A protocol to impute haptoglobin haplotypes from surrounding genotypes computed from genotype array or whole genome sequence data using the reference panel from:
Boettger L., McCarroll S., et al. Recurring exon deletions in the HP (haptoglobin)
gene contribute to lower blood cholesterol levels. Nat. Genet. 48, 359–366 (2016)
This reference panel was generated as explained here. For any feedback, send an email to giulio.genovese@gmail.com or mccarroll@genetics.med.harvard.edu
Install basic tools (Debian/Ubuntu specific):
sudo apt install wget gzip samtools bcftools plink1.9 openjdk-11-jre-headless
Preparation steps
mkdir -p $HOME/res
Download Beagle binary
wget -P $HOME/res/ https://faculty.washington.edu/browning/beagle/beagle.25Nov19.28d.jar
Download reference panels
wget -P $HOME/res/ https://personal.broadinstitute.org/giulio/panels/HP_Euroref_1kgOMNI_HM3_merged.GRCh3{7,8}.vcf.gz
Run imputation using Beagle
vcf="..."
out="..."
build=38 # build=37
declare -A reg=( ["37"]="16:71070878-73097663" ["38"]="chr16:71036975-73063764" )
bcftools view --no-version "$vcf" -r ${reg[$build]} | \
java -Xmx8g -jar $HOME/res/beagle.25Nov19.28d.jar gt=/dev/stdin \
ref=$HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh$build.vcf.gz out="$out" \
map=<(bcftools query -f "%CHROM\t%POS\n" $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh$build.vcf.gz | \
awk '{print $1"\t.\t"$2/1e7"\t"$2}')
Extract imputed haptoglobin alleles into a table
out="..."
build=38 # build=37
declare -A reg=( ["37"]="16:72092044-72092044" ["38"]="chr16:72058145-72058145" )
bcftools index -ft "$out.vcf.gz" && \
bcftools query -f "[%SAMPLE\t%ALT\t%GT\n]" "$out.vcf.gz" -r ${reg[$build]} | tr -d '[<>]' | \
awk -F"\t" -v OFS="\t" '{split($2,a,","); a["0"]="NA"; split($3,b,"|"); \
print $1,a[b[1]],a[b[2]]}' > "$out.tsv"
This section is only in case you want to build the reference panels yourself, you can skip it otherwise
Download GRCh37 human genome reference
wget -O- ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/human_g1k_v37.fasta.gz | \
gzip -d > $HOME/res/human_g1k_v37.fasta
samtools faidx $HOME/res/human_g1k_v37.fasta
Download GRCh38 human genome reference
wget -O- ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz | \
gzip -d > $HOME/res/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna
samtools faidx $HOME/res/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna
Download haptoglobin reference panel in Beagle 3 format with marker positions for the GRCh37 human genome reference
wget -P $HOME/res/ https://raw.githubusercontent.com/freeseek/imputehp/master/HP_Euroref_1kgOMNI_HM3_merged.bgl.phased
Liftover marker positions for the GRCh38 human genome reference
wget http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/liftOver
chmod a+x liftOver
wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/liftOver/hg19ToHg38.over.chain.gz
tail -n+2 $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.bgl.phased | grep -v HP | \
awk '{print "16\t"$2}' > $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh37
tail -n+2 $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.bgl.phased | grep -v HP | \
awk '{print "chr16\t"$2-1"\t"$2}' | \
./liftOver /dev/stdin hg19ToHg38.over.chain.gz /dev/stdout /dev/stderr | \
awk '{print $1"\t"$3}' > $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh38
Generate haptoglobin reference panels in VCF format for both the GRCh37 and GRCh38 human genome references
declare -A fasta=( ["37"]="$HOME/res/human_g1k_v37.fasta" ["38"]="$HOME/res/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna" )
declare -A chr=( ["37"]="16" ["38"]="chr16" )
declare -A pos=( ["37"]="72092044" ["38"]="72058145" )
grep HP $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.bgl.phased | \
awk 'BEGIN {x["AAAAABAAA"]=1; x["AABBBAAAB"]=4; x["BAABBAABA"]=3; x["BBAAAABAA"]=2}
{for (i=3; i<=NF; i++) y[i]=y[i]$i}
END {printf "<HP1A>,<HP1B>,<HP2A>,<HP2B>\t.\t.\t.\tGT";
for (i in y) printf "\t"x[y[i]]}' | \
sed 's/\t\([1-9]\)\t\([1-9]\)/\t\1|\2/g' > tmp
for build in 37 38; do
tail -n+2 $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.bgl.phased | grep -v HP | tr ' ' '\t' | cut -f3- | \
paste $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh$build - | \
sed 's/\t\([ACGT]\)\t\([ACGT]\)/\t\1\2/g' | \
bcftools convert --no-version --tsv2vcf /dev/stdin -c CHROM,POS,AA -f ${fasta[$build]} --samples \
$(head -n1 $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.bgl.phased | tr ' ' '\n' | \
tail -n+3 | uniq | tr '\n' ',' | sed 's/,$//') | tr '/' '|' | \
awk -v chr=${chr[$build]} -v pos=${pos[$build]} 'NR==FNR {line=$0}
NR>FNR {if (line && $0!~"^#" && $2>pos) {print chr"\t"pos"\t.\tG\t"line; line=""} print}' tmp - | \
bcftools view --no-version -Oz \
-o $HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh$build.vcf.gz
done
/bin/rm tmp
Convert the haptoglobin and 1000 Genomes project reference panels to plink and then merge to compute consistency
url="ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase1/analysis_results/integrated_call_sets/ALL.chr16.integrated_phase1_v3.20101123.snps_indels_svs.genotypes.vcf.gz"
bcftools view --no-version $url -r 16:71070878-73097663 | \
awk 'NF==2 {print "##contig=<ID=16,length=90354753>"} {print}' | \
bcftools view --no-version -v snps | \
bcftools annotate --no-version -x ID -I +'%CHROM:%POS:%REF:%ALT' | \
$HOME/bin/plink --vcf /dev/stdin --keep-allele-order --const-fid --make-bed \
--out ALL.chr16.integrated_phase1_v3.20101123.snps_indels_svs.genotypes
bcftools annotate --no-version -x ID -I +'%CHROM:%POS:%REF:%ALT' \
$HOME/res/HP_Euroref_1kgOMNI_HM3_merged.GRCh37.vcf.gz | \
$HOME/bin/plink --vcf /dev/stdin --biallelic-only --keep-allele-order --const-fid --make-bed \
--out HP_Euroref_1kgOMNI_HM3_merged.GRCh37
plink --bfile HP_Euroref_1kgOMNI_HM3_merged.GRCh37 \
--bmerge ALL.chr16.integrated_phase1_v3.20101123.snps_indels_svs.genotypes --merge-mode 6
You should get the following result
241696 overlapping calls, 241696 nonmissing in both filesets.
240611 concordant, for a concordance rate of 0.995511.