%\VignetteIndexEntry{A Tutorial for the R Package SNPRelate}
%\VignetteDepends{gdsfmt, SNPRelate}
%\VignetteKeywords{GWAS, SNP}
%\VignettePackage{SNPRelate}

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\begin{document}

\title{A Tutorial for the R Package SNPRelate}
\author{Xiuwen Zheng\\
	GENEVA Coordinating Center\\
	Department of Biostatistics\\
	University of Washington}
\date{Jan 28, 2013}


\maketitle
\tableofcontents

\SweaveOpts{keep.source=TRUE, eps=FALSE}

% ---- Overview ----
\section{Overview}

Genome-wide association studies (GWAS) are widely used to help determine the genetic basis of diseases and traits, but they pose many computational challenges. We developed \hn{gdsfmt} and \hn{SNPRelate} (high-performance computing R packages for multi-core symmetric multiprocessing computer architectures) to accelerate two key computations in GWAS: principal component analysis (PCA) and relatedness analysis using identity-by-descent (IBD) measures$^1$. The kernels of our algorithms are written in C/C++ and have been highly optimized. The calculations of the genetic covariance matrix in PCA and pairwise IBD coefficients are split into non-overlapping parts and assigned to multiple cores for performance acceleration, as shown in Figure~1. Benchmarks show the uniprocessor implementations of PCA and IBD are $\sim$8 to 50 times faster than the implementations provided in the popular \hn{EIGENSTRAT} (v3.0) and \hn{PLINK} (v1.07) programs respectively, and can be sped up to 30$\sim$300 folds by utilizing multiple cores.

GDS is also used by an R/Bioconductor package \hn{GWASTools} as one of its data storage formats$^{2,3}$. \hn{GWASTools} provides many functions for quality control and analysis of GWAS, including statistics by SNP or scan, batch quality, chromosome anomalies, association tests, etc.


% -------- BEGIN FIGURE --------
\begin{figure}[!tpb]
	\centering
	\includegraphics[width=0.9\columnwidth]{SNPRelate-Flowchart.png}
\caption{Flowchart of parallel computing for principal component analysis and identity-by-descent analysis.}
\end{figure}
% -------- END FIGURE --------

R is the most popular statistical programming environment, but one not typically optimized for high performance or parallel computing which would ease the burden of large-scale GWAS calculations. To overcome these limitations we have developed a project named CoreArray (\url{http://corearray.sourceforge.net/}) that includes two R packages: \hn{gdsfmt} to provide efficient, platform independent memory and file management for genome-wide numerical data, and \hn{SNPRelate} to solve large-scale, numerically intensive GWAS calculations (i.e., PCA and IBD) on multi-core symmetric multiprocessing (SMP) computer architectures.

This vignette takes the user through the relatedness and principal component analysis used for genome wide association data. The methods in these vignettes have been introduced in the paper of Zheng {\it et al.} (2012)$^1$. For replication purposes the data used here are taken from the HapMap Phase II project. These data were kindly provided by the Center for Inherited Disease Research (CIDR) at Johns Hopkins University and the Broad Institute of MIT and Harvard University (Broad). The data supplied here should not be used for any purpose other than this tutorial.



% ---- Preparing Data ----
\section{Preparing Data}

% -------- Data formats used in SNPRelate --------
\subsection{Data formats used in SNPRelate}

To support efficient memory management for genome-wide numerical data, the \hn{gdsfmt} package provides the genomic data structure (GDS) file format for array-oriented bioinformatic data, which is a container for storing annotation data and SNP genotypes. In this format each byte encodes up to four SNP genotypes thereby reducing file size and access time. The GDS format supports data blocking so that only the subset of data that is being processed needs to reside in memory. GDS formatted data is also designed for efficient random access to large data sets.

<<>>=
# load the R packages: gdsfmt and SNPRelate
library(gdsfmt)
library(SNPRelate)
@

Here is a typical GDS file:
<<>>=
snpgdsSummary(snpgdsExampleFileName())
@
\fn{snpgdsExampleFileName()} returns the file name of a GDS file used as an example in \hn{SNPRelate}, and it is a subset of data from the HapMap project and the samples were genotyped by the Center for Inherited Disease Research (CIDR) at Johns Hopkins University and the Broad Institute of MIT and Harvard University (Broad). \fn{snpgdsSummary()} summarizes the genotypes stored in the GDS file. ``Individual-major mode'' indicates listing all SNPs for an individual before listing the SNPs for the next individual, etc. Conversely, ``SNP-major mode'' indicates listing all individuals for the first SNP before listing all individuals for the second SNP, etc. Sometimes ``SNP-major mode'' is more computationally efficient than ``individual-major model''. For example, the calculation of genetic covariance matrix deals with genotypic data SNP by SNP, and then ``SNP-major mode'' should be more efficient.
<<>>=
# open a GDS file
(genofile <- openfn.gds(snpgdsExampleFileName()))
@

The output lists all variables stored in the GDS file. At the first level, it stores variables \vr{sample.id}, \vr{snp.id}, etc. The additional information are displayed in the square brackets indicating data type, size, compressed or not + compression ratio. The second-level variables \vr{sex} and \vr{pop.group} are both stored in the folder of \vr{sample.annot}. All of the functions in \hn{SNPRelate} require a minimum set of variables in the annotation data.  The minimum required variables are
\begin{itemize}
	\item{\vr{sample.id}, a unique identifier for each sample.}
	\item{\vr{snp.id}, a unique identifier for each SNP.}
	\item{\vr{snp.position}, the base position of each SNP on the chromosome, and 0 for unknown position; it does not allow NA.}
	\item{\vr{snp.chromosome}, an integer mapping for each chromosome, with values 1-26, mapped in order from 1-22, 23=X,24=XY (the pseudoautosomal region), 25=Y, 26=M (the mitochondrial probes), and 0 for probes with unknown positions; it does not allow NA.}
	\item{\vr{genotype}, a SNP genotypic matrix. SNP-major mode: $n_\text{sample} \times n_\text{snp}$, individual-major mode: $n_\text{snp} \times n_\text{sample}$.}
\end{itemize}

Users can define the numeric chromosome codes which are stored with the variable \vr{snp.chromosome} as attributes. For example, \vr{snp.chromosome} has the attributes of chromosome coding:
<<>>=
# get the attributes of chromosome coding
get.attr.gdsn(index.gdsn(genofile, "snp.chromosome"))
@
\vr{autosome.start} is the starting numeric code of autosomes, and \vr{autosome.end} is the last numeric code of autosomes. \fn{put.attr.gdsn} can be used to add a new attribute or modify an existing attribute.

There are four possible values stored in the variable \vr{genotype}: 0, 1, 2 and 3. For bi-allelic SNP sites, ``0'' indicates two B alleles, ``1'' indicates one A allele and one B allele, ``2'' indicates two A alleles, and ``3'' is a missing genotype. For multi-allelic sites, it is a count of the reference allele (3 meaning no call). ``Bit2'' indicates that each byte encodes up to four SNP genotypes since one byte consists of eight bits.
<<>>=
# Take out genotype data for the first 3 samples and the first 5 SNPs
(g <- read.gdsn(index.gdsn(genofile, "genotype"), start=c(1,1), count=c(5,3)))

# Get the attribute of genotype
get.attr.gdsn(index.gdsn(genofile, "genotype"))
@
The returned value could be either ``snp.order'' or ''sample.order'', indicating individual-major mode (snp is the first dimension) and SNP-major mode (sample is the first dimension) respectively.

<<>>=
# Take out snp.id
head(read.gdsn(index.gdsn(genofile, "snp.id")))
# Take out snp.rs.id
head(read.gdsn(index.gdsn(genofile, "snp.rs.id")))
@
There are two additional variables:
\begin{itemize}
	\item{\vr{snp.rs.id}, a character string for reference SNP ID that may not be unique.}
	\item{\vr{snp.allele}, it is not necessary for the analysis, but it is necessary when merging genotypes from different platforms. The format of \vr{snp.allele} is ``A allele/B allele'', like ``T/G'' where T is A allele and G is B allele.}
\end{itemize}

The information of sample annotation can be obtained by the same function \fn{read.gdsn}. For example, population information. ``FStr8'' indicates a character-type variable.
<<>>=
# Read population information
pop <- read.gdsn(index.gdsn(genofile, path="sample.annot/pop.group"))
table(pop)

# close the GDS file
closefn.gds(genofile)
@



% -------- Create a GDS File of Your Own --------
\subsection{Create a GDS File of Your Own}

%%%%%%%%
\subsubsection{snpgdsCreateGeno}

The function \fn{snpgdsCreateGeno} can be used to create a GDS file. The first argument should be a numeric matrix for SNP genotypes. There are possible values stored in the input genotype matrix: 0, 1, 2 and other values. ``0'' indicates two B alleles, ``1'' indicates one A allele and one B allele, ``2'' indicates two A alleles, and other values indicate a missing genotype. The SNP matrix can be either $n_\text{sample} \times n_\text{snp}$ (snpfirstdim=FALSE, the argument in \fn{snpgdsCreateGeno}) or $n_\text{snp} \times n_\text{sample}$ (snpfirstdim=TRUE).

For example,

<<>>=
# load data
data(hapmap_geno)

# create a gds file
snpgdsCreateGeno("test.gds", genmat = hapmap_geno$genotype,
    sample.id = hapmap_geno$sample.id, snp.id = hapmap_geno$snp.id,
    snp.chromosome = hapmap_geno$snp.chromosome,
    snp.position = hapmap_geno$snp.position,
    snp.allele = hapmap_geno$snp.allele, snpfirstdim=TRUE)

# open the gds file
(genofile <- openfn.gds("test.gds"))

# close the genotype file
closefn.gds(genofile)
@


%%%%%%%%
\subsubsection{Uses of the Functions in the gdsfmt Package}

In the following code, the functions \fn{createfn.gds}, \fn{add.gdsn}, \fn{put.attr.gdsn}, \fn{write.gdsn}, \fn{index.gdsn}, \fn{closefn.gds} are defined in the gdsfmt package:

\begin{Schunk}
\begin{Soutput}
# create a new GDS file
newfile <- createfn.gds("your_gds_file.gds")

# add variables
add.gdsn(newfile, "sample.id", sample.id)
add.gdsn(newfile, "snp.id", snp.id)
add.gdsn(newfile, "snp.position", snp.position)
add.gdsn(newfile, "snp.chromosome", snp.chromosome)
add.gdsn(newfile, "snp.allele", c("A/G", "T/C", ...))


#####################################################################
# create a snp-by-sample genotype matrix

# add genotypes
var.geno <- add.gdsn(newfile, "genotype",
	valdim=c(length(snp.id), length(sample.id)), storage="bit2")

# indicate the SNP matrix is snp-by-sample
put.attr.gdsn(var.geno, "snp.order")

# write SNPs into the file sample by sample
for (i in 1:length(sample.id))
{
	g <- ...
	write.gdsn(var.geno, g, start=c(1,i), count=c(-1,1))
}


#####################################################################
# OR, create a sample-by-snp genotype matrix

# add genotypes
var.geno <- add.gdsn(newfile, "genotype",
	valdim=c(length(sample.id), length(snp.id)), storage="bit2")

# indicate the SNP matrix is sample-by-snp
put.attr.gdsn(var.geno, "sample.order")

# write SNPs into the file sample by sample
for (i in 1:length(snp.id))
{
	g <- ...
	write.gdsn(var.geno, g, start=c(1,i), count=c(-1,1))
}



# get a description of chromosome codes
#   allowing to define a new chromosome code, e.g., snpgdsOption(Z=27)
option <- snpgdsOption()
var.chr <- index.gdsn(newfile, "snp.chromosome")
put.attr.gdsn(var.chr, "autosome.start", option$autosome.start)
put.attr.gdsn(var.chr, "autosome.end", option$autosome.end)
for (i in 1:length(option$chromosome.code))
{
	put.attr.gdsn(var.chr, names(option$chromosome.code)[i],
		option$chromosome.code[[i]])
}

# add your sample annotation
samp.annot <- data.frame(sex = c("male", "male", "female", ...),
	pop.group = c("CEU", "CEU", "JPT", ...), ...)
add.gdsn(newfile, "sample.annot", samp.annot)

# close the GDS file
closefn.gds(newfile)
@
\end{Soutput}
\end{Schunk}




% -------- Format conversion from PLINK binary files --------
\subsection{Format conversion from PLINK binary files}

The \hn{SNPRelate} package provides a function \fn{snpgdsBED2GDS} for converting a PLINK binary file to a GDS file:

<<>>=
# the PLINK BED file, using the example in the SNPRelate package
bed.fn <- system.file("extdata", "plinkhapmap.bed", package="SNPRelate")
bim.fn <- system.file("extdata", "plinkhapmap.bim", package="SNPRelate")
fam.fn <- system.file("extdata", "plinkhapmap.fam", package="SNPRelate")
@
Or, uses your own PLINK files:
<<eval=FALSE>>=
bed.fn <- "C:/your_folder/your_plink_file.bed"
bim.fn <- "C:/your_folder/your_plink_file.bim"
fam.fn <- "C:/your_folder/your_plink_file.fam"
@
<<>>=
# convert
snpgdsBED2GDS(bed.fn, fam.fn, bim.fn, "test.gds")

# summary
snpgdsSummary("test.gds")
@


% -------- Format conversion from VCF files --------
\subsection{Format conversion from VCF files}

The \hn{SNPRelate} package provides a function \fn{snpgdsVCF2GDS} to reformat a VCF file. There are two options for extracting markers from a VCF file for downstream analyses:  (1) to extract and store dosage of the reference allele only for biallelic SNPs and (2) to extract and store dosage of the reference allele for all variant sites, including bi-allelic SNPs, multi-allelic SNPs, indels and structural variants.

<<>>=
# the VCF file, using the example in the SNPRelate package
vcf.fn <- system.file("extdata", "sequence.vcf", package="SNPRelate")
@
Or, uses your own VCF file:
<<eval=FALSE>>=
vcf.fn <- "C:/your_folder/your_vcf_file.vcf"
@
<<>>=
# reformat
snpgdsVCF2GDS(vcf.fn, "test.gds", method="biallelic.only")

# summary
snpgdsSummary("test.gds")
@



% ---- Data Analysis ----
\section{Data Analysis}

We developed \hn{gdsfmt} and \hn{SNPRelate} (high-performance computing R packages for multi-core symmetric multiprocessing computer architectures) to accelerate two key computations in GWAS: principal component analysis (PCA) and relatedness analysis using identity-by-descent (IBD) measures.

<<>>=
# open the GDS file
genofile <- openfn.gds(snpgdsExampleFileName())
@

<<>>=
# get population information
#   or pop_code <- scan("pop.txt", what=character()), if it is stored in a text file "pop.txt"
pop_code <- read.gdsn(index.gdsn(genofile, path="sample.annot/pop.group"))

# display the first six values
head(pop_code)
@


% -------- LD-based SNP pruning --------
\subsection{LD-based SNP pruning}

It is suggested to use a pruned set of SNPs which are in approximate linkage equilibrium with each other to avoid the strong influence of SNP clusters in principal component analysis and relatedness analysis.

<<>>=
set.seed(1000)

# try different LD thresholds for sensitivity analysis
snpset <- snpgdsLDpruning(genofile, ld.threshold=0.2)
names(snpset)
head(snpset$chr1)  # snp.id

# get all selected snp id
snpset.id <- unlist(snpset)
@


% -------- Principal Component Analysis --------
\subsection{Principal Component Analysis}

The functions in \hn{SNPRelate} for PCA include calculating the genetic covariance matrix from genotypes, computing the correlation coefficients between sample loadings and genotypes for each SNP, calculating SNP eigenvectors (loadings), and estimating the sample loadings of a new dataset from specified SNP eigenvectors.

<<>>=
# uses different colors with respect to ethnicities
race <- as.factor(pop_code)
@

<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
# run PCA
pca <- snpgdsPCA(genofile, maf=0.05, missing.rate=0.05,
	snp.id=snpset.id, num.thread=2)
plot(pca$eigenvect[,2], pca$eigenvect[,1], xlab="Principal Component 2",
	ylab="Principal Component 1", type="n")

# draw points
points(pca$eigenvect[,2], pca$eigenvect[,1], col=race)
legend("topleft", legend=levels(race), text.col=1:nlevels(race))
@

The code below shows how to calculate the percent of variation is accounted for by the principal component for the first 16 PCs. It is clear to see the first two eigenvectors hold the largest percentage of variance among the population, although the total variance accounted for is still less the one-quarter of the total.
<<>>=
pc.percent <- 100 * pca$eigenval[1:16]/sum(pca$eigenval)
pc.percent
@

Plot the principal component pairs for the first four PCs:
<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
lbls <- paste("PC", 1:4, "\n", format(pc.percent[1:4], digits=2), "%", sep="")
pairs(pca$eigenvect[,1:4], col=race, labels=lbls)
@

To calculate the SNP correlations between eigenvactors and SNP genotypes:
<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
# get chromosome index
chr <- read.gdsn(index.gdsn(genofile, "snp.chromosome"))
CORR <- snpgdsPCACorr(pca, genofile, eig.which=1:4)

par( mfrow=c(3,1))
for (i in 1:3)
{
	plot(abs(CORR$snpcorr[i,]), ylim=c(0,1), xlab="SNP Index",
		ylab=paste("PC", i), col=chr, pch="+")
}
@



% -------- Relatedness Analysis --------
\subsection{Relatedness Analysis}

For relatedness analysis, identity-by-descent (IBD) estimation in \hn{SNPRelate} can be done by either the method of moments (MoM) (Purcell et al., 2007) or maximum likelihood estimation (MLE) (Milligan, 2003; Choi et al., 2009). Although MLE estimates are more reliable than MoM, MLE is significantly more computationally intensive. For both of these methods it is preffered to use a LD pruned SNP set.

<<>>=
# YRI samples
sample.id <- read.gdsn(index.gdsn(genofile, "sample.id"))
YRI.id <- sample.id[pop_code == "YRI"]
@

\subsubsection{Estimating IBD Using PLINK method of moments (MoM)}

<<>>=
# estimate IBD coefficients
ibd <- snpgdsIBDMoM(genofile, sample.id=YRI.id, snp.id=snpset.id,
	maf=0.05, missing.rate=0.05)

# make a data.frame
ibd.coeff <- snpgdsIBDSelection(ibd)
head(ibd.coeff)
@

<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
plot(ibd.coeff$k0, ibd.coeff$k1, xlim=c(0,1), ylim=c(0,1),
	xlab="k0", ylab="k1", main="YRI samples (MoM)")
lines(c(0,1), c(1,0), col="red", lty=2)
@

\subsubsection{Estimating IBD Using Maximum Likelihood Estimation (MLE)}

<<eval=FALSE>>=
# estimate IBD coefficients
set.seed(1000)
snp.id <- sample(snpset.id, 5000)  # random 5000 SNPs
ibd <- snpgdsIBDMLE(genofile, sample.id=YRI.id, snp.id=snp.id,
	maf=0.05, missing.rate=0.05)
@

<<eval=FALSE>>=
# make a data.frame
ibd.coeff <- snpgdsIBDSelection(ibd)
@

<<eval=FALSE>>=
plot(ibd.coeff$k0, ibd.coeff$k1, xlim=c(0,1), ylim=c(0,1),
	xlab="k0", ylab="k1", main="YRI samples (MLE)")
lines(c(0,1), c(1,0), col="red", lty=2)
@
\includegraphics{SNPRelateTutorial-IBDMLE.png}



% -------- Identity-By-State Analysis --------
\subsection{Identity-By-State Analysis}

For the $n$ individuals in a sample, \fn{snpgdsIBS} can be used to create a $n \times n$ matrix of genome-wide average IBS pairwise identities:

<<>>=
ibs <- snpgdsIBS(genofile, num.thread=2)
@
The heat map is shown:
<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
library(lattice)

L <- order(pop_code)
levelplot(ibs$ibs[L, L], col.regions = terrain.colors)
@

To perform multidimensional scaling analysis on the $n \times n$ matrix of genome-wide IBS pairwise distances:
<<>>=
loc <- cmdscale(1 - ibs$ibs, k = 2)
x <- loc[, 1]; y <- loc[, 2]
race <- as.factor(pop_code)
@

<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
plot(x, y, col=race, xlab = "", ylab = "",
	main = "Multidimensional Scaling Analysis (IBS Distance)")
legend("topleft", legend=levels(race), text.col=1:nlevels(race))
@

To perform cluster analysis on the $n \times n$ matrix of genome-wide IBS pairwise distances, and determine the groups by a permutation score:

<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
set.seed(100)
ibs.hc <- snpgdsHCluster(snpgdsIBS(genofile, num.thread=2))

# to determine groups of individuals automatically
rv <- snpgdsCutTree(ibs.hc)
plot(rv$dendrogram, leaflab="none", main="HapMap Phase II")
table(rv$samp.group)
@

Here is the population information we have known:
<<>>=
# to determine groups of individuals by population information
rv2 <- snpgdsCutTree(ibs.hc, samp.group=as.factor(pop_code))
@

<<fig=TRUE, pdf=FALSE, png=TRUE, resolution=100>>=
plot(rv2$dendrogram, leaflab="none", main="HapMap Phase II")
legend("topright", legend=levels(race), col=1:nlevels(race), pch=19, ncol=4)
@

<<>>=
# close the GDS file
closefn.gds(genofile)
@



% ---- Resources ----
\section{Resources}

\begin{enumerate}
	\item \hn{CoreArray} project: \url{http://corearray.sourceforge.net/}
	\item \hn{gdsfmt} R package: \url{http://cran.r-project.org/web/packages/gdsfmt/index.html}
	\item \hn{SNPRelate} R package: \url{http://cran.r-project.org/web/packages/SNPRelate/index.html}
	\item \hn{GENEVA} R package: \url{https://www.genevastudy.org/Accomplishments/software}
	\item \hn{GWASTools}: an R/Bioconductor package for quality control and analysis of Genome-Wide Association Studies \url{http://www.bioconductor.org/packages/2.11/bioc/html/GWASTools.html}
\end{enumerate}



% ---- References ----
\section{References}

\begin{enumerate}
	\item {\bf A High-performance Computing Toolset for Relatedness and Principal Component Analysis of SNP Data.} 
Xiuwen Zheng; David Levine; Jess Shen; Stephanie M. Gogarten; Cathy Laurie; Bruce S. Weir. Bioinformatics 2012; doi: 10.1093/bioinformatics/bts606.
	\item {\bf GWASTools: an R/Bioconductor package for quality control and analysis of Genome-Wide Association Studies.} Stephanie M. Gogarten, Tushar Bhangale, Matthew P. Conomos, Cecelia A. Laurie, Caitlin P. McHugh, Ian Painter, Xiuwen Zheng, David R. Crosslin, David Levine, Thomas Lumley, Sarah C. Nelson, Kenneth Rice, Jess Shen, Rohit Swarnkar, Bruce S. Weir, and Cathy C. Laurie. Bioinformatics 2012; doi:10.1093/bioinformatics/bts610.
	\item {\bf Quality control and quality assurance in genotypic data for genome-wide association studies.} Laurie CC, Doheny KF, Mirel DB, Pugh EW, Bierut LJ, Bhangale T, Boehm F, Caporaso NE, Cornelis MC, Edenberg HJ, Gabriel SB, Harris EL, Hu FB, Jacobs KB, Kraft P, Landi MT, Lumley T, Manolio TA, McHugh C, Painter I, Paschall J, Rice JP, Rice KM, Zheng X, Weir BS; GENEVA Investigators. Genet Epidemiol. 2010 Sep;34(6):591-602.
\end{enumerate}



\section{Acknowledgements}
The author would like to thank members of the GENEVA consortium (\url{http://www.genevastudy.org}) for access to the data used for testing the \hn{gdsfmt} and \hn{SNPRelate} packages.


\end{document}
