rna_gatk

Run GATK best practices for RNAseq short variant discovery (SNPs + Indels).

The RNA GATK pipeline processes the input FASTQ files, performing the steps show below. The output is in VCF format.

../../_images/rna_gatk.png

Quick Start

# The command line below will run the RNA GATK pipeline.
$ pbrun rna_gatk \
    --ref Ref/Homo_sapiens_assembly38.fasta \
    --in-fq Data/sample_1.fq.gz Data/sample_2.fq.gz \
    --read-files-command  zcat \
    --genome-lib-dir Ref/ \
    --out-variants output.vcf \
    --out-bam tumor.bam \
    --output-dir output

rna_gatk Reference

Run the GATK Best Practices pipeline for RNA-seq data from 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 group with quotes (Example: "@RGtID:footLB:lib1tPL:bartSM:sampletPU:foo"). Files must be in fastq or fastq.gz format. If no read group is provided, one will be automatically added by the pipeline. Example 1: --in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz . Example 2: --in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz "@RGtID:footLB:lib1tPL:bartSM:sampletPU:unit1" (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: "@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)

--genome-lib-dir GENOME_LIB_DIR

Path to a genome resource library directory. We assume that the indexing required to run star has been completed by the user. (default: None)

Option is required.

--knownSites KNOWNSITES

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

--interval-file INTERVAL_FILE

Path to an interval file with possible 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)

--output-dir OUTPUT_DIR

Path to the directory that will contain all of the generated files. (default: None)

Option is required.

--out-recal-file OUT_RECAL_FILE

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

--out-bam OUT_BAM

Path of output BAM file. (default: None)

Option is required.

--out-variants OUT_VARIANTS

Path of VCF/g.vcf/gVCF.gz file after variant calling. The argument can also be a local folder in batch mode. (default: None)

Option is required.

Options specific to this tool

--num-cpu-threads NUM_CPU_THREADS

Number of CPU threads to traverse separate chromosomes in splitncigar. (default: 6)

--no-ignore-mark

Do not ignore marked reads in sorted output. (default: None)

--num-threads NUM_THREADS

Number of running worker threads per GPU. (default: 4)

--out-prefix OUT_PREFIX

Prefix filename for output data. (default: None)

--read-files-command READ_FILES_COMMAND

Command line to execute for each of the input files. This command should generate FASTA or FASTQ text and send it to stdout. For example: zcat - to uncompress .gz files, bzcat - to uncompress .bz2 files, etc. (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)

--two-pass-mode TWO_PASS_MODE

2-pass mapping mode. The string can be "None" for 1-pass mapping or "Basic" for basic 2-pass mapping with all 1st pass junctions inserted into the genome indices on the fly. (default: Basic)

--read-length READ_LENGTH

Input read length used to determine sjdbOverhang. (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)

--ploidy PLOIDY

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

-L INTERVAL, --interval INTERVAL

Interval within which to call the variants from the bam/cram file. 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)

-ip INTERVAL_PADDING, --interval-padding INTERVAL_PADDING

Amount of padding (in base pairs) to add to each interval you are including. (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.