Splice Variant Caller
DRAGEN calls splice variants by taking advantage of its fast and highly accurate splice-aware read mapper/aligner that aligns reads to the whole genome to identify novel alternative Splice Junction (SJ) candidates. These candidates can be filtered by additional information provided such as a "normals list" and a "target regions list", or whitelisted with a "knowns list". The "splice fusion genes" file allows intergenic or intragenic splice variants to be reported in the RNA Gene Fusion output.
During the read sorting phase, evidence for these alternative splice variant candidates vs. reference splicing is accumulated. Then, the candidates are scored based on the accumulated read evidence and the results are written to TSV and VCF files for downstream tertiary analysis.
To use the RNA Splice Variant caller, use the option --enable-rna-splice-variant=true. Following is an example command line for a WTS dataset.
dragen \
-r <HASHTABLE> \
-1 <FASTQ1> \
-2 <FASTQ2> \
-a <GTF_FILE> \
--output-dir <OUT_DIRECTORY> \
--output-file-prefix <PREFIX> \
--RGID <READ_GROUP_ID> \
--RGSM <SAMPLE_NAME> \
--enable-rna true \
--enable-rna-splice-variant true \
--enable-duplicate-marking true For RNA panels, use rna-enriched-regions (bed format) or rna-enriched-genes (text file with one gene per line) to set the targeted gene information.
Splice Variant Optional Input Files
In addition to the required inputs listed in the above example (i.e. paired fastq reads, reference hashtable, and annotation), the following three optional input resource files can be provided to help provide better precision by reducing FP count.
Normals List
A list of Normal splice variants that will be filtered out of the final output (i.e. operating as a blacklist), as long as they are not in the "knowns" list, using the --rna-splice-variant-normals option.
The format of this file should be a tab separated file in the same format as the SJ.out.tab, except only the first 4 columns are used, i.e.
contig name
first base of the splice junction (1-based)
last base of the splice junction (1-based)
strand (0: undefined, 1: +, 2: -)
To create a Normals list file, a collection of DRAGEN RNA mapper output SJ.out.tab files for at least 30 samples can be used along with a simple script to process all the SJs in these files. The pseudo code block below describes the function of this script:
Knowns List
A list of known splice variants that are exempt from being filtered out of the final output (i.e. operating as a whitelist), using the --rna-splice-variant-knowns option. The format of the file should be a tab separated file in the same format as the SJ.out.tab with 9 columns present, except only the first 4 columns are evaluated, i.e.
contig name
first base of the splice junction (1-based)
last base of the splice junction (1-based)
strand (0: undefined, 1: +, 2: -)
By default, the caller will not consider any splice variant candidates that are found in the input annotation file since it is looking for denovo variants, unless it is included in the knowns list which directs it not to discard the specified candidate. Note that some newer gene annotation models have added alt transcripts that contain clinically relevant splice variants, which causes DRAGEN to skip reporting them.
To ensure these are reported, the user may want to pass these in with a knowns file containing these common variants if they are found in the annotation that is used. An example is shown below using hg38 coordinates specifying the MET exon 14 skip, EGFRv3, and ARv7 alt splicing events, respectively.
Splice Fusion Genes
A list of genes or gene pairs can be defined with the option --rna-splice-variant-fusion-genes. These genes or gene pairs will be passed to the RNA Gene Fusion output if detected by the splice variant caller. Note that the gene names should be present in the annotation file. Lines starting with the # symbol will be ignored. The default list of splice fusions are:
The file above was made based on clinical recurrent splice fusions and indicates that:
any intragenic splice variant in the EGFR gene or any intergenic splice fusion with one gene EGFR will be passed to the RNA Gene Fusion component;
any intragenic splice variant in the MET gene or any intergenic splice fusion with one gene MET will be passed to the RNA Gene Fusion component;
all intergenic splice fusions between BC039389 and GATM will be passed to the RNA Gene Fusion component; and so on.
For more details see Section Merging Splice Variants with the Gene Fusion Caller.
Target Regions BED or text file
If the RNA dataset consists of a gene panel, the gene names or amplified regions can be passed using --rna-enriched-genes or --rna-enriched-regions. Both options cannot be set together. To pass a list of genes (one per line), use --rna-enriched-genes. Only splice variants within these genes will be reported. For intergenic splice variants, at least one gene must be enriched to be reported. To pass a list of regions, use the --rna-enriched-regions option. Any splice variant candidates will be excluded if they are not within these regions. This file should be in BED file format with the following info, except that the regions are 1-based.
chromosome id
start position (1-based)
end position (1-based)
region (i.e. gene) name
Splice Variant Output Files
The detected splice variants are output as two separate TSV files for the intragenic (within one gene) and intergenic (between two genes) candidates, and as a VCF for the intragenic candidates. The number of reads supporting the reference vs. the variant SJ are reported and used to score the candidate.
For a read to be considered as support for a SJ candidate it must meet the following criteria:
Must contain a splice junction (i.e. an alignment gap in the CIGAR containing skip ops).
Must have overhangs on either side of the skip that are at least 6 base pairs.
Reads are classified by whether they are marked as PCR duplicates or not, and whether they are uniquely mapping (NH=1) or multi-mapping (NH>1). (See RNA-Seq BAM Tags for more information.) For a splice variant to be reported, at least one deduplicated uniquely mapping read supporting it must be found.
Splice Variant TSV Files
The two TSV output files are named:
output-file-prefix.splice_variants.tsv which contains the intragenic alt splice junctions that result in transcript variants
output-file-prefix.splice_variant_fusions.tsv which contains the intergenic alt splice junctions that cause fusions across genes
Each detected splice junction contains the following columns:
gene_start - Gene name(s) at the start of the SJ. Multiple genes are separated by a semicolon
gene_end - Gene name(s) at the end of the SJ. Multiple genes are separated by a semicolon
chromosome - Chromosome containing the SJ
start - SJ's start position (1-based genomic coordinate)
end - SJ's end position (1-based genomic coordinate)
filter - A list of filters separated by semicolon. See Section Splice Variant Filters.
strand - Detected strand of the SJ (+ or -)
motif - Intron motif, 0: noncanonical, 1: GT/AG, 2: CT/AC, 3: GC/AG, 4: CT/GC, 5: AT/AC, 6: GT/AT
annotated - "TRUE" if annotated in the reference GTF, otherwise "FALSE"
unique_dedup_ref_reads - The number of deduplicated uniquely mapping reads that support the reference SJ. A read is considered to be "uniquely mapping" only if NH=1.
unique_total_ref_reads - Total number of uniquely mapping reads that support the reference SJ, both duplicate and deduplicated.
multi_dedup_ref_reads - The number of deduplicated multi-mapping reads that support the reference SJ. Reads are considered multi-mapping if NH>1.
multi_total_ref_reads - Total number of multi-mapping reads that support the reference SJ, both duplicate and deduplicated.
unique_dedup_alt_reads - The number of deduplicated uniquely mapping reads that support the candidate SJ.
unique_total_alt_reads - Total number of uniquely mapping reads that support the candidate SJ, both duplicate and deduplicated.
multi_dedup_alt_reads - The number of deduplicated multi-mapping reads that support the candidate SJ.
multi_total_alt_reads - Total number of multi-mapping reads that support the candidate SJ, both duplicate and deduplicated.
high_qual_unique_dedup_alt_reads - Number of uniquely mapping deduplicated reads that support the candidate SJ and have MAPQ higher than a threshold determined by the option
rna-splice-variant-min-mapq. The default value for the MAPQ threshold is 35.max_mapQ_ref - Maximum MAPQ of deduplicated reads uniquely mapping to the reference SJ. If no reads, the value will be zero.
max_mapQ_alt - Maximum MAPQ of deduplicated reads uniquely mapping to the candidate SJ. If no reads, the value will be zero.
avg_mapQ_ref - Average MAPQ of deduplicated reads uniquely mapping to the reference SJ. If no reads, the value will be zero.
avg_mapQ_alt - Average MAPQ of deduplicated reads uniquely mapping to the candidate SJ. If no reads, the value will be zero.
max_spliced_alignment_overhang - Maximum spliced alignment overhang from all uniquely mapping reads supporting the candidate SJ.
normalized_overhang -
max_spliced_alignment_overhangnormalized by maximum read length.score - The candidate SJ score (ranging from 0 to 1). This score is calculated from a pre-trained ML model.
read_through - Only for intergenic output - This column will have value "1", if the splice variant is read through (adjacent genes on the same strand) or "0", otherwise.
Note:
In the intragenic output file containing transcript variant splice junctions, the gene_start and gene_end columns must match.
In the intergenic output file containing fusions from splice junctions, the gene_start and gene_end columns must be different.
Splice Variant VCF File
The candidate intragenic splice variants are reported in a zipped VCF file titled <output-file-prefix>.splice_variants.vcf.gz, where each splice variant candidate is written as a one-line VCF record of SV DEL event. The Splice Variant VCF output contains the following fields:
CHROM - Chromosome of the splice
POS - SJ start position (1-based) i.e. first base of intron
ID - "." (unused)
REF - Base from the reference genome FASTA at the SJ start position
ALT - Always "DEL"
QUAL - The junction score from in Phred scale
FILTER - Semicolon separated list of filters
INFO - See the possible Info fields below
FORMAT - SR (supporting reads for ref and alt)
SAMPLE - Counts for {unique_dedup_alt_reads},{unique_dedup_ref_reads}
The VCF header is given below:
For example:
Splice Variant Filters
The following filters are applied for confidence or informative only on splice variant candidates.
Filter
Type
Description
Option to adjust
LOW_SCORE
intragenic and intergenic
Score < minimum threshold (by default 0.5)
--rna-splice-variant-min-score
MIN_SUPPORT
intragenic and intergenic
Supporting reads (unique_dedup_alt_reads) is <3 if motif=0, or <2 if dist>50000, or <3 if dist>100000, or <4 if dist>200000 where distance is the distance between the breakpoints.
LOW_UNIQUE_ALIGNMENTS
intragenic and intergenic
Unique supporting read count <2
--rna-splice-variant-min-support-known for known and annotated splice variants and --rna-splice-variant-min-support-novel for novel splice variants.
ANCHOR_SUPPORT
Intragenic and Intergenic
if sj.motif == 0, requires at least 30bp overhang. Otherwise requires at least 12bp overhang
-
NON_PROTEIN_CODING
Intragenic and Intergenic
If genes have non protein coding genes.
To enforce this filter for intergenic splice variants, set --rna-gf-restrict-genes=true
LOW_ALT_TO_REF
Intergenic only
REF support is 200x or more than the ALT support
--rna-splice-fusion-max-ref-alt-ratio
READ_THROUGH
Intergenic only
Whether the 5' gene is downstream of the 3' gene
--rna-gf-report-read-through
Merging Splice Variants with the Gene Fusion Caller
When the splice variant caller and gene fusion caller are both enabled, the passing intergenic splice variants will be passed to the gene fusion caller to be merged into the relevant fusion output VCF and TSV files. 
The following splice variant calls will be passed to the gene fusion caller:
All passing intergenic splice variants. By default intergenic splice variants on adjacent genes on the same strand are not passed to the gene fusion caller. In order to enable read-through splice fusions, use
--rna-gf-report-read-through=true. Many read through fusions are present in mRNA and only a number of them are clinically relevant. The Splice Fusion Genes input file lists clinically relevant readthrough fusions to be reported.All intergenic and intragenic splice variants matching the gene patterns in the Splice Fusion Genes input.
The passing calls are reported in the fusion caller's <output-file-prefix>.fusion_candidates.final and <output-file-prefix>.fusion_candidates.vcf.gz file. In the <output-file-prefix>.fusion_candidates.vcf.gz the variant has the "SPLICE_VARIANT" flag in the info field. In the fusion_candidates.final file, the tab separated fields are described below.
Field Names
Description
FusionGene
Left and Right gene names (separated by "--")
Score
Value between 0 and 1
LeftBreakpoint, RightBreakpoint
The location for left and right sides of the splice with three colon separated fields: chromosome:coordinate:strand(+/-)
Gene1Location, Gene2Location
Splice Variant caller always outputs "SpliceVar" here instead of Exon/Intron location
Gene1Sense, Gene2Sense
Always TRUE by design
Gene1Id, Gene2Id
Long form ID (i.e. for Gencode it is usually "ENSG.version")
NumSplitReads
Taken from the dedupUniqueSupportingReads count (i.e. split_unique_reads_alt column value)
NumSoftClippedReads, NumPairedReads
These values are not used by RSV caller and are set to '0'
ReadNames
Not provided by this caller and set to 'N/A'
List of All RNA Splice Variant Options
enable-rna-splice-variant
Enable rna splice variant caller
true/false
false
rna-splice-variant-knowns
Expected splice junctions, call even if they are annotated
string (path to file)
None
rna-splice-variant-normals
Normal (non-variant) splice junctions, do not report if found
string (path to file)
None
rna-splice-variant-fusion-genes
List of hotspot genes that may contain splice fusions
string (path to file)
None
rna-enriched-regions
List of regions that splice junctions must overlap (used for panels)
string (path to file)
None
rna-enriched-genes
List of genes that splice junctions must overlap (used for panels)
string (path to file)
None
rna-splice-variant-min-score
Score threshold for filtering RNA splice variant candidates.
number between 0 and 1
0.5
rna-splice-variant-min-support-known
Minimum number of unique reads supporting known and annotated splice variant required to PASS.
Integer >= 0
2
rna-splice-variant-min-support-novel
Minimum number of unique reads supporting novel splice variant required to PASS.
Integer >= 0
2
rna-splice-fusion-max-ref-alt-ratio
Maximum ref to alt ratio for the spliced fusion required to PASS.
number >= 0
200
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