RNA Panel

DRAGEN Recipe - RNA Panel Sequencing

Overview

This recipe is for processing panel data for RNA workflows.

Example Command Line

For most scenarios, simply creating the union of the command line options from the single caller scenarios will work.

• Configure the INPUT options

• Configure the OUTPUT options

• Configure the RNA MAP/ALIGN options

• Configure the QUANT options

• Configure the SPLICE options

• Configure the FUSION options

• Configure the VARIANT options

The following are partial templates that can be used as starting points. Adjust them accordingly for your specific use case.

#!/bin/bash
set -euo pipefail

# Path to DRAGEN hashtable
DRAGEN_HASH_TABLE=<REF_DIR>

# Path to output directory for the DRAGEN run
OUTPUT=<OUT_DIR>

# File prefix for DRAGEN output files
PREFIX=<OUT_PREFIX>

# Define the input sources, select fastq list, fastq files, bam, or cram.
INPUT_FASTQ_LIST="
  --fastq-list $FASTQ_LIST \
  --fastq-list-sample-id $FASTQ_LIST_SAMPLE_ID \
"

INPUT_FASTQ="
  --fastq-file1 $FASTQ1 \
  --fastq-file2 $FASTQ2 \
  --RGSM $RGSM \
  --RGID $RGID \
"

# You could use the tumor fastq options to provide the FASTQ files.
INPUT_TUMOR_FASTQ="
  --tumor-fastq1 $FASTQ1 \
  --tumor-fastq2 $FASTQ2 \
  --RGSM $RGSM \
  --RGID $RGID \
"

INPUT_BAM="
  --bam-input $BAM \
"

INPUT_CRAM="
  --cram-input $CRAM \
"

# Select input source, here in this example we use INPUT_FASTQ_LIST.
INPUT_OPTIONS="
  --ref-dir $DRAGEN_HASH_TABLE \
  $INPUT_FASTQ_LIST \
"


OUTPUT_OPTIONS="
  --output-directory $OUTPUT \
  --output-file-prefix $PREFIX \
"

# When running a panel you need to set the target regions, accordingly. 
# This file should be in BED format with the region name in the fourth column.
INPUT_PANEL_BED=<TARGET_REGIONS_PATH>

# RNA aligner requires an annotation file in GTF or GFF3 format.
GTF=<GTF_PATH>

# RNA pipeline requires map-align to be true.
RNA_MAP_OPTIONS="
  --enable-rna true \
  --enable-map-align true \
  --annotation-file $GTF \
"

# You should set the library according to the read orientations.
# The options for orientation are IU, ISR, ISF, U, SR, or SF. To automatically detect the correct read orientation, set this option to A.
QUANT_OPTIONS="
  --enable-rna-quantification true \
  --rna-library-type IU \
  --rna-quantification-gc-bias true \
"

# For panels, you can set a target region bed file. 
# This BED file has the name of the region in the fourth column.
SPLICE_OPTIONS="
  --enable-rna-splice-variant \
  --rna-splice-variant-regions $INPUT_PANEL_BED
"

# For panels, the list of enriched genes should be set. 
# You can select a list of genes or a list of regions in BED format. 
FUSION_OPTIONS="
  --enable-rna-gene-fusion true \
  --rna-gf-enriched-regions $INPUT_PANEL_BED \
"

VARIANT_OPTIONS="
  --enable-variant-caller true \
  --vc-target-bed $INPUT_PANEL_BED
"

# Construct final command line
CMD="
  dragen \
  $INPUT_OPTIONS \
  $OUTPUT_OPTIONS \
  $RNA_MAP_OPTIONS \
  $QUANT_OPTIONS \
  $SPLICE_OPTIONS \
  $FUSION_OPTIONS \
  $VARIANT_OPTIONS 
"

# Execute
echo $CMD
bash -c $CMD

Additional Notes and Options

Amplicon data

If you are running amplicon, you need to set --enable-rna-amplicon true --amplicon-target-bed <AMPLICON_BED_PATH>.

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

SPLICE options

You can provide a list of normal slice variants to reduce noisy calls. The file should be a tab separated file with the following first four columns:

• contig name

• first base of the splice junction (1-based)

• last base of the splice junction (1-based)

• strand (0: undefined, 1: +, 2: -) Use the optional option --rna-splice-variant-normals <SPLICE_NORMAL_FILE_PATH> to provide the normal splice variants.