5 Base DNA Somatic Tumor-Only ctDNA Panel UMI
A DRAGEN recipe, like this one, is a predefined set of analysis parameters and workflow settings tailored to a specific type of genomic analysis. For clarity, some default parameters are explicitly included and annotated with comments.
/opt/dragen/$VERSION/bin/dragen #DRAGEN install path
--ref-dir $REF_DIR #path to DRAGEN linear hashtable
--output-directory $OUTPUT
--intermediate-results-dir $PATH #e.g. SSD /staging
--output-file-prefix $PREFIX
# Inputs
--tumor-fastq-list $PATH #see 'Input Options' for FQ, BAM or CRAM
--tumor-fastq-list-sample-id $STRING
# Mapper
--enable-map-align true #optional with BAM/CRAM input
--enable-map-align-output true #optionally save the output BAM
--enable-sort true #default=true
# UMI
--umi-enable true
# 5-Base
--methylation-conversion illumina
--methylation-generate-cytosine-report true
--methylation-compress-cx-report true
# Small variant caller
--enable-variant-caller true
--vc-target-bed $VC_TARGET_BED
--vc-systematic-noise $PATH #Required
--vc-enable-umi-liquid true #>= 0.1% VAF
# Annotation
--variant-annotation-data PATH
--vc-enable-germline-tagging true Notes and additional options
Hashtable
For DRAGEN somatic runs it is recommended to use the linear hashtable.
See: Product Files
Input options
DRAGEN input sources include: fastq list, fastq, bam, or cram. For BCL input, first create FASTQs using BCL conversion.
FQ list Input
--tumor-fastq-list $PATH
--tumor-fastq-list-sample-id $STRING FQ Input
--tumor-fastq1 $PATH
--tumor-fastq2 $PATH
--RGSM-tumor $STRING
--RGID-tumor $STRING BAM Input
--tumor-bam-input $PATH CRAM Input
--tumor-cram-input $PATH Mapping and Aligning
--enable-map-align true
Optionally disable map & align (default=true).
--enable-map-align-output true
Optionally save the output BAM (default=false).
--Aligner.clip-pe-overhang 2
Clean up any unwanted UMI indexes. Only use when reads contain UMIs, but UMI collapsing was not run.
Fractional (Raw Reads) Downsampling
DRAGEN can subsample a random, fractional percentage of reads from an input file using the fractional downsampler. You can use downsampling to subsample data sets in order to simulate different amounts of sequencing. DRAGEN randomly subsamples reads from primary analysis without any modification (e.g. no trimming, no filtering, etc.).
Downsampling may be useful to reduce runtime on very deep samples. For Tumor-Normal analyses it is also recommended to use a normal sample with coverage that is less than the tumor sample. If the matched normal has deeper coverage than the tumor sample, then the fractional samples may be used to reduce coverage on the normal sample.
--enable-fractional-down-sampler
Set to true to enable fractional downsampling. The default value is false.
--down-sampler-normal-subsample
Specify the fraction of reads to keep as a subsample of normal input data. The default value is 1.0 (100%).
--down-sampler-tumor-subsample
Specify the fraction of reads to keep as a subsample of tumor input data. The default value is 1.0 (100%).
--down-sampler-random-seed
Specify the random seed for different runs of the same input data. The default value is 42.
UMI
--umi-nonrandom-whitelist $PATH
If UMI is nonrandom, either a whitelist or correction table is required. The whitelist includes a valid UMI sequence per line.
--umi-correction-table $PATH
If UMI is nonrandom, either a whitelist or correction table is required. The correction table defaults to the table used by TruSight Oncology: <INSTALL_PATH>/resources/umi/umi_correction_table.txt.gz.
--umi-min-supporting-reads INT
Specify the number of matching UMI input reads required to generate a consensus read. Any family with insufficient supporting reads is discarded. Most pipelines perform better with a setting of 1. A setting of 2 may potentially be relevant for samples with ultra deep coverage (e.g. ctDNA). (Default since DRAGEN V4.5 is 1)
--umi-metrics-interval-file $BED
Target region in BED format.
--umi-emit-multiplicity both
Set the consensus sequence type to output. DRAGEN UMI allows collapsing duplex sequences from the two strands of the original molecules. For more information, see Merge Duplex UMIs.
--umi-start-mask-length INT
Number of additional bases to ignore from start of read. The default is 0. To reduce FP optionally set to 1.
--umi-end-mask-length INT
Number of additional bases to ignore from end of read. The default is 0. To reduce FP optionally set to 3.
For more information see: UMI Options.
5-Base Methylation
--methylation-conversion STRING
Library conversion for methylation analysis. Options: none, c_t, mc_t, illumina (default=none).
--methylation-protocol STRING
Library protocol for methylation analysis. Options: none, directional, non-directional, directional-complement, pbat. The default value for methylation-conversion=illumina is directional, otherwise it is none.
--methylation-mapq-threshold INT
Only reads with MAPQ greater or equal than the threshold will be included in methyl-seq analysis (default=0).
--methylation-generate-mbias-report true
Whether to generate a per-sequencer-cycle methylation bias report (default=true).
--mbias-report-include-overlaps
Calculate methylation stats for overlapping bases between mates (default=false).
--methylation-generate-cytosine-report true
Whether to generate a genome-wide cytosine methylation CX_report file (default=false).
--methylation-compress-cx-report true
Set to true to enable compression of the CX_report (default=true).
--methylation-keep-ref-cytosine true
Set to true to keep all reference cytosines in the CX_report file, even if they don't appear in the input reads (default=false).
--enable-cpg-methylated-mapping true
Enable methylated mapping with base conversions restricted to CpG context (default=true). When false, runs DRAGEN Methylation 3-base map/align instead.
--methylation-report-to-vcf
Specify methylation type (none, cg, or c) which is reported in VCF files (default=c).
--methylation-report-to-gvcf
Specify methylation type (none, cg, or c) which is reported in gVCF files (default=cg).
For more information see: 5-Base Pipeline.
SNV
--vc-target-bed
Limit variant calling to region of interest.
--vc-combine-phased-variants-distance INT
Maximum distance in base pairs (BP) over which phased variants will be combined. Set to 0 to disable. Valid range is [0; 15] BP (Default=2)
--vc-systematic-noise $PATH
Systematic noise file. This filter is recommended for removing systematic noise observed in normal samples (i.e. systematic alignment errors, sequencing errors, etc.). When working with panels it is recommended that a custom systematic noise file be created for each assay.
--vc-somatic-hotspots $PATH
DRAGEN has a default set of hotspot variants (positions and alleles) where it will assign an increased prior probability. Use this option to override with a custom hotspots file.
--vc-enable-liquid-tumor-mode true
Tumor-in-normal contamination. Only use if there is some tumor leakage in the normal control.
--vc-override-tumor-pcr-params-with-normal false
Mixed sample preparation. Only use if the tumor and normal samples exhibit different PCR (indel) noise patterns, e.g., due to using different sample preparation.
--vc-sq-filter-threshold $NUM
Threshold for sensitivity-specificity tradeoff using SQ score. The pipeline specific default threshold is 2. Raise this value to improve specificity at the cost of sensitivity, or lower it to improve sensitivity at the cost of specificity.
--vc-systematic-noise-filter-threshold $INT
Threshold for sensitivity-specificty tradeoff using AQ score for non-hotspot variants. This is only used when supplying a systematic noise file. Pipeline specific default value = 60. Raise this value to improve specificity at the cost of sensitivity, or lower it to improve sensitivity at the cost of specificity.
--vc-systematic-noise-filter-threshold-in-hotspot $INT
Threshold for sensitivity-specificty tradeoff using AQ score for hotspot variants. This is only used when supplying a systematic noise file. Pipeline specific default value = 20. Raise this value to improve specificity at the cost of sensitivity, or lower it to improve sensitivity at the cost of specificity.
--vc-excluded-regions-bed $BED
Hard filter variants that overlap with this region. ALU regions comprise approximately 11% of the genome, and are often exceptionally noisy regions in FFPE samples. Optionally filter out ALU regions using the DRAGEN excluded regions filter. ALU bed files can be downloaded as part of the Bed File Collection: Bed File Collection
High-coverage sequencing panels allow for the detection of low-frequency alleles. DRAGEN supports 3 main settings for improved sensitivity on low VAF variant calls.
--vc-target-vaf FLOAT
The default is 0.03 (3%). Set to e.g. 0.01 to improve SNV sensitivity on 1% VAF variants (assuming sufficient coverage).
--vc-enable-umi-solid true
Optimized for 1% and higher VAFs on UMI (or read position collapsed) samples with approx 300-1000X coverage.
--vc-enable-umi-liquid true
Optimized for 0.1% and higher VAFs on UMI samples with 1000X or higher coverage as expected in liquid biopsies.
For more detail on the small variant caller in somatic mode please refer to Somatic Mode
Annotation
For instructions on how to download the Nirvana annotation database, please refer to Nirvana
Resource Files
DRAGEN requires resource files for components such as SNV, SV, and CNV. The following notes provide references for downloading these files or generating them for custom workflows or assays.
SNV Systematic Noise
Systematic noise files are considered essential in Tumor-Only workflows. It is also recommended for Tumor-Normals workflows.
DRAGEN has pre-build systematic noise files for WES/WGS. These files should also be used in 5-Base workflows. The 5-Base workflows have not been tested with custom noise files.
Prebuild
Prebuilt systematic noise BED files (WES and WGS) can be downloaded here: Product Files.
WGS_hg38_v2.0.0_systematic_noise.snv.bed.gz
For WGS FF
FFPE_WGS_hg38_v2.0.0_systematic_noise.snv.bed.gz
For WGS FFPE (only hg38)
WES_hg38_v2.0.0_systematic_noise.snv.bed.gz
For WES FF and FFPE
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