ONT_variant_calling.md

Oxford Nanopore R9.4.1 Guppy 5 Sup variant calling workflow

PEPPER-Margin-DeepVariant is a haplotype-aware variant calling pipeline for long reads.

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HG002 chr20 case-study

We evaluated this pipeline on ~90x HG002 data. The data is publicly available, please feel free to download, run and evaluate the pipeline.

Sample:     HG002
Coverage:   ~25-90x
Basecaller: Guppy 5.0.7 "SUP"
Region:     chr20
Reference:  GRCh38_no_alt

Command-line instructions

Step 1: Install docker

Expand to see docker installation guide.

Please install docker and wget if you don't have it installed already. You can install docker for other distros from here:

• CentOS docker installation guide
• Debian/Raspbian docker installation guide
• Fedora installation guide
• Ubuntu installation guide

We show the installation instructions for Ubuntu here:

# Install wget to download data files.
sudo apt-get -qq -y update
sudo apt-get -qq -y install wget

# Install docker using instructions on:
# https://docs.docker.com/install/linux/docker-ce/ubuntu/
sudo apt-get -qq -y install apt-transport-https ca-certificates curl gnupg-agent software-properties-common
curl -fsSL https://download.docker.com/linux/ubuntu/gpg | sudo apt-key add -

sudo add-apt-repository \
"deb [arch=amd64] https://download.docker.com/linux/ubuntu \
$(lsb_release -cs) \
stable"

sudo apt-get -qq -y update
sudo apt-get -qq -y install docker-ce
docker --version

# To add the user to avoid running docker with sudo:
# Details: https://docs.docker.com/engine/install/linux-postinstall/

sudo groupadd docker
sudo usermod -aG docker $USER

# Log out and log back in so that your group membership is re-evaluated.

# After logging back in.
docker run hello-world

# If you can run docker without sudo then change the following commands accordingly.

Step 2: Download and prepare input data

BASE="${HOME}/ont-case-study"

# Set up input data
INPUT_DIR="${BASE}/input/data"
REF="GRCh38_no_alt.chr20.fa"
BAM="HG002_guppy_507_2_GRCh38_pass.chr20.bam"

# Set the number of CPUs to use
THREADS="64"

# Set up output directory
OUTPUT_DIR="${BASE}/output"
OUTPUT_PREFIX="HG002_ONT_R941_2_GRCh38_PEPPER_Margin_DeepVariant.chr20"
OUTPUT_VCF="HG002_ONT_R941_2_GRCh38_PEPPER_Margin_DeepVariant.chr20.vcf.gz"

## Create local directory structure
mkdir -p "${OUTPUT_DIR}"
mkdir -p "${INPUT_DIR}"

# Download the data to input directory
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/usecase_data/HG002_guppy_507_2_GRCh38_pass.chr20.bam
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/usecase_data/HG002_guppy_507_2_GRCh38_pass.chr20.bam.bai
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/usecase_data/GRCh38_no_alt.chr20.fa
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/usecase_data/GRCh38_no_alt.chr20.fa.fai

Step 3: Run PEPPER-Margin-DeepVariant

## Pull the docker image.
sudo docker pull kishwars/pepper_deepvariant:r0.8

# Run PEPPER-Margin-DeepVariant
sudo docker run \
-v "${INPUT_DIR}":"${INPUT_DIR}" \
-v "${OUTPUT_DIR}":"${OUTPUT_DIR}" \
kishwars/pepper_deepvariant:r0.8 \
run_pepper_margin_deepvariant call_variant \
-b "${INPUT_DIR}/${BAM}" \
-f "${INPUT_DIR}/${REF}" \
-o "${OUTPUT_DIR}" \
-p "${OUTPUT_PREFIX}" \
-t "${THREADS}" \
--ont_r9_guppy5_sup

Evaluation using hap.py (Optional)

You can evaluate the variants using hap.py.

Download benchmarking data:

# Set up input data
TRUTH_VCF="HG002_GRCh38_1_22_v4.2.1_benchmark.vcf.gz"
TRUTH_BED="HG002_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.bed"

# Download truth VCFs
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/usecase_data/HG002_GRCh38_1_22_v4.2.1_benchmark.vcf.gz
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/usecase_data/HG002_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.bed

Run hap.py:

# Pull the docker image
sudo docker pull jmcdani20/hap.py:v0.3.12

# Run hap.py
sudo docker run -it \
-v "${INPUT_DIR}":"${INPUT_DIR}" \
-v "${OUTPUT_DIR}":"${OUTPUT_DIR}" \
jmcdani20/hap.py:v0.3.12 /opt/hap.py/bin/hap.py \
${INPUT_DIR}/${TRUTH_VCF} \
${OUTPUT_DIR}/${OUTPUT_VCF} \
-f "${INPUT_DIR}/${TRUTH_BED}" \
-r "${INPUT_DIR}/${REF}" \
-o "${OUTPUT_DIR}/happy.output" \
--pass-only \
-l chr20 \
--engine=vcfeval \
--threads="${THREADS}"

Expected output:

Type	Truth total	True positives	False negatives	False positives	Recall	Precision	F1-Score
INDEL	11256	8981	2275	837	0.797886	0.916692	0.853172
SNP	71333	71257	94	68	0.998682	0.999047	0.998864

Authors:

This pipeline is developed in a collaboration between UCSC genomics institute and the genomics team at Google health.