DRAGEN Multi-region Joint Detection (MRJD) is a de novo germline small variant caller for paralogous regions. In DRAGEN v4.4, MRJD covers regions that include six clinically relevant genes: NEB, TTN, SMN1/2, PMS2, STRC, and IKBKG. MRJD is compatible with hg38, hg19 and GRCh37 reference genome. The table below includes hg38 region coordinates covered by MRJD.
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Description
MRJD is a variant calling method that is designed to detect de novo germline small variants in paralogous regions of the genome. A conventional variant caller relies on the read aligner to determine which reads likely originated from a given location. This method works well when the region of interest does not resemble any other region of the genome over the span of a single read (or a pair of reads for paired-end sequencing). However, a significant fraction of the human genome does not meet this criterion. At least 5% of the human genome consists of segmental duplications. Many regions of the genome have near-identical copies elsewhere, and as a result, the true source location of a read might be subject to considerable uncertainty. If a group of reads is mapped with low confidence, a conventional variant caller might ignore the reads, even though they contain useful information. If a read is mismapped (i.e., the primary alignment is not the true source of the read), it can result in variant detection errors.
MRJD is designed in an attempt to tackle the complexities raised by segmental duplication regions. Instead of considering each region in isolation, MRJD considers all locations from which a group of reads may have originated and attempts to detect the underlying sequences jointly across all paralogous regions in the sample of interest.
Below is a diagram showing the general workflow of MRJD in a pair of paralogous regions. MRJD takes primary alignments in all paralogous regions, regardless of mapping quality, builds and places all copies in a pair of paralogous regions based on reads and prior knowledge, call small variants based on the placed copies, and output final genotypes.
Figure 1. MRJD Caller workflow.
Two modes of the MRJD Caller
There are two modes of the DRAGEN MRJD Caller, default mode and high sensitivity mode. Here are details on the differences between the two modes.
With --enable-mrjd=true, the MRJD Caller will report the following two types of variants:
Uniquely placed variants, which means the variant is found and placed in one of the paralogous regions without ambiguity. These variants will be labeled as "UNIQUELY_PLACED" in the VCF INFO field.
Region-ambiguous variants. In this case, the aggregated genotype contains a variant allele with high confidence, but MRJD Caller is unable to place the variant allele in one of the paralogous regions with high confidence. The MRJD Caller will report the variant allele in all paralogous regions. These variants will be labeled as "REGION_AMBIGUOUS" in the VCF INFO field.
High Sensitivity mode
With both --enable-mrjd=true and --mrjd-enable-high-sensitivity-mode=true, the MRJD Caller reports the same variants as from the default mode, plus two other types of variants.
Positions where the reference alleles in all paralogous regions are not the same. It is well established that gene conversion, including reciprocal crossover, is a common event between paralogous regions (such as PMS2 and PMS2CL). When reciprocal crossover event occurs, the prior model, without nearby information on phasing, might end up placing the converted haplotype in the source region instead of the destination region, resulting in no variant. The high sensitivity mode compensates for this event by reporting the variant in corresponding positions in all paralogous regions. These variants will be labeled as "MRJD_HS;REF_DIFF_SITE" in the VCF INFO field.
Variants that have been placed uniquely in one of the paralogous regions and no variant in the corresponding position in the other region. The high sensitivity mode reports the variant in the rest of the paralogous regions. This is to compensate the fact that sometimes the prior knowledge that is used to help place the variant is not sufficient or is estimated incorrectly. In those cases, the variant allele still exists but is placed in the wrong paralog region. Therefore, reporting the variant in the other paralogous regions can help maximize sensitivity even with the lack of prior. These variants will be labeled as
Running DRAGEN MRJD
The MRJD Caller is disabled by default and requires WGS data aligned to a human reference genome build 38, 19, or GRCh37.
Here is the list of options related to MRJD.
--enable-mrjd If set to true, MRJD is enabled for the DRAGEN pipeline.
--mrjd-enable-high-sensitivity-mode If set to true, MRJD high sensitivity mode is enabled for the DRAGEN pipeline. See previous section on what variant types are reported in MRJD default mode and high sensitivity mode (default = ‘false’).
The following command-line example uses FASTQ input and runs MRJD Caller with high sensitivity mode:
The following command-line example uses BAM input that has already been aligned and runs MRJD Caller with high sensitivity mode:
Example WGS workflow that includes both DRAGEN Small Variant Caller and MRJD
Starting from DRAGEN v4.4, MRJD can run together with the DRAGEN Small Variant Caller in the same DRAGEN run. Here are the example command lines to run DNA Mapping using FASTQ files as input, followed by Small Variant Calling and MRJD.
Using this workflow, two VCF files will be created (<sample>.hard-filtered.vcf.gz by DRAGEN Small Variant Caller and <sample>.mrjd.hard-filtered.vcf.gz by DRAGEN MRJD). To help user get a single VCF file for downstream analysis, we prepared an utility tool that replaces the DRAGEN Small Variant Caller output in the homology region of the six medically relevant and challenging genes with MRJD caller output. The tool also annotates the calls made by MRJD (with "MRJD" tag in the INFO column). Please refer to the to download the utility tool.
The MRJD Caller generates a <sample>.mrjd.hard-filtered.vcf.gz file in the output directory. The output file is a compressed VCFv4.2 formatted file that contains the VCF representation of the small variants from the identified genotype.
Uniquely placed call
The following are example output format for uniquely placed variant. The DRAGENHardQual filter is applied to the records if the variant has a QUAL < 3.00.
Figure 2. VCF output format example for uniquely placed call.
Non-uniquely-placed call
For variants that are not uniquely placed, including region-ambiguous variants from default mode, and all variants from high sensitivity mode, the MRJD Caller will also report variants under diploid genotype format, which can be interpreted the same way as uniquely placed variant (the genotype is region-specific instead of being an aggregate across all regions). Under this format, the QUAL represents phred-scaled quality score for the assertion made in ALT (i.e. −10log10 prob(GT==0/0)). Note that the QUAL score will be equal to or less than 3 (if the QUAL > 3, then the call should be uniquely placed).
The QUAL, GT, GQ and PL will be reported similarly to the DRAGEN germline VC. To avoid losing information about the aggregated genotype across paralogous regions, the MRJD Caller reports genotype, phred-scaled quality score, and the phred-scaled genotype likelihoods for aggregated genotype using JGT, JQL, and JPL in the FORMAT column.
Figure 3. VCF output format example for region-ambiguous call.
