# DRAGEN v4.4.6 T/N Recipe

The following DRAGEN recipes are specific for Illumina FFPE DNA Prep with Exome 2.5 Enrichment **tumor and normal libraries** (do not use for [tumor only](https://help.ffpe-exomev2p5.illumina.com/additional-information/tumor-only-analysis) analysis or for a different library prep method). A general recipe for Somatic analysis of Tumor Normal samples with UMI is provided on [DRAGEN v4.4 support pages](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-recipes/dna-somatic-tumor-normal-solid-wes-umi).

```bash
/opt/dragen/$VERSION/bin/dragen        #DRAGEN install path 
--ref-dir $REF_DIR                     #path to DRAGEN linear hashtable 
--output-directory $OUTPUT 
--intermediate-results-dir /staging    #tmp dir on fast SDD 
--output-file-prefix $PREFIX 
# Inputs
--tumor-fastq-list $PATH                #see 'Input Options' for FQ, BAM or CRAM 
--tumor-fastq-list-sample-id $STRING 
--fastq-list $PATH                      #see 'Input Options' for FQ, BAM or CRAM 
--fastq-list-sample-id $STRING 
# Mapper
--enable-map-align true
--enable-map-align-output true         #save the output BAM (default=false)
--qc-coverage-ignore-overlaps true     #do not double-count overlapping mates
# UMI
--umi-enable true 
--umi-library-type nonrandom-duplex
--umi-min-supporting-reads 1
--umi-start-mask-length 1
--umi-end-mask-length 3
--tumor-normal-has-umi both
# Small variant caller 
--enable-variant-caller true 
--vc-target-bed $VC_TARGET_BED         #see Resource Files page
--vc-systematic-noise $PATH            #see Resource Files page
--vc-enable-umi-solid true 
--vc-sq-call-threshold 3 
--vc-sq-filter-threshold 15
--vc-enable-non-primary-allelic-filter true 
--vc-enable-triallelic-filter false 
--vc-skip-germline-tagging true        
# CNV 
--enable-cnv true
--cnv-use-somatic-vc-baf true
--cnv-target-bed $VC_TARGET_BED 
--cnv-combined-counts $PATH            #see Resource Files page
# Annotation                           #annotation is required if enabling TMB
--enable-variant-annotation true
--variant-annotation-data $PATH        #see notes below
--variant-annotation-assembly GRCh37/8
# TMB 
--enable-tmb true
# HRD Scoring 
--enable-hrd true                       #requires CNV 
# Microsatellite Instability (MSI) 
--msi-command tumor-only
--max-base-quality 63                                    #default if UMI is enabled
--msi-coverage-threshold 40                              #see notes below 
--msi-microsatellites-file ${microsatellite_file}        #see Resource Files page
--msi-ref-normal-dir ${normal_reference_directory}       #see Resource Files page
```

## Notes and additional options

### Hashtable

For DRAGEN somatic runs it is recommended to use the linear (non-graph) hashtable.

Please see: [DRAGEN references](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html)

### Inputs

[Here are support pages for the formats of different input file types](https://support-docs.illumina.com/SW/DRAGEN_v39/Content/SW/DRAGEN/Inputfiles_fDG.htm).

FQ list Input

```
--fastq-list $PATH 
--fastq-list-sample-id $STRING 
```

or

```
--tumor-fastq-list $PATH 
--tumor-fastq-list-sample-id $STRING 
```

FQ Input

```
--fastq-file1 $PATH 
--fastq-file2 $PATH 
--RGSM $STRING 
--RGID $STRING 
```

or

```
--tumor-fastq1 $PATH 
--tumor-fastq2 $PATH 
--RGSM-tumor $STRING 
--RGID-tumor $STRING 
```

BAM Input

```
--bam-input $PATH
```

or

```
--tumor-bam-input $PATH
```

CRAM Input

```
--cram-input $PATH
```

or

```
--tumor-cram-input $PATH
```

### Mapper

<table><thead><tr><th width="300.26513671875">Option</th><th>Description</th></tr></thead><tbody><tr><td>--qc-coverage-ignore-overlaps true</td><td>resolve all of the alignments for each fragment and avoid double-counting any overlapping bases</td></tr></tbody></table>

### UMI

The above recipe details UMI options specific for the Illumina FFPE DNA Prep with Exome 2.5 Enrichment Library kit.

### SNV

The `vc-sq-filter-threshold` flag can be used to fine-tune the "somatic quality" value at which variants are called in order to balance sensitivity and specificity. Variants with values between the `vc-sq-call-threshold` and `vc-sq-filter-threshold` values are labeled as "weak\_evidence" in the output VCF file. The [default SQ Filter threshold in DRAGEN](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-recipes/dna-somatic-tumor-normal-solid-wes-umi) for tumor-normal analysis is 17.5.

### Annotation

Top directory containing Nirvana data file. Instructions on how to download the resource are at <https://support.illumina.com/content/dam/illumina-support/help/Illumina_DRAGEN_Bio_IT_Platform_v3_7_1000000141465/Content/SW/Informatics/Dragen/Nirvana_DownloadData_fDG.htm>. DRAGEN is expecting a top directory containing the sub-directories <kbd>Cache</kbd>, <kbd>References</kbd>, <kbd>Supplementary Annotation</kbd>.

### Microsatellite Instability (MSI)

Microsatellite Instability (MSI) is run in tumor-only mode, but other variants and biomarkers are calculated in tumor-normal mode. <kbd>msi-coverage-threshold</kbd> is a required parameter for both tumor-only and tumor-normal, and should be set according to the sample coverage. The sites in the <kbd>msi-microsatellites-file</kbd> need to match those in the dist files within the <kbd>msi-ref-normal-dir</kbd> .