variant_calling_singularity_quickstart.md

Variant calling quickstart (Using singularity)

Here we show a quickstart for using Singularity in your system.

Install docker

Expand to see singularity installation guide.

Please install Singularity. This must be installed by the system admin.

Follow these installation instructions to install Singularity 3.7, if you want to install a newer version please follow instructions from the singulaity website.

# Install dependencies
sudo apt-get update && sudo apt-get install -y \
build-essential \
libssl-dev \
uuid-dev \
libgpgme11-dev \
squashfs-tools \
libseccomp-dev \
wget \
pkg-config \
git \
cryptsetup

# Install Go on linux: https://golang.org/doc/install
export VERSION=1.14.12 OS=linux ARCH=amd64 && \
wget https://dl.google.com/go/go$VERSION.$OS-$ARCH.tar.gz && \
sudo tar -C /usr/local -xzvf go$VERSION.$OS-$ARCH.tar.gz && \
rm go$VERSION.$OS-$ARCH.tar.gz

# Set environment variable
echo 'export PATH=/usr/local/go/bin:$PATH' >> ~/.bashrc && \
source ~/.bashrc

# Download and install singularity
export VERSION=3.7.0 && # adjust this as necessary \
wget https://github.com/hpcng/singularity/releases/download/v${VERSION}/singularity-${VERSION}.tar.gz && \
tar -xzf singularity-${VERSION}.tar.gz && \
cd singularity

# install sigularity
./mconfig && \
make -C builddir && \
sudo make -C builddir install  

# After installation is complete log out and log in
singularity help

Quickstart: Nanopore variant calling

BASE="${HOME}/singularity-quickstart"

# Set up input data
INPUT_DIR="${BASE}/input/data"
REF="GRCh38_no_alt.chr20.fa"
BAM="HG002_ONT_2_GRCh38.chr20.quickstart.bam"

# Set the number of CPUs to use
THREADS="1"

# Set up output directory
OUTPUT_DIR="${BASE}/output"
OUTPUT_PREFIX="HG002_ONT_2_GRCh38_PEPPER_Margin_DeepVariant.chr20"
OUTPUT_VCF="HG002_ONT_2_GRCh38_PEPPER_Margin_DeepVariant.chr20.vcf.gz"
TRUTH_VCF="HG002_GRCh38_1_22_v4.2.1_benchmark.quickstart.vcf.gz"
TRUTH_BED="HG002_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.quickstart.bed"

## 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/quickstart_data/HG002_ONT_2_GRCh38.chr20.quickstart.bam
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/quickstart_data/HG002_ONT_2_GRCh38.chr20.quickstart.bam.bai
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/quickstart_data/GRCh38_no_alt.chr20.fa
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/quickstart_data/GRCh38_no_alt.chr20.fa.fai
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/quickstart_data/HG002_GRCh38_1_22_v4.2.1_benchmark.quickstart.vcf.gz
wget -P ${INPUT_DIR} https://storage.googleapis.com/pepper-deepvariant-public/quickstart_data/HG002_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.quickstart.bed

# Pull the docker images
singularity pull docker://jmcdani20/hap.py:v0.3.12
singularity pull docker://kishwars/pepper_deepvariant:r0.8

# Run PEPPER-Margin-DeepVariant
singularity exec --bind /usr/lib/locale/ \
pepper_deepvariant_r0.8.sif \
run_pepper_margin_deepvariant call_variant \
-b "${INPUT_DIR}/${BAM}" \
-f "${INPUT_DIR}/${REF}" \
-o "${OUTPUT_DIR}" \
-p "${OUTPUT_PREFIX}" \
-t ${THREADS} \
-r chr20:1000000-1020000 \
--ont_r9_guppy5_sup

# Run hap.py
singularity exec --bind /usr/lib/locale/ \
hap.py_v0.3.12.sif \
/opt/hap.py/bin/hap.py \
${INPUT_DIR}/${TRUTH_VCF} \
${OUTPUT_DIR}/${OUTPUT_VCF} \
-f "${INPUT_DIR}/${TRUTH_BED}" \
-r "${INPUT_DIR}/${REF}" \
-o "${OUTPUT_DIR}/happy.output" \
--pass-only \
-l chr20:1000000-1020000 \
--engine=vcfeval \
--threads="${THREADS}"

Expected output:

Type	Truth total	True positives	False negatives	False positives	Recall	Precision	F1-Score
INDEL	2	2	0	0	1.0	1.0	1.0
SNP	39	39	39	39	1.0	1.0	1.0

Authors:

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