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A protocol to impute C4 alleles from MHC genotypes computed from genotype array or whole genome sequence data using the HapMap3 CEU reference panel from:

Sekar A., McCarroll S., et al. Schizophrenia risk from complex
variation of complement component 4. Nature 530, 177–183 (2016)

This reference panel was generated using droplet digital PCR as explained here. For non-European populations and lower-frequency alleles efforts are underway to create a more advanced reference panel from whole genome sequencing data: check here for updates. For any feedback, send an email to or


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/

Download reference panels

wget -P $HOME/res/{7,8}.vcf.gz

Run C4 imputation from an input VCF

Run imputation using Beagle

build=38 # build=37
declare -A reg=( ["37"]="6:24894177-33890574" ["38"]="chr6:24893949-33922797" )

bcftools view --no-version "$vcf" -r ${reg[$build]} | \
  java -Xmx8g -jar $HOME/res/beagle.25Nov19.28d.jar gt=/dev/stdin \
  ref=$HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build.vcf.gz out="$out" \
  map=<(bcftools query -f "%CHROM\t%POS\n" $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build.vcf.gz | \
  awk '{print $1"\t.\t"$2/1e7"\t"$2}')

Notice that, due to the special nature of the MHC region, we select a genetic map with almost no recombination allowed

Extract imputed C4 alleles into a table

build=38 # build=37
declare -A reg=( ["37"]="6:31948000-31948000" ["38"]="chr6:31980223-31980223" )

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"

Notice that due to the location of the C4 gene in the MHC locus, the imputed C4 alleles are particularly susceptible to potential confounding due to: (i) linkage disequilibrium mediated correlation with genotypes at other MHC variants, including HLA variants; (ii) population stratification as MHC haplotypes are prone to high allele frequency differences across ethnic groups due to strong natural selection at the MHC locus.

Build the reference panels yourself

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- | \
  gzip -d > $HOME/res/human_g1k_v37.fasta
samtools faidx $HOME/res/human_g1k_v37.fasta

Download GRCh38 human genome reference

wget -O- | \
  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 C4 reference panel in Beagle 3 format and an additional file with marker positions for the GRCh37 human genome reference

wget -P $HOME/res/
wget -P $HOME/res/

Liftover marker positions for the GRCh38 human genome reference

chmod a+x liftOver
awk 'NR>1 {print "chr6\t"$2-1"\t"$2"\t"$1}' $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh37 | \
  ./liftOver /dev/stdin hg19ToHg38.over.chain.gz /dev/stdout /dev/stderr | \
  awk 'BEGIN {print "id\tpos"} {print $4"\t"$3}' > $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh38

Generate C4 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 chrom=( ["37"]="6" ["38"]="chr6" )
declare -A length=( ["37"]="171115067" ["38"]="170805979" )

for build in 37 38; do
  (echo "##fileformat=VCFv4.2"; \
  echo "##FORMAT=<ID=GT,Number=1,Type=String,Description=\"Genotype\">"; \
  echo "##contig=<ID=${chrom[$build]},length=${length[$build]}>"; \
  tr '\r' '\n' < $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.bgl | \
    grep C4 | cut -f3- | tr '\t' '\n' | sort | uniq | awk '{print "##ALT=<ID="$0">"}'; \
  echo "##reference=${fasta[$build]}"; \
  tr '\r' '\n' < $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.bgl | \
    paste $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build - | \
    awk -v chr="${chrom[$build]}" 'NR==1 {for (i=5; i<NF; i+=2) printf "\t"$i; printf "\n"} \
    NR>1 {printf chr"\t"$2"\t"$4; delete x; delete y; j=0; \
    for (i=5; i<=NF; i++) if (!($i in x)) {x[$i]=j; y[j++]=$i} \
    if ($4=="C4") {printf "\tG\t<"y[0]">"; for (i=1; i<j; i++) printf ",<"y[i]">" } \
    else { printf "\t"y[0]"\t"y[1]; for (i=2; i<j; i++) printf ","y[i] } \
    printf "\t.\t.\t.\tGT"; \
    if ($4=="C4") for (i=5; i<NF; i+=2) printf "\t"1+x[$i]"|"1+x[$(i+1)]; \
    else for (i=5; i<NF; i+=2) printf "\t"x[$i]"|"x[$(i+1)]; \
    printf "\n"}') | \
    bcftools +fixref --no-version -Ov \
    -o $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build.vcf -- \
    --fasta-ref ${fasta[$build]} --mode flip

  bcftools norm --no-version --check-ref w --fasta-ref ${fasta[$build]} \
    $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build.vcf -o /dev/null 2>&1 | \
    grep REF_MISMATCH | cut -f2,3 | awk -F"\t" -v OFS="\t" 'NR==FNR {x[$2]++} \
    NR>FNR && $2 in x {alt=$4; ref=$5; $4=ref; $5=alt; \
    for (i=10; i<=NF; i++) $i=1-substr($i,1,1)"|"1-substr($i,3,1)} NR>FNR' \
    - $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build.vcf | \
    bcftools view --no-version -Oz \
    -o $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh$build.vcf.gz

Check for consistency of the C4 reference panel

Convert the C4 and 1000 Genomes project reference panels to plink and then merge to compute consistency

bcftools view --no-version $url -r 6:24894177-33890574 | \
  awk 'NF==2 {print "##contig=<ID=6,length=171115067>"} {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.chr6.integrated_phase1_v3.20101123.snps_indels_svs.genotypes

bcftools annotate --no-version -x ID -I +'%CHROM:%POS:%REF:%ALT' \
  $HOME/res/MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh37.vcf.gz | \
  $HOME/bin/plink --vcf /dev/stdin --biallelic-only --keep-allele-order --const-fid --make-bed \
  --out MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh37

plink --bfile MHC_haplotypes_CEU_HapMap3_ref_panel.GRCh37 \
  --bmerge ALL.chr6.integrated_phase1_v3.20101123.snps_indels_svs.genotypes --merge-mode 6

You should get the following result

577275 overlapping calls, 577275 nonmissing in both filesets.
575472 concordant, for a concordance rate of 0.996877.


A protocol to impute C4 alleles from MHC genotypes



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