# 5 Base DNA Somatic Tumor-Only Solid 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. This recipe includes the recommended commands for solid samples. These settings support fresh frozen samples, as well as some optional settings for FFPE 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 # 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 --umi-min-supporting-reads 1 #Default=2 # 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-excluded-regions-bed $BED #FFPE: optionally mask ALUs --vc-enable-umi-solid true #>= 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](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.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/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. | ### 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. | ### UMI | Option | Description | | ---------------------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `--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: \/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. The default is 2, but most pipelines perform better with this setting set to 1. A setting of 2 may potentially be relevant for samples with ultra deep coverage (e.g. ctDNA). | | `--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](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-dna-pipeline/unique-molecular-identifiers#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](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-dna-pipeline/unique-molecular-identifiers#umi-options). ### 5-Base Methylation | Option | Description | | --------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `--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](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-methylation-pipeline/dragen-5base-pipeline). ### 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-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 4. 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. 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. 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) | 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. | High Sensitivity Option | Description | | ----------------------------- | ------------------------------------------------------------------------------------------------------------------------ | | `--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](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-dna-pipeline/small-variant-calling/somatic-mode) ### Annotation For instructions on how to download the Nirvana annotation database, please refer to [Nirvana](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/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](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 |