Clair3-Nova: Accurate Nanopore long-read de novo variant calling in family trios with deep neural networks
Contact: Ruibang Luo, Junhao Su
Email: [email protected], [email protected]
Clair3-Nova is the 2nd generation of Clair3-Trio. Nova supports de novo variant calling in trio. To cite Clair3-Nova, please cite Clair3-trio (Briefing in Bioinformatics, 2022).
- Latest Updates
- Pre-trained Models
- Quick Demo
- Installation
- General Usage
- Options
- Output Files
- How to get high-quality de novo variants from the output
- Model Training
v0.3.2 (Sep 24, 2024):
- add "INFO/DNP" score for all child's "0/1" output;
- add "INFO/HDN" tag for "high quality de novo variant", default configured as DNP > 0.8;
- add reference call in gVCF output;
- add "INFO/RPL" tag for saving reference PL score for gvcf output.
v0.3.1 (July 9, 2024): fix bug in multiple alternative sites.
v0.3 (June 23, 2024): add r10.4.1 hac model and add base_err feature.
- add r10 HAC model trained at HG002 trio
- add
--base_errflag for reducing "./." in gvcf output - add
--keep_iupac_basesflag to showing iupac char.
v0.2.1 (May 15, 2024): fix bugs of SelectCandidates_Trio.
v0.2 (Apr 2, 2024): added models for r10.4.1 and r9.4.1
v0.1 (Feb 6, 2024): Initial release.
Download models from here or click on the links below.
| Model name | Platform | Training samples | Date | Basecaller | File | Link |
|---|---|---|---|---|---|---|
| r1041_e82_400bps_sup_nova | ONT r10.4.1 E8.2 (5kHz) | HG002,3,4 | 20240206 | Dorado v4.0.0 SUP | r1041_e82_400bps_sup_nova.tar.gz | Download |
| r1041_e82_400bps_hac_nova | ONT r10.4.1 E8.2 (5kHz) | HG002,3,4 | 20240723 | Dorado v4.0.0 HAC | r1041_e82_400bps_hac_nova.tar.gz | Download |
| r941_prom_sup_g5014_nova | ONT r9.4.1 | HG002,3,4 | 20240330 | Guppy5 sup | r941_prom_sup_g5014_nova.tar.gz | Download |
When using the Clair3-Nova model, please use a corresponding Clair3 model for Pileup calling. Check here or here for more information about Clair3 pretrained model.
| Model name | Platform | Training samples | Date | Basecaller | File | Link |
|---|---|---|---|---|---|---|
| r1041_e82_400bps_sup_v430 | ONT r10.4.1 E8.2 (5kHz) | - | - | Dorado v4.3.0 SUP | r1041_e82_400bps_sup_v430.tar.gz | Download |
| r1041_e82_400bps_hac_v430 | ONT r10.4.1 E8.2 (5kHz) | - | - | Dorado v4.3.0 HAC | r1041_e82_400bps_hac_v430.tar.gz | Download |
| r941_prom_sup_g5014 | ONT r9.4.1 | - | - | Guppyu5 sup | r941_prom_sup_g5014.tar.gz | Download |
A pre-built docker image is available here. With it you can run Clair3-Nova using a single command.
Caution: Absolute path is needed for both INPUT_DIR and OUTPUT_DIR.
INPUT_DIR="[YOUR_INPUT_FOLDER]" # e.g. /input
REF=${_INPUT_DIR}/ref.fa # change your reference file name here
OUTPUT_DIR="[YOUR_OUTPUT_FOLDER]" # e.g. /output
THREADS="[MAXIMUM_THREADS]" # e.g. 8
MODEL_C3="[Clair3 MODEL NAME]" # e.g. Clair3 model, e.g. r1041_e82_400bps_sup_v430
MODEL_C3D="[Clair3-Trio MODEL NAME]" # e.g. Clair3-Nova model, r1041_e82_400bps_sup_nova
docker run -it \
-v ${INPUT_DIR}:${INPUT_DIR} \
-v ${OUTPUT_DIR}:${OUTPUT_DIR} \
hkubal/clair3-nova:latest \
/opt/bin/run_clair3_nova.sh \
--ref_fn=${INPUT_DIR}/ref.fa \ ## change your reference file name here
--bam_fn_c=${INPUT_DIR}/child_input.bam \ ## change your child's bam file name here
--bam_fn_p1=${INPUT_DIR}/parent1_input.bam \ ## change your parent-1's bam file name here
--bam_fn_p2=${INPUT_DIR}/parenet2_input.bam \ ## change your parent-2's bam file name here
--sample_name_c=${SAMPLE_C} \ ## change your child's name here
--sample_name_p1=${SAMPLE_P1} \ ## change your parent-1's name here
--sample_name_p2=${SAMPLE_P2} \ ## change your parent-2's name here
--threads=${THREADS} \ ## maximum threads to be used
--model_path_clair3="/opt/models/clair3_models/${MODEL_C3}" \
--model_path_clair3_nova="/opt/models/clair3_nova_models/${MODEL_C3D}" \
--output=${OUTPUT_DIR} ## absolute output path prefix
Anaconda install:
Please install anaconda using the official guide or using the commands below:
wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
chmod +x ./Miniconda3-latest-Linux-x86_64.sh
./Miniconda3-latest-Linux-x86_64.shInstall Clair3 env and Clair3-Nova using anaconda step by step:
# create and activate an environment named clair3
conda create -n clair3 python=3.9.0 -y
source activate clair3
# install pypy and packages in the environemnt
conda install -c conda-forge pypy3.6 -y
pypy3 -m ensurepip
pypy3 -m pip install mpmath==1.2.1
# install python packages in environment
conda install -c conda-forge tensorflow==2.8.0 -y
conda install -c conda-forge pytables -y
conda install -c anaconda pigz cffi==1.14.4 -y
conda install -c conda-forge parallel=20191122 zstd -y
conda install -c conda-forge -c bioconda samtools=1.15.1 -y
conda install -c conda-forge -c bioconda whatshap=1.7 -y
conda install -c conda-forge xz zlib bzip2 automake curl -y
# tensorflow-addons is required in training
pip install tensorflow-addons
# clone Clair3-Nova
git clone https://github.com/HKU-BAL/Clair3-Nova.git
cd Clair3-Nova
# download Clair3's pre-trained models
mkdir -p models/clair3_models
wget http://www.bio8.cs.hku.hk/clair3_trio/clair3_models/clair3_models.tar.gz
tar -zxvf clair3_models.tar.gz -C ./models/clair3_models
# download Clair3-Nova's pre-trained models
mkdir -p models/clair3_nova_models
wget http://www.bio8.cs.hku.hk/clair3_trio/clair3_nova_models/clair3_nova_models.tar.gz
tar -zxvf clair3_nova_models.tar.gz -C ./models/clair3_nova_models
# run clair3-nova
_INPUT_DIR="[YOUR_INPUT_FOLDER]" # e.g. ./input
_BAM_C=${_INPUT_DIR}/input_child.bam # chnage your child's bam file name here
_BAM_P1=${_INPUT_DIR}/input_parent1.bam # chnage your parent-1's bam file name here
_BAM_P2=${_INPUT_DIR}/input_parent2.bam # chnage your parent-2's bam file name here
_SAMPLE_C="[Child sample ID]" # child sample ID, e.g. HG002
_SAMPLE_P1="[Parent1 sample ID]" # parent1 sample ID, e.g. HG003
_SAMPLE_P2="[Parent2 sample ID]" # parent2 sample ID, e.g. HG004
_REF=${_INPUT_DIR}/ref.fa # change your reference file name here
_OUTPUT_DIR="[YOUR_OUTPUT_FOLDER]" # e.g. ./output
_THREADS="[MAXIMUM_THREADS]" # e.g. 8
_MODEL_DIR_C3="[Clair3 MODEL NAME]" # e.g. ./models/clair3_models/r1041_e82_400bps_sup_v430
_MODEL_DIR_C3D="[Clair3-Nova MODEL NAME]" # e.g. ./models/clair3_nova_models/r1041_e82_400bps_sup_nova
./run_clair3_nova.sh \
--bam_fn_c=${_BAM_C} \
--bam_fn_p1=${_BAM_P1} \
--bam_fn_p2=${_BAM_P2} \
--output=${_OUTPUT_DIR} \
--ref_fn=${_REF} \
--threads=${_THREADS} \
--model_path_clair3="${_MODEL_DIR_C3}" \
--model_path_clair3_nova="${_MODEL_DIR_C3D}" \
--sample_name_c=${_SAMPLE_C} \
--sample_name_p1=${_SAMPLE_P1} \
--sample_name_p2=${_SAMPLE_P2}
Building a docker image.
# clone Clair3-Nova
git clone https://github.com/hku-bal/Clair3-Nova.git
cd Clair3-Nova
# build a docker image named hkubal/clair3-nova:latest
# might require docker authentication to build docker image
docker build -f ./Dockerfile -t hkubal/clair3-nova:latest .
# run clair3-docker image like
docker run -it hkubal/clair3-nova:latest /opt/bin/run_clair3_nova.sh --help
Caution: Use =value for optional parameters, e.g. --bed_fn=fn.bed instead of --bed_fn fn.bed.
./run_clair3_nova.sh \
--bam_fn_c=${_BAM_C} \
--bam_fn_p1=${_BAM_P1} \
--bam_fn_p2=${_BAM_P2} \
--output=${_OUTPUT_DIR} \
--ref_fn=${_REF} \
--threads=${_THREADS} \
--model_path_clair3="${_MODEL_DIR_C3}" \
--model_path_clair3_nova="${_MODEL_DIR_C3D}" \
--sample_name_c=${_SAMPLE_C} \
--sample_name_p1=${_SAMPLE_P1} \
--sample_name_p2=${_SAMPLE_P2}
Required parameters:
--bam_fn_c=FILE BAM file input, for child. The input file must be samtools indexed.
--bam_fn_p1=FILE BAM file input, for parent1. The input file must be samtools indexed.
--bam_fn_p2=FILE BAM file input, for parent1. The input file must be samtools indexed.
--ref_fn=FILE FASTA reference file input. The input file must be samtools indexed.
--model_path_clair3=STR The folder path containing a Clair3 model (requiring six files in the folder, including pileup.data-00000-of-00002, pileup.data-00001-of-00002 pileup.index, full_alignment.data-0000
0-of-00002, full_alignment.data-00001-of-00002 and full_alignment.index).
--model_path_clair3_nova=STR The folder path containing a Clair3-Nova model (files structure same as Clair3).
-t, --threads=INT Max #threads to be used. The full genome will be divided into small chunks for parallel processing. Each chunk will use 4 threads. The #chunks being processed simultaneously is ceil
(#threads/4)*3. 3 is the overloading factor.
-o, --output=PATH VCF/GVCF output directory.
Other parameters:
Caution: Use =value for optional parameters, e.g., --bed_fn=fn.bed instead of --bed_fn fn.bed
-v, --version Check Clair3-Nova version
-h, --help Check Clair3-Nova help page
--bed_fn=FILE Call variants only in the provided bed regions.
--vcf_fn=FILE Candidate sites VCF file input, variants will only be called at the sites in the VCF file if provided.
--ctg_name=STR The name of the sequence to be processed.
--pileup_only Use the pileup model only when calling, default: disable.
--pileup_phasing Use the pileup model calling and phasing, default: disable.
--sample_name_c=STR Define the sample name for Child to be shown in the VCF file.[Child]
--sample_name_p1=STR Define the sample name for Parent1 to be shown in the VCF file.[Parent1]
--sample_name_p2=STR Define the sample name for Parent2 to be shown in the VCF file.[Parent2]
--gvcf Enable GVCF output, default: disable.
--qual=INT If set, variants with >=$qual will be marked PASS, or LowQual otherwise.
--samtools=STR Path of samtools, samtools version >= 1.10 is required.
--python=STR Path of python, python3 >= 3.6 is required.
--pypy=STR Path of pypy3, pypy3 >= 3.6 is required.
--parallel=STR Path of parallel, parallel >= 20191122 is required.
--whatshap=STR Path of whatshap, whatshap >= 1.0 is required.
--chunk_size=INT The size of each chuck for parallel processing, default: 5000000.
--print_ref_calls Show reference calls (0/0) in VCF file, default: disable.
--include_all_ctgs Call variants on all contigs, otherwise call in chr{1..22,X,Y} and {1..22,X,Y}, default: disable.
--snp_min_af=FLOAT Minimum SNP AF required for a candidate variant. Lowering the value might increase a bit of sensitivity in trade of speed and accuracy, default: ont:0.08.
--indel_min_af=FLOAT Minimum Indel AF required for a candidate variant. Lowering the value might increase a bit of sensitivity in trade of speed and accuracy, default: ont:0.15.
--nova_model_prefix=STR Model prefix in trio & nova calling, including $prefix.data-00000-of-00002, $prefix.data-00001-of-00002 $prefix.index, default: nova.
--enable_output_phasing Output phased variants using whatshap, default: disable.
--enable_output_haplotagging Output enable_output_haplotagging BAM variants using whatshap, default: disable.
--enable_phasing It means `--enable_output_phasing`. The option is retained for backward compatibility.
--var_pct_full=FLOAT EXPERIMENTAL: Specify an expected percentage of low quality 0/1 and 1/1 variants called in the pileup mode for full-alignment mode calling, default: 0.3.
--ref_pct_full=FLOAT EXPERIMENTAL: Specify an expected percentage of low quality 0/0 variants called in the pileup mode for full-alignment mode calling, default: 0.1 .
--var_pct_phasing=FLOAT EXPERIMENTAL: Specify an expected percentage of high quality 0/1 variants used in WhatsHap phasing, default: 0.8 for ont guppy5 and 0.7 for other platforms.
--pileup_model_prefix=STR EXPERIMENTAL: Model prefix in pileup calling, including $prefix.data-00000-of-00002, $prefix.data-00001-of-00002 $prefix.index. default: pileup.
--keep_iupac_bases EXPERIMENTAL: Keep IUPAC reference and alternate bases, default: convert all IUPAC bases to N.
--base_err=FLOAT EXPERIMENTAL: Estimated base error rate when enabling gvcf option, default: 0.05 (set smaller will increase reduce the number of ./. output).
--gq_bin_size=INT EXPERIMENTAL: Default gq bin size for merge non-variant block when enabling gvcf option, default: 5.
Clair3-Nova outputs files in VCF/GVCF format for the trio & de novo genotype. The output files (for a trio [C ], [P1], [P2]) including:
.
├── run_clair3_nova.log # Clair3-Nova running log
├── [C ].vcf.gz # Called variants in vcf format for [C ]
├── [P1].vcf.gz # Called variants in vcf format for [P1]
├── [P2].vcf.gz # Called variants in vcf format for [P2]
├── [C ].gvcf.gz # Called variants in gvcf format for [C ] (when enabled `--gvcf`)
├── [P1].gvcf.gz # Called variants in gvcf format for [P2] (when enabled `--gvcf`)
├── [P2].gvcf.gz # Called variants in gvcf format for [P2] (when enabled `--gvcf`)
├── phased_[C ].vcf.gz # Called phased variants for [C ] (when enabled `--enable_output_phasing`)
├── phased_[P1].vcf.gz # Called phased variants for [P1] (when enabled `--enable_output_phasing`)
├── phased_[P2].vcf.gz # Called phased variants for [P2] (when enabled `--enable_output_phasing`)
├── phased_[C ].bam # alignment tagged with phased variants info. for [C ] (when enabled `--enable_output_haplotagging`)
├── phased_[P1].bam # alignment tagged with phased variants info. for [P1] (when enabled `--enable_output_haplotagging`)
├── phased_[P2].bam # alignment tagged with phased variants info. for [P2] (when enabled `--enable_output_haplotagging`)
├── /log # folder for detailed running log
└── /tmp # folder for all running temporary files
# input: clair3-nova's output of ${SAMPLE[0]}.vcf.gz, ${SAMPLE[1]}.vcf.gz, ${SAMPLE[2]}.vcf.gz files
# output: merged vcf and de novo variants
# requires bcftools and rtg tools
# install bcftools: https://github.com/samtools/bcftools
# install rtg tools: https://github.com/RealTimeGenomics/rtg-tools
BCFTOOLS=bcftools
RTG=rtg
# input files
# requires trio's ped file, reference sdf file
# example input
_TRIO_PED=/autofs/bal31/jhsu/home/data/giab/trio.ped
cat $_TRIO_PED
#PED format pedigree
#
#fam-id/ind-id/pat-id/mat-id: 0=unknown
#sex: 1=male; 2=female; 0=unknown
#phenotype: -9=missing, 0=missing; 1=unaffected; 2=affected
#
#fam-id ind-id pat-id mat-id sex phen
1 HG002 HG003 HG004 1 0
1 HG003 0 0 1 0
1 HG004 0 0 2 0
# your reference sdf file path
REF_SDF_FILE_PATH=./GCA_000001405.15_GRCh38_no_alt_analysis_set.sdf
# output files
# merged and de novo vcfs
M_VCF=trio_m.vcf.gz
M_VCF_annotated=trio_m_ann.vcf.gz
denovo_VCF=trio_all_denovo.vcf.gz
denovo_VCF_sf=trio_high_quality_denovo.vcf.gz
# merge trio vcfs
${BCFTOOLS} merge ${SAMPLE[0]}.vcf.gz \
${SAMPLE[1]}.vcf.gz \
${SAMPLE[2]}.vcf.gz \
--threads 32 -f PASS -0 -m all| ${BCFTOOLS} view -O z -o ${M_VCF}
# index
${BCFTOOLS} index ${M_VCF}
#${BCFTOOLS} view ${M_VCF} -H | wc -l
# annotate with Mendelian inherrtance pattern
${RTG} mendelian -i ${M_VCF} -o ${M_VCF_annotated} --pedigree ${_TRIO_PED} -t ${REF_SDF_FILE_PATH} |& tee MDL.log
# get de novo variants
${BCFTOOLS} view -i 'INFO/MCV ~ "0/0+0/0->0/1"' ${M_VCF_annotated} -O z -o ${denovo_VCF}
${BCFTOOLS} index ${denovo_VCF}
# get high quality de novo variants
${BCFTOOLS} view -i "INFO/DNP>0.85" ${denovo_VCF} -s ${SAMPLE[0]} -O z -o ${denovo_VCF_sf}
${BCFTOOLS} index ${denovo_VCF_sf}
# high quality de novo variants set is in ${denovo_VCF_sf}