# 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](https://www.illumina.com/science/genomics-research/articles/HBA-targeted-caller.html). ## 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 `.targeted.json` that also contains calls from other targets (see [Targeted JSON File](https://help.connected.illumina.com/dragen/dragen-v4.3/product-guide/dragen-v4.3/dragen-dna-pipeline/targeted-caller/..#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](https://help.connected.illumina.com/dragen/dragen-v4.3/product-guide/dragen-v4.3/dragen-dna-pipeline/targeted-caller/..#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 `.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" } ] } ``` --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. 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