@DIB-LAB/The Great Genotyper

📖 Table of Contents

➤ Table of Contents

• ➤ Table of Contents
• ➤ Introduction
• ➤ Quick Installation
• ➤ Build from source
  • Clone
  • Install dependencies
  • Build
    • CMake options
    • Build The kProcessor Library
    • Build Everything
• ➤ Manually build the Python bindings
  • Generate bindings
• ➤ Contributors
• ➤ License

➤ Introduction

The Great Genotyper is a population genotyping workflow. The workflow begins by preprocessing 4.2K short-read samples of 183TB raw data to create an 867GB Counting Colored De Bruijn Graph. The Great Genotyper uses the succinct CCDG to genotype any list of phased or unphased variants, leveraging the population information to increase both precision and recall. The Great Genotyper offers the same accuracy as the state-of-the-art with unprecedented performance. It took 100 hours to genotype 4.5M variants in the 4.2K samples using one server with 32 cores and 145GB of memory. A similar task would take months or even years using single-sample genotypers with the same computational resources.

➤ Build from source

Clone

git clone https://github.com/dib-lab/TheGreatGenotyper.git
cd TheGreatGenotyper/

Install dependencies

conda env create -f environment.yml
conda activate gg
conda env config vars set CPATH=${CONDA_PREFIX}/include:${CPATH}
conda activate
conda activate gg

Build

# Run CMake configure
cmake -Bbuild

# Run make with parallel execution.
cmake --build build -j4 # -j4 = execute 4 recipes simultaneously.

Download Beagle

wget https://faculty.washington.edu/browning/beagle/beagle.22Jul22.46e.jar
wget https://bochet.gcc.biostat.washington.edu/beagle/genetic_maps/plink.GRCh38.map.zip
unzip plink.GRCh38.map.zip 

➤ Run

Prepare Input Data

uncompress test_data

gzip -d test_data/GRCh38_chr21.fa.gz test_data/test.vcf.gz  test_data/test.unphased.vcf.gz

Download index(50G) for Simons Genome Diversity population(SGDP).

mkdir -p index/SGDP/
cd index/SGDP/
wget https://farm.cse.ucdavis.edu/~mshokrof/indexes/SGDP/graph.dbg
wget https://farm.cse.ucdavis.edu/~mshokrof/indexes/SGDP/graph.desc.tsv
wget https://farm.cse.ucdavis.edu/~mshokrof/indexes/SGDP/annotation.relaxed.row_diff_int_brwt.annodbg
wget https://farm.cse.ucdavis.edu/~mshokrof/indexes/SGDP/samples.csv
cd -
echo "index/SGDP/" > indexes

Running

The figure provides an overview of the various workflows utilized in the Great Genotyper. This includes three distinct workflows to create reference panels, each illustrated with a different color of the arrow: The Red workflow creates a high-quality reference panel from phased variants, the green workflow creates the panel from variants without phasing information, and the blue workflow enhances the green panel by phasing the input variants then following the red workflow. Each of these workflows utilizes three specific processes: Unique k-mer Extractor, Extract Phasing Information, and High-Quality Genotype. These procedures are based on a scaled-up version of the Pangenie model, which enables the simultaneous processing of thousands of samples. “Population Genotype correction and phasing” scrutinize genotypes by evaluating the genotyping quality across all samples. Once completed, the removed genotypes are re-estimated using a statistical imputation process, implemented by Beagle. Beagle also uses the results from the population genotype to phase all variants, thereby generating a reference panel based on the input variants. Lastly, “Fast genotyping” produces initial genotypes for all the samples in the population database by comparing the k-mer counts of unique k-mers to the average sample coverage.

High Quality Genotyping for phased variants(red arrow):

Run The great genotyper. It took 12 mins using 32 core and 85GB ram.

mkdir test_output

./build/pangenie/src/TheGreatGenotyper -g  -i indexes  -j 32 -t 32 -r test_data/GRCh38_chr21.fa  -y  emissions -v test_data/test.vcf -o - 2> log | bgzip > test_output/test.vcf.gz
tabix -p vcf test_output/test.vcf.gz

java -Xmx40G -jar beagle.22Jul22.46e.jar gt=test_output/test.vcf.gz out=test_output/test.phased nthreads=32  map=plink.chr21.GRCh38.map
tabix -p vcf test_output/test.phased.vcf.gz

bcftools +fill-tags test_output/test.phased.vcf.gz -Oz  -o test_output/test.phased.tagged.vcf.bgz -- -t all
tabix -p vcf test_output/test.phased.tagged.vcf.bgz
 

Fast Genotyping for variants without phasing(green arrow):

Note: "-a" parameter is added to use kmer-only genotyping model.

mkdir test_output

./build/pangenie/src/TheGreatGenotyper -a -g  -i indexes  -j 32 -t 32 -r test_data/GRCh38_chr21.fa  -y  emissions -v test_data/test.unphased.vcf -o - 2> log | bgzip > test_output/test.vcf.gz
tabix -p vcf test_output/test.vcf.gz

java -Xmx40G -jar beagle.22Jul22.46e.jar gt=test_output/test.vcf.gz out=test_output/test.phased nthreads=32  map=plink.chr21.GRCh38.map
tabix -p vcf test_output/test.phased.vcf.gz

bcftools +fill-tags test_output/test.phased.vcf.gz -Oz  -o test_output/test.phased.tagged.vcf.bgz -- -t all
tabix -p vcf test_output/test.phased.tagged.vcf.bgz
 

Second pass Genotyping for variants without phasing(blue arrow):

After running the Green workflow, Use the workflow output to phase the input variants and rerun the great genotyper as in the red workflow

java -Xmx40G -jar beagle.22Jul22.46e.jar gt=test_data/test.unphased.vcf ref=test_output/test.phased.vcf.gz out=test_data/test.GG.phased nthreads=32  map=plink.chr21.GRCh38.map
gzip -d test_data/test.GG.phased.vcf.gz

./build/pangenie/src/TheGreatGenotyper -g  -i indexes  -j 32 -t 32 -r test_data/GRCh38_chr21.fa  -y  emissions -v test_data/test.GG.phased.vcf -o - 2> log | bgzip > test_output/secondpass.vcf.gz
tabix -p vcf test_output/secondpass.vcf.gz

java -Xmx40G -jar beagle.22Jul22.46e.jar gt=test_output/secondpass.vcf.gz out=test_output/secondpass.phased nthreads=32  map=plink.chr21.GRCh38.map
tabix -p vcf test_output/secondpass.phased.vcf.gz

bcftools +fill-tags test_output/secondpass.phased.vcf.gz -Oz  -o test_output/secondpass.phased.tagged.vcf.bgz -- -t all
tabix -p vcf test_output/secondpass.phased.tagged.vcf.bgz

➤ Running on the Full 4.2K samples index

The previous examples utilize a small index of 276 samples. The full index, which contains 4.2K samples encompassing the 1000 Genome Samples, Human Diversity Project, and Simons Diversity Project, is available at Full Index. This index comprises 29 sub-indexes, each stored in a separate folder. A script is provided to assist with downloading all the indexes. Metadata for the samples in the index can be found at index_metadata.csv.

The full index may require a significant amount of memory when dealing with a large number of variants. We have developed a Snakemake workflow that operates on each sub-index independently, thereby avoiding any population-wide filtering. Following this, it integrates the results from each sub-index, performs population-level filtering, and carries out Beagle imputation. The Snakemake script is available at this link.

You will need to specify the input in the configuration file, an example of which can be found here. You can run the workflow using the following command:

snakemake  -j 5 --configfile config.yaml --use-conda

➤ Postprocessing

Query the frequent variants

bcftools view  -q 0.9 test_output/test.phased.tagged.vcf.bgz |grep -vP "^#" |head

Query the rare variants

bcftools view  -Q 0.1 test_output/test.phased.tagged.vcf.bgz |grep -vP "^#" |head

Advanced post-processing

Manipulating VCF files containing genotypes for hundreds or thousands of samples can be a challenging task. To effectively navigate and analyze such data, we recommend utilizing Hail as a tool for exploring the resulting VCF file. I prepared a jupyter notebook containing examples of tasks to be done using hail like: stratifying allele frequencies by population, and plotting the PCA of the genotypes. The following figure is the PCA plot generated by Hail using "test_output/merged.vcf.bgz"

To run the notebook, You need to install :

1. jupyter: https://jupyterlab.readthedocs.io/en/stable/getting_started/installation.html
2. Hail: https://hail.is/docs/0.2/getting_started.html

The notebook needs two input files: vcf file("test_output/test.phased.tagged.vcf.bgz") and samples description.

➤ Contributors

Moustafa Shokrof

C.Titus Brown

Tamer Mansour

➤ License

Licensed under BSD-3-Clause.