Multi-Region Joint Detection
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.
chr2
151578759
151588523
NEB exon 98-105
chr2
151589318
151599076
NEB exon 90-97
chr2
151599871
151609628
NEB exon 82-89
chr2
178653238
178654995
TTN exon 172-180
chr2
178657498
178659255
TTN exon 181-189
chr2
178661759
178663516
TTN exon 190-198
chr5
70049522
70077596
SMN2
chr5
70924940
70953013
SMN1
chr7
5970924
5980896
PMS2 exon 13-15
chr7
5980968
5987689
PMS2 exon 11-12
chr7
6737007
6743712
PMS2CL exon 2-3
chr7
6743880
6753867
PMS2CL exon 4-6
chr15
43599563
43602630
STRC exon 24-29
chr15
43602982
43611000
STRC exon 14-23
chr15
43611040
43618800
STRC exon 1-13
chr15
43699379
43702452
STRCP1 exon 23-28
chr15
43702488
43710472
STRCP1 exon 13-22
chr15
43710502
43718262
STRCP1 exon 1-12
chrX
154555884
154565047
IKBKG exon 3-10
chrX
154639390
154648553
IKBKGP1
MRJD method
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.
Default mode
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 "MRJD_HS;ALT_LOCATION" in the VCF INFO field.
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-mrjdIf set to true, MRJD is enabled for the DRAGEN pipeline.--mrjd-enable-high-sensitivity-modeIf 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.
Output format
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.
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