germline (GATK Germline Pipeline)

NVIDIA Parabricks 4.2.1

GATK, the Genome Analysis Toolkit, is an industry standard software package developed by the Broad Institute of MIT and Harvard and designed to be used for a wide range of genomic analyses, including variant discovery, genotyping, and more. GATK is one of the most popular tools used in bioinformatics for analyzing next-generation sequencing datasets and is an industry standard for calling single nucleotide variants (SNVs) and insertions/deletions (InDels) from sequencing data in germline samples.

GATK offers robust, accurate analysis of sequencing data and is frequently updated to include the latest best practices for variant discovery. With high reliability and the ability to be used for a number of use cases, GATK is a gold standard tool for any researcher working with next-generation sequencing data.

The GATK germline workflow for variant calling can be deployed within NVIDIA’s Parabricks software suite, which is designed for accelerated secondary analysis in genomics, bringing industry standard tools and workflows from CPU to GPU and delivering the same results at up to 60x faster runtimes. A 30x whole genome can be analyzed in under 25 minutes on an NVIDIA DGX system, compared to over 30 hours on a CPU instance (m5.24xlarge, 96 x vCPU), and exomes can be analyzed in just 4 minutes. This means Parabricks, running on one NVIDIA DGX A100, can analyze up to 25,000 whole genomes per year. The NVIDIA team collaborated with the GATK team at the Broad Institute to evaluate the accuracy of germline workflows. Through this rigorous process, they verified that the Parabricks workflows produce results that are functionally equivalent to the CPU-native GATK versions.

As a specific example, benchmarking on publicly available Genome in a Bottle (GIAB) samples with the fq2bam and germline caller workflows from the Parabricks suite produced variant calling results that were >0.9999 equivalent in both precision and recall to those produced by the BWA, MarkDuplicates, BQSR, and HaplotypeCaller commands in the GATK’s Whole Genome Germline Single Sample variant calling workflow.

Given one or more pairs of FASTQ files, you can run the germline variant tool to generate BAM, variants, duplicate metrics and recal.

The germline pipeline shown below resembles the GATK4 best practices pipeline. The inputs are BWA-indexed reference files, pair-ended FASTQ files, and knownSites for BQSR calculation. The outputs of this pipeline are as follows:

  • Aligned, co-ordinate sorted, duplicated marked BAM

  • BQSR report

  • Variants in vcf/g.vcf/g.vcf.gz format

germline.png

Running the germline pipeline:

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# This command assumes all the inputs are in INPUT_DIR and all the outputs go to OUTPUT_DIR. docker run --rm --gpus all --volume INPUT_DIR:/workdir --volume OUTPUT_DIR:/outputdir \ --workdir /workdir \ nvcr.io/nvidia/clara/clara-parabricks:4.2.1-1 \ pbrun germline \ --ref /workdir/${REFERENCE_FILE} \ --in-fq /workdir/${INPUT_FASTQ_1} /workdir/${INPUT_FASTQ_2} \ --knownSites /workdir/${KNOWN_SITES_FILE} \ --out-bam /outputdir/${OUTPUT_BAM} \ --out-variants /outputdir/${OUTPUT_VCF} \ --out-recal-file /outputdir/${OUT_RECAL_FILE}

Several original HaplotypeCaller options are supported by Parabricks. To specify the inclusion or exclusion of several haplotype caller annotations, use the --haplotypecaller-options option:

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$ # This command assumes all the inputs are in INPUT_DIR and all the outputs go to OUTPUT_DIR. docker run --rm --gpus all --volume INPUT_DIR:/workdir --volume OUTPUT_DIR:/outputdir \ --workdir /workdir \ nvcr.io/nvidia/clara/clara-parabricks:4.2.1-1 \ pbrun haplotypecaller \ ... --haplotypecaller-options '-min-pruning 4 -A AS_BaseQualityRankSumTest -A TandemRepeat' ...

Annotations may be excluded in the same manner using the -AX option. There should be a space between the -A/-AX flag and its value.

The following are supported options and their allowed values:

  • -A
    • AS_BaseQualityRankSumTest

    • AS_FisherStrand

    • AS_InbreedingCoeff

    • AS_MappingQualityRankSumTest

    • AS_QualByDepth

    • AS_RMSMappingQuality

    • AS_ReadPosRankSumTest

    • AS_StrandOddsRatio

    • BaseQualityRankSumTest

    • ChromosomeCounts

    • ClippingRankSumTest

    • Coverage

    • DepthPerAlleleBySample

    • DepthPerSampleHC

    • ExcessHet

    • FisherStrand

    • InbreedingCoeff

    • MappingQualityRankSumTest

    • QualByDepth

    • RMSMappingQuality

    • ReadPosRankSumTest

    • ReferenceBases

    • StrandBiasBySample

    • StrandOddsRatio

    • TandemRepeat

  • -AX
    • (same as for the -A option)

  • --output-mode
    • EMIT_VARIANTS_ONLY

    • EMIT_ALL_CONFIDENT_SITES

    • EMIT_ALL_ACTIVE_SITES

  • -max-reads-per-alignment-start
    • a positive integer

  • -min-dangling-branch-length
    • a positive integer

  • -min-pruning
    • a positive integer

  • -pcr-indel-model
    • NONE

    • HOSTILE

    • AGGRESSIVE

    • CONSERVATIVE

  • -standard-min-confidence-threshold-for-calling
    • a positive integer

The commands below are the bwa-0.7.12 and GATK4 counterpart of the 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 bwa-mem and pipe output to create sorted BAM $ bwa mem \ -t 32 \ -K 10000000 \ -R '@RG\tID:sample_rg1\tLB:lib1\tPL:bar\tSM:sample\tPU:sample_rg1' \ <INPUT_DIR>/${REFERENCE_FILE} <INPUT_DIR>/${INPUT_FASTQ_1} <INPUT_DIR>/${INPUT_FASTQ_2} | \ gatk SortSam \ --java-options -Xmx30g \ --MAX_RECORDS_IN_RAM 5000000 \ -I /dev/stdin \ -O cpu.bam \ --SORT_ORDER coordinate # Mark Duplicates $ gatk MarkDuplicates \ --java-options -Xmx30g \ -I cpu.bam \ -O mark_dups_cpu.bam \ -M metrics.txt # Generate BQSR Report $ gatk BaseRecalibrator \ --java-options -Xmx30g \ --input mark_dups_cpu.bam \ --output <OUTPUT_DIR>/${OUT_RECAL_FILE} \ --known-sites <INPUT_DIR>/${KNOWN_SITES_FILE} \ --reference <INPUT_DIR>/${REFERENCE_FILE} # Run ApplyBQSR Step $ gatk ApplyBQSR \ --java-options -Xmx30g \ -R <INPUT_DIR>/${REFERENCE_FILE} \ -I mark_dups_cpu.bam \ --bqsr-recal-file <OUTPUT_DIR>/${OUT_RECAL_FILE} \ -O cpu_nodups_BQSR.bam #Run Haplotype Caller $ gatk HaplotypeCaller \ --java-options -Xmx30g \ --input cpu_nodups_BQSR.bam \ --output <OUTPUT_DIR>/${OUTPUT_VCF} \ --reference <INPUT_DIR>/${REFERENCE_FILE} \ --native-pair-hmm-threads 16

Run Germline pipeline to convert FASTQ to VCF.

Input/Output file options

--ref REF

Path to the reference file. (default: None)

Option is required.

--in-fq [IN_FQ [IN_FQ ...]]

Path to the pair-ended FASTQ files followed by optional read groups with quotes (Example: "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:foo"). The files must be in fastq or fastq.gz format. All sets of inputs should have a read group; otherwise, none should have a read group, and it will be automatically added by the pipeline. This option can be repeated multiple times. Example 1: --in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz. Example 2: --in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1" --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz "@RG\tID:foo2\tLB:lib1\tPL:bar\tSM:sample\tPU:unit2". For the same sample, Read Groups should have the same sample name (SM) and a different ID and PU. (default: None)

--in-se-fq [IN_SE_FQ [IN_SE_FQ ...]]

Path to the single-ended FASTQ file followed by optional read group with quotes (Example: "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:foo"). The file must be in fastq or fastq.gz format. Either all sets of inputs have a read group, or none should have one, and it will be automatically added by the pipeline. This option can be repeated multiple times. Example 1: --in-se-fq sampleX_1.fastq.gz --in-se-fq sampleX_2.fastq.gz . Example 2: --in-se-fq sampleX_1.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1" --in-se-fq sampleX_2.fastq.gz "@RG\tID:foo2\tLB:lib1\tPL:bar\tSM:sample\tPU:unit2" . For the same sample, Read Groups should have the same sample name (SM) and a different ID and PU. (default: None)

--knownSites KNOWNSITES

Path to a known indels file. The file must be in vcf.gz format. This option can be used multiple times. (default: None)

--interval-file INTERVAL_FILE

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. (default: None)

--out-recal-file OUT_RECAL_FILE

Path of the report file after Base Quality Score Recalibration. (default: None)

--out-bam OUT_BAM

Path of BAM file after Marking Duplicates. (default: None)

Option is required.

--htvc-bam-output HTVC_BAM_OUTPUT

File to which assembled haplotypes should be written in HaplotypeCaller. (default: None)

--out-variants OUT_VARIANTS

Path of the vcf/gvcf/gvcf.gz file after variant calling. (default: None)

Option is required.

--out-duplicate-metrics OUT_DUPLICATE_METRICS

Path of duplicate metrics file after Marking Duplicates. (default: None)

Tool Options:

-L INTERVAL, --interval INTERVAL

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". (default: None)

--bwa-options BWA_OPTIONS

Pass supported bwa mem options as one string. The current original bwa mem supported options are -M, -Y and -T e.g. --bwa-options="-M -Y" (default: None)

--no-warnings

Suppress warning messages about system thread and memory usage. (default: None)

--filter-flag FILTER_FLAG

Don't generate SAM entries in the output if the entry's flag's meet this criteria. Criteria: (flag & filter != 0) (default: 0)

--skip-multiple-hits

Filter SAM entries whose length of SA is not 0. (default: None)

--min-read-length MIN_READ_LENGTH

Skip reads below minimum read length. They will not be part of the output. (default: None)

--align-only

Generate output BAM after bwa-mem. The output will not be co-ordinate sorted or duplicates will not be marked. (default: None)

--no-markdups

Do not perform the Mark Duplicates step. Return BAM after sorting. (default: None)

--fix-mate

Add mate cigar (MC) and mate quality (MQ) tags to the output file. (default: None)

--markdups-assume-sortorder-queryname

Assume the reads are sorted by queryname for Marking Duplicates. This will mark secondary, supplementary, and unmapped reads as duplicates as well. This flag will not impact variant calling while increasing processing times. (default: None)

--markdups-picard-version-2182

Assume marking duplicates to be similar to Picard version 2.18.2. (default: None)

--optical-duplicate-pixel-distance OPTICAL_DUPLICATE_PIXEL_DISTANCE

The maximum offset between two duplicate clusters in order to consider them optical duplicates. Ignored if --out-duplicate-metrics is not passed. (default: None)

--read-group-sm READ_GROUP_SM

SM tag for read groups in this run. (default: None)

--read-group-lb READ_GROUP_LB

LB tag for read groups in this run. (default: None)

--read-group-pl READ_GROUP_PL

PL tag for read groups in this run. (default: None)

--read-group-id-prefix READ_GROUP_ID_PREFIX

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. (default: None)

-ip INTERVAL_PADDING, --interval-padding INTERVAL_PADDING

Amount of padding (in base pairs) to add to each interval you are including. (default: None)

--standalone-bqsr

Run standalone BQSR. (default: None)

--max-read-length-fq2bamfast MAX_READ_LENGTH_FQ2BAMFAST

Maximum read length/size (i.e., sequence length) used for bwa and filtering FASTQ input (Argument only applies to --fq2bamfast) (default: 480)

--min-read-length-fq2bamfast MIN_READ_LENGTH_FQ2BAMFAST

Minimum read length/size (i.e., sequence length) used for bwa and filtering FASTQ input (Argument only applies to --fq2bamfast) (default: 10)

--haplotypecaller-options HAPLOTYPECALLER_OPTIONS

Pass supported haplotype caller options as one string. The following are currently supported original haplotypecaller options: -A <AS_BaseQualityRankSumTest, AS_FisherStrand, AS_InbreedingCoeff, AS_MappingQualityRankSumTest, AS_QualByDepth, AS_RMSMappingQuality, AS_ReadPosRankSumTest, AS_StrandOddsRatio, BaseQualityRankSumTest, ChromosomeCounts, ClippingRankSumTest, Coverage, DepthPerAlleleBySample, DepthPerSampleHC, ExcessHet, FisherStrand, InbreedingCoeff, MappingQualityRankSumTest, QualByDepth, RMSMappingQuality, ReadPosRankSumTest, ReferenceBases, StrandBiasBySample, StrandOddsRatio, TandemRepeat>,-AX <same options as -A>,--output-mode <EMIT_VARIANTS_ONLY, EMIT_ALL_CONFIDENT_SITES, EMIT_ALL_ACTIVE_SITES> ,-max-reads-per-alignment-start <int>, -min-dangling-branch-length <int>, -min-pruning <int>, -pcr-indel-model <NONE, HOSTILE, AGGRESSIVE, CONSERVATIVE>, -standard-min-confidence-threshold-for-calling <int>(e.g. --haplotypecaller-options="-min-pruning 4 -standard-min-confidence-threshold-for-calling 30"). (default: None)

--static-quantized-quals STATIC_QUANTIZED_QUALS

Use static quantized quality scores to a given number of levels. Repeat this option multiple times for multiple bins. (default: None)

--gvcf

Generate variant calls in .gvcf Format. (default: None)

--disable-read-filter DISABLE_READ_FILTER

Disable the read filters for BAM entries. Currently, the supported read filters that can be disabled are MappingQualityAvailableReadFilter, MappingQualityReadFilter, NotSecondaryAlignmentReadFilter, and WellformedReadFilter. (default: None)

--max-alternate-alleles MAX_ALTERNATE_ALLELES

Maximum number of alternate alleles to genotype. (default: None)

-G ANNOTATION_GROUP, --annotation-group ANNOTATION_GROUP

The groups of annotations to add to the output variant calls. Currently supported annotation groups are StandardAnnotation, StandardHCAnnotation, and AS_StandardAnnotation. (default: None)

-GQB GVCF_GQ_BANDS, --gvcf-gq-bands GVCF_GQ_BANDS

Exclusive upper bounds for reference confidence GQ bands. Must be in the range [1, 100] and specified in increasing order. (default: None)

--rna

Run haplotypecaller optimized for RNA data. (default: None)

--dont-use-soft-clipped-bases

Don't use soft clipped bases for variant calling. (default: None)

--no-alt-contigs

Get rid of output records for alternate contigs. (default: None)

--ploidy PLOIDY

Ploidy assumed for the BAM file. Currently only haploid (ploidy 1) and diploid (ploidy 2) are supported. (default: 2)

--sample-sex SAMPLE_SEX

Sex of the sample input. This option will override the sex determined from any X/Y read ratio range. Must be either male or female. (default: None)

--range-male RANGE_MALE

Inclusive male range for the X/Y read ratio. The sex is declared male if the actual ratio falls in the specified range. Syntax is "<min>-<max>" (e.g. "--range-male 1-10"). (default: None)

--range-female RANGE_FEMALE

Inclusive female range for the X/Y read ratio. The sex is declared female if the actual ratio falls in the specified range. Syntax is "<min>-<max>" (e.g. "--range-female 150-250"). (default: None)

--use-GRCh37-regions

Use the pseudoautosomal regions for GRCh37 reference types. This flag should be used for GRCh37 and UCSC hg19 references. By default, GRCh38 regions are used.

(default: None)

Performance Options:

--fq2bamfast

Use fq2bamfast as the alignment tool instead of fq2bam (default: None)

--gpuwrite

Use one GPU to accelerate writing final BAM. (default: None)

--gpuwrite-deflate-algo GPUWRITE_DEFLATE_ALGO

Choose the nvCOMP DEFLATE algorithm to use with --gpuwrite. Note these options do not correspond to CPU DEFLATE options. Valid options are 0 and 3. Option 0 is faster while option 3 provides a better compression ratio. (default=0) (default: None)

--gpusort

Use GPUs to accelerate sorting and marking. (default: None)

--use-gds

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. (default: None)

--memory-limit MEMORY_LIMIT

System memory limit in GBs during sorting and postsorting. By default, the limit is half of the total system memory. (default: 62)

--low-memory

Use low memory mode (default: None)

--num-cpu-threads-per-stage NUM_CPU_THREADS_PER_STAGE

Number of CPU threads to use per stage. (default: 8)

--bwa-nstreams BWA_NSTREAMS

Number of streams per GPU to use; note: more streams increases device memory usage (Argument only applies to --fq2bamfast) (default: 4)

--bwa-cpu-thread-pool BWA_CPU_THREAD_POOL

Number of threads to devote to CPU thread pool per GPU (Argument only applies to --fq2bamfast) (default: 16)

--htvc-low-memory

Use low memory mode in htvc. (default: None)

--num-htvc-threads NUM_HTVC_THREADS

Number of CPU threads. (default: 5)

--run-partition

Divide the whole genome into multiple partitions and run multiple processes at the same time, each on one partition. (default: None)

--gpu-num-per-partition GPU_NUM_PER_PARTITION

Number of GPUs to use per partition. (default: None)

--read-from-tmp-dir

Running variant caller reading from bin files generated by Aligner and sort. Run postsort in parallel. This option will increase device memory usage. (default: None)

Common options:

--logfile LOGFILE

Path to the log file. If not specified, messages will only be written to the standard error output. (default: None)

--tmp-dir TMP_DIR

Full path to the directory where temporary files will be stored.

--with-petagene-dir WITH_PETAGENE_DIR

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 (default: None)

--keep-tmp

Do not delete the directory storing temporary files after completion.

--no-seccomp-override

Do not override seccomp options for docker (default: None).

--version

View compatible software versions.

GPU options:

--num-gpus NUM_GPUS

Number of GPUs to use for a run. GPUs 0..(NUM_GPUS-1) will be used.

Note

The --in-fq option takes the names of two FASTQ files, optionally followed by a quoted read group. The FASTQ filenames must not start with a hyphen.

Note

In the values provided to --haplotypecaller-options --output-mode requires two leading hyphens, while all other values take a single hyphen.


© Copyright 2023, Nvidia. Last updated on Dec 13, 2023.