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Clara Parabricks v4.5.1

ont_germline

Beta

Note that the Parabricks GPU-accelerated ont_germline tool is currently in beta.

Run the germline variant tool to generate BAM and variants on long read ONT sequences using minimap2 for alignment as well as the DeepVariant variant caller.

See the ont_germline Reference section for a detailed listing of all available options.

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# This command assumes all the inputs are in the current working directory and all the outputs go to the same place. docker run --rm --gpus all --volume $(pwd):/workdir --volume $(pwd):/outputdir \ --workdir /workdir \ nvcr.io/nvidia/clara/clara-parabricks:4.5.1-1 \ pbrun ont_germline \ --ref /workdir/${REFERENCE_FILE} \ --in-fq /workdir/${INPUT_FASTQ} \ --out-bam /outputdir/${OUTPUT_BAM} \ --out-variants /outputdir/${OUTPUT_VCF}

The commands below are the minimap2-v2.26, GATK4, and Google DeepVariant counterpart of the Clara Parabricks command above. The output from these commands will be identical to the output from the above command. See the Output Comparison page for comparing the results.

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# Run minimap2 and pipe the output to create a sorted BAM. $ minimap2 -ax map-ont \ <INPUT_DIR>/${REFERENCE_FILE} \ <INPUT_DIR>/${INPUT_FASTQ} | \ gatk SortSam \ --java-options -Xmx30g \ --MAX_RECORDS_IN_RAM 5000000 \ -I /dev/stdin \ -O cpu.bam \ --SORT_ORDER coordinate # Run deepvariant BIN_VERSION="1.6.1" sudo docker run \ -v "${PWD}":"/input" \ -v "${PWD}/output":"/output" \ -v "${PWD}/Ref":"/reference" \ google/deepvariant:"${BIN_VERSION}" \ /opt/deepvariant/bin/run_deepvariant \ --model_type ONT_R104 \ --ref /reference/${REFERENCE_FILE} \ --reads cpu.bam \ --output_vcf /output/"${OUTPUT_VCF_FILE}" \ --num_shards $(nproc) \ --make_examples_extra_args "ws_use_window_selector_model=true"

Please note that a change must be made to the baseline minimap2 code in order to match the results exactly:

A fix must be made to the baseline KSW2 code to round the loop fission start and end points by changing them to st and en respectively. If the start point (st0) is a number below 16, but greater than 0, its scoring values will not be initialized correctly, but will still be used later when computing the actual alignment. This can be fixed by rounding the start and end points to multiples of 16.

To make this fix, change the following code in ksw2_extd2_sse.c:

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// loop fission: set scores first if (!(flag & KSW_EZ_GENERIC_SC)) { for (t = st0; t <= en0; t += 16) { __m128i sq, st, tmp, mask; sq = _mm_loadu_si128((__m128i*)&sf[t]); st = _mm_loadu_si128((__m128i*)&qrr[t]); mask = _mm_or_si128(_mm_cmpeq_epi8(sq, m1_), _mm_cmpeq_epi8(st, m1_)); tmp = _mm_cmpeq_epi8(sq, st); #ifdef __SSE4_1__ tmp = _mm_blendv_epi8(sc_mis_, sc_mch_, tmp); tmp = _mm_blendv_epi8(tmp, sc_N_, mask); #else tmp = _mm_or_si128(_mm_andnot_si128(tmp, sc_mis_), _mm_and_si128(tmp, sc_mch_)); tmp = _mm_or_si128(_mm_andnot_si128(mask, tmp), _mm_and_si128(mask, sc_N_)); #endif _mm_storeu_si128((__m128i*)((int8_t*)s + t), tmp); } } else { for (t = st0; t <= en0; ++t) ((uint8_t*)s)[t] = mat[sf[t] * m + qrr[t]]; }

Fixed version that uses lf_start and lf_en:

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// loop fission: set scores first int lf_start = st, lf_en = en; if (!(flag & KSW_EZ_GENERIC_SC)) { for (t = lf_start; t <= lf_en; t += 16) { __m128i sq, st, tmp, mask; sq = _mm_loadu_si128((__m128i*)&sf[t]); st = _mm_loadu_si128((__m128i*)&qrr[t]); mask = _mm_or_si128(_mm_cmpeq_epi8(sq, m1_), _mm_cmpeq_epi8(st, m1_)); tmp = _mm_cmpeq_epi8(sq, st); #ifdef __SSE4_1__ tmp = _mm_blendv_epi8(sc_mis_, sc_mch_, tmp); tmp = _mm_blendv_epi8(tmp, sc_N_, mask); #else tmp = _mm_or_si128(_mm_andnot_si128(tmp, sc_mis_), _mm_and_si128(tmp, sc_mch_)); tmp = _mm_or_si128(_mm_andnot_si128(mask, tmp), _mm_and_si128(mask, sc_N_)); #endif _mm_storeu_si128((__m128i*)((int8_t*)s + t), tmp); } } else { for (t = lf_start; t <= lf_en; ++t) ((uint8_t*)s)[t] = mat[sf[t] * m + qrr[t]]; }

See the DeepVariant Models for additional GPUs section for instructions on downloading and using model files for additional GPUs.

Run the germline pipeline from FASTQ/BAM to VCF by aligning long read ONT sequences with minimap2 and using a deep neural network analysis.


Type

Name

Required?

Description

I/O ‑‑ref REF Yes Path to the reference file.
I/O ‑‑index INDEX No Path to a minimizer index file generated by vanilla minimap2 to reduce indexing time.
I/O ‑‑in‑fq IN_FQ No Path to a query sequence file in fastq or fastq.gz format.
I/O ‑‑in‑bam IN_BAM No Path to the input BAM/CRAM file.
I/O ‑‑knownSites KNOWNSITES No Path to a known indels file. The file must be in vcf.gz format. This option can be used multiple times.
I/O ‑‑interval‑file INTERVAL_FILE No Path to an interval file in one of these formats: Picard-style (.interval_list or .picard), GATK-style (.list or .intervals), or BED file (.bed). This option can be used multiple times.
I/O ‑‑pb‑model‑file PB_MODEL_FILE No Path to a non-default parabricks model file for deepvariant.
I/O ‑‑out‑recal‑file OUT_RECAL_FILE No Path of the report file after Base Quality Score Recalibration.
I/O ‑‑out‑bam OUT_BAM Yes Path of BAM file after marking duplicates.
I/O ‑‑out‑variants OUT_VARIANTS Yes Path of the vcf/vcf.gz/gvcf/gvcf.gz file after variant calling.
I/O ‑‑out‑duplicate‑metrics OUT_DUPLICATE_METRICS No Path of a duplicate metrics file after marking duplicates.
I/O ‑‑proposed‑variants PROPOSED_VARIANTS No Path of the VCF file, which has proposed variants for the make examples stage.
Tool ‑k MINIMIZER_KMER_LEN, ‑‑minimizer‑kmer‑len MINIMIZER_KMER_LEN No Minimizer k-mer length.
Tool ‑uf, ‑‑forward‑transcript‑strand No Force minimap2 to consider the forward transcript strand only when finding canonical splicing sites GT-AG.
Tool ‑‑eqx No Write =/X CIGAR operators.
Tool ‑L INTERVAL, ‑‑interval INTERVAL No Interval within which to call bqsr from the input reads. All intervals will have a padding of 100 to get read records, and overlapping intervals will be combined. Interval files should be passed using the --interval-file option. This option can be used multiple times (e.g. "-L chr1 -L chr2:10000 -L chr3:20000+ -L chr4:10000-20000").
Tool ‑ip INTERVAL_PADDING, ‑‑interval‑padding INTERVAL_PADDING No Amount of padding (in base pairs) to add to each interval you are including.
Tool ‑‑standalone‑bqsr No Run standalone BQSR after generating sorted BAM. This option requires both --knownSites and --out-recal-file input parameters.
Tool ‑‑read‑group‑sm READ_GROUP_SM No SM tag for read groups in this run.
Tool ‑‑read‑group‑lb READ_GROUP_LB No LB tag for read groups in this run.
Tool ‑‑read‑group‑pl READ_GROUP_PL No PL tag for read groups in this run.
Tool ‑‑read‑group‑id‑prefix READ_GROUP_ID_PREFIX No Prefix for the ID and PU tags for read groups in this run. This prefix will be used for all pairs of FASTQ files in this run. The ID and PU tags will consist of this prefix and an identifier, that will be unique for a pair of FASTQ files.
Tool ‑‑disable‑use‑window‑selector‑model No Change the window selector model from Allele Count Linear to Variant Reads. This option will increase the accuracy and runtime.
Tool ‑‑gvcf No Generate variant calls in .gvcf format.
Tool ‑‑norealign‑reads No Do not locally realign reads before calling variants. Reads longer than 500 bp are never realigned.
Tool ‑‑sort‑by‑haplotypes No Reads are sorted by haplotypes (using HP tag).
Tool ‑‑keep‑duplicates No Keep reads that are duplicate.
Tool ‑‑keep‑legacy‑allele‑counter‑behavior No If specified, the behavior in this commit is reverted: 'https://github.com/google/deepvariant/commit/fbde0674639a28cb9e8004c7a01bbe25240c7d46'. We do not recommend setting this flag to True.
Tool ‑‑vsc‑min‑count‑snps VSC_MIN_COUNT_SNPS No SNP alleles occurring at least this many times in the AlleleCount will be advanced as candidates. (default: 2)
Tool ‑‑vsc‑min‑count‑indels VSC_MIN_COUNT_INDELS No Indel alleles occurring at least this many times in the AlleleCount will be advanced as candidates. (default: 2)
Tool ‑‑vsc‑min‑fraction‑snps VSC_MIN_FRACTION_SNPS No SNP alleles occurring at least this fraction of all counts in the AlleleCount will be advanced as candidates. (default: 0.12)
Tool ‑‑vsc‑min‑fraction‑indels VSC_MIN_FRACTION_INDELS No Indel alleles occurring at least this fraction of all counts in the AlleleCount will be advanced as candidates.
Tool ‑‑min‑mapping‑quality MIN_MAPPING_QUALITY No By default, reads with any mapping quality are kept. Setting this field to a positive integer i will only keep reads that have a MAPQ >= i. Note this only applies to aligned reads. (default: 5)
Tool ‑‑min‑base‑quality MIN_BASE_QUALITY No Minimum base quality. This option enforces a minimum base quality score for alternate alleles. Alternate alleles will only be considered if all bases in the allele have a quality greater than min_base_quality. (default: 10)
Tool ‑‑alt‑aligned‑pileup ALT_ALIGNED_PILEUP No Value can be one of [none, diff_channels]. Include alignments of reads against each candidate alternate allele in the pileup image.
Tool ‑‑variant‑caller VARIANT_CALLER No Value can be one of [VERY_SENSITIVE_CALLER, VCF_CANDIDATE_IMPORTER]. The caller to use to make examples. If you use VCF_CANDIDATE_IMPORTER, it implies force calling. Default is VERY_SENSITIVE_CALLER.
Tool ‑‑add‑hp‑channel No Add another channel to represent HP tags per read.
Tool ‑‑parse‑sam‑aux‑fields No Auxiliary fields of the BAM/CRAM records are parsed. If either --sort-by-haplotypes or --add-hp-channel is set, then this option must also be set.
Tool ‑‑use‑wes‑model No If specified, the WES model file will be used. Only used in shortread mode.
Tool ‑‑include‑med‑dp No If specified, include MED_DP in the output gVCF records.
Tool ‑‑normalize‑reads No If specified, allele counter left align INDELs for each read.
Tool ‑‑pileup‑image‑width PILEUP_IMAGE_WIDTH No Pileup image width. Only change this if you know your model supports this width. (default: 221)
Tool ‑‑channel‑insert‑size No If specified, add insert_size channel into the pileup image. By default, this parameter is true in WGS and WES mode.
Tool ‑‑no‑channel‑insert‑size No If specified, don't add insert_size channel into the pileup image.
Tool ‑‑max‑read‑size‑512 No Allow deepvariant to run on reads of size 512bp. The default size is 320 bp.
Tool ‑‑prealign‑helper‑thread No Use an extra thread for the pre-align step. This parameter is more useful when --max-reads-size-512 is set.
Tool ‑‑track‑ref‑reads No If specified, allele counter keeps track of reads supporting ref. By default, allele counter keeps a simple count of the number of reads supporting ref.
Tool ‑‑phase‑reads No Calculate phases and add HP tag to all reads automatically.
Tool ‑‑dbg‑min‑base‑quality DBG_MIN_BASE_QUALITY No Minimum base quality in a k-mer sequence to consider. (default: 15)
Tool ‑‑ws‑min‑windows‑distance WS_MIN_WINDOWS_DISTANCE No Minimum distance between candidate windows for local assembly. (default: 80)
Tool ‑‑channel‑gc‑content No If specified, add gc_content channel into the pileup image.
Tool ‑‑channel‑hmer‑deletion‑quality No If specified, add hmer deletion quality channel into the pileup image.
Tool ‑‑channel‑hmer‑insertion‑quality No If specified, add hmer insertion quality channel into the pileup image.
Tool ‑‑channel‑non‑hmer‑insertion‑quality No If specified, add non-hmer insertion quality channel into the pileup image.
Tool ‑‑skip‑bq‑channel No If specified, ignore base quality channel.
Tool ‑‑aux‑fields‑to‑keep AUX_FIELDS_TO_KEEP No Comma-delimited list of auxiliary BAM fields to keep. Values can be [HP, tp, t0]. (default: HP)
Tool ‑‑vsc‑min‑fraction‑hmer‑indels VSC_MIN_FRACTION_HMER_INDELS No Hmer Indel alleles occurring at least this be advanced as candidates. Use this threshold if hmer and non-hmer indels should be treated differently (Ultima reads)Default will use the same threshold for hmer and non-hmer indels, as defined in vsc_min_fraction_indels.
Tool ‑‑vsc‑turn‑on‑non‑hmer‑ins‑proxy‑support No Add read-support from soft-clipped reads and other non-hmer insertion alleles,to the most frequent non-hmer insertion allele.
Tool ‑‑consider‑strand‑bias No If specified, expect SB field in calls and write it to the VCF file.
Tool ‑‑p‑error P_ERROR No Basecalling error for reference confidence model. (default: 0.001)
Tool ‑‑channel‑ins‑size No If specified, add another channel to represent size of insertions (good for flow-based sequencing).
Tool ‑‑max‑ins‑size MAX_INS_SIZE No Max insertion size for ins_size_channel, larger insertions will look like max (have max intensity). (default: 10)
Tool ‑‑disable‑group‑variants No If using vcf_candidate_importer and multi-allelic sites are split across multiple lines in VCF, add this flag so that variants are not grouped when transforming CallVariantsOutput to Variants.
Tool ‑‑filter‑reads‑too‑long No Ignore all input BAM reads with size > 512bp.
Tool ‑‑haploid‑contigs HAPLOID_CONTIGS No Optional list of non autosomal chromosomes. For all listed chromosomes HET probabilities are not considered.
Performance ‑‑num‑threads NUM_THREADS No Number of processing threads. (default: 28)
Performance ‑‑nstreams NSTREAMS No Number of streams to use per GPU. (default: 4)
Performance ‑‑gpuwrite No Use one GPU to accelerate writing final BAM/CRAM.
Performance ‑‑gpuwrite‑deflate‑algo GPUWRITE_DEFLATE_ALGO No Choose the nvCOMP DEFLATE algorithm to use with --gpuwrite. Note these options do not correspond to CPU DEFLATE options. Valid options are 1, 2, and 4. Option 1 is fastest, while options 2 and 4 have progressively lower throughput but higher compression ratios. The default value is 1 when the user does not provide an input (i.e., None).
Performance ‑‑gpusort No Use GPUs to accelerate sorting.
Performance ‑‑use‑gds No Use GPUDirect Storage (GDS) to enable a direct data path for direct memory access (DMA) transfers between GPU memory and storage. Must be used concurrently with --gpuwrite. Please refer to Parabricks Documentation > Best Performance for information on how to set up and use GPUDirect Storage.
Performance ‑‑max‑queue‑reads MAX_QUEUE_READS No Max number of reads to allow in the alignment processing stage. Increasing this value may result in faster processing, but it will use more host memory. (default: 500000)
Performance ‑‑low‑memory No Use low memory mode.
Performance ‑‑num‑cpu‑threads‑per‑stream NUM_CPU_THREADS_PER_STREAM No Number of CPU threads to use per stream. (default: 6)
Performance ‑‑num‑streams‑per‑gpu NUM_STREAMS_PER_GPU No Number of streams to use per GPU. (default: 2)
Performance ‑‑run‑partition No Divide the whole genome into multiple partitions and run multiple processes at the same time, each on one partition.
Performance ‑‑gpu‑num‑per‑partition GPU_NUM_PER_PARTITION No Number of GPUs to use per partition.
Performance ‑‑max‑reads‑per‑partition MAX_READS_PER_PARTITION No The maximum number of reads per partition that are considered before following processing such as sampling and realignment. (default: 1500)
Performance ‑‑partition‑size PARTITION_SIZE No The maximum number of basepairs allowed in a region before splitting it into multiple smaller subregions. (default: 1000)
Performance ‑‑use‑tf32 No Enable inference optimization using Tensor Float 32(TF32) on ampere+ gpu. Note that this might introduce a few mismatches in the output VCF.
Performance ‑‑read‑from‑tmp‑dir No Running variant caller reading from bin files generated by Aligner and sort. Run postsort in parallel. This option will increase device memory usage.
Runtime ‑‑verbose No Enable verbose output.
Runtime ‑‑x3 No Show full command line arguments.
Runtime ‑‑logfile LOGFILE No Path to the log file. If not specified, messages will only be written to the standard error output.
Runtime ‑‑tmp‑dir TMP_DIR No Full path to the directory where temporary files will be stored. (default: .)
Runtime ‑‑with‑petagene‑dir WITH_PETAGENE_DIR No Full path to the PetaGene installation directory. By default, this should have been installed at /opt/petagene. Use of this option also requires that the PetaLink library has been preloaded by setting the LD_PRELOAD environment variable. Optionally set the PETASUITE_REFPATH and PGCLOUD_CREDPATH environment variables that are used for data and credentials. Optionally set the PetaLinkMode environment variable that is used to further configure PetaLink, notably setting it to "+write" to enable outputting compressed BAM and .fastq files.
Runtime ‑‑keep‑tmp No Do not delete the directory storing temporary files after completion.
Runtime ‑‑no‑seccomp‑override No Do not override seccomp options for docker.
Runtime ‑‑version No View compatible software versions.
Runtime ‑‑num‑gpus NUM_GPUS No Number of GPUs to use for a run. (default: 1)
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