> For the complete documentation index, see [llms.txt](https://help.dragen.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.dragen.illumina.com/dragen-v4.5/product-guides/dragen-v4.5/dragen-recipes/rna-wts.md).

# 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](/dragen-v4.5/product-guides/dragen-v4.5/bcl-conversion.md).

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](/dragen-v4.5/product-guides/dragen-v4.5/nirvana.md)


---

# Agent Instructions
This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com.

## Querying This Documentation
If you need additional information that is not directly available in this page, you can query the documentation by asking a question.

Perform an HTTP GET request on the following URL with the `ask` and `goal` query parameters:

```
GET https://help.dragen.illumina.com/dragen-v4.5/product-guides/dragen-v4.5/dragen-recipes/rna-wts.md?ask=<question>&goal=<user_goal>
```

`ask` is the immediate question: it should be specific, self-contained, and written in natural language.
`goal` is what the user is ultimately trying to achieve, the reason they need the answer. Sharing it helps GitBook give you a better, more relevant answer. A goal is most helpful when it describes the outcome the user wants rather than restating the question. For example, with `ask=how do I create an API token`, a goal like `automate deployments from our CI pipeline` lets GitBook tailor the answer to that use case.

The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.