# RNA Amplicon

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 
# RNA amplicon 
--enable-rna-amplicon true 
--amplicon-target-bed $PATH 
# 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 
# RNA Splice Variants 
--enable-rna-splice-variant true 
# RNA Gene Fusions 
--enable-rna-gene-fusion true 
--rna-gf-enriched-regions $PATH         #see 'RNA Fusion' auto-generated from amplicon target bed
# RNA 3'/5' imbalance-ratio             #optional for panels that support 3'/5' imbalance-ratio  
--amplicon-enable-imbalance-ratio true  
```

## DRAGEN Amplicon Pillar Panel Specific Settings

To support the varied designs of amplicon panels and the specific requirements of different analysis types (e.g., SNV, CNV, SV, MSI, RNA fusion, RNA splice variants, and RNA 3'/5' imbalance ratio), panel-specific parameter settings have been integrated into the command-line options. Each supported Pillar panel has a dedicated option, and the details for these RNA panels are listed in the table below:

|             **Panel Name**            |      **Short Name**      | **Panel Code** | **Sample Type** |             **Default variant caller enabled**            |      **Command Line Options**     |
| :-----------------------------------: | :----------------------: | :------------: | :-------------: | :-------------------------------------------------------: | :-------------------------------: |
|            oncoReveal Heme            |           Heme           |    P-HFU-01    |       RNA       |                         RNA fusion                        |     --amplicon-enable-rna-heme    |
|         oncoReveal Fusion LBx         |        Fusion LBx        |    P-LBX-03    |      cfRNA      |               RNA fusion, RNA splice-variant              | --amplicon-enable-cfrna-lbxfusion |
| oncoReveal Multi-Cancer RNA Fusion v2 | Multi-Cancer with Fusion |      SF-V2     |       RNA       | RNA fusion, RNA splice-variant, RNA 3'/5' imbalance-ratio | --amplicon-enable-rna-multicancer |

For more detail on the amplicon pipeline, please refer to [DRAGEN Amplicon Pipeline](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-amplicon-pipeline)

## Notes and additional options

### Hashtable

For DRAGEN RNA amplicon 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/dragen-v4.4/product-guide/dragen-v4.4/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). |

### Duplicate Marking

| Option                             | Description                                                                                                                                                                                               |
| ---------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| `--enable-duplicate-marking false` | The Amplicon Pipeline disables duplicate marking. In amplicon assays, fragments originate from a limited number of unique start and end positions, making conventional duplicate detection inappropriate. |

### RNA Fusion

| Option                            | Description                                                                                                                                                                                                                                             |
| --------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| `--rna-gf-enriched-regions $PATH` | Fusion calling parameters are automatically set in RNA amplicon mode but can be overridden in the command line. If fusion targets are not listed in the amplicon BED file, users can explicitly set it to a file containing fusion gene IDs or symbols. |