The Rh Caller is capable of identifying a common gene conversion between RHD and RHCE genes from whole-genome sequencing (WGS) data, that is referred to as RHCE Exon2 gene conversion. Due to high sequence similarity between the genes, a specialized caller is necessary to resolve the gene conversion between the pair of genes. We consider 798 loci, called differentiating sites, that represents differences between the RHD and RHCE genes, that are well preserved in the population.
The Rh Caller performs the following steps:
Determines total copy number from read depth of the RHD and RHCE regions.
Detect RHD -> RHCE breakpoints that are consistent with the RHCE Exon2 gene conversion.
The Rh Caller requires WGS data aligned to a human reference genome with at least 30x coverage. Reference genome builds must be based on hg19, GRCh37, or hg38.
The Rh Caller is run by default when the small variant caller is enabled, the sample is a not a tumor sample, and the sample is detected as WGS by the Ploidy Estimator.
The first step of Rh calling is to determine the copy number of RHD and RHCE regions. Reads aligned to the RHD and RHCE regions are counted according to their support of the differentiating sits. The counts in each region are corrected for GC-bias, and then normalized to a diploid baseline. The GC-bias correction and normalization factors are determined from read counts in 3000 preselected 2 kb regions across the genome. These 3000 normalization regions were randomly selected from the portion of the reference genome having stable coverage across population samples.
A collection of 4 differentiating sites in the exon 2 region of RHD and RHCE are used to detect the presence of the RHCE Exon2 gene conversion in the sample. An iterative phasing algorithm is used to build up haplotypes that are supported by the read data. The phasing algorithm starts with candidate haplotypes formed from all possible bases at the first differentiating site. The haplotypes are then extended at the next differentiating site by considering all reads that can be uniquely assigned to a single candidate haplotype. If these reads support only a single base at the next differentiating site for a given candidate haplotype, then the haplotype is extended with that base. When a candidate haplotype can be extended by both bases at the next differentiating site then both possible extended haplotypes are included in the set of candidate haplotypes, growing the set by 1. Subsequent extension steps are performed at neighboring differentiating sites until all sites have been processed. Some haplotypes may have sites that are unresolved (i.e. ambiguous), but these haplotypes can still participate in RHD -> RHCE breakpoint detection.
When the phased haplotypes support the RHCE Exon2 gene conversion. We visit all the differentiating sites ad report them as variants in the output VCF file with ploidy identified using the copy number estimated from the read depth of the differentiating site.
The Rh Caller generates a <output-file-prefix>.targeted.json file in the output directory. The output file is a JSON formatted file containing the fields below.
The rh fields are defined as below.
For the variants the fields are defined as below.
Examples of the Rh Caller content in the output json file are shown below.
The Rh Caller also generates a <output-file-prefix>.targeted.vcf[.gz] file in the output directory. The output file is a VCFv4.2 formatted file, possibly compressed.
The following are example output files:
| Fields in JSON | Explanation | Type and Possible Values |
|---|---|---|
| Fields in JSON | Explanation | Type and Possible Values |
|---|---|---|
| Fields in JSON | Explanation | Type and Possible Values |
|---|---|---|
sample
The sample name.
string
dragenVersion
The version of DRAGEN.
string
rh
The RH targeted caller specific fields.
dictionary
totalCopyNumber
Total RHD/RHCE copy number
integer
rhdCopyNumber
RHD gene copy number
integer
rhceCopyNumber
RHCE copy number
integer
variants
List of known variants from recombination that were detected in RHD/RHCE.
list of variants
hgvs
HGVS identifier of the variant
string, "NC_000001.11g.25405596_25409676con25283766_25287797"
qual
Phred QUAL score of the variant
double
altCopyNumber
Copy number of the ALT variant
double
altCopyNumberQuality
Phred QUAL copy number of the ALT variant
double