CYP21A2 Caller

The CYP21A2 Caller is capable of genotyping the CYP21A2 gene from whole-genome sequencing (WGS) data. Due to high sequence similarity with its pseudogene paralog CYP21A1P and a wide variety of common structural variants (SVs), a specialized caller is necessary to resolve variants.

The CYP21A2 calling workflow is broken up into the following major stages:

1. Loading input configuration

2. Processing read data

3. Analyzing read data

Read data analysis is further split into the following steps:

1. Determine total CYP21A2 and CYP21A1P copy number from read depth.

2. Call small variants in CYP21A2 copies.

3. Phase reads to detect common variants and recombination events.

4. Identify most likely haplotypes.

The CYP21A2 Caller requires WGS data aligned to a human reference genome with at least 30x coverage.

Total CYP21A2 and CYP21A1P Copy Number

The first step of CYP21A2 calling is to determine the combined copy number of CYP21A2 and CYP21A1P. Reads aligned to regions in either CYP21A2 or CYP21A1P are counted. 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 2kb regions across the genome. These 3000 normalization regions were randomly selected from the portion of the reference genome having stable coverage across population samples. The combined CYP21A2 and CYP21A1P copy number is then calculated from the average sequencing depth across the CYP21A2 and CYP21A1P regions.

Nonrecombinant-like Variant Calling

Of the known nonrecombinant-like variants, some are in unique (nonhomologous) regions of CYP21A2 with high mapping quality. Only reads mapping to CYP21A2 are used for calling variants in nonhomologous regions. The other variants occur in homologous regions of CYP21A2/CYP21A1P where reads mapping to either are used for variant calling.

For each variant, reads containing either the variant allele or the nonvariant allele is counted. A binomial model that incorporates the sequencing error rate is then used to determine the most likely variant copy number (0 for nonvariant).

For a list of the supported nonrecombinant-like variants, refer to the targeted/cyp21a2/target_variants_*.tsv files located in the resources directory of the DRAGEN install location.

Nonallelic Homologous Recombination Variant Calling

To analyze the homologous region even further, DRAGEN phases reads covering differentiating sites and known variant sites. Whenever a detected haplotype has a CYP21A2->CYP21A1P or CYP21A1P->CYP21A2 transition that is consistent with one of the known recombinant-like variants, the transition is considered as a candidate breakpoint for calling those variants. Reads containing phasing information for the two sites flanking each candidate breakpoint are used for variant calling. When the read data supports the hypothesis that the sample contains at least one copy of a candidate breakpoint, the associated haplotype is a recombinant haplotype candidate. Recombinant haplotype candidates are sorted by likelihood and the number of variant sites. If no wild type haplotype was detected, DRAGEN reports any detected homozygous recombinant haplotype, or up to two different recombinant haplotypes (i.e. compound het) if detected. If any wild type haplotype was found, DRAGEN reports a maximum of one recombinant haplotype. When no recombinant haplotypes are detected two wild type haplotypes are reported.

For a list of recombinant variant sites, refer to the targeted/cyp21a2/recombinant_variants_*.tsv files located in the resources directory of the DRAGEN install location.

Note that NM_000500.9:c.710_719delinsACGAGGAGAA will be reported as the following three variants on the same haplotype: NM_000500.9:c.710T>A NM_000500.9:c.713T>A NM_000500.9:c.719T>A

CYP21A2 Output File

The CYP21A2 Caller generates its output in the targeted caller output file <output-file-prefix>.targeted.json that also contains calls from other targets (see Targeted JSON File).

Fields in JSON	Explanation	Type and Possible Values
totalCopyNumber	Total copy number of CYP21A2 and CYP21A1P genes including hybrids	nonnegative integer
deletionBreakpointInGene	null (i.e. unknown) if totalCopyNumber > 3	true, false, null
	true if CN <= 3 and a deletion-like recombinant variant haplotype is detected
	false if CN <=3 and no deletion-like recombinant variant is detected
recombinantHaplotypes	List of detected haplotypes arising from nonallelic homologous recombination variant calling	Array of two strings. Each string consists of all associated allele IDs (if any) within the haplotype. Consecutive IDs in the same haplotype are separated by a '+'.
variants	List of single site, nonrecombinant-like variants (i.e. not arising from nonallelic homologous recombination). An empty list if no variants are detected.	Array of nonrecombinant-like variants.

Note: A deletion-like recombinant variant haplotype (as opposed to a gene conversion-like recombinant variant haplotype) is defined as a haplotype with one or fewer switch sites (transitions from a CYP21A1P allele to a CYP21A2 allele) after excluding some sites with common gene conversions in CYP21A1P.

Each nonrecombinant-like variant reported in the variants array will have the fields below.

Fields in JSON	Explanation	Type and Possible Values
alleleId	HGVS identifier of the variant allele	string
alleleCopyNumber	Copy number of the allele in the called genotype	nonnegative integer
genotypeQuality	Phred-scaled quality for the called genotype	nonnegative integer
filter	Filter for the called genotype	string. "PASS" when not filtered

Recombinant-like and nonrecombinant-like variants are reported in VCF format. See Targeted VCF File for details about how these variants are reported in VCF.

Output File Example

An example of the CYP21A2 caller content in the <output-file-prefix>.targeted.json output file is shown below.

{        
    "cyp21a2": {
            "totalCopyNumber": 2,
            "deletionBreakpointInGene": true,
            "recombinantHaplotypes": [
                "NM_000500.9:c.92C>T+NM_000500.9:c.293-13C>G+NM_000500.9:c.332_339del+NM_000500.9:c.518T>A+NM_000500.9:c.710T>A+NM_000500.9:c.713T>A+NM_000500.9:c.719T>A+NM_000500.9:c.923dupT",
                "NM_000500.9:c.92C>T+NM_000500.9:c.293-13C>G+NM_000500.9:c.332_339del+NM_000500.9:c.518T>A+NM_000500.9:c.710T>A+NM_000500.9:c.713T>A+NM_000500.9:c.719T>A+NM_000500.9:c.923dupT+NM_000500.9:c.955C>T"
            ],
            "variants": [
                {
                    "alleleId": "NM_000500.9:c.1360C>T",
                    "alleleCopyNumber": 2,
                    "genotypeQuality": 18,
                    "filter": "PASS"
                }
            ]
    }
}