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 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 
# Amplicon 
--enable-rna-amplicon true 
--amplicon-target-bed $PATH 
--amplicon-enable-imbalance-ratio true  #optional for panels that support 3'/5' imbalance-ratio 
--enable-duplicate-marking false        #default=false 
# 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

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 enriched option (rna-enriched-regions or rna-enriched-genes), since DRAGEN will read the enriched genes names from the amplicon BED file (fifth column).

Pillar Specific Amplicon 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

Input options

DRAGEN input sources include: fastq list, fastq, bam, or cram. For BCL input, first create FASTQs using 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 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 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 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