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".
During the read sorting phase, evidence for these alternative splice variant candidates vs. reference splicing are accumulated. Then, each of 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.
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.
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.
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:
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.
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 the 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.
Target Regions BED
A list of regions that called splice variants must fall within using the --rna-splice-variant-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.
Splice Variant Output Files
The detected splice variants are output as two separate TSV files for the intragenic and intergenic 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 for mor 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
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
This file contains the detected intra-genic splice junction variants that are not filtered out, and are written into 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 containing the fields below:
CHROM - Chromosome of the splice
POS - SJ start position (1-based) i.e. first base of intron
The following lines of the VCF header describe columns 5 to 10 (last 6 columns)
All splice variants are reported as SV DEL events. For example:
Note on Filter Thresholds The passing thresholds for the LowQ and LowUniqueAlignments filters are fixed to the settings below.
* Panel is determined by setting the rna-splice-variant-regions option.
Merging Splice Variants with the Gene Fusion Caller
When the splice variant caller and gene fusion caller are both enabled, the passing and failed intergenic splice variants will be passed to the gene fusion caller to be scored and merged into the relevant fusion output VCF and TSV files.
The passing calls get added to the fusion caller's <output-file-prefix>.fusion_candidates.final and <output-file-prefix>.fusion_candidates.vcf.gz file. In the 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.
By default intergenic splice variants on adjacent genes are not passed to the gene fusion caller. In order to enable read through splice variant fusions, use rna-splice-variant-enable-readthrough=true.
A list of known splice variant fusions can be given to the splice variant caller to be passed to the gene fusion caller. To pass intergenic fusions, enter two gene names on one line and for intra-genic splice variants, enter one gene on one line. Pass the file with the option rna-splice-variant-fusion-genes. Below is an example of a file with intragenic splice variant fusions on EGFR and an intergenic splice variant fusion between AJM1 and PHPT1. Note that the gene names should be the present in the annotation file. Lines starting with the # symbol will be ignored.
List of RNA Splice Variant Options
Option
Description
Type
Default Value