# 5 Base DNA Germline WGS 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 pangenome hashtable --output-directory $OUTPUT --intermediate-results-dir $PATH #e.g. SSD /staging --output-file-prefix $PREFIX # Inputs --fastq-list $PATH #see 'Input Options' for FQ, BAM or CRAM --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 # Annotation --variant-annotation-data PATH --enable-variant-annotation true # SV --enable-sv true # CNV --enable-cnv true --cnv-enable-self-normalization true ``` ## Notes and additional options ### Hashtable For DRAGEN germline runs, it is recommended to use the pangenome 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 ``` --fastq-list $PATH --fastq-list-sample-id $STRING ``` FQ Input ``` --fastq-file1 $PATH --fastq-file2 $PATH --RGSM $STRING --RGID $STRING ``` BAM Input ``` --bam-input $PATH ``` CRAM Input ``` --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. | ### 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) | 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) ### 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. | For more information, see [CNV Calling](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-dna-pipeline/cnv-calling).