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DeepVariant whole genome sequencing case study

In this case study, we describe applying DeepVariant to a real WGS sample. Then we assess the quality of the DeepVariant variant calls with hap.py.

To make it faster to run over this case study, we run only on chromosome 20.

Prepare environment

Tools

Docker will be used to run DeepVariant and hap.py,

Download Reference

We will be using GRCh38 for this case study.

mkdir -p reference

FTPDIR=ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids

curl ${FTPDIR}/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz | gunzip > reference/GRCh38_no_alt_analysis_set.fasta
curl ${FTPDIR}/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.fai > reference/GRCh38_no_alt_analysis_set.fasta.fai

Download Genome in a Bottle Benchmarks

We will benchmark our variant calls against v4.2.1 of the Genome in a Bottle small variant benchmarks for HG003.

mkdir -p benchmark

FTPDIR=ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/release/AshkenazimTrio/HG003_NA24149_father/NISTv4.2.1/GRCh38

curl ${FTPDIR}/HG003_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.bed > benchmark/HG003_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.bed
curl ${FTPDIR}/HG003_GRCh38_1_22_v4.2.1_benchmark.vcf.gz > benchmark/HG003_GRCh38_1_22_v4.2.1_benchmark.vcf.gz
curl ${FTPDIR}/HG003_GRCh38_1_22_v4.2.1_benchmark.vcf.gz.tbi > benchmark/HG003_GRCh38_1_22_v4.2.1_benchmark.vcf.gz.tbi

Download HG003 chr20 BAM

We'll use HG003 Illumina WGS reads publicly available from the PrecisionFDA Truth v2 Challenge.

mkdir -p input
HTTPDIR=https://storage.googleapis.com/deepvariant/case-study-testdata

curl ${HTTPDIR}/HG003.novaseq.pcr-free.35x.dedup.grch38_no_alt.chr20.bam > input/HG003.novaseq.pcr-free.35x.dedup.grch38_no_alt.chr20.bam
curl ${HTTPDIR}/HG003.novaseq.pcr-free.35x.dedup.grch38_no_alt.chr20.bam.bai > input/HG003.novaseq.pcr-free.35x.dedup.grch38_no_alt.chr20.bam.bai

Running DeepVariant with one command

DeepVariant pipeline consists of 3 steps: make_examples, call_variants, and postprocess_variants. You can now run DeepVariant with one command using the run_deepvariant script.

Running on a CPU-only machine

mkdir -p output
mkdir -p output/intermediate_results_dir

BIN_VERSION="1.6.1"

sudo docker run \
  -v "${PWD}/input":"/input" \
  -v "${PWD}/output":"/output" \
  -v "${PWD}/reference":"/reference" \
  google/deepvariant:"${BIN_VERSION}" \
  /opt/deepvariant/bin/run_deepvariant \
  --model_type WGS \
  --ref /reference/GRCh38_no_alt_analysis_set.fasta \
  --reads /input/HG003.novaseq.pcr-free.35x.dedup.grch38_no_alt.chr20.bam \
  --output_vcf /output/HG003.output.vcf.gz \
  --output_gvcf /output/HG003.output.g.vcf.gz \
  --num_shards $(nproc) \
  --regions chr20 \
  --intermediate_results_dir /output/intermediate_results_dir

By specifying --model_type WGS, you'll be using a model that is best suited for Illumina Whole Genome Sequencing data.

NOTE: If you want to run each of the steps separately, add --dry_run=true to the command above to figure out what flags you need in each step. Based on the different model types, different flags are needed in the make_examples step.

--intermediate_results_dir flag is optional. By specifying it, the intermediate outputs of make_examples and call_variants stages can be found in the directory. After the command, you can find these files in the directory:

call_variants_output.tfrecord.gz
gvcf.tfrecord-?????-of-?????.gz
make_examples.tfrecord-?????-of-?????.gz

For running on GPU machines, or using Singularity instead of Docker, see Quick Start.

Benchmark on chr20

mkdir -p happy

sudo docker pull jmcdani20/hap.py:v0.3.12

sudo docker run \
  -v "${PWD}/benchmark":"/benchmark" \
  -v "${PWD}/input":"/input" \
  -v "${PWD}/output":"/output" \
  -v "${PWD}/reference":"/reference" \
  -v "${PWD}/happy:/happy" \
  jmcdani20/hap.py:v0.3.12 /opt/hap.py/bin/hap.py \
  /benchmark/HG003_GRCh38_1_22_v4.2.1_benchmark.vcf.gz \
  /output/HG003.output.vcf.gz \
  -f /benchmark/HG003_GRCh38_1_22_v4.2.1_benchmark_noinconsistent.bed \
  -r /reference/GRCh38_no_alt_analysis_set.fasta \
  -o /happy/happy.output \
  --engine=vcfeval \
  --pass-only \
  -l chr20

Output:

Benchmarking Summary:
Type Filter  TRUTH.TOTAL  TRUTH.TP  TRUTH.FN  QUERY.TOTAL  QUERY.FP  QUERY.UNK  FP.gt  FP.al  METRIC.Recall  METRIC.Precision  METRIC.Frac_NA  METRIC.F1_Score  TRUTH.TOTAL.TiTv_ratio  QUERY.TOTAL.TiTv_ratio  TRUTH.TOTAL.het_hom_ratio  QUERY.TOTAL.het_hom_ratio
INDEL    ALL        10628     10588        40        21099        19      10036     15      3       0.996236          0.998283        0.475662         0.997258                     NaN                     NaN                   1.748961                   2.318182
INDEL   PASS        10628     10588        40        21099        19      10036     15      3       0.996236          0.998283        0.475662         0.997258                     NaN                     NaN                   1.748961                   2.318182
  SNP    ALL        70166     69917       249        84796        59      14782     13      3       0.996451          0.999157        0.174324         0.997802                2.296566                2.085786                   1.883951                   1.920577
  SNP   PASS        70166     69917       249        84796        59      14782     13      3       0.996451          0.999157        0.174324         0.997802                2.296566                2.085786                   1.883951                   1.920577