# DNA Somatic Tumor-Only Heme WGS 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 --enable-duplicate-marking true #default=true # Small variant caller --enable-variant-caller true --vc-systematic-noise $PATH #Required --vc-target-vaf $NUM #Default = 0.03 (>= 3% VAF) # SV --heme-sv true --sv-systematic-noise $PATH #Recommended --enable-oncovirus-detection true #Optional --oncovirus-detection-db $PATH #Optional # DUX4 --enable-dux4-caller true # CNV --heme-cnv true --cnv-population-b-allele-vcf $POP_VCF --cnv-enable-self-normalization true # Annotation --variant-annotation-data $NIRVANA_PATH --vc-enable-germline-tagging true --vc-germline-tag-hotspots false #When germline tagging is enabled, disable it only for somatic hotspot variants ``` ## Notes and additional options ### Hashtable For DRAGEN somatic runs it is recommended to use the linear hashtable. See: [Product Files](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html) ### Input options DRAGEN input sources include: fastq list, fastq, bam, or cram. For BCL input, first create FASTQs using [BCL conversion](https://help.dragen.illumina.com/product-guides/dragen-v4.5/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 | Option | Description | | -------------------------------- | ---------------------------------------------------------------------------------------------------- | | `--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. | ### Duplicate Marking | Option | Description | | --------------------------------- | ------------------------------------------------------------------------------- | | `--enable-duplicate-marking true` | By default, DRAGEN marks duplicate reads and exclude them from variant calling. | ### 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. | Option | Description | | ---------------------------------- | ----------------------------------------------------------------------------------------------------------- | | `--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. | ### SNV | Option | Description | | -------------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `--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-emit-ref-confidence GVCF` | To enable gVCF output. | | `--vc-enable-vcf-output` | To enable VCF file output during a gVCF run, set to true. The default value is false. | | `--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.). | | `--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-sq-filter-threshold $NUM` | Threshold for sensitivity-specificity tradeoff using SQ score. The pipeline specific default threshold is 3. 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-specificity 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-specificity 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](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html) | For more detail on the small variant caller in somatic mode please refer to [Somatic Mode](https://help.dragen.illumina.com/product-guides/dragen-v4.5/dragen-dna-pipeline/small-variant-calling/somatic-mode) ### CNV | Option | Description | | ---------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------- | | `--cnv-enable-gcbias-correction true` | Enable or disable GC bias correction when generating target counts. | | `--cnv-segmentation-mode $SEG_MODE` | Option to override the default segmentation algorithm. Defaults include `slm` for germline WGS, `aslm` for somatic WGS, and `hslm` for targeted analysis. | | `--cnv-segmentation-bed $PATH` | Specify a segmentation bed file to add pre-defined segments to be called. | | `--cnv-population-b-allele-vcf $POP_VCF` | Specify a population SNP VCF. This option can be used when a matched normal sample is not available and analysis must be performed in tumor-only mode. | | `--cnv-enable-cyto-output true` | Enable Cytogenetics-compatible output (default false). | | `--heme-cnv true` | Configures DRAGEN to use CNV settings for HEME. | ### Annotation For instructions on how to download the Nirvana annotation database, please refer to [Nirvana](https://help.dragen.illumina.com/product-guides/dragen-v4.5/nirvana) ### SV | Option | Description | | -------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `--sv-call-regions-bed` | Specifies a BED file containing the set of regions to call. Optionally gzip or bgzip format. | | `--sv-exclusion-bed` | Specifies a BED file containing the set of regions to exclude for the SV calling. Optionally, you can compress the file in gzip or bgzip format. | | `--enable-variant-deduplication true` | Relevant when both SV and SNV callers are enabled in somatic workflows. Can increase sensitivity and prevent the occurrence of replicated variants within genes such as FLT3 and KMT2A. Filter all small indels in the structural variant VCF that appear and are passing in the small variant VCF. DRAGEN will create a new VCF that contains variants in SV VCF that are not matching a variant from SNV VCF file. The new deduplicated SV VCF file will have the same prefix passed by `--output-file-prefix` followed by `sv.small_indel_dedup`. DRAGEN normalizes variants by trimming and left shifting by up to 500 bases. | | `--sv-systematic-noise $BEDPE` | Systematic noise BEDPE file containing the set of noisy paired regions (optionally gzip or bzip compressed). Optional for WGS Tumor-Normal, but strongly recommended for WGS Tumor-Only. Has not been validated in WES, enrichment and amplicon panels. | | `--sv-somatic-ins-tandup-hotspot-regions-bed $BED` | Specify a custom BED of ITD hotspot regions to increase sensitivity for calling ITDs in somatic variant analysis. The default file includes FLT3, ARHGEF7, KMT2A, and UBTF exonic regions with some padding on both sides (300 bps) | | `--sv-min-candidate-variant-size` | Run SV caller and report all SVs/indels at or above this size. The default value is set to 10. | | `--sv-min-scored-variant-size` | After candidate identification, only score and report SVs/indels at or above this size. The default value is set to 50. This parameter doesn't affect the somatic hotspot region. | | `--enable-oncovirus-detection` | Set to enable detection of oncoviral integration. See [Oncovirus Detection](https://help.dragen.illumina.com/product-guides/dragen-v4.5/dragen-dna-pipeline/oncovirus-detection) for more information. | | `--oncovirus-detection-db` | Specifies the directory containing oncovirus detection resource files. See [Oncovirus Detection](https://help.dragen.illumina.com/product-guides/dragen-v4.5/dragen-dna-pipeline/oncovirus-detection) for more information. | | Option | Recommended Value for Liquid Tumors (e.g. AML/MLL) | | ----------------------------------- | ----------------------------------------------------------------------- | | `--heme-sv true` | Configures DRAGEN to use SV settings for Liquid Tumors (e.g., AML/MLL). | | `--sv-min-scored-variant-size $INT` | 100000 | For more information, see [Structural Variant Calling](https://help.dragen.illumina.com/product-guides/dragen-v4.5/dragen-dna-pipeline/sv-calling). ### DUX4 | Option | Description | | ------------------------------- | --------------------------------------------------------------------------------------------------------- | | `--dux4-skip-sanity-check true` | Bypass the requirements checks if the input datasets don't comply with parameters listed in prerequisites | For more information, see [DUX4-rearrangement Calling](https://help.dragen.illumina.com/product-guides/dragen-v4.5/dragen-dna-pipeline/dux4-rearrangement-caller). ## 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-built systematic noise files for WGS, WES and for Pillar Amplicons. For high sensitivity applications, including panels or clinical WES/WGS assays, it is recommended to create your own systematic noise file as described under Custom. #### Prebuilt Prebuilt systematic noise BED files (WES and WGS) can be downloaded here: [Product Files](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html). | Prebuilt WES/WGS noise files | Description | | -------------------------------------------------- | ------------------------ | | `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 | #### Custom This section describes how to generate systematic noise files from phenotypically normal (non-tumor) samples to optimize the performance of a specific assay. For best accuracy, the normal samples should ideally closely match the sequencer, sample type, library prep, and coverage of the tumor samples of interest. It is typically recommended to use 30-70 normals when building a noise file, but fewer can be used. **Step 1. Run DRAGEN somatic tumor-only on each of approximately 30-70 normal samples.** ``` /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 --tumor-fastq-list $PATH #see 'Input Options' for FQ, BAM or CRAM --tumor-fastq-list-sample-id $STRING --vc-detect-systematic-noise=true --vc-enable-germline-tagging=true --vc-germline-tag-hotspots=false #When germline tagging is enabled, disable it only for somatic hotspot variants --variant-annotation-data $NIRVANA_PATH --intermediate-results-dir $PATH --output-directory $PATH --output-file-prefix $STRING ``` For WES and WGS pipelines gather the full paths to the small variant hard filtered VCFs (not GVCFs) from step 1 and create a lines file `${VCF_LIST}` by specifying 1 file per line. **Step 2. Generate the final noise file.** This step generates a bed file containing mean and max noise estimates per position. This can be used directly during variant calling (argument --vc-systematic-noise). The distribution of noise per position can also be plotted to identify particularly noisy positions that could be troubleshooted (e.g. modify assay settings or DRAGEN settings) or blocklisted ``` /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 --build-sys-noise-vcfs-list ${VCF_LIST} ``` The SNV systematic noise files can also be built in the cloud using the [DRAGEN Baseline Builder App on BaseSpace](https://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub/apps/dragen-baseline-builder.html) or the DRAGEN Systematic Noise File Builder Pipeline on [ICA](https://www.illumina.com/products/by-type/informatics-products/connected-analytics.html). ### SV Systematic Noise SV systematic noise files are recommended for WGS workflows. Prebuilt WGS noise files are available. #### Prebuilt Prebuilt WGS SV systematic noise files can be downloaded here: [Product Files](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html). | Prebuilt WGS noise files | Description | | --------------------------------------------------- | ---------------- | | `WGS_hg38_v3.0.0_systematic_noise.sv.bedpe.gz` | For WGS, FF/FFPE | | `IDPF_WGS_hg38_v3.0.0_systematic_noise.sv.bedpe.gz` | For WGS, HEME | #### Custom Custom systematic noise files can be generated for WES, Panels or Amplicon. For best accuracy the normal samples should ideally closely match the sequencer, sample type, library prep and coverage of the tumor samples of interest. It is typically recommended to use 30 - 100 normals when building a noise file, but fewer can be used. **Step 1. Run DRAGEN somatic tumor-only on normal samples with `--sv-detect-systematic-noise` set to true to generate VCF output per normal sample.** ``` /opt/dragen/$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 --tumor-fastq-list $PATH #see 'Input Options' for FQ, BAM or CRAM --tumor-fastq-list-sample-id $STRING --sv-detect-systematic-noise true ``` **Step 2. Build the BEDPE file using input VCFs from previous step.** ``` /opt/dragen/$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 --sv-build-systematic-noise-vcfs-list $VCF_LIST#one VCF per line. ``` Systematic noise BEDPE files can also be built in the cloud using the [DRAGEN Baseline Builder App on BaseSpace](https://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub/apps/dragen-baseline-builder.html) or the DRAGEN Systematic Noise File Builder Pipeline on [ICA](https://www.illumina.com/products/by-type/informatics-products/connected-analytics.html).