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Background

Haplotype inference based on unphased SNP markers is an important task in population genetics. Although there are different approaches to the inference of haplotypes in diploid species, the existing software is not suitable for inferring haplotypes from unphased SNP data in polyploid species, such as the cultivated potato (Solanum tuberosum). Potato species are tetraploid and highly heterozygous.

Results

Here we present the software SATlotyper which is able to handle polyploid and polyallelic data. SATlo-typer uses the Boolean satisfiability problem to formulate Haplotype Inference by Pure Parsimony. The software excludes existing haplotype inferences, thus allowing for calculation of alternative inferences. As it is not known which of the multiple haplotype inferences are best supported by the given unphased data set, we use a bootstrapping procedure that allows for scoring of alternative inferences. Finally, by means of the bootstrapping scores, it is possible to optimise the phased genotypes belonging to a given haplotype inference. The program is evaluated with simulated and experimental SNP data generated for heterozygous tetraploid populations of potato. We show that, instead of taking the first haplotype inference reported by the program, we can significantly improve the quality of the final result by applying additional methods that include scoring of the alternative haplotype inferences and genotype optimisation. For a sub-population of nineteen individuals, the predicted results computed by SATlotyper were directly compared with results obtained by experimental haplotype inference via sequencing of cloned amplicons. Prediction and experiment gave similar results regarding the inferred haplotypes and phased genotypes.

Conclusion

Our results suggest that Haplotype Inference by Pure Parsimony can be solved efficiently by the SAT approach, even for data sets of unphased SNP from heterozygous polyploids. SATlotyper is freeware and is distributed as a Java JAR file. The software can be downloaded from the webpage of the GABI Primary Database at . The application of SATlotyper will provide haplotype information, which can be used in haplotype association mapping studies of polyploid plants.

Background

Analyses of genetic data at the level of haplotypes provide increased accuracy and power to infer genotype-phenotype correlations and evolutionary history of a locus. However, empirical determination of haplotypes is expensive and laborious. Therefore, several methods of inferring haplotypes from unphased genotypic data have been proposed, but it is unclear how accurate each of the methods is or which methods are superior. The accuracy of some of the leading methods of computational haplotype inference (PL-EM, Phase, SNPHAP, Haplotyper) are compared using a large set of 308 empirically determined haplotypes based on 15 SNPs, among which 36 haplotypes were observed to occur. This study presents several advantages over many previous comparisons of haplotype inference methods: a large number of subjects are included, the number of known haplotypes is much smaller than the number of chromosomes surveyed, a range in values of linkage disequilibrium, presence of rare SNP alleles, and considerable dispersion in the frequencies of haplotypes.

Results

In contrast to some previous comparisons of haplotype inference methods, there was very little difference in the accuracy of the various methods in terms of either assignment of haplotypes to individuals or estimation of haplotype frequencies. Although none of the methods inferred all of the known haplotypes, the assignment of haplotypes to subjects was about 90% correct for individuals heterozygous for up to three SNPs and was about 80% correct for up to five heterozygous sites. All of the methods identified every haplotype with a frequency above 1%, and none assigned a frequency above 1% to an incorrect haplotype.

Conclusions

All of the methods of haplotype inference have high accuracy and one can have confidence in inferences made by any one of the methods. The ability to identify even rare (≥ 1%) haplotypes is reassuring for efforts to identify haplotypes that contribute to disease in a significant proportion of a population. Assignment of haplotypes is relatively accurate among subjects heterozygous for up to 5 sites, and this might be the largest number of SNPs for which one should define haplotype blocks or have confidence in haplotype assignments.

Background

Since the completion of the HapMap project, huge numbers of individual genotypes have been generated from many kinds of laboratories. The efforts of finding or interpreting genetic association between disease and SNPs/haplotypes have been on-going widely. So, the necessity of the capability to analyze huge data and diverse interpretation of the results are growing rapidly.

Results

We have developed an advanced tool to perform linkage disequilibrium analysis, and genetic association analysis between disease and SNPs/haplotypes in an integrated web interface. It comprises of four main analysis modules: (i) data import and preprocessing, (ii) haplotype estimation, (iii) LD blocking and (iv) association analysis. Hardy-Weinberg Equilibrium test is implemented for each SNPs in the data preprocessing. Haplotypes are reconstructed from unphased diploid genotype data, and linkage disequilibrium between pairwise SNPs is computed and represented by D', r2 and LOD score. Tagging SNPs are determined by using the square of Pearson's correlation coefficient (r2). If genotypes from two different sample groups are available, diverse genetic association analyses are implemented using additive, codominant, dominant and recessive models. Multiple verified algorithms and statistics are implemented in parallel for the reliability of the analysis.

Conclusion

SNPAnalyzer 2.0 performs linkage disequilibrium analysis and genetic association analysis in an integrated web interface using multiple verified algorithms and statistics. Diverse analysis methods, capability of handling huge data and visual comparison of analysis results are very comprehensive and easy-to-use.

Motivation

Recent advances in genotyping technology has made data acquisition for whole-genome association study cost effective, and a current active area of research is developing efficient methods to analyze such large-scale data sets. Most sophisticated association mapping methods that are currently available take phased haplotype data as input. However, phase information is not readily available from sequencing methods and inferring the phase via computational approaches is time-consuming, taking days to phase a single chromosome.

Results

In this paper, we devise an efficient method for scanning unphased whole-genome data for association. Our approach combines a recently found linear-time algorithm for phasing genotypes on trees with a recently proposed tree-based method for association mapping. From unphased genotype data, our algorithm builds local phylogenies along the genome, and scores each tree according to the clustering of cases and controls. We assess the performance of our new method on both simulated and real biological data sets.

Motivation: Recent advances in genotyping technology has made data acquisition for whole-genome association study cost effective, and a current active area of research is developing efficient methods to analyze such large-scale datasets. Most sophisticated association mapping methods that are currently available take phased haplotype data as input. However, phase information is not readily available from sequencing methods and inferring the phase via computational approaches is time-consuming, taking days to phase a single chromosome.

Results: In this article, we devise an efficient method for scanning unphased whole-genome data for association. Our approach combines a recently found linear-time algorithm for phasing genotypes on trees with a recently proposed tree-based method for association mapping. From unphased genotype data, our algorithm builds local phylogenies along the genome, and scores each tree according to the clustering of cases and controls. We assess the performance of our new method on both simulated and real biological datasets.

Availability The software described in this article is available at http://www.daimi.au.dk/~mailund/Blossoc and distributed under the GNU General Public License.

Contact:mailund@birc.au.dk

Typically locus specific genotype data do not contain information regarding the gametic phase of haplotypes, especially when an individual is heterozygous at more than one locus among a large number of linked polymorphic loci. Thus, studying disease-haplotype association using unphased genotype data is essentially a problem of handling a missing covariate in a case-control design. There are several methods for estimating a disease-haplotype association parameter in a matched case-control study. Here we propose a conditional likelihood approach for inference regarding the disease-haplotype association using unphased genotype data arising from a matched case-control study design. The proposed method relies on a logistic disease risk model and a Hardy-Weinberg equilibrium (HWE) among the control population only. We develop an expectation and conditional maximization (ECM) algorithm for jointly estimating the haplotype frequency and the disease-haplotype association parameter(s). We apply the proposed method to analyze the data from the Alpha-Tocopherol, Beta-Carotene Cancer prevention study, and a matched case-control study of breast cancer patients conducted in Israel. The performance of the proposed method is evaluated via simulation studies.

Accounting for interactions with environmental factors in association studies may improve the power to detect genetic effects and may help identifying important environmental effect modifiers. The power of unphased genotype-versus haplotype-based methods in regions with high linkage disequilibrium (LD), as measured by D', for analyzing gene × environment (gene × sex) interactions was compared using the Genetic Analysis Workshop 15 (GAW15) simulated data on rheumatoid arthritis with prior knowledge of the answers. Stepwise and regular conditional logistic regression (CLR) was performed using a matched case-control sample for a HLA region interacting with sex. Haplotype-based analyses were performed using a haplotype-sharing-based Mantel statistic and a test for haplotype-trait association in a general linear model framework. A step-down minP algorithm was applied to derive adjusted p-values and to allow for power comparisons. These methods were also applied to the GAW15 real data set for PTPN22.

For markers in strong LD, stepwise CLR performed poorly because of the correlation/collinearity between the predictors in the model. The power was high for detecting genetic main effects using simple CLR models and haplotype-based methods and for detecting joint effects using CLR and Mantel statistics. Only the haplotype-trait association test had high power to detect the gene × sex interaction.

In the PTPN22 region with markers characterized by strong LD, all methods indicated a significant genotype × sex interaction in a sample of about 1000 subjects. The previously reported R620W single-nucleotide polymorphism was identified using logistic regression, but the haplotype-based methods did not provide any precise location information.

Background

Haplotypes extracted from human DNA can be used for gene mapping and other analysis of genetic patterns within and across populations. A fundamental problem is, however, that current practical laboratory methods do not give haplotype information. Estimation of phased haplotypes of unrelated individuals given their unphased genotypes is known as the haplotype reconstruction or phasing problem.

Results

We define three novel statistical models and give an efficient algorithm for haplotype reconstruction, jointly called HaploRec. HaploRec is based on exploiting local regularities conserved in haplotypes: it reconstructs haplotypes so that they have maximal local coherence. This approach – not assuming statistical dependence for remotely located markers – has two useful properties: it is well-suited for sparse marker maps, such as those used in gene mapping, and it can actually take advantage of long maps.

Conclusion

Our experimental results with simulated and real data show that HaploRec is a powerful method for the large scale haplotyping needed in association studies. With sample sizes large enough for gene mapping it appeared to be the best compared to all other tested methods (Phase, fastPhase, PL-EM, Snphap, Gerbil; simulated data), with small samples it was competitive with the best available methods (real data). HaploRec is several orders of magnitude faster than Phase and comparable to the other methods; the running times are roughly linear in the number of subjects and the number of markers. HaploRec is publicly available at .

Background

The completion of the HapMap project has stimulated further development of haplotype-based methodologies for disease associations. A key aspect of such development is the statistical inference of individual diplotypes from unphased genotypes. Several methodologies for inferring haplotypes have been developed, but they have not been evaluated extensively to determine which method not only performs well, but also can be easily incorporated in downstream haplotype-based association analyses. In this paper, we attempt to do so. Our evaluation was carried out by comparing the two leading Bayesian methods, implemented in PHASE and HAPLOTYPER, and the two leading empirical methods, implemented in PL-EM and HPlus. We used these methods to analyze real data, namely the dense genotypes on X-chromosome of 30 European and 30 African trios provided by the International HapMap Project, and simulated genotype data. Our conclusions are based on these analyses.

Results

All programs performed very well on X-chromosome data, with an average similarity index of 0.99 and an average prediction rate of 0.99 for both European and African trios. On simulated data with approximation of coalescence, PHASE implementing the Bayesian method based on the coalescence approximation outperformed other programs on small sample sizes. When the sample size increased, other programs performed as well as PHASE. PL-EM and HPlus implementing empirical methods required much less running time than the programs implementing the Bayesian methods. They required only one hundredth or thousandth of the running time required by PHASE, particularly when analyzing large sample sizes and large umber of SNPs.

Conclusion

For large sample sizes (hundreds or more), which most association studies require, the two empirical methods might be used since they infer the haplotypes as accurately as any Bayesian methods and can be incorporated easily into downstream haplotype-based analyses such as haplotype-association analyses.

Background

The haplotypes of the X chromosome are accessible to direct count in males, whereas the diplotypes of the females may be inferred knowing the haplotype of their sons or fathers. Here, we investigated: 1) the possible large-scale haplotypic structure of the X chromosome in a Caucasian population sample, given the single-nucleotide polymorphism (SNP) maps and genotypes provided by Illumina and Affimetrix for Genetic Analysis Workshop 14, and, 2) the performances of widely used programs in reconstructing haplotypes from population genotypic data, given their known distribution in a sample of unrelated individuals.

Results

All possible unrelated mother-son pairs of Caucasian ancestry (N = 104) were selected from the 143 families of the Collaborative Study on the Genetics of Alcoholism pedigree files, and the diplotypes of the mothers were inferred from the X chromosomes of their sons. The marker set included 313 SNPs at an average density of 0.47 Mb. Linkage disequilibrium between pairs of markers was computed by the parameter D', whereas for measuring multilocus disequilibrium, we developed here an index called D*, and applied it to all possible sliding windows of 5 markers each. Results showed a complex pattern of haplotypic structure, with regions of low linkage disequilibrium separated by regions of high values of D*. The following programs were evaluated for their accuracy in inferring population haplotype frequencies: 1) ARLEQUIN 2.001; 2) PHASE 2.1.1; 3) SNPHAP 1.1; 4) HAPLOBLOCK 1.2; 5) HAPLOTYPER 1.0. Performances were evaluated by Pearson correlation (r) coefficient between the true and the inferred distribution of haplotype frequencies.

Conclusion

The SNP haplotypic structure of the X chromosome is complex, with regions of high haplotype conservation interspersed among regions of higher haplotype diversity. All the tested programs were accurate (r = 1) in reconstructing the distribution of haplotype frequencies in case of high D* values. However, only the program PHASE realized a high correlation coefficient (r > 0.7) in conditions of low linkage disequilibrium.

The genome-wide association study (GWAS) has become a routine approach for mapping disease risk loci with the advent of large-scale genotyping technologies. Multi-allelic haplotype markers can provide superior power compared with single-SNP markers in mapping disease loci. However, the application of haplotype-based analysis to GWAS is usually bottlenecked by prohibitive time cost for haplotype inference, also known as phasing. In this study, we developed an efficient approach to haplotype-based analysis in GWAS. By using a reference panel, our method accelerated the phasing process and reduced the potential bias generated by unrealistic assumptions in phasing process. The haplotype-based approach delivers great power and no type I error inflation for association studies. With only a medium-size reference panel, phasing error in our method is comparable to the genotyping error afforded by commercial genotyping solutions.

Background

In many contexts, pedigrees for individuals are known even though not all individuals have been fully genotyped. In one extreme case, the genotypes for a set of full siblings are known, with no knowledge of parental genotypes. We propose a method for inferring phased haplotypes and genotypes for all individuals, even those with missing data, in such pedigrees, allowing a multitude of classic and recent methods for linkage and genome analysis to be used more efficiently.

Results

By artificially removing the founder generation genotype data from a well-studied simulated dataset, the quality of reconstructed genotypes in that generation can be verified. For the full structure of repeated matings with 15 offspring per mating, 10 dams per sire, 99.89% of all founder markers were phased correctly, given only the unphased genotypes for offspring. The accuracy was reduced only slightly, to 99.51%, when introducing a 2% error rate in offspring genotypes. When reduced to only 5 full-sib offspring in a single sire-dam mating, the corresponding percentage is 92.62%, which compares favorably with 89.28% from the leading Merlin package. Furthermore, Merlin is unable to handle more than approximately 10 sibs, as the number of states tracked rises exponentially with family size, while our approach has no such limit and handles 150 half-sibs with ease in our experiments.

Conclusions

Our method is able to reconstruct genotypes for parents when genotype data is only available for offspring individuals, as well as haplotypes for all individuals. Compared to the Merlin package, we can handle larger pedigrees and produce superior results, mainly due to the fact that Merlin uses the Viterbi algorithm on the state space to infer the genotype sequence. Tracking of haplotype and allele origin can be used in any application where the marker set does not directly influence genotype variation influencing traits. Inference of genotypes can also reduce the effects of genotyping errors and missing data. The cnF2freq codebase implementing our approach is available under a BSD-style license.

With the widespread availability of SNP genotype data, there is great interest in analyzing pedigree haplotype data. Intermarker linkage disequilibrium for micro-satellite markers is usually low due to their physical distance; however, for dense maps of SNP markers, there can be strong linkage disequilibrium between marker loci. Linkage analysis (parametric and nonparametric) and family-based association studies are currently being carried out using dense maps of SNP marker loci. Monte Carlo methods are often used for both linkage and association studies; however, to date there are no programs available which can generate haplotype and/or genotype data consisting of a large number of loci for pedigree structures. SimPed is a program that quickly generates haplotype and/or genotype data for pedigrees of virtually any size and complexity. Marker data either in linkage disequilibrium or equilibrium can be generated for greater than 20,000 diallelic or multiallelic marker loci. Haplotypes and/or genotypes are generated for pedigree structures using specified genetic map distances and haplotype and/or allele frequencies. The simulated data generated by SimPed is useful for a variety of purposes, including evaluating methods that estimate haplotype frequencies for pedigree data, evaluating type I error due to intermarker linkage disequilibrium and estimating empirical p values for linkage and family-based association studies.

Dense SNP maps can be highly informative for linkage studies. But when parental genotypes are missing, multipoint linkage scores can be inflated in regions with substantial marker-marker linkage disequilibrium (LD). Such regions were observed in the Affymetrix SNP genotypes for the Genetic Analysis Workshop 14 (GAW14) Collaborative Study on the Genetics of Alcoholism (COGA) dataset, providing an opportunity to test a novel simulation strategy for studying this problem. First, an inheritance vector (with or without linkage present) is simulated for each replicate, i.e., locations of recombinations and transmission of parental chromosomes are determined for each meiosis. Then, two sets of founder haplotypes are superimposed onto the inheritance vector: one set that is inferred from the actual data and which contains the pattern of LD; and one set created by randomly selecting parental alleles based on the known allele frequencies, with no correlation (LD) between markers. Applying this strategy to a map of 176 SNPs (66 Mb of chromosome 7) for 100 replicates of 116 sibling pairs, significant inflation of multipoint linkage scores was observed in regions of high LD when parental genotypes were set to missing, with no linkage present. Similar inflation was observed in analyses of the COGA data for these affected sib pairs with parental genotypes set to missing, but not after reducing the marker map until r2 between any pair of markers was ≤ 0.05. Additional simulation studies of affected sib pairs assuming uniform LD throughout a marker map demonstrated inflation of significance levels at r2 values greater than 0.05. When genotypes are available only from two affected siblings in many families in a sample, trimming SNP maps to limit r2 to 0–0.05 for all marker pairs will prevent inflation of linkage scores without sacrificing substantial linkage information. Simulation studies on the observed pedigree structures and map can also be used to determine the effect of LD on a particular study.

In genetic mapping of complex traits, scored haplotypes are likely to represent only a subset of all causal polymorphisms. At the extreme of this scenario, observed polymorphisms are not themselves functional, and only linked to causal ones via linkage disequilibrium (LD). We will demonstrate that due to such incomplete knowledge regarding the underlying genetic mechanism, the variance of a trait may become different between the scored haplotypes. Thus, unequal variances between haplotypes may be indicative of additional functional polymorphisms affecting the trait. Methods accounting for such haplotype-specific variance may also provide an increased power to detect complex associations. We suggest ways to estimate and test these haplotypic variance contrasts, and incorporate them into the haplotypic tests for association. We further extend this approach to data with unknown gametic phase via likelihood-based simultaneous estimation of haplotypic effects and their frequencies. We find our approach to provide additional power, especially under the following types of models: (a) where scored and unobserved variants are epistatically interacting with each other; and (b) under heterogeneity models, where multiple unobserved mutations are linked to nonfunctional observed polymorphisms via LD. An illustrative example of usefulness of the method is discussed, utilizing analysis of multilocus effects within the catechol-O-methyl transferase (COMT) gene.

Linkage disequilibrium (LD) is a major concern in many genetic studies because of the markedly increased density of SNP (Single Nucleotide Polymorphism) genotype markers. This dramatic increase in the number of SNPs may cause problems in statistical analyses, such as by introducing multiple comparisons in hypothesis testing and colinearity in logistic regression models, because of the presence of complex LD structures. Inferences must be made about the underlying genetic variation through the LD structure before applying statistical models to the data. Therefore, we introduced the textile plot to provide a visualization of LD to improve the analysis of the genetic variation present in multiple-SNP genotype data. The plot can accentuate LD by displaying specific geometrical shapes, and allowing for the underlying haplotype structure to be inferred without any haplotype-phasing algorithms. Application of this technique to simulated and real data sets illustrated the potential usefulness of the textile plot as an aid to the interpretation of LD in multiple-SNP genotype data. The initial results of LD mapping and haplotype analyses of disease genes are encouraging, indicating that the textile plot may be useful in disease association studies.

In the past two years, tracking the explosion in data due to ever-improving single nucleotide polymorphism (SNP) maps and cheaper high-throughput genotyping technologies, a bewildering array of new algorithms and relevant software have appeared for haplotype phase inference. The alternatives to haplotype inference are to resolve haplotypes completely, either by in vitro methods or by typing close pedigrees, which is expensive and is not guaranteed in pedigrees, or to ignore haplotype-level analysis in favour of genotype-level analysis, which avoids the danger of treating inferred haplotypes as real but denies the researcher, potentially, any valuable analytic insights. This review attempts a snapshot of this rapidly moving field as it stands at present, and is mainly restricted, given the current predominance of SNP genotyping, to the consideration of diallelic data. For completeness, the review will occasionally refer to algorithms for which no software exists.

Background

Genetic association studies, especially genome-wide studies, make use of linkage disequilibrium(LD) information between single nucleotide polymorphisms (SNPs). LD is also used for studying genome structure and has been valuable for evolutionary studies. The strength of LD is commonly measured by r2, a statistic closely related to the Pearson's χ2 statistic. However, the computation and testing of linkage disequilibrium using r2 requires known haplotype counts of the SNP pair, which can be a problem for most population-based studies where the haplotype phase is unknown. Most statistical genetic packages use likelihood-based methods to infer haplotypes. However, the variability of haplotype estimation needs to be accounted for in the test for linkage disequilibrium.

Findings

We develop a Monte Carlo based test for LD based on the null distribution of the r2 statistic. Our test is based on r2 and can be reported together with r2. Simulation studies show that it offers slightly better power than existing methods.

Conclusions

Our approach provides an alternative test for LD and has been implemented as a R program for ease of use. It also provides a general framework to account for other haplotype inference methods in LD testing.

Background

Genetic isolates such as the Ashkenazi Jews (AJ) potentially offer advantages in mapping novel loci in whole genome disease association studies. To analyze patterns of genetic variation in AJ, genotypes of 101 healthy individuals were determined using the Affymetrix EAv3 500 K SNP array and compared to 60 CEPH-derived HapMap (CEU) individuals. 435,632 SNPs overlapped and met annotation criteria in the two groups.

Results

A small but significant global difference in allele frequencies between AJ and CEU was demonstrated by a mean FST of 0.009 (P < 0.001); large regions that differed were found on chromosomes 2 and 6. Haplotype blocks inferred from pairwise linkage disequilibrium (LD) statistics (Haploview) as well as by expectation-maximization haplotype phase inference (HAP) showed a greater number of haplotype blocks in AJ compared to CEU by Haploview (50,397 vs. 44,169) or by HAP (59,269 vs. 54,457). Average haplotype blocks were smaller in AJ compared to CEU (e.g., 36.8 kb vs. 40.5 kb HAP). Analysis of global patterns of local LD decay for closely-spaced SNPs in CEU demonstrated more LD, while for SNPs further apart, LD was slightly greater in the AJ. A likelihood ratio approach showed that runs of homozygous SNPs were approximately 20% longer in AJ. A principal components analysis was sufficient to completely resolve the CEU from the AJ.

Conclusion

LD in the AJ versus was lower than expected by some measures and higher by others. Any putative advantage in whole genome association mapping using the AJ population will be highly dependent on regional LD structure.

We compared the accuracy of haplotype inferences at a 6 Mb region on chromosome 7 where significant linkage between a brain oscillation phenotype and a cholinergic muscarinic receptor gene was previously reported. Individual haplotype assignments and haplotype frequencies were estimated using 5, 10, and 14 consecutive Illumina single-nucleotide polymorphisms (SNPs) within the 1-LOD unit support interval of the chromosome 7 linkage peak. Initially, haplotypes were constructed incorporating phase information provided by relatives using the pedigree analysis package MERLIN. Population-based haplotypes were inferred using the haplotype estimation software HAPLO.STATS and PHASE, using unrelated individuals.

The 14 SNPs within this region exhibited markedly low linkage disequilibrium, and the average D' estimate between SNPs was 0.18 (range: 0.01–0.97). In comparison to the family-based haplotypes calculated in MERLIN, the computational inferences of individual haplotype assignments were most accurate when considering 5 consecutive SNPs, but decayed dramatically when considering 10 or 14 SNPs in both PHASE and HAPLO.STATS. When comparing the two haplotype inference methods, both PHASE and HAPLO.STATS performed poorly. These analyses underscore the difficulties of haplotype estimation in the presence of low linkage disequilibrium and stress the importance of careful consideration of confidence measures when using estimated haplotype frequencies and individual assignments in biomedical research.

Given the increasing size of modern genetic data sets and, in particular, the move towards genome-wide studies, there is merit in considering analyses that gain computational efficiency by being more heuristic in nature. With this in mind, we present results of cladistic analyses methods on the Genetic Analysis Workshop 15 Problem 3 simulated data (answers known). Our analysis attempts to capture similarities between individuals using a series of trees, and then looks for regions in which mutations on those trees can successfully explain a phenotype of interest. Existing varieties of such algorithms assume haplotypes are known, or have been inferred, an assumption that is often unrealistic for genome-wide data. We therefore present an extension of these methods that can successfully analyze genotype, rather than haplotype, data.

Reconstruction of haplotypes, or the allelic phase, of single nucleotide polymorphisms (SNPs) is a key component of studies aimed at the identification and dissection of genetic factors involved in complex genetic traits. In humans, this often involves investigation of SNPs in case/control or other cohorts in which the haplotypes can only be partially inferred from genotypes by statistical approaches with resulting loss of power. Moreover, alternative statistical methodologies can lead to different evaluations of the most probable haplotypes present, and different haplotype frequency estimates when data are ambiguous. Given the cost and complexity of SNP studies, a robust and easy-to-use molecular technique that allows haplotypes to be determined directly from individual DNA samples would have wide applicability. Here, we present a reliable, automated and high-throughput method for molecular haplotyping in 2 kb, and potentially longer, sequence segments that is based on the physical determination of the phase of SNP alleles on either of the individual paternal haploids. We demonstrate that molecular haplotyping with this technique is not more complicated than SNP genotyping when implemented by matrix-assisted laser desorption/ionisation mass spectrometry, and we also show that the method can be applied using other DNA variation detection platforms. Molecular haplotyping is illustrated on the well-described β2-adrenergic receptor gene.

Background

Genotyping technologies enable us to genotype multiple Single Nucleotide Polymorphisms (SNPs) within selected genes/regions, providing data for haplotype association analysis. While haplotype-based association analysis is powerful for detecting untyped causal alleles in linkage-disequilibrium (LD) with neighboring SNPs/haplotypes, the inclusion of extraneous SNPs could reduce its power by increasing the number of haplotypes with each additional SNP.

Methods

Here, we propose a haplotype-based stepwise procedure (HBSP) to eliminate extraneous SNPs. To evaluate its properties, we applied HBSP to both simulated and real data, generated from a study of genetic associations of the bactericidal/permeability-increasing (BPI) gene with pulmonary function in a cohort of patients following bone marrow transplantation.

Results

Under the null hypothesis, use of the HBSP gave results that retained the desired false positive error rates when multiple comparisons were considered. Under various alternative hypotheses, HBSP had adequate power to detect modest genetic associations in case-control studies with 500, 1,000 or 2,000 subjects. In the current application, HBSP led to the identification of two specific SNPs with a positive validation.

Conclusion

These results demonstrate that HBSP retains the essence of haplotype-based association analysis while improving analytic power by excluding extraneous SNPs. Minimizing the number of SNPs also enables simpler interpretation and more cost-effective applications.

The Genetic Analysis Workshop 16 rheumatoid arthritis data include a set of 868 cases and 1194 controls genotyped at 545,080 single-nucleotide polymorphisms (SNPs) from the Illumina 550 k chip. We focus on investigating chromosomes 6 and 18, which have 35,574 and 16,450 SNPs, respectively. Association studies, including single SNP and haplotype-based analyses, were applied to the data on those two chromosomes. Specifically, we conducted a generalized linear model with regularization (rGLM) approach for detecting disease-haplotype association using unphased SNP data. A total of 444 and 43 four-SNP tests were found to be significant at the Bonferroni corrected 5% significance level on chromosome 6 and 18, respectively.

Uncertainty about linkage phases of multiple single nucleotide polymorphisms (SNPs) in heterozygous diploids challenges the identification of specific DNA sequence variants that encode a complex trait. A statistical technique implemented with the EM algorithm has been developed to infer the effects of SNP haplotypes from genotypic data by assuming that one haplotype (called the risk haplotype) performs differently from the rest (called the non-risk haplotype). This assumption simplifies the definition and estimation of genotypic values of diplotypes for a complex trait, but will reduce the power to detect the risk haplotype when non-risk haplotypes contain substantial diversity. In this article, we incorporate general quantitative genetic theory to specify the differentiation of different haplotypes in terms of their genetic control of a complex trait. A model selection procedure is deployed to test the best number and combination of risk haplotypes, thus providing a precise and powerful test of genetic determination in association studies. Our method is derived on the maximum likelihood theory and has been shown through simulation studies to be powerful for the characterization of the genetic architecture of complex quantitative traits.