Genome Wide Association analysis (GWAS)¶

To test the association of of a genome-wide set of genetic variants with a given trait.

install PLINK 1.9 ¶

  cd /usr/local/bin/
  #wget https://www.cog-genomics.org/static/bin/plink170627/plink_linux_x86_64.zip
  cp /media/sf_shared_VM/plink_linux_x86_64.zip .
  unzip plink_linux_x86_64.zip
  rm -f plink_linux_x86_64.zip

install vcftools ¶

  cd  
  #git clone https://github.com/vcftools/vcftools.git
  cp -R /media/sf_shared_VM/vcftools .     
  cd vcftools
  ./autogen.sh
  ./configure
  make
  make install

Make a working directory for the GWAS analysis¶

  mkdir ~/GWAS && cd ~/GWAS

Download the sample VCF file and phenotype data¶

Genotyping of 476840 SNPs in 53 dogs (24 yellow coat and 29 dark coat)

  #wget https://de.cyverse.org/dl/d/E0A502CC-F806-4857-9C3A-BAEAA0CCC694/pruned_coatColor_maf_geno.vcf.gz
  cp /media/sf_shared_VM/pruned_coatColor_maf_geno.vcf.gz .      
  gunzip pruned_coatColor_maf_geno.vcf.gz
  #wget https://de.cyverse.org/dl/d/3B5C1853-C092-488C-8C2F-CE6E8526E96B/coatColor.pheno
  cp /media/sf_shared_VM/coatColor.pheno .

convert VCF into Plink readable format (map,ped) then Plink binary format (fam,bed,bim)¶

  vcftools --vcf pruned_coatColor_maf_geno.vcf --plink --out coatColor
  plink --file coatColor --allow-no-sex --dog --make-bed --out coatColor.binary

create list of alternative alleles¶

  cat pruned_coatColor_maf_geno.vcf | awk 'BEGIN{FS="\t";OFS="\t";}/#/{next;}{{if($3==".")$3=$1":"$2;}print $3,$5;}'  > alt_alleles

Run a simple association analysis¶

–assoc performs a standard case/control association analysis which is a chi-square test of allele frequency.

By default, the minor allele is coded A1 and tested for being the risk allele. The minor allele is expected to be the alternative allele but could happen to be the reference one. –reference-allele allow you to use your list of A1 alleles

–adjust enables correction for multiple analysis and automatically calculates the genomic inflation factor

  plink --bfile coatColor.binary --make-pheno coatColor.pheno "yellow" --assoc --reference-allele alt_alleles --allow-no-sex --adjust --dog --out coatColor

Create Manhattan plot¶

Install qqman package

Rscript -e "install.packages('qqman',  contriburl=contrib.url('http://cran.r-project.org/'))"

Identify statistical cutoffs

unad_cutoff_sug=$(tail -n+2 coatColor.assoc.adjusted | awk '$10>=0.05' | head -n1 | awk '{print $3}')
unad_cutoff_conf=$(tail -n+2 coatColor.assoc.adjusted | awk '$10>=0.01' | head -n1 | awk '{print $3}')

Run the plotting function

Rscript -e 'args=(commandArgs(TRUE));library(qqman);'\
'data=read.table("coatColor.assoc", header=TRUE); data=data[!is.na(data$P),];'\
'bitmap("coatColor_man.bmp", width=20, height=10);'\
'manhattan(data, p = "P", col = c("blue4", "orange3"),'\
'suggestiveline = 12,'\
'genomewideline = 15,'\
'chrlabs = c(1:38, "X"), annotateTop=TRUE, cex = 1.2);'\
'graphics.off();' $unad_cutoff_sug $unad_cutoff_conf

The top associated mutation is a nonsense SNP in MC1R (c.916C>T) known to control pigment production

Table Of Contents