In smgogarten/GWASTools: Tools for Genome Wide Association Studies

Overview

This vignette describes how to prepare Affymetrix data for use in r Biocpkg("GWASTools"). After following the examples here for preparing the data, the analysis procedure is the same as presented in the GWAS Data Cleaning vignette. For a description of the file formats (NetCDF and GDS), see the Data Formats vignette.

Creating the SNP Annotation Data Object {#create_snpann}

All of the functions in r Biocpkg("GWASTools") require a minimum set of variables in the SNP annotation data object. The minimum required variables are

• snpID, a unique integer identifier for each SNP

• chromosome, an integer mapping for each chromosome, with values 1-27, mapped in order from 1-22, 23=X, 24=XY (the pseudoautosomal region), 25=Y, 26=M (the mitochondrial probes), and 27=U (probes with unknown positions)

• position, the base position of each SNP on the chromosome.

We create the integer chromosome mapping for a few reasons. The chromosome is stored as an integer in the NetCDF or GDS files, so in order to link the SNP annotation with the NetCDF/GDS file, we use the integer values in the annotation as well. For convenience when using GWASTools functions, the chromosome variable is most times assumed to be an integer value. Thus, for the sex chromosomes, we can simply use the chromosome values. For presentation of results, it is important to have the mapping of the integer values back to the standard designations for the chromosome names, thus the getChromosome() functions in the GWASTools objects have a char=TRUE option to return the characters 1-22, X, XY, Y, M, U. The position variable should hold all numeric values of the physical position of a probe. The SNP annotation file is assumed to list the probes in order of chromosome and position within chromosome.

Note that for Affymetrix data, the rs ID is often not unique, as there may be multiple probes for a given SNP. The probe ID is usually the unique SNP identifier. Also, Affymetrix annotation generally has a separate column or columns to indicate pseudoautosomal regions; some manipulation is usually required to include this information within the chromosome column itself.

library(GWASTools)
library(GWASdata)
# Load the SNP annotation (simple data frame)
data(affy_snp_annot)
# Create a SnpAnnotationDataFrame
snpAnnot <- SnpAnnotationDataFrame(affy_snp_annot)
# names of columns
varLabels(snpAnnot)
# data
head(pData(snpAnnot))
# Add metadata to describe the columns
meta <- varMetadata(snpAnnot)
meta[c("snpID", "chromosome", "position", "rsID", "probeID"), 
  "labelDescription"] <- c("unique integer ID for SNPs",                      
  paste("integer code for chromosome: 1:22=autosomes,",
   "23=X, 24=pseudoautosomal, 25=Y, 26=Mitochondrial, 27=Unknown"), 
  "base pair position on chromosome (build 36)",
  "RS identifier",
  "unique ID from Affymetrix")
varMetadata(snpAnnot) <- meta

Creating the Scan Annotation Data Object {#create_sampann}

The scan annotation file holds attributes for each genotyping scan that are relevant to genotypic data cleaning. These data include processing variables such as tissue type, DNA extraction method, and genotype processing batch. They also include individual characteristics such as gender and race. The initial sample annotation file is created from the raw data supplied by the genotyping center and/or study investigator, providing a mapping from the raw data file(s) for each sample scan to other sample information such as sex, coded as M and F, ethnicity, unique scan identifier, called scanID, and unique subject identifier. Since a single subject may have been genotyped multiple times as a quality control measure, it is important to distinguish between the scanID (unique genotyping instance) and subjectID (person providing a DNA sample).

# Load the scan annotation (simple data frame)
data(affy_scan_annot)
# Create a ScanAnnotationDataFrame
scanAnnot <- ScanAnnotationDataFrame(affy_scan_annot)
# names of columns
varLabels(scanAnnot)
# data
head(pData(scanAnnot))
# Add metadata to describe the columns
meta <- varMetadata(scanAnnot)
meta[c("scanID", "subjectID", "family", "father", "mother",
  "CoriellID", "race", "sex", "status", "genoRunID", "plate",
  "alleleFile", "chpFile"), "labelDescription"] <- 
   c("unique integer ID for scans", 
  "subject identifier (may have multiple scans)",
  "family identifier", 
  "father identifier as subjectID",
  "mother identifier as subjectID",
  "Coriell subject identifier",
  "HapMap population group",
  "sex coded as M=male and F=female",
  "simulated case/control status" ,
  "genotyping instance identifier",
  "plate containing samples processed together for genotyping chemistry",
  "data file with intensities",
  "data file with genotypes and quality scores")
varMetadata(scanAnnot) <- meta

Creating the Data Files

The data for genotype calls, allelic intensities and other variables such as BAlleleFrequency are stored as NetCDF or GDS files.

Genotype Files

The genotype files store genotypic data in 0, 1, 2 format indicating the number of "A" alleles in the genotype (i.e. AA=2, AB=1, BB=0 and missing=-1). The conversion from AB format and forward strand (or other) allele formats can be stored in the SNP annotation file.

Creating the Genotype file

geno.file <- "tmp.geno.gds"

# first 3 samples only
scan.annotation <- affy_scan_annot[1:3, c("scanID", "genoRunID", "chpFile")]
names(scan.annotation) <- c("scanID", "scanName", "file")

# indicate which column of SNP annotation is referenced in data files
snp.annotation <- affy_snp_annot[,c("snpID", "probeID", "chromosome", "position")]
names(snp.annotation)[2] <- "snpName"

# col.nums is an integer vector indicating which columns of the raw text file 
# contain variables for input
col.nums <- as.integer(c(2,3))
names(col.nums) <- c("snp", "geno")

# Define a path to the raw data CHP text files
path <- system.file("extdata", "affy_raw_data", package="GWASdata")

diag.geno.file <- "diag.geno.RData"
diag.geno <- createDataFile(path = path,
  filename = geno.file, 
  file.type = "gds",
  variables = "genotype",
  snp.annotation = snp.annotation,
  scan.annotation = scan.annotation, 
  sep.type = "\t",
  skip.num = 1,
  col.total = 6,
  col.nums = col.nums,
  scan.name.in.file = -1,
  diagnostics.filename = diag.geno.file,
  verbose = FALSE)
# Look at the values included in the "diag.geno" object which holds 
#   all output from the function call
names(diag.geno)
# `read.file' is a vector indicating whether (1) or not (0) each file 
#   specified in the `files' argument was read successfully
table(diag.geno$read.file)
# `row.num' is a vector of the number of rows read from each file
table(diag.geno$row.num) 
# `sample.match' is a vector indicating whether (1) or not (0) 
#   the sample name inside the raw text file matches that in the 
#   sample annotation data.frame
table(diag.geno$sample.match)
# `snp.chk' is a vector indicating whether (1) or not (0) 
#   the raw text file has the expected set of SNP names
table(diag.geno$snp.chk)
# `chk' is a vector indicating whether (1) or not (0) all previous
#   checks were successful and the data were written to the file
table(diag.geno$chk)

# open the file we just created
(genofile <- GdsGenotypeReader(geno.file))
# Take out genotype data for the first 3 samples and 
#   the first 5 SNPs
(genos <- getGenotype(genofile, snp=c(1,5), scan=c(1,3)))
close(genofile)

# check that the data file matches the raw text files
check.geno.file <- "check.geno.RData"
check.geno <- checkGenotypeFile(path = path,
  filename = geno.file, 
  file.type = "gds",
  snp.annotation = snp.annotation,
  scan.annotation = scan.annotation, 
  sep.type = "\t",
  skip.num = 1,
  col.total = 6,
  col.nums = col.nums,
  scan.name.in.file = -1,
  check.scan.index = 1:3,
  n.scans.loaded = 3,
  diagnostics.filename = check.geno.file,
  verbose = FALSE)
# Look at the values included in the "check.geno" object which holds 
#   all output from the function call
names(check.geno)
# 'geno.chk' is a vector indicating whether (1) or not (0) the genotypes 
#   match the text file
table(check.geno$geno.chk)

Intensity Files

The intensity files store quality scores and allelic intensity data for each SNP. Affymetrix data are provided in two files per genotyping scan. The CHP file holds the genotype calls, used to create the genotype file, as well as the confidence score, which is written to the quality file. The normalized X and Y intensity data are stored in the allele_summary files in the format of two rows per SNP, one for each allelic probe. These are written to the intensity file.

Creating the quality file

qual.file <- "tmp.qual.gds"

scan.annotation <- affy_scan_annot[1:3, c("scanID", "genoRunID", "chpFile")]
names(scan.annotation) <- c("scanID", "scanName", "file")

snp.annotation <- affy_snp_annot[,c("snpID", "probeID", "chromosome", "position")]
names(snp.annotation)[2] <- "snpName"

col.nums <- as.integer(c(2,4))
names(col.nums) <- c("snp", "quality")

path <- system.file("extdata", "affy_raw_data", package="GWASdata")

diag.qual.file <- "diag.qual.RData"
diag.qual <- createDataFile(path = path,
  filename = qual.file, 
  file.type = "gds",
  variables = "quality",
  snp.annotation = snp.annotation,
  scan.annotation = scan.annotation, 
  sep.type = "\t",
  skip.num = 1,
  col.total = 6,
  col.nums = col.nums,
  scan.name.in.file = -1,
  diagnostics.filename = diag.qual.file,
  verbose = FALSE)

# Open the GDS file we just created
(qualfile <- GdsIntensityReader(qual.file))
# Take out the quality scores for the first 
#     5 SNPs for the first 3 samples
(qual <- getQuality(qualfile, snp=c(1,5), scan=c(1,3)))
close(qualfile)

# check
check.qual.file <- "check.qual.RData"
check.qual <- checkIntensityFile(path = path,
  filename = qual.file, 
  file.type = "gds",
  snp.annotation = snp.annotation,
  scan.annotation = scan.annotation, 
  sep.type = "\t",
  skip.num = 1,
  col.total = 6,
  col.nums = col.nums,
  scan.name.in.file = -1,
  check.scan.index = 1:3,
  n.scans.loaded = 3,
  affy.inten = FALSE,
  diagnostics.filename = check.qual.file,
  verbose = FALSE)

Creating the Intensity file

xy.file <- "tmp.xy.gds"

# we need the allele files instead of CHP files
scan.annotation <- affy_scan_annot[1:3, c("scanID", "genoRunID", "alleleFile")]
names(scan.annotation) <- c("scanID", "scanName", "file")

snp.annotation <- affy_snp_annot[,c("snpID", "probeID", "chromosome", "position")]
names(snp.annotation)[2] <- "snpName"

diag.xy.file <- "diag.xy.RData"
diag.xy <- createAffyIntensityFile(path = path,
  filename = xy.file, 
  file.type = "gds",
  snp.annotation = snp.annotation, 
  scan.annotation = scan.annotation, 
  diagnostics.filename = diag.xy.file,
  verbose = FALSE)

# Open the file we just created
(intenfile <- GdsIntensityReader(xy.file))
# Take out the normalized X intensity values for the first 
#     5 SNPs for the first 3 samples
(xinten <- getX(intenfile, snp=c(1,5), scan=c(1,3)))
close(intenfile)

# check the intensity files - note affy.inten=TRUE
check.xy.file <- "check.xy.RData"
check.xy <- checkIntensityFile(path = path,
  filename = xy.file,
  file.type = "gds", 
  snp.annotation = snp.annotation,
  scan.annotation = scan.annotation, 
  sep.type = "\t",
  skip.num = 1,
  col.total = 2,
  col.nums = setNames(as.integer(c(1,2,2)), c("snp", "X", "Y")),
  scan.name.in.file = -1,
  check.scan.index = 1:3,
  n.scans.loaded = 3,
  affy.inten = TRUE,
  diagnostics.filename = check.xy.file,
  verbose = FALSE)

BAlleleFrequency and LogRRatio Files

The BAlleleFrequency and LogRRatio file stores these values for every sample by SNP. For Affymetrix data, these values must be calculated by the user. For a thorough explanation and presentation of an application of these values, please refer to Peiffer, Daniel A., et al. (2006).[^1]

For a given sample and SNP, R and $\theta$ are calculated using the X and Y intensities, where $$\label{rthe} R=X+Y$$ $$\theta = \frac{2\arctan(Y/X)}{\pi}$$ $\theta$ corresponds to the polar coordinate angle and R is the sum of the normalized X and Y intensities (not, as one might assume, the magnitude of the polar coordinate vector). It is from these values that we calculate the LogRRatio and BAlleleFrequency.

The LogRRatio is given below. The expected value of R is derived from a plot of $\theta$ versus R for a given SNP. It is the predicted value of R derived from a line connecting the centers of the two nearest genotype clusters. $${\rm LogRRatio} =\log \left( \frac{R_{\text{observed values}}}{R_{\text{expected values}}}\right)$$ Variation in the LogRRatio across a single chromosome indicates possible duplication or deletion, and is an indication of overall sample quality.

The BAlleleFrequency is the frequency of the B allele in the population of cells from which the DNA is extracted. Each sample and SNP combination has a BAlleleFrequency value. Note the BAlleleFrequency values vary for a subject with each DNA extraction and tissue used. After all SNPs have been read and all samples have been clustered for a probe, the mean $\theta$ "cluster" value is calculated for each probe, for each of the three genotype clusters, resulting in $\theta_{AA}, \theta_{AB} \text{ and }\theta_{BB}$ for every probe. Then the $\theta$ value for each sample, call it $\theta_{n}$, is compared to $\theta_{AA}, \theta_{AB}\text{ and }\theta_{BB}$. The BAlleleFrequency is calculated $$\text{BAlleleFrequency}= \begin{cases} 0 & \text{if } \theta_{n} < \theta_{AA}\ \mbox{}\ \frac{{\textstyle (1/2)(\theta_{n}-\theta_{AA})}}{{\textstyle \theta_{AB}-\theta_{AA}}} & \text{if } \theta_{AA} \leq \theta_{n} < \theta_{AB}\ \mbox{}\ \frac{{\textstyle 1}}{{\textstyle 2}}+\frac{{\textstyle (1/2)(\theta_{n}-\theta_{AB})}} {{\textstyle \theta_{BB}-\theta_{AB}}} & \text{if } \theta_{AB} \leq \theta_{n} < \theta_{BB}\ \mbox{}\ 1 & \text{if } \theta_{n} \geq \theta_{BB} \end{cases}$$ A $\theta_{n}$ value of 0 or 1 corresponds to a homozygote genotype for sample $n$ at that particular probe, and a $\theta_{n}$ value of 1/2 indicates a heterozygote genotype. Thus, $BAlleleFrequency \in[0,1]$ for each probe. Across a chromosome, three bands are expected, one hovering around 0, one around 1 and one around 0.5, and any deviation from this is considered aberrant.

We use the BAlleleFrequency and LogRRatio values to detect mixed samples or samples of low quality, as well as chromosomal duplications and deletions. Samples that have a significantly large (partial or full chromosome) aberration for a particular chromosome as detected from the BAlleleFrequency values are recommended to be filtered out, for the genotype data are not reliable in these situations. Because of these applications, the BAlleleFrequency and LogRRatio values are a salient part of the data cleaning steps.

Because we have already completed the creation of both the genotype and intensity files, we simply use those files to access the data. The BAlleleFrequency and LogRRatio values are calculated in subsets for efficiency and written to the corresponding subset indices in the file.

Creating the BAlleleFrequency and LogRRatio file

We calculate the BAlleleFrequency and LogRRatio values for each sample by SNP and write these values to a data file by calling the function BAFfromGenotypes. We will also select "by.study" as the call method, so all 3 samples have their genotype clusters called together. In normal usage, we recommend calling Affymetrix genotypes "by.plate" (in which case the plate.name argument is passed to the function). For more detail regarding the BAFfromGenotypes function, please see the function documentation. After the function is complete, we will look at a few values to ensure the file was created successfully.

bl.file <- "tmp.bl.gds"
xyData <- IntensityData(GdsIntensityReader(xy.file),
                        snpAnnot=snpAnnot)
genoData <- GenotypeData(GdsGenotypeReader(geno.file),
                         snpAnnot=snpAnnot)
BAFfromGenotypes(xyData, genoData,
                 filename = bl.file, 
                 file.type = "gds",
                 min.n.genotypes = 0,
                 call.method ="by.study",
                 verbose = FALSE)
close(xyData)
close(genoData)
# Open the file we just created
(blfile <- GdsIntensityReader(bl.file))
# Look at the BAlleleFrequency values for the first 5 SNPs
(baf <- getBAlleleFreq(blfile, snp=c(1,5), scan=c(1,3)))
close(blfile)

file.remove(geno.file, qual.file, xy.file, bl.file)
file.remove(diag.geno.file, diag.qual.file, diag.xy.file)
file.remove(check.geno.file, check.qual.file, check.xy.file)

[^1]: Peiffer, Daniel A., et al. High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping. Genome Research 16, 1136-1148 (September 2006).

smgogarten/GWASTools documentation built on July 4, 2023, 2:32 a.m.