This vignette provides a description of how to use the GENESIS package to run genetic association tests. GENESIS uses mixed models for genetic association testing, as PC-AiR PCs can be used as fixed effect covariates to adjust for population stratification, and a kinship matrix (or genetic relationship matrix) estimated from PC-Relate can be used to account for phenotype correlation due to genetic similarity among samples.
The fitNullMM
function in the GENESIS
package reads sample data from either a standard data.frame
class object or a ScanAnnotationDataFrame
class object as created by the GWASTools
package. This object must contain all of the outcome and covariate data for all samples to be included in the mixed model analysis. Additionally, this object must include a variable called "scanID" which contains a unique identifier for each sample in the analysis. While a standard data.frame
can be used, we recommend using a ScanAnnotationDataFrame
object, as it can be paired with the genotype data (see below) to ensure matching of sample phenotype and genotype data. Through the use of GWASTools
, a ScanAnnotationDataFrame
class object can easily be created from a data.frame
class object. Example R code for creating a ScanAnnotationDataFrame
object is presented below. Much more detail can be found in the GWASTools
package reference manual.
library(GENESIS) library(GWASTools) # file path to GDS file gdsfile <- system.file("extdata", "HapMap_ASW_MXL_geno.gds", package="GENESIS") # read in GDS data HapMap_geno <- GdsGenotypeReader(filename = gdsfile) # create a GenotypeData class object HapMap_genoData <- GenotypeData(HapMap_geno) # load saved matrix of KING-robust estimates data("HapMap_ASW_MXL_KINGmat") # run PC-AiR mypcair <- pcair(genoData = HapMap_genoData, kinMat = HapMap_ASW_MXL_KINGmat, divMat = HapMap_ASW_MXL_KINGmat) # run PC-Relate mypcrel <- pcrelate(genoData = HapMap_genoData, pcMat = mypcair$vectors[,1], training.set = mypcair$unrels) # generate a phenotype set.seed(4) pheno <- 0.2*mypcair$vectors[,1] + rnorm(mypcair$nsamp, mean = 0, sd = 1)
# mypcair contains PCs from a previous PC-AiR analysis # mypcrel contains Kinship Estimates from a previous PC-Relate analysis # pheno is a vector of Phenotype values # make a data.frame mydat <- data.frame(scanID = mypcrel$sample.id, pc1 = mypcair$vectors[,1], pheno = pheno) head(mydat) # make ScanAnnotationDataFrame scanAnnot <- ScanAnnotationDataFrame(mydat) scanAnnot
The assocTestMM
function in the GENESIS
package reads genotype data from a GenotypeData
class object as created by the GWASTools
package. Through the use of GWASTools
, a GenotypeData
class object can easily be created from:
Example R code for creating a GenotypeData
object is presented below. Much more detail can be found in the GWASTools
package reference manual.
geno <- MatrixGenotypeReader(genotype = genotype, snpID = snpID, chromosome = chromosome, position = position, scanID = scanID) genoData <- GenotypeData(geno)
genotype
is a matrix of genotype values coded as 0 / 1 / 2, where rows index SNPs and columns index samplessnpID
is an integer vector of unique SNP IDschromosome
is an integer vector specifying the chromosome of each SNPposition
is an integer vector specifying the position of each SNPscanID
is a vector of unique individual IDsgeno <- GdsGenotypeReader(filename = "genotype.gds") genoData <- GenotypeData(geno)
filename
is the file path to the GDS objectThe SNPRelate
package provides the snpgdsBED2GDS
function to convert binary PLINK files into a GDS file.
snpgdsBED2GDS(bed.fn = "genotype.bed", bim.fn = "genotype.bim", fam.fn = "genotype.fam", out.gdsfn = "genotype.gds")
bed.fn
is the file path to the PLINK .bed filebim.fn
is the file path to the PLINK .bim filefam.fn
is the file path to the PLINK .fam fileout.gdsfn
is the file path for the output GDS fileOnce the PLINK files have been converted to a GDS file, then a GenotypeData
object can be created as described above.
To demonstrate association testing with the GENESIS
package, we analyze SNP data from the Mexican Americans in Los Angeles, California (MXL) and African American individuals in the southwestern USA (ASW) population samples of HapMap 3. Mexican Americans and African Americans have a diverse ancestral background, and familial relatives are present in these data. Genotype data at a subset of 20K autosomal SNPs for 173 individuals are provided as a GDS file.
# read in GDS data gdsfile <- system.file("extdata", "HapMap_ASW_MXL_geno.gds", package="GENESIS") HapMap_geno <- GdsGenotypeReader(filename = gdsfile)
# create a GenotypeData class object with paired ScanAnnotationDataFrame HapMap_genoData <- GenotypeData(HapMap_geno, scanAnnot = scanAnnot) HapMap_genoData
A mixed model for genetic association testing typically includes a genetic relationship matrix (GRM) to account for genetic similarity among sample individuals. If we are using kinship coefficient estimates from PC-Relate to construct this GRM, then the function pcrelateMakeGRM
should be used to provide the matrix in the appropriate format for fitNullMM
.
myGRM <- pcrelateMakeGRM(mypcrel) myGRM[1:5,1:5]
Note that both the row and column names of this matrix are the same scanIDs as used in the scan annotation data.
There are two steps to performing genetic association testing with GENESIS. First, the null model (i.e. the model with no SNP genotype term) is fit using the fitNullMM
function. Second, the output of the null model fit is used in conjunction with the genotype data to quickly run SNP-phenotype association tests using the assocTestMM
function. There is a computational advantage to splitting these two steps into two function calls; the null model only needs to be fit once, and SNP association tests can be paralelized by chromosome or some other partitioning to speed up analyses (details below).
The first step for association testing with GENESIS
is to fit the mixed model under the null hypothesis that each SNP has no effect. This null model contains all of the covariates, including ancestry representative PCs, as well as any random effects, such as a polygenic effect due to genetic relatedness, but it does not include any SNP genotype terms as fixed effects.
Using the fitNullMM
function, random effects in the null model are specified via their covariance structures. This allows for the inclusion of a polygenic random effect using a kinship matrix or genetic relationship matrix (GRM).
A linear mixed model (LMM) should be fit when analyzing a quantitative phenotype. The example R code below fits a basic null mixed model.
# fit the null mixed model nullmod <- fitNullMM(scanData = scanAnnot, outcome = "pheno", covars = "pc1", covMatList = myGRM, family = gaussian)
scanData
is the class ScanAnnotationDataFrame
or data.frame
object containing the sample dataoutcome
specifies the name of the outcome variable in scanData
covars
specifies the names of the covariates in scanData
covMatList
specifies the covariance structures for the random effects included in the modelfamily
should be gaussian for a quantitative phenotype, specifying a linear mixed modelThe Average Information REML (AIREML) procedure is used to estimate the variance components of the random effects. When verbose = TRUE
, the variance component estimates, the log-likelihood, and the residual sum of squares in each iteration are printed to the R console (shown above). In this example, Sigma^2_A
is the variance component for the random effect specified in covMatList
, and Sigma^2_E
is the residual variance component.
The model can be fit with multiple fixed effect covariates by setting covars
equal to vector of covariate names. For example, if we wanted to include the variables "pc1", "pc2", "sex", and "age" all as covariates in the model:
nullmod <- fitNullMM(scanData = scanAnnot, outcome = "pheno", covars = c("pc1","pc2","sex","age"), covMatList = myGRM, family = gaussian)
The model also can be fit with multiple random effects. This is done by setting covMatList
equal to a list of matrices. For example, if we wanted to include a polygenic random effect with covariance structure given by the matrix "myGRM" and a household random effect with covariance structure specified by the matrix "H":
nullmod <- fitNullMM(scanData = scanAnnot, outcome = "pheno", covars = "pc1" covMatList = list("GRM" = myGRM, "House" = H), family = gaussian)
The names of the matrices in covMatList
determine the names of the variance component parameters. Therefore, in this example, the output printed to the R console will include Sigma^2_GRM
for the random effect specified by "myGRM", Sigma^2_House
for the random effect specified by "H", and Sigma^2_E
for the residual variance component.
Note: the row and column names of each matrix used to specify the covariance structure of a random effect in the mixed model must be the unique scanIDs for each sample in the analysis.
LMMs are typically fit under an assumption of constant (homogeneous) residual variance for all observations. However, for some outcomes, there may be evidence that different groups of observations have different residual variances, in which case the assumption of homoscedasticity is violated. group.var
can be used in order to fit separate (heterogeneous) residual variance components by some grouping variable. For example, if we have a categorical variable "race" in our scanData
, then we can estimate a different residual variance component for each unique value of "race" by using the following code:
nullmod <- fitNullMM(scanData = scanAnnot, outcome = "pheno", covars = "pc1", covMatList = myGRM, family = gaussian, group.var = "race")
In this example, the residual variance component Sigma^2_E
is replaced with group specific residual variance components Sigma^2_race1
, Sigma^2_race2
, ..., where "race1", "race2", ... are the unique values of the "race" variable.
Ideally, a generalized linear mixed model (GLMM) would be fit for a binary phenotype; however, fitting a GLMM is much more computationally demanding than fitting an LMM. To provide a compuationally efficient approach to fitting such a model, fitNullMM
uses the penalized quasi-likelihood (PQL) approximation to the GLMM (Breslow and Clayton). The implementation of this procedure in GENESIS
is the same as in GMMAT (Chen et al.), and more details can be found in that manuscript. If our outcome variable, "pheno", were binary, then the same R code could be used to fit the null model, but with family = binomial
.
nullmod <- fitNullMM(scanData = scanAnnot, outcome = "pheno", covars = "pc1", covMatList = myGRM, family = binomial)
Multiple fixed effect covariates and multiple random effects can be specified for binary phenotypes in the same way as they are for quantitative phenotypes. group.var
does not apply here.
The second step for association testing with GENESIS
is to use the fitted null model to test the SNPs in the GenotypeData
object for association with the specified outcome variable. This is done with the assocTestMM
function. Both (approximate) Wald and score tests are available, but the Wald test can only be performed when family = gaussian
in the null model. Otherwise, the use of assocTestMM
for running association tests with a quantitative or binary phenotype is identical.
The example R code below runs the association analyses using the null model we fit using fitNullMM
in the previous section.
assoc <- assocTestMM(genoData = HapMap_genoData, nullMMobj = nullmod, test = "Wald")
genoData
is a GenotypeData
class objectnullMMobj
is the output from fitNullMM
test
specifies whether to use a "Wald" or "Score" testBy default, the function will perform association tests at all SNPs in the genoData
object. However, for computational reasons it may be practical to parallelize this step, partitioning SNPs by chromosome or some other pre-selected grouping. If we only want to test a pre-specified set of SNPs, this can be done by passing a vector of snpID values to the snp.include
argument.
# mysnps is a vector of snpID values for the SNPs we want to test assoc <- assocTestMM(genoData = HapMap_genoData, nullMMobj = nullmod, test = "Wald", snp.include = mysnps)
If we only want to test SNPs on chromosome 22, this can be done by specifying the chromosome
argument.
assoc <- assocTestMM(genoData = HapMap_genoData, nullMMobj = nullmod, test = "Wald", chromosome = 22)
Multiple chromosomes can be specified at once by setting chromosome
equal to a vector of integer values.
Note: if snp.include
is specified, then the chromosome
argument is ignored.
The fitNullMM
function will return a list with a large amount of data. Some of the more useful output for the user includes:
varComp
: the variance component estimates for the random effectsfixef
: a data.frame
with point estimates, standard errors, test statistics, and p-values for each of the fixed effect covariatesfitted.values
: the fitted values from the modelresid.marginal
and resid.conditional
: the marginal and conditional residuals from the modelThere are also metrics assessing model fit such as the log-likelihood (logLik
), restricted log-likelihood (logLikR
), and the Akaike information criterion (AIC
). Additionally, there are some objects such as the working outcome vector (workingY
) and the Cholesky decomposition of the inverse of the estimated phenotype covariance matrix (cholSigmaInv
) that are used by the assocTestMM
function for association testing. Further details describing all of the output can be found with the command help(fitNullMM)
.
The assocTestMM
function will return a data.frame
with summary information from the association test for each SNP. Each row corresponds to a different SNP.
head(assoc)
snpID
: the unique snpIDchr
: the chromosomen
: the number of samples analyzed at that SNPMAF
: the estimated minor allele frequencyminor.allele
: which allele is the minor allele (either "A" or "B")Est
: the effect size estimate (beta) for that SNPSE
: the estimated standard error of the effect size estimateWald.Stat
: the chi-squared Wald test statisticWald.pval
: the p-value based on the Wald test statisticNote: when test = "Score"
in assocTestMM
(rather than test = "Wald"
), then Est
, SE
, Wald.Stat
, and Wald.pval
are replaced by:
Score
: the value of the score functionVar
: the variance of the score Score.Stat
: the chi-squared score test statisticScore.pval
: the p-value based on the score test statisticFurther details describing all of the output can be found with the command help(assocTestMM)
.
It is often of interest to estimate the proportion of the total phenotype variability explained by the entire set of genotyped SNPs avaialable; this provides an estimate of the narrow sense heritability of the trait. One method for estimating heritability is to use the variance component estimates from the null mixed model. GENESIS
includes the varCompCI
function for computing the proportion of variance explained by each random effect along with 95% confidence intervals.
varCompCI(nullMMobj = nullmod, prop = TRUE)
close(HapMap_genoData)
nullMMobj
is the output from fitNullMM
prop
is a logical indicator of whether the point estimates and confidence intervals should be returned as the proportion of total variability explained (TRUE) or on the orginal scale (FALSE)When additional random effects are included in the model (e.g. a shared household effect), varCompCI
will also return the proportion of variability explained by each of these components.
Note: varCompCI
can not compute proportions of variance explained when heterogeneous residual variances are used in the null model (i.e. group.var
is used in fitNullMM
). Confidence intervals can still be computed for the variance component estimates on the original scale by setting prop = FALSE
.
Note: variance component estimates are not interpretable for binary phenotypes when fit using the PQL method implemented in fitNullMM
; proportions of variance explained should not be calculated for these models.
Breslow NE and Clayton DG. (1993). Approximate Inference in Generalized Linear Mixed Models. Journal of the American Statistical Association 88: 9-25.
Chen H, Wang C, Conomos MP, Stilp AM, Li Z, Sofer T, Szpiro AA, Chen W, Brehm JM, Celedon JC, Redline S, Papanicolaou GJ, Thornton TA, Laurie CC, Rice K and Lin X. Control for Population Structure and Relatedness for Binary Traits in Genetic Association Studies Using Logistic Mixed Models. (Submitted).
Gogarten, S.M., Bhangale, T., Conomos, M.P., Laurie, C.A., McHugh, C.P., Painter, I., ... & Laurie, C.C. (2012). GWASTools: an R/Bioconductor package for quality control and analysis of Genome-Wide Association Studies. Bioinformatics, 28(24), 3329-3331.
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