HBA Caller

The HBA Caller is capable of genotyping the HBA1 and HBA2 genes from whole-genome sequencing (WGS) data. Due to high sequence similarity between the genes, a specialized caller is necessary to resolve the possible genotypes of the pair of genes. We consider regions surrounding the HBA1 and HBA2 sites to resolve the possible HBA1 and HBA2 genotypes.

The HBA Caller performs the following steps:

1. Determines total copy number from read depth of the regions surrounding the HBA1 and HBA2 sites.

2. Determines HBA genotypes based on the copy number of the regions surrounding the HBA1 and HBA2 sites.

3. Calls small variants in the HBA1 and HBA2 regions based on the region copy number derived from the genotype along with allele counts from read information.

The HBA 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.

For a comprehensive evaluation of the HBA caller, see HBA targeted caller blog post.

Total Copy Number of the regions sorrounding the HBA1 and HBA2 sites

The first step of HBA calling is to determine the copy number of the regions sorrounding the HBA1 and HBA2 sites. Reads aligned to the regions 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 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. Finally, a Gaussian Mixture Model (GMM) is used to obtain the integer region copy number from the region normalized counts.

Genotyping

The genotyping step attempts to identify the two likely haplotypes described in the following table, where "a" stands for a functional copy of either HBA1 or HBA2, "-" stands for a nonfunctional/missing copy of either HBA1 or HBA2, while "3.7" and "4.2" describe the recombinant event that likely caused the deletion/duplication of the functional HBA copy. The second column of the following table reports the interpretation of the genotype.

Genotype	Interpretation
aaa3.7/aa	alpha-globin triplication
aaa4.2/aa	alpha-globin triplication
aa/aa	Normal
-a3.7/aa	Silent Carrier
-a4.2/aa	Silent Carrier
--/aaa3.7	Carrier
--/aaa4.2	Carrier
-a3.7/-a3.7	Carrier
-a4.2/-a4.2	Carrier
-a3.7/-a4.2	Carrier
--/aa	Carrier
--/-a3.7	HbH
--/-a4.2	HbH
--/--	Hb Bart's

If none of the previous genotype is identified, then no call is made and the caller reports a "None" genotype.

Small Variant Calling

18 small variants are detected from the read alignments. These variants occur in homologous regions of HBA1 and HBA2 where reads mapping to either HBA1 or HBA2 are used for variant calling.

For each variant, reads containing either the variant allele or the nonvariant allele are counted and a binomial model is used to determine the likelihood for each possible variant allele copy number up to the maximum possible as determined from the HBA1/HBA2 genotyping.

HBA Output File

The HBA 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
genotype	The HBA genotype.	string
genotypeFilter	The HBA genotype filter.	string, [PASS, HBALowGQ, HBALowPValue, No_call]
genotypeQual	The HBA Phred genotype quality.	double
minPValue	The minimum copy number p-value of regions used to determine copy number genotype of the HBA locus.	double
variants	List of detected homology region variants in HBA1/HBA2.	Array of variants

Each 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

Structural variant and homology region 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 HBA caller content in the <output-file-prefix>.targeted.json output file is shown below.

  "hba": {
    "totalCopyNumber": 4,
    "genotype": "aa/aa",
    "genotypeQuality": 93,
    "genotypeFilter": "PASS",
    "variants": [
      {
        "alleleId": "NM_000517.6:c.95+1G>A",
        "alleleCopyNumber": 1,
        "genotypeQuality": 26,
        "filter": "PASS"
      }
    ]
  }