# RNA Panel

The DRAGEN recipe includes the recommended pipeline specific commands. A DRAGEN recipe is a predefined set of analysis parameters and workflow settings tailored for a specific type of genomic analysis. Some default parameters are included for clarity and are marked 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. SDD /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-splice-variant-regions $PATH 
# RNA Gene Fusions 
--enable-rna-gene-fusion true 
--rna-gf-enriched-regions $PATH         #see 'RNA Fusion' 
```

## 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.

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 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. For WTS, a bed file specifying the gene-coding regions should be provided to avoid calling erroneous variants in non-coding 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 setting list of normal splice variants that will be used 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-regions $PATH` | Target region bed file. Required for panels. The name of the region must be specified in the fourth column.                                                                                                                                                                                                                            |

### RNA Fusion

| Option                            | Description                                                                                                         |
| --------------------------------- | ------------------------------------------------------------------------------------------------------------------- |
| `--rna-gf-enriched-regions $PATH` | For panels, the list of enriched genes should be set, either as a list of genes or a list of regions in BED format. |

### RNA Amplicon

To enable RNA amplicon, set:

* `--enable-rna-amplicon true`, and
* `--amplicon-target-bed $PATH`.

If RNA amplicon mode is enabled and the amplicon bed file already includes the gene name, then it is not required to set the ENRICH options option, since DRAGEN will read the enriched genes names from the amplicon BED file (fifth column).
