserting proxy-SNPs for missing markers to avoid unnecessary power loss. MA with YAMAS can be readily conducted as YAMAS provides a generic parser for heterogeneous tabulated file formats within the GWAS field and avoids cumbersome setups. In this way, it supplements the meta-analysis process.
doi:10.1186/1471-2105-13-231
PMCID: PMC3472171  PMID: 22971100
17.  1000 Genomes-based imputation identifies novel and refined associations for the Wellcome Trust Case Control Consortium phase 1 Data 
We hypothesize that imputation based on data from the 1000 Genomes Project can identify novel association signals on a genome-wide scale due to the dense marker map and the large number of haplotypes. To test the hypothesis, the Wellcome Trust Case Control Consortium (WTCCC) Phase I genotype data were imputed using 1000 genomes as reference (20100804 EUR), and seven case/control association studies were performed using imputed dosages. We observed two ‘missed' disease-associated variants that were undetectable by the original WTCCC analysis, but were reported by later studies after the 2007 WTCCC publication. One is within the IL2RA gene for association with type 1 diabetes and the other in proximity with the CDKN2B gene for association with type 2 diabetes. We also identified two refined associations. One is SNP rs11209026 in exon 9 of IL23R for association with Crohn's disease, which is predicted to be probably damaging by PolyPhen2. The other refined variant is in the CUX2 gene region for association with type 1 diabetes, where the newly identified top SNP rs1265564 has an association P-value of 1.68 × 10−16. The new lead SNP for the two refined loci provides a more plausible explanation for the disease association. We demonstrated that 1000 Genomes-based imputation could indeed identify both novel (in our case, ‘missed' because they were detected and replicated by studies after 2007) and refined signals. We anticipate the findings derived from this study to provide timely information when individual groups and consortia are beginning to engage in 1000 genomes-based imputation.
doi:10.1038/ejhg.2012.3
PMCID: PMC3376268  PMID: 22293688
genome-wide association study; the 1000 Genomes project; imputation
18.  GACT: a Genome build and Allele definition Conversion Tool for SNP imputation and meta-analysis in genetic association studies 
BMC Genomics  2014;15:610.
Background
Genome-wide association studies (GWAS) have successfully identified genes associated with complex human diseases. Although much of the heritability remains unexplained, combining single nucleotide polymorphism (SNP) genotypes from multiple studies for meta-analysis will increase the statistical power to identify new disease-associated variants. Meta-analysis requires same allele definition (nomenclature) and genome build among individual studies. Similarly, imputation, commonly-used prior to meta-analysis, requires the same consistency. However, the genotypes from various GWAS are generated using different genotyping platforms, arrays or SNP-calling approaches, resulting in use of different genome builds and allele definitions. Incorrect assumptions of identical allele definition among combined GWAS lead to a large portion of discarded genotypes or incorrect association findings. There is no published tool that predicts and converts among all major allele definitions.
Results
In this study, we have developed a tool, GACT, which stands for Genome build and Allele definition Conversion Tool, that predicts and inter-converts between any of the common SNP allele definitions and between the major genome builds. In addition, we assessed several factors that may affect imputation quality, and our results indicated that inclusion of singletons in the reference had detrimental effects while ambiguous SNPs had no measurable effect. Unexpectedly, exclusion of genotypes with missing rate > 0.001 (40% of study SNPs) showed no significant decrease of imputation quality (even significantly higher when compared to the imputation with singletons in the reference), especially for rare SNPs.
Conclusion
GACT is a new, powerful, and user-friendly tool with both command-line and interactive online versions that can accurately predict, and convert between any of the common allele definitions and between genome builds for genome-wide meta-analysis and imputation of genotypes from SNP-arrays or deep-sequencing, particularly for data from the dbGaP and other public databases.
GACT software
http://www.uvm.edu/genomics/software/gact
doi:10.1186/1471-2164-15-610
PMCID: PMC4223508  PMID: 25038819
Allele definition (nomenclature); Genome build; Genome-wide association study (GWAS); Imputation; Meta-analysis
19.  Analyses and Comparison of Imputation-Based Association Methods 
PLoS ONE  2010;5(5):e10827.
Genotype imputation methods have become increasingly popular for recovering untyped genotype data. An important application with imputed genotypes is to test genetic association for diseases. Imputation-based association test can provide additional insight beyond what is provided by testing on typed tagging SNPs only. A variety of effective imputation-based association tests have been proposed. However, their performances are affected by a variety of genetic factors, which have not been well studied. In this study, using both simulated and real data sets, we investigated the effects of LD, MAF of untyped causal SNP and imputation accuracy rate on the performances of seven popular imputation-based association methods, including MACH2qtl/dat, SNPTEST, ProbABEL, Beagle, Plink, BIMBAM and SNPMStat. We also aimed to provide a comprehensive comparison among methods. Results show that: 1). imputation-based association tests can boost signals and improve power under medium and high LD levels, with the power improvement increasing with strengthening LD level; 2) the power increases with higher MAF of untyped causal SNPs under medium to high LD level; 3). under low LD level, a high imputation accuracy rate cannot guarantee an improvement of power; 4). among methods, MACH2qtl/dat, ProbABEL and SNPTEST perform similarly and they consistently outperform other methods. Our results are helpful in guiding the choice of imputation-based association test in practical application.
doi:10.1371/journal.pone.0010827
PMCID: PMC2877082  PMID: 20520814
20.  Comparison of imputation methods for missing laboratory data in medicine 
BMJ Open  2013;3(8):e002847.
Objectives
Missing laboratory data is a common issue, but the optimal method of imputation of missing values has not been determined. The aims of our study were to compare the accuracy of four imputation methods for missing completely at random laboratory data and to compare the effect of the imputed values on the accuracy of two clinical predictive models.
Design
Retrospective cohort analysis of two large data sets.
Setting
A tertiary level care institution in Ann Arbor, Michigan.
Participants
The Cirrhosis cohort had 446 patients and the Inflammatory Bowel Disease cohort had 395 patients.
Methods
Non-missing laboratory data were randomly removed with varying frequencies from two large data sets, and we then compared the ability of four methods—missForest, mean imputation, nearest neighbour imputation and multivariate imputation by chained equations (MICE)—to impute the simulated missing data. We characterised the accuracy of the imputation and the effect of the imputation on predictive ability in two large data sets.
Results
MissForest had the least imputation error for both continuous and categorical variables at each frequency of missingness, and it had the smallest prediction difference when models used imputed laboratory values. In both data sets, MICE had the second least imputation error and prediction difference, followed by the nearest neighbour and mean imputation.
Conclusions
MissForest is a highly accurate method of imputation for missing laboratory data and outperforms other common imputation techniques in terms of imputation error and maintenance of predictive ability with imputed values in two clinical predicative models.
doi:10.1136/bmjopen-2013-002847
PMCID: PMC3733317  PMID: 23906948
21.  Recombination locations and rates in beef cattle assessed from parent-offspring pairs 
Background
Recombination events tend to occur in hotspots and vary in number among individuals. The presence of recombination influences the accuracy of haplotype phasing and the imputation of missing genotypes. Genes that influence genome-wide recombination rate have been discovered in mammals, yeast, and plants. Our aim was to investigate the influence of recombination on haplotype phasing, locate recombination hotspots, scan the genome for Quantitative Trait Loci (QTL) and identify candidate genes that influence recombination, and quantify the impact of recombination on the accuracy of genotype imputation in beef cattle.
Methods
2775 Angus and 1485 Limousin parent-verified sire/offspring pairs were genotyped with the Illumina BovineSNP50 chip. Haplotype phasing was performed with DAGPHASE and BEAGLE using UMD3.1 assembly SNP (single nucleotide polymorphism) coordinates. Recombination events were detected by comparing the two reconstructed chromosomal haplotypes inherited by each offspring with those of their sires. Expected crossover probabilities were estimated assuming no interference and a binomial distribution for the frequency of crossovers. The BayesB approach for genome-wide association analysis implemented in the GenSel software was used to identify genomic regions harboring QTL with large effects on recombination. BEAGLE was used to impute Angus genotypes from a 7K subset to the 50K chip.
Results
DAGPHASE was superior to BEAGLE in haplotype phasing, which indicates that linkage information from relatives can improve its accuracy. The estimated genetic length of the 29 bovine autosomes was 3097 cM, with a genome-wide recombination distance averaging 1.23 cM/Mb. 427 and 348 windows containing recombination hotspots were detected in Angus and Limousin, respectively, of which 166 were in common. Several significant SNPs and candidate genes, which influence genome-wide recombination were localized in QTL regions detected in the two breeds. High-recombination rates hinder the accuracy of haplotype phasing and genotype imputation.
Conclusions
Small population sizes, inadequate half-sib family sizes, recombination, gene conversion, genotyping errors, and map errors reduce the accuracy of haplotype phasing and genotype imputation. Candidate regions associated with recombination were identified in both breeds. Recombination analysis may improve the accuracy of haplotype phasing and genotype imputation from low- to high-density SNP panels.
doi:10.1186/1297-9686-46-34
PMCID: PMC4071795  PMID: 24885305
22.  Imputation of Variants from the 1000 Genomes Project Modestly Improves Known Associations and Can Identify Low-frequency Variant - Phenotype Associations Undetected by HapMap Based Imputation 
PLoS ONE  2013;8(5):e64343.
Genome-wide association (GWA) studies have been limited by the reliance on common variants present on microarrays or imputable from the HapMap Project data. More recently, the completion of the 1000 Genomes Project has provided variant and haplotype information for several million variants derived from sequencing over 1,000 individuals. To help understand the extent to which more variants (including low frequency (1% ≤ MAF <5%) and rare variants (<1%)) can enhance previously identified associations and identify novel loci, we selected 93 quantitative circulating factors where data was available from the InCHIANTI population study. These phenotypes included cytokines, binding proteins, hormones, vitamins and ions. We selected these phenotypes because many have known strong genetic associations and are potentially important to help understand disease processes. We performed a genome-wide scan for these 93 phenotypes in InCHIANTI. We identified 21 signals and 33 signals that reached P<5×10−8 based on HapMap and 1000 Genomes imputation, respectively, and 9 and 11 that reached a stricter, likely conservative, threshold of P<5×10−11 respectively. Imputation of 1000 Genomes genotype data modestly improved the strength of known associations. Of 20 associations detected at P<5×10−8 in both analyses (17 of which represent well replicated signals in the NHGRI catalogue), six were captured by the same index SNP, five were nominally more strongly associated in 1000 Genomes imputed data and one was nominally more strongly associated in HapMap imputed data. We also detected an association between a low frequency variant and phenotype that was previously missed by HapMap based imputation approaches. An association between rs112635299 and alpha-1 globulin near the SERPINA gene represented the known association between rs28929474 (MAF = 0.007) and alpha1-antitrypsin that predisposes to emphysema (P = 2.5×10−12). Our data provide important proof of principle that 1000 Genomes imputation will detect novel, low frequency-large effect associations.
doi:10.1371/journal.pone.0064343
PMCID: PMC3655956  PMID: 23696881
23.  Utilizing Genotype Imputation for the Augmentation of Sequence Data 
PLoS ONE  2010;5(6):e11018.
Background
In recent years, capabilities for genotyping large sets of single nucleotide polymorphisms (SNPs) has increased considerably with the ability to genotype over 1 million SNP markers across the genome. This advancement in technology has led to an increase in the number of genome-wide association studies (GWAS) for various complex traits. These GWAS have resulted in the implication of over 1500 SNPs associated with disease traits. However, the SNPs identified from these GWAS are not necessarily the functional variants. Therefore, the next phase in GWAS will involve the refining of these putative loci.
Methodology
A next step for GWAS would be to catalog all variants, especially rarer variants, within the detected loci, followed by the association analysis of the detected variants with the disease trait. However, sequencing a locus in a large number of subjects is still relatively expensive. A more cost effective approach would be to sequence a portion of the individuals, followed by the application of genotype imputation methods for imputing markers in the remaining individuals. A potentially attractive alternative option would be to impute based on the 1000 Genomes Project; however, this has the drawbacks of using a reference population that does not necessarily match the disease status and LD pattern of the study population. We explored a variety of approaches for carrying out the imputation using a reference panel consisting of sequence data for a fraction of the study participants using data from both a candidate gene sequencing study and the 1000 Genomes Project.
Conclusions
Imputation of genetic variation based on a proportion of sequenced samples is feasible. Our results indicate the following sequencing study design guidelines which take advantage of the recent advances in genotype imputation methodology: Select the largest and most diverse reference panel for sequencing and genotype as many “anchor” markers as possible.
doi:10.1371/journal.pone.0011018
PMCID: PMC2882389  PMID: 20543988
24.  Family-based association analysis to finemap bipolar linkage peak on chromosome 8q24 using 2,500 genotyped SNPs and 15,000 imputed SNPs 
Bipolar Disorders  2010;12(8):786-792.
Objectives
Multiple linkage and association studies have suggested chromosome 8q24 as a promising candidate region for bipolar disorder (BP). We performed a detailed association analysis assessing the contribution of common genetic variation in this region to the risk of BP.
Methods
We analyzed 2,756 single nucleotide polymorphism (SNP) markers in the chromosome 8q24 region of 3,512 individuals from 737 families. In addition, we extended genotype imputation methods to family-based data and imputed 22,725 HapMap SNPs in the same region on 8q24. We applied a family-based method to test 15,552 high-quality genotyped or imputed SNPs for association with BP.
Results
Our association analysis identified the most significant marker (p = 4.80 × 10−5), near the gene encoding potassium voltage-gated channel KQT-like protein (KCNQ3). Other marginally significant markers were located near adenylate cyclase 8 (ADCY8) and ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1).
Conclusions
We developed an approach to apply MACH imputation to family-based data, which can increase the power to detect association signals. Our association results showed suggestive evidence of association of BP with loci near KCNQ3, ADCY8, and ST3GAL1. Consistent with genes identified by genome-wide association studies for BP, our results are consistent with the involvement of ion channelopathy in BP pathogenesis. However, common variants are insufficient to explain linkage findings in 8q24; other genetic variations should be explored.
doi:10.1111/j.1399-5618.2010.00883.x
PMCID: PMC3290916  PMID: 21176025
8q24; bipolar disorder; imputation; ion channelopathy
25.  Genotype Imputation for African Americans using data from HapMap Phase II versus 1000 Genomes Projects 
Genetic epidemiology  2012;36(5):508-516.
Genotype imputation provides imputation of untyped SNPs that are present on a reference panel such as those from the HapMap Project. It is popular for increasing statistical power and comparing results across studies using different platforms. Imputation for African American populations is challenging because their LD blocks are shorter and also because no ideal reference panel is available due to admixture. In this paper, we evaluated three imputation strategies for African Americans. The intersection strategy used a combined panel consisting of SNPs polymorphic in both CEU and YRI. The union strategy used a panel consisting of SNPs polymorphic in either CEU or YRI. The merge strategy merged results from two separate imputations, one using CEU and the other using YRI. Because recent investigators are increasingly using the data from the 1000 Genomes (1KG) Project for genotype imputation, we evaluated both 1KG-based imputations and HapMap-based imputations. We used 23,707 SNPs from chromosomes 21 and 22 on Affymetrix SNP Array 6.0 genotyped for 1,075 HyperGEN African Americans. We found that 1KG-based imputations provided a substantially larger number of variants than HapMap-based imputations, about three times as many common variants and eight times as many rare and low frequency variants. This higher yield is expected because the 1KG panel includes more SNPs. Accuracy rates using 1KG data were slightly lower than those using HapMap data before filtering, but slightly higher after filtering. The union strategy provided the highest imputation yield with next highest accuracy. The intersection strategy provided the lowest imputation yield but the highest accuracy. The merge strategy provided the lowest imputation accuracy. We observed that SNPs polymorphic only in CEU had much lower accuracy, reducing the accuracy of the union strategy. Our findings suggest that 1KG-based imputations can facilitate discovery of significant associations for SNPs across the whole MAF spectrum. Because the 1KG Project is still underway, we expect that later versions will provide better imputation performance.
doi:10.1002/gepi.21647
PMCID: PMC3703942  PMID: 22644746

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