> 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-panel.md).

# RNA 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 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 
--rna-enriched-genes $PATH              #for RNA panels 
--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.                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
| `--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. |

### 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)


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