# 5 Base DNA Germline Panel 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 --enable-duplicate-marking true #default=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 # Annotation --variant-annotation-data $NIRVANA_PATH --enable-variant-annotation 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.dragen.illumina.com/product-guides/dragen-v4.5/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. | ### Duplicate Marking | Option | Description | | ------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `--enable-duplicate-marking true` | By default, DRAGEN marks duplicate reads and exclude them from variant calling. | | `--enable-positional-collapsing true` | Alternative to `--enable-duplicate-marking=true`. Instead of discarding duplicate reads, DRAGEN can optionally perform positional collapsing, merging them into higher-quality consensus reads. This is beneficial for small panels without UMIs and coverage between 300X and 1000X. However, it's slower than standard duplicate marking and less effective on samples with coverage lower than 300X. For very high coverage (1000X+), avoid it due to potential read collisions. For high-sensitivity panels with 1000X+ coverage, consider using UMIs. | ### 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-vcf` | Specify methylation type (none, cg, or c) which is reported in gVCF files (default=cg). | For more information see: [5-Base Pipeline](https://help.dragen.illumina.com/product-guides/dragen-v4.5/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) | ### 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)