human_par

Given one or more pairs of FASTQ files, you can run the human_par pipeline workflow to generate output, including BAM, recal, and variants called with proper pseudoautosomal region ploidy values.

The human_par pipeline shown below resembles the GATK4 best practices pipeline. The inputs are BWA-indexed reference files, pair-ended FASTQ files, knownSites for BQSR calculation, and specifications to determine which sex to run variant calling. Male samples run Haplotype Caller first on non-X/Y regions and the pseudoautosomal region with ploidy 1, then on X/Y regions without the pseudoautosomal region with ploidy 2. Female samples run Haplotype Caller on all regions with ploidy 2. 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

Three options are used to determine the sex of the input sample. The --sample-sex manually sets the sex as male or female, overriding the sex detected from the number of X and Y reads. Additionally, two X/Y ratio range options can be used to automatically detect the sex of the sample based on the number of X and Y reads. Both --range-male and --range-female options provide a range for the possible value of the X/Y ratio. If the X/Y ratio falls in any of the given ranges, that sex will be used for Haplotype Caller; however, if the X/Y ratio is not in any range, the pipeline relies on the --sample-sex option to continue. At least one of these three options must be provided.

Quick Start

Run a human_par pipeline:

$ pbrun human_par \
    --ref Ref/Homo_sapiens_assembly38.fasta \
    --in-fq Data/sample_1.fq.gz Data/sample_2.fq.gz  \
    --knownSites Ref/Homo_sapiens_assembly38.known_indels.vcf.gz \
    --range-male 1-10 \
    --range-female 150-250 \
    --sample-sex male \
    --out-bam output.bam \
    --out-variants output.vcf \
    --out-recal-file report.txt

Compatible CPU-based BWA-MEM, GATK4 Commands

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.

# 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' \
Ref/Homo_sapiens_assembly38.fasta S1_1.fastq.gz S1_2.fastq.gz | gatk \
SortSam --java-options -Xmx30g --MAX_RECORDS_IN_RAM=5000000 -I=/dev/stdin \
-O=cpu.bam --SORT_ORDER=coordinate --TMP_DIR=/raid/myrun

# Mark Duplicates
$ gatk MarkDuplicates --java-options -Xmx30g -I=cpu.bam -O=mark_dups_cpu.bam \
-M=metrics.txt --TMP_DIR=/raid/myrun

# Generate BQSR Report
$ gatk BaseRecalibrator --java-options -Xmx30g --input mark_dups_cpu.bam --output \
recal_cpu.txt --known-sites Ref/Homo_sapiens_assembly38.known_indels.vcf.gz \
--reference Ref/Homo_sapiens_assembly38.fasta

# Run ApplyBQSR Step
$ gatk ApplyBQSR --java-options -Xmx30g -R Ref/Homo_sapiens_assembly38.fasta \
-I=mark_dups_cpu.bam --bqsr-recal-file=recal_file.txt -O=cpu_nodups_BQSR.bam

#Run Haplotype Caller on the non-X/Y regions and the pseudoautosomal region
$ gatk HaplotypeCaller --java-options -Xmx30g --input cpu_nodups_BQSR.bam --output \
result_cpu_non_xy.vcf --reference Ref/Homo_sapiens_assembly38.fasta \
-L non_xy_regions_with_par.list --native-pair-hmm-threads 16

#Run Haplotype Caller on the X/Y regions without the pseudoautosomal region
$ gatk HaplotypeCaller --java-options -Xmx30g --input cpu_nodups_BQSR.bam --output \
result_cpu_xy.vcf --reference Ref/Homo_sapiens_assembly38.fasta \
-L xy_regions_without_par.list --native-pair-hmm-threads 16 \
(--ploidy 1 for male samples)

#Merge the variants from both Haplotype Caller runs
$ gatk MergeVcfs -I result_cpu_non_xy.vcf -I result_cpu_xy.vcf -O result_cpu.vcf

human_par Reference

Run the germline pipeline from FASTQ to VCF with correct ploidy values for human sex chromosome handling.

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 group with quotes (Example: "@RGtID:footLB:lib1tPL:bartSM:sampletPU:foo"). Files must be in fastq/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 ID:footLB:lib1tPL:bartSM:sampletPU:unit1" --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz "@RG ID:foo2tLB:lib1tPL:bartSM:sampletPU:unit2" . For same sample, Read Groups should have same sample name (SM) and different ID and PU. (default: None)

Option is required.

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

Path to the single ended FASTQ file followed by optional read group with quotes (Example: "@RGtID:footLB:lib1tPL:bartSM:sampletPU:foo"). The file must be in fastq/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-se-fq sampleX_1.fastq.gz --in-se-fq sampleX_2.fastq.gz . Example 2: --in-se-fq sampleX_1.fastq.gz "@RGtID:footLB:lib1tPL:bartSM:sampletPU:unit1" --in-se-fq sampleX_2.fastq.gz "@RGtID:foo2tLB:lib1tPL:bartSM:sampletPU:unit2" . For same sample, Read Groups should have same sample name (SM) and different ID and PU. (default: None)

--knownSites KNOWNSITES

Path to a known indels file. Must be in vcf/vcf.gz format. 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.

--out-variants OUT_VARIANTS

Path of a 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)

Options specific to this tool

-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. Current original bwa mem supported options, -M, -Y, -T. e.g. --bwa-options="-M -Y" (default: None)

--no-warnings

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

--no-markdups

Do not perform 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 ID and PU tag for read groups in this run. This prefix will be used for all pair of fastq files in this run. The ID and PU tag will consist of this prefix and an identifier which 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 after generating sorted bam. This option requires both --knownSites and --out-recal-file input parameters. (default: None)

--haplotypecaller-options HAPLOTYPECALLER_OPTIONS

Pass supported haplotype caller options as one string. Currently supported original haplotypecaller options: -min-pruning <int>, -standard-min-confidence-threshold-for-calling <int>, -max-reads-per-alignment-start <int>, -min-dangling-branch-length <int>, -pcr-indel-model <NONE, HOSTILE, AGGRESSIVE, CONSERVATIVE>, --output-mode <EMIT_VARIANTS_ONLY, EMIT_ALL_CONFIDENT_SITES, EMIT_ALL_ACTIVE_SITES>. 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)

--batch

Given an input list of BAMs, run the variant calling of each BAM using one GPU, and process BAMs in parallel based on how many GPUs the system has. (default: None)

--disable-read-filter DISABLE_READ_FILTER

Disable the read filters for bam entries. Currently supported read filters that can be disabled: MappingQualityAvailableReadFilter, MappingQualityReadFilter, NotSecondaryAlignmentReadFilter, 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

Which groups of annotations to add to the output variant calls. Currently supported annotation groups: StandardAnnotation, StandardHCAnnotation, 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)

--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)

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.

--license-file LICENSE_FILE

Path to license file license.bin if not in the installation directory.

--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.

--gpu-devices GPU_DEVICES

GPU devices to use for a run. By default, all GPU devices will be used. To use specific GPU devices, enter a comma-separated list of GPU device numbers. Possible device numbers can be found by examining the output of the nvidia-smi command. For example, using --gpu-devices 0,1 would only use the first two GPUs.

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.