Create a Reference Panel

This tutorial will help you to create your own reference panel and integrate it into Michigan Imputation Server.

Required Software

  • To create the m3vcf files for imputation, please use Minimac3.
  • To create the bcf files for phasing, please use bcftools and tabix.
  • To create the legend files for QC, please use vcftools or bcftools.

Folder Structure

We recommend the following folder structure:

├── cloudgene.yaml
├── bcfs
|   ├── chr1.bcf
    ├── chr1.bcf.csi
|   ├── ...
    ├── chr22.bcf
|   └── chr22.bcf.csi
├── legends
|   ├── chr1.legend.gz
|   ├── ...
|   └── chr22.legend.gz
├── m3vcfs
|   ├── chr1.m3vcf.gz
|   ├── ...
|   └── chr22.m3vcf.gz
├── map
|   └── genetic_map_hg19_withX.txt.gz

Init (build GRCh37/hg19)

Create a new folder and add a cloudgene.yaml file.

name:  My Reference Panel name
id: unique-id
description: a short description
category: RefPanel
version: 1.0.0

  hdfs: ${hdfs_app_folder}/m3vcfs/chr$chr.m3vcf.gz
  legend: ${local_app_folder}/legends/chr$chr.legend.gz
  mapEagle: ${hdfs_app_folder}/map/genetic_map_hg19_withX.txt.gz
  refEagle: ${hdfs_app_folder}/bcfs/chr$chr.bcf
  build: hg19
    all: 2504
    mixed: -1
    all: ALL
    mixed: Other/Mixed


  - import:
      source: ${local_app_folder}/bcfs
      target: ${hdfs_app_folder}/bcfs

  - import:
      source: ${local_app_folder}/m3vcfs
      target: ${hdfs_app_folder}/m3vcfs

  - import:
      source: ${local_app_folder}/map
      target: ${hdfs_app_folder}/map

Adaptions for build GRCh38/hg38

For a reference panel build 38, the following options must be added to properties in the cloudgene.yaml file:

    mapMinimac: ${app_hdfs_folder}/map/   
    mapEagle: ${app_hdfs_folder}/map/genetic_map_hg38_withX.txt.gz
    build: hg38

Prepare VCF files

Michigan Imputaiton Server requires each chromosome in a seperated file. Chromosome X must be split into three parts: chrX.PAR1, chrX.PAR2 and chrX.nonPAR. Use bcftools to split by region:

bcftools view <vcf-input> -r <region> -o <vcf-out> -O z

Chromosome X regions GRCh37/hg19

Use the following regions for the -r option:

X:60001-2699520 (chrX.PAR1)
X:2699521-154931043 (chrX.nonPAR)
X:154931044-155260560 (chrX.PAR2)

Chromosome X regions GRCh38/hg38

chrX:10001-2781479 (chrX.PAR1)
chrX:2781480-155701382 (chrX.nonPAR)
chrX:155701383-156030895 (chrX.PAR2)

Create bcf files

BCF files are required for phasing with eagle.

for chr in `seq 1 22` X.nonPAR X.PAR1 X.PAR2
    bcftools view chr${chr}.vcf.gz -O b -o chr${chr}.bcf
    bcftools index chr${chr}.bcf

Create m3vcf files

m3vcf files are used to store large reference panels in a compact way. Learn more about the file format here. For GRCh38/hg38, --mychromosome must be added, since chromosomes are coded as chr1 - chr22.

for chr in `seq 1 22` X.nonPAR X.PAR1 X.PAR2
    Minimac3 --refHaps chr${chr}.vcf.gz --processReference --prefix m3vcfs/chr${chr} --rsid

Create legend files

A legend file is a tab-delimited file consisting of 5 columns (id, position, a0, a1, all.aaf).

echo "id position a0 a1 all.aaf" > header
    for chr in `seq 1 22` X
    bcftools query -f '%CHROM %POS %REF %ALT %AC %AN\n' chr${chr}.bcf |  awk -F" " 'BEGIN { OFS = " " } {print $1":"$2 " " $2 " " $3 " "$4  " "  $5/$6}' | cat header - | bgzip > chr${chr}.legend.gz 

or in case AC / AN is not defined:

echo "id position a0 a1 all.aaf" > header
for chr in `seq 1 22`
    vcftools --gzvcf chr${chr}.vcf.gz --freq --out chr${chr}
    sed 's/:/\t/g' chr${chr}.frq | sed 1d | awk '{print $1":"$2" "$2" "$5" "$7" "$8}' > chr${chr}.legend
    cat header chr${chr}.legend | bgzip > chr${chr}.legend.gz
    rm chr${chr}.legend

Reference genetic maps

The genetic maps for eagle (hg19/hg38) can be found here.

Integrate your new reference panel

The created folder structure must be compressed to a zip archive and can now be integrated into Michigan Imputation Server. Please see here to start a Docker container and integrate the panel. A full working zip archive for Hapmap can be found here.