# RNA WTS

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 
--fastq-list $PATH                      #see 'Input Options' for FQ, BAM or CRAM 
--fastq-list-sample-id $STRING 
# Mapper 
--enable-rna true 
--annotation-file $GTF                  #GTF or GFF3 format 
--enable-map-align true                 #required for RNA/scRNA 
--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-target-bed $VC_TARGET_BED 
# RNA Quantification 
--enable-rna-quantification true 
--rna-library-type A                    #see 'RNA Quant' 
--rna-quantification-gc-bias true 
# RNA Splice Variants 
--enable-rna-splice-variant true 
# RNA Gene Fusions 
--enable-rna-gene-fusion true 
# Annotation 
--variant-annotation-data $NIRVANA_PATH 
--enable-variant-annotation true 
```

## Notes and additional options

### Hashtable

For DRAGEN RNA/scRNA 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/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`        | For RNA/scRNA pipelines, map-align should always be turned on. |
| `--enable-map-align-output true` | Optionally save the output BAM (default=false).                |

### Duplicate Marking

| Option                            | Description                                                                     |
| --------------------------------- | ------------------------------------------------------------------------------- |
| `--enable-duplicate-marking true` | By default, DRAGEN marks duplicate reads and exclude them from variant calling. |

### RNA Variant Calling

| Option                  | Description                                                                                                                                                                                               |
| ----------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| `--vc-target-bed $PATH` | Restrict the variants called to a target bed. A bed file specifying the gene-coding regions should be provided to avoid calling erroneous variants in unenriched or noncoding regions due to noisy reads. |

### RNA Quant

| Option               | Description                                                                                                                                                              |
| -------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| `--rna-library-type` | Set the library according to the read orientations. Set to 'A' to auto detect the correct read orientation. Alternatively select 'IU', 'ISR', 'ISF', 'U', 'SR', or 'SF'. |

### RNA Splice

| Option                                    | Description                                                                                                                                                                                                                                                                                                                       |
| ----------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| `--rna-splice-variant-normals $PATH`      | Optional list of normal splice variants that will be used to filter false positive calls. The file should be a tab separated file with the following first four columns: (1) contig name, (2) first base of the splice junction (1-based), (3) last base of the splice junction (1-based), (4) strand (0: undefined, 1: +, 2: -). |
| `--rna-splice-variant-knowns $PATH`       | File with a list of expected splice junctions, these will be passed                                                                                                                                                                                                                                                               |
| `--rna-splice-variant-fusion-genes $PATH` | List of hotspot genes that may contain spliced fusions                                                                                                                                                                                                                                                                            |

### RNA Fusion

| Option                            | Description                                                                                                              |
| --------------------------------- | ------------------------------------------------------------------------------------------------------------------------ |
| `--rna-gf-restrict-genes`         | Ignore genes that are not protein coding for gene fusions (Default=true)                                                 |
| `--rna-gf-enable-post-filters`    | Enable stringent post-filtering of RNA gene fusion candidates by quality flags to reduce false positives (Default=false) |
| `--rna-gf-output-fusion-sequence` | Output assembled gene fusion sequence in fusion\_candidates.final file (Default=true)                                    |
| `--rna-gf-report-intronic`        | Report fusion calls with intronic breakpoints (Default=true)                                                             |
| `--rna-gf-report-antisense`       | Report fusion calls with antisense and intronic breakpoints (Default=false)                                              |
| `--rna-gf-report-intergenic`      | Report fusion calls with intergenic, antisense and intronic breakpoints (Default=false)                                  |
| `--rna-gf-report-read-through`    | Report read-through fusion calls (Default=false)                                                                         |
| `--rna-gf-ptd-genes`              | List of gene names where we allow PTD/ITD associated self fusions                                                        |

### Annotation

For instructions on how to download the Nirvana annotation database, please refer to [Nirvana](https://help.connected.illumina.com/dragen/product-guides/dragen-v4.5/nirvana)