Assay for hemoglobin a (HBA) detection and genotyping

A computer-implemented method comprising:

• obtaining nucleic acid from a plurality of samples;
• performing a Hemoglobin A (HBA) assay on the nucleic acid to obtain raw data, wherein the HBA assay is performed using a plurality of probes capable of detection of copy number losses or gains in an α-globin gene cluster region of each sample of the plurality of samples, and the raw data comprises HBA copy number data for the plurality of probes;
• selecting, by a computing system, a reference sample from the plurality of samples, wherein the reference sample is selected based on variability among the plurality of probes and a determination of an absence of a copy number variation based on the raw data for the plurality of probes for the reference sample;
• calculating, by the computing system, a first set of probe ratios for each sample of the plurality of samples based on the raw data from the HBA assay and the reference sample;
• identifying, by the computing system, a predetermined number of reference samples to be combined as a synthetic reference sample for the plurality of samples based on the first set of probe ratios;
• generating, by the computing system, a list of the predetermined number of reference samples;
• generating, by the computing system, the synthetic reference sample as a concatenation of the predetermined number of reference samples in the list;
• calculating, by the computing system, a second set of probe ratios for each sample of the plurality of samples based on the raw data from the HBA assay and the synthetic reference sample;
• iteratively inputting, by the computing system, the second set of probe ratios for each sample into a decision tree matrix;
• determining, by the computing system using the decision tree matrix, an HBA genotype for each sample based on the second set of probe ratios for each sample and copy number calling thresholds for sample probe/reference probe ratios associated with each probe of the plurality of probes;
• determining, by the computing system, one or more particular copy number variations are assigned to one or more samples of the plurality of samples based on the HBA genotype determined for each of the one or more samples;
• and
• performing a confirmation test by multiplex assay methodology and gel electrophoresis or Sanger sequencing for a subset of samples, wherein each sample in the subset of samples is determined to have the one or more particular copy number variations.