Assay for hemoglobin a (HBA) detection and genotyping

Abstract

The present disclosure relates to a Hemoglobin A (HBA) assay and a HBA genotyping decision tree matrix for implementation into clinical testing. Particularly, aspects are directed to obtaining raw data from the HBA assay performed on a plurality of samples, calculating a first set of probe ratios for each sample based on the raw data, identifying a number of reference samples to be combined as a synthetic reference sample based on the first set of probe ratios, calculating a second set of probe ratios for each sample of the plurality of samples based on the raw data and the synthetic reference sample, and determining, by the decision tree matrix, a 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.

Description

(19) *EP004693299A2* (11) EP 4 693 299 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 11.02.2026 Bulletin 2026 / 07 (21) Application number: 25210108.4 (22) Date of filing: 05.03.2021 (51) International Patent Classification (IPC): G16B 20 / 00 (2019.01) (52) Cooperative Patent Classification (CPC): G16B 20 / 20; C12Q 1 / 6883; C12Q 2600 / 156; Y02A 50 / 30 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 06.03.2020 US 202062986152 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 21715371.7 / 4 114 976 (71) Applicant: Laboratory Corporation of America Holdings Burlington, North Carolina 27215 (US) (72) Inventors: • WALKER, Andrew Burlington, NC 27215 (US) • ROBINSON, Matthew Craig Burlington, NC 27215 (US) (74) Representative: Forresters IP LLP Skygarden Erika-Mann-Straße 11 80636 München (DE) Remarks: •This application was filed on 21.10.2025 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application / after the date of receipt of the divisional application (Rule 68(4) EPC). (54) ASSAY FOR HEMOGLOBIN A (HBA) DETECTION AND GENOTYPING (57) The present disclosure relates to a Hemoglobin A (HBA) assay and a HBA genotyping decision tree matrix for implementation into clinical testing. Particu- larly, aspects are directed to obtaining raw data from the HBA assay performed on a plurality of samples, calculat- ing a first set of probe ratios for each sample basedon the rawdata, identifying anumber of reference samples to be combined as a synthetic reference sample based on the first set of probe ratios, calculating a second set of probe ratios for each sample of the plurality of samples based on the raw data and the synthetic reference sample, and determining, by the decision treematrix, aHBAgenotype 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. EP 4 69 3 29 9 A 2 Processed by Luminess, 75001 PARIS (FR) Description PRIORITY CLAIM

[0001] This application claims the benefit of and priority to U.S. Provisional Application Number 62 / 986,152, filed on March 6, 2020, which is hereby incorporated by reference in its entirety for all purposes. FIELD

[0002] The present disclosure relates to Hemoglobin A (HBA) clinical testing, and in particular to techniques for a HBA assay and a HBA genotyping decision tree matrix for implementation into clinical testing. BACKGROUND

[0003] Hemoglobin isan iron-richprotein in redbloodcells that carriesoxygen tocells throughout thebody.Theprotein is composed of two α-globin and two β-globin subunits, the former coded for by the HBA1 and HBA2 genes, and the latter coded for by the HBB gene. When these genes are altered (changed) or missing, thalassemia occurs. The hemoglobin protein subunit affected in alpha thalassemia (α-thalassemia) is the alpha globin. People who inherit defective α- thalassemia gene that from one parent but and normal α-thalassemia genes from the other parent are carriers for α- thalassemia.Carriers ofα-thalassemia usually have no signs or symptoms.However, they canpass the faulty genes on to their children. People who have moderate to severe forms of α-thalassemia have inherited multiple faulty α-thalassemia genes from both parents. These are inherited in an autosomal recessive pattern. α-Thalassemia is the most common inherited disorder of hemoglobin synthesis in the world, with mutant allele frequencies varying between 1% and 98% throughout the tropics and subtropics, wheremalaria is endemic.α-Thalassemia canoccur in all ethnic groups but ismore common in thoseof Southeast Asian descent. The high prevalence ofα-thalassemia in specific populations is likely due to the protection from infection by the malaria parasite (multiple species of Plasmodium) that is offered by 1‑2 defective copies of HBA genes. The American College of Obstetricians and Gynecologists recommends hemoglobinopathy screening for those of African, Southeast Asian, Mediterranean, Middle Eastern or West Indian descent, though hemoglobinopathies are becoming more prevalent in admixed populations.

[0004] Alpha globin coded for by two genes (α-globin genes,HBA1 andHBA2) on chromosome16. Each person needs four functional HBA genes (two from each parent) to make enough α-globin for the body’s hemoglobin to work normally. Different formsofα-thalassemiaoccur if oneormoreof thesegenesaredefective. If onegene is defective, thenaperson is a "silent" carrier of theα-thalassemia trait and usually has no signs or symptoms. If two genes are defective, then a person hasα-thalassemia trait (alsocalledalpha thalassemiaminor) andmayhavemild anemia. If threegenesaredefective, then a person has hemoglobin H disease. This can cause moderate to severe anemia. If all four genes are missing, then a person has α-thalassemia major (also called hemoglobin Bart’s or hydrops fetalis). This is the most severe type of α- thalassemia. A fetus with this disorder will usually die in the womb or the baby will die soon after birth because the child is unable to make normal hemoglobin to carry oxygen throughout the body.

[0005] More than 90%ofα-thalassemia results from the deletion of twoormore copies of theα-globin genes (HBA1and HBA2) onchromosome16.TheHBA1andHBA2genesare locatedwithinan~30kbα-globingenecluster onchromosome 16, that includes the following alpha globin genes and (pseudogenes) from telomere to centromere in this order: HBZ, (HBZP1) .HBM, (HBAP1),HBA2,HBA1,HBQ1 (see, e.g., FIG. 1). The coding sequences ofHBA1andHBA2are identical with divergent sequences located in the introns and 5’‑ and 3’-untranslated regions. In addition, the deletion of the HS‑40 major hypersensitive site,which is located40kbupstreamof theHBZgene in thepromoter region, affectsRNAexpression of bothHEA1andHBA2, thereby causinganα-thalassemia trait in heterozygotes.. TheHbConstantSpringpointmutation at the first base of the termination codon inHBA2 affectsRNAexpression ofHBA2, and causing amore severe phenotype than aHBA2 deletion allele.. Lastly, gene conversions are common betweenHBA1 andHBA2 due to their close proximity andhighhomology, but havenoclinical significance. In viewof these factors, itmaybedesirable todevelopassays forHBA detection that are capable of genotyping multiple distinct loci of the α-globin region to easily screen for α-thalassemia. SUMMARY

[0006] In various embodiments, a computer-implemented method is provided that includes: obtaining raw data from a Hemoglobin A (HBA) assay performed on a plurality of samples, wherein the HBA assay is performed using a plurality of probes capable of detectionof copynumber lossesor gains in aα-globin genecluster regionof eachsample of theplurality of samples, and the raw data comprises HBA copy number data for the plurality of probes resolved by capillary electrophoresis for each sample of the plurality of samples; selecting a reference sample from the plurality of samples; calculating a first set of probe ratios for each sample of the plurality of samples based on the raw data from theHBA assay 2 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 and the reference sample; identifying 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 the synthetic reference sample based on the predetermined number of reference samples; calculating 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 the second set of probe ratios for each sample into a decision treematrix; determining, by the decision tree matrix, a 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; and providing the HBA genotype for each sample.

[0007] In someembodiments, wherein the calculating the first set of probe ratios comprises: (i) comparing control probe peakheights, or signals, in each sampleof theplurality of samples to corresponding control probepeakheights, or signals, in the reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals, in the reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metrics are greater than a predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metricsaregreater than thepredetermined threshold, and (v) for eachsample that doesnot fail, comparing test probepeak heights, or signals, in the sample to corresponding test probe peak heights in the reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the reference sample.

[0008] In some embodiments, the calculating the second set of probe ratios comprises: (i) comparing the control probe peakheights, or signals, in each sampleof theplurality of samples to corresponding control probepeakheights, or signals, in the synthetic referencesample, (ii) calculating variability in signals between the control probepeakheights, or signals, in each sample and the corresponding control probe peak heights, or signals in the synthetic reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metrics are greater than the predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the synthetic reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the synthetic reference sample.

[0009] In some embodiments, the determining the HBA genotype for each sample comprises: (i) determining aberrant probe ratio patterns for each sample based on the second set of probe ratios for each sample and the copy number calling thresholds for the sample probe / reference probe ratios associated with each probe of the plurality of probes, and (ii) identify the HBA genotype for each sample based on the aberrant probe ratio patterns.

[0010] In someembodiments, thedetermining theaberrant probe ratiopatternsand the identifying theHBAgenotype for each sample comprises: classifying each sample as normal, having a copy number variation (CNV), or as polymorphic basedon theaberrantprobe ratiopatterns; andsubclassifyinganysampleclassifiedashaving theCNVasa large targeted deletion, duplication or ’other’ based on the aberrant probe ratio patterns.

[0011] In someembodiments, thedetermining theaberrant probe ratiopatternsand the identifying theHBAgenotype for each sample further comprises: sub classifying any sample classified as having the large targeted deletion as a large heterozygousdeletionor ahomozygousdeletion; subclassifyinganysampleclassifiedas the largeheterozygousdeletion or the homozygous deletion for one ormore of the following deletions: SEA, FIL / THAI,MEDorα20.5 based on the aberrant probe ratio patterns; and sub classifying any sample classified as having the large heterozygous deletion and one ormore of the following: α3.7 deletion, , α4.2 deletion, or α3.7 duplication based on the aberrant probe ratio patterns.

[0012] In someembodiments, thedetermining theaberrant probe ratiopatternsand the identifying theHBAgenotype for each sample further comprises: sub classifying any sample classified as having ’other’; sub classifying any sample classified ’other’ as having α3.7 deletions, α4.2 deletions, and / or α3.7 duplications based on the aberrant probe ratio patterns.

[0013] In some embodiments, the method further comprises triggering performance of a confirmatory test on each sample of the plurality of samples that has the HBA genotype that is not normal or does not require manual review.

[0014] In some embodiments, a system is provided that includes one or more data processors and a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform part or all of one or more methods or processes disclosed herein.

[0015] In some embodiments, a computer-program product is provided that is tangibly embodied in a non-transitory machine-readable storage medium and that includes instructions configured to cause one or more data processors to perform part or all of one or more methods disclosed herein.

[0016] Some embodiments of the present disclosure include a system including one or more data processors. In some embodiments, the system includes a non-transitory computer readable storage medium containing instructions which, when executed on the one ormore data processors, cause the one ormore data processors to perform part or all of one or moremethods and / or part or all of one ormore processes disclosed herein. Some embodiments of the present disclosure 3 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 include a computer-program product tangibly embodied in a non-transitory machine-readable storagemedium, including instructionsconfigured tocauseoneormoredataprocessors toperformpart or all of oneormoremethodsand / orpart or all of one or more processes disclosed herein.

[0017] The termsandexpressionswhichhavebeenemployedare usedas termsof description andnot of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodi- ments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be better understood in view of the following non-limiting figures, in which: FIG. 1 shows gene data for chromosome 16 in accordance with various embodiments; FIG. 2 shows a block diagram of an HBA assay platform in accordance with various embodiments; FIG. 3A shows an overview of MLPA assay chemistry in accordance with various embodiments; FIG. 3B shows locations of 34 α-globin gene cluster region probes and common HBA deletions in accordance with various embodiments; FIG. 4 shows an exemplary flow for anHBAassay andHBAgenotyping using anHBAassay platform and genotyping techniques in accordance with various embodiments; FIG. 5 shows an exemplary flow for HBA genotyping using an HBA assay platform and genotyping techniques in accordance with various embodiments; FIG. 6 shows an exemplary computing device in accordance with various embodiments; and FIG. 7 shows a probe-by-probe plot of fold change relative to reference for an α3.7 HET deletion (sample B09) in accordance with various embodiments.

[0019] In theappendedfigures, similar components and / or features canhave the same reference label. Further, various components of the same type can be distinguished by following the reference label by a dash and a second label that distinguishes among the similar components. If only the first reference label is used in the specification, the description is applicable to any one of the similar components having the same first reference label irrespective of the second reference label. DETAILED DESCRIPTION

[0020] The ensuing description provides preferred exemplary embodiments only, and is not intended to limit the scope, applicability or configuration of the disclosure. Rather, the ensuing description of the preferred exemplary embodiments will provide thoseskilled in theartwithanenablingdescription for implementing variousembodiments. It is understood that various changesmay bemade in the function and arrangement of elementswithout departing from the spirit and scope as set forth in the appended claims.

[0021] Specific details are given in the following description to provide a thorough understanding of the embodiments. However, itwill beunderstood that theembodimentsmaybepracticedwithout these specific details. For example, circuits, systems, networks, processes, and other componentsmay be shownas components in block diagram form in order not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, struc- tures, and techniques may be shown without unnecessary detail in order to avoid obscuring the embodiments.

[0022] Also, it is noted that individual embodiments may be described as a process which is depicted as a flowchart, a flow diagram, a data flow diagram, a structure diagram, or a block diagram. Although a flowchart or diagrammay describe the operations as a sequential process, many of the operations may be performed in parallel or concurrently. In addition, theorder of theoperationsmaybe re-arranged.Aprocess is terminatedwhen its operationsare completed, but could have additional steps not included in a figure. A process may correspond to a method, a function, a procedure, a subroutine, a 4 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 subprogram, etc. When a process corresponds to a function, its termination may correspond to a return of the function to the calling function or the main function. I. Introduction

[0023] DNA analysis of the α-globin region can be performed by targeting multiple distinct loci using multiplex ligation- dependent probe amplification (MLPA). This methodology, developed by MRC Holland (Product Description SALSA® MLPA® probemix P140-C1 HBA), detects genomic deletions and duplications involving this locus, including the seven most common types of α-thalassemia deletions (α3.7, α4.2, SEA, MED, THAI, FIL, and α20.5), as well the Constant Spring point mutation and the HBAx HS‑40 promoter deletion. Conventionally the HBA MLPA copy number data is manually reviewed in order to determine genotype, due to the complexity of the α-globin region and number of loci analyzed by the MLPA assay. Confirmation testing may be performed bymultiplex PCR and gel electrophoresis or Sanger sequencing. A problem associated with conventional HBAMLPA assays andmanual genotyping is that they generate a large amount of MLPAcopy number data from theα-globin region and utilize complex tables that guide themanual evaluation of theMLPA copy number data, which require extensive training experience and time. Moreover, conventional HBA MLPA assays typically demonstrate higher error rates for evaluation steps taken duringmanual genotyping. This error rate trickles down andundesirably impacts thequality of thescreening test (e.g., falsepositivesor overdiagnosis) andmay incorrectly trigger costly confirmation testing.

[0024] Toaddress these limitations andproblems, various embodiments described herein are directed to anHBAassay and genotyping technique capable of reducing ambiguity in decision-making and providing a comprehensive analysis of the consequences of each possible decision while achieving minimal error rate, with limited processing, memory and power resources. In some instances, processes were developed that include gating whether or not confirmation testing should be performed on a sample based on whether a decision tree concludes the samples includes a HBA genotype indicative of α-thalassemia. For example, various embodiments of the present disclosure include a system including one or more processors and a memory coupled to the one or more processors. The memory is encoded with a set of instructions configured to perform a process including: obtaining raw data from a HBA assay performed on a plurality of samples, wherein the HBA assay is performed using a plurality of probes capable of detection of copy number losses or gains in a α-globin gene cluster region of each sample of the plurality of samples, and the raw data comprises HBA copy number data (e.g., MLPA copy number data) for the plurality of probes resolved by capillary electrophoresis for each sampleof theplurality of samples; selectinga referencesample from theplurality of samples; calculatingafirst 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 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 the synthetic reference sample based on the predetermined number of reference samples; calculating a second set of probe ratios for each sample of the plurality of samplesbasedon the rawdata from theHBAassay and the synthetic reference sample; iteratively inputting the second set of probe ratios for each sample into a decision treematrix; determining, by the decision treematrix, a 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; and providing the HBA genotype for each sample. In some instances the technique further includes triggering performance of a confirmatory test on each sample of the plurality of samples that has a HBA genotype that is not normal or does not require manual review.

[0025] Advantageously, these approaches provide an HBA assay and genotyping technique that are capable of achieving minimal error rate in an environment such as a sequencing system with limited processing, memory and power resources. For example, the decision tree can be implemented with a low power budget (e.g., a simple tree of conditionals), andprovidespowerful non-linear classificationcapabilitiesof amulti-dimensional searchspace.Further, the non-linear classification may be used to gate whether or not confirmation testing should be performed on a sample, and consequently saves on costs of erroneously performing confirmation testing and increases robustness of the overall screening HBA assay.

[0026] As used herein, the terms "substantially," "approximately" and "about" are defined as being largely but not necessarilywhollywhat is specified (and includewhollywhat is specified)asunderstoodbyoneofordinaryskill in theart. In any disclosed embodiment, the term "substantially," "approximately," or "about" may be substituted with "within [a percentage] of" what is specified, where the percentage includes 0.1, 1, 5, and 10 percent. As used herein, when an action is "based on" something, this means the action is based at least in part on at least a part of the something.

[0027] It will be appreciated that the HBA genotyping techniques disclosed herein can be applied to assess other types of sequencing raw data as compared to the MLPA copy number data specifically described herein. It will also be appreciated that other assay methodologies and types of polymerase chain reaction (PCR) or multiplex (PCR) are contemplated to identify one or more loci within a given chromosome or gene region (e.g., chromosome 16 or α-globin region). For example, alternatively or additionally, reverse Dot-Blot Hybridisation (RDB), Southern blot (SB), or Gap-PCR may be used to identify one or more loci within chromosome 16 or the α-globin region. 5 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 II. HBA Assay Techniques

[0028] One or more embodiments described herein can be implemented using programmatic modules, engines, or components.Aprogrammaticmodule, engine, or component can includeaprogram, asub-routine, a portionof a program, or a software component or a hardware component capable of performing one or more stated tasks or functions. As used herein, a module or component can exist on a hardware component independently of other modules or components. Alternatively, amodule or component canbeasharedelement or processof othermodules, programsormachines. FIG. 2 shows a block diagram of an a HBA assay platform 100 for detection of deletion(s) and / or duplication(s) in the α-globin region located on chromosome 16 as a potential cause for, screening tool, and / or clinical diagnosis ofα-thalassemia, and illustrates modules, engines, or components (e.g., program, code, or instructions) executable by one or more processors that may be used to implement the various subsystems of a analyzer system 205 according to various embodiments. The modules, engines, or components may be stored on a non-transitory computer medium. As needed, one or more of the modules, engines, or components may be loaded into system memory (e.g., RAM) and executed by one or more processors the analyzer system 205. In the example depicted in FIG. 2, modules, engines, or components are shown implementing gene analyzer subsystem 210 and HBA genotyping subsystem 215.

[0029] FIG. 2also illustratesawet lab subsystem220 includinga laboratorywhere chemicals, drugs, or othermaterial or biologicalmatter are tested andanalyzed requiringwater, direct ventilation, and specializedpipedutilities. TheHBAassay platform 200 includes obtaining one or more samples 225 at block 230 within the wet lab subsystem 220. In some instances, the samples 225 comprise nucleic acid extracted from human cell lines. In some instances, the samples 225 comprise nucleic acid obtained from a male or female patient. In some instances, the samples 225 are nucleic acid extracted fromwhole blood, amniotic fluid, amniotic fluid cell cultures, chorionic villus sampling, or chorionic villus sample cell cultures obtained from a male or female patient. In certain instances, the sample or samples 225 have one or more genomic deletion(s), point mutation(s) and / or duplication(s) within the α-globin gene cluster region of chromosome 16.

[0030] Atblock235within thewet labsubsystem220,anHBAAssay isperformed includingDNAanalysisof theα-globin gene cluster region (HBA1 / HBA2,OMIM 141800 / 141850,16pter‑16p13.3) is performed by targeting multiple distinct loci using a multiplex assay methodology 250 (e.g., multiplex PCR methodology). The multiplex assay methodology 250 detects genomic deletions andduplications involving this locus, including the sevenmost common types ofα-thalassemia deletions (α3.7, α4.2, SEA, MED, THAI, FIL, and α20.5), as well the Constant Spring point mutation and the HBAx HS‑40 promoter deletion. In certain instances, the HBA assay is used for: (i) a screening carriers of the α3.7, α4.2, SEA, MED, THAI,FIL,α20.5andHS‑40deletions, (ii) identificationofat-risk couplesbyscreeningpartnersof carriers for anyof theHBA mutations, and / or (iii) targeted familial testing and prenatal testing for HBA deletions in which one or both parents have been determined to carry a pathogenicmutation as described herein. Confirmation testingmay be performed bymultiplex assay methodology and gel electrophoresis or Sanger sequencing.

[0031] In various embodiments, the multiplex assay methodology 250 is MPLA technology (e.g., the MPLA methodol- ogy developed by MRC Holland - Product Description SALSA® MLPA® probemix P140-C1 HBA), which is a semi- quantitative, ligation-dependentmultiplexPCRmethod that candeterminecopynumberdifferences in the target regionby measuring the relative signal intensity between the sample or samples 225 and a synthetic reference sample 240. The HBAassayusesapool ofMLPAoligonucleotides245such that a probe is formedwhen twooligonucleotides that hybridize immediately adjacent to each other at a target sequence are ligated together. As shown in FIG. 3A, each ligated probe 300 may be made up of two oligonucleotides 305 (e.g., from the pool of oligonucleotides 245) of which one end comprises a hybridization sequence 310 specific for a target sequence and another end comprises a PCR primer 315 (e.g., a labeled universal PCR primer) sequence for multiplexing. In certain instances, stuffer sequences 320 of different lengths may be used to allow for distinct probe sizes. After sample and reference denaturation, the oligonucleotides 305 are hybridized to the target sequences for a predetermined amount of time (e.g., 16‑20 hours). Following hybridization, the two oligonu- cleotides 305 that hybridize to immediately adjacent sites are ligated together to form uniquely sized ligated probes 300. This reaction ishighly specificandoccurswhen therearenogapsbigger thananickbetween the twooligonucleotides305. The ligated probes 300 are PCR amplified using the PCR primers 315. Ligated probes 300 that bind poorly to their target sequence or not at all due to SNPs or absence of target sequence will not amplify.

[0032] In some embodiments, theHBAassay includes the use of 45 target-specific probes (amplification sizes 131‑481 bases) that include 33 probes for detection of deletions and duplications in the α-globin gene cluster region and surrounding sequences and 1 probe for presence of the Hb Constant Spring point mutation. Of the 33 probes, there are 5 single probes that are specific for eitherHBA1orHBA2and3probes that target sequences in that are present in both genes; i.e., in a normal sample, 2 or 4 copies would be detected by a probe mapping to a unique sequence or a probe mapping to a sequencepresent in bothHBA1andHBA2, respectively. Due to thehighhomology and closeproximity of the twogenes, therearealso5probepairs that bind to different sequences in thesame locationsof theHEA1andHBA2genes such that one probe in a pair is specific forHBA1 and the other probe is specific forHBA2. These probe pairs are used to detect gene conversions, or polymorphisms, that have no clinical significance but could confound the genotyping call. When a polymorphism is present, the fold change for one probe in the pair would indicate a duplication, while the other, a 6 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 deletion. The 45 target-specific probes further include 11 control probes that target reference sequences on other autosomal chromosomes besides chromosome 16, and are used for normalization of the 33 probes for detection of copy number changes in the HBA region. In some instances, quality control (Q‑ and D-fragments) and gender-specific fragments, which are single oligonucleotides, may be included to ensure that the MLPA chemistry worked as expected. Locations of probes that detect variants in the α-globin gene cluster region and surrounding sequences as well as the locations for the targeted deletions in this test are schematically shown in FIG. 3B. Shown are locations for the paired probes (P) that candetect polymorphismsdue togeneconversionsbetweenHBA1andHBA2, theprobesmapping to both HEA1 andHBA2, (bold arrows), and the probe for Hb Constant Spring (CS). The FIL and THAI deletions are detected by thesameprobesand requiremultiplexPCR fordifferentiation. In some instances, thedeletionboundaries forα3.7,α4.2 and MED can vary and their detection may involve different probes.

[0033] At optional block 255, post-PCR cleanup may be performed on the PCR products (e.g., the amplified ligated probes). In some instances, thepost-PCRcleanup includesmixing thePCRproductswithmagnetic beads,washingwitha wash solution such as 70% ethanol, air drying, and eluting purified PCR products 260 to boost the signal-to-noise ratio. After amplification andpost-PCRcleanup, thepurifiedPCRproducts 260maybe loadedonto analyzer system205 (e.g., a fluorescence-based separation instrument system) for downstream processing.

[0034] At block 265, the multiplex assay products or optional purified PCR products 260 are resolved using capillary electrophoresis, detectedusing the label, e.g. a fluorescent dye, andoutput as rawdata 270comprisingHBAcopynumber data.. In some instances, the raw data 270 may be generated, collected, and stored on the analyzer system 205 in a memory storage device. At block 275, the analyzer subsystem 210 obtains the raw data 270 for each sample from the analyzer system 205, determines sizing of the amplicons, and automatically selects a normalized sample with the least variability in peak height ratios and no detected deletions / duplications as a reference sample. The analyzer subsystem 210 normalizes the raw peak signals for each test probe (normal probe) to that of control probes in order to minimize any preferential amplification of smaller fragments. For example, the control probe peak heights, or signals, in a test sample maybecompared to thecorrespondingcontrol probepeakheights in the referencesample.Variability in the ratioof signals between the twoare calculatedas the control probe standard deviation, and valuesof the control probe standard deviation greater than a predetermined threshold (e.g., 0.125 threshold)may cause the sample to fail. The analyzer subsystem210 may also check the quality control D‑ and Q-fragments to ensure that the PCR reaction meets predetermined quality criteria and anything outside the predetermined quality criteria may cause the sample to fail.

[0035] The normalized probe peak signals of test probes for the samples that did not fail are then compared to the peak signals for the corresponding test probes in the reference sample. Relative probe ratios, or fold changes between the sample and reference probe signals are then calculated by the analyzer subsystem 210, and any changes to the sample probe / reference probe ratio that are outside a normal range (e.g., copy number calling threshold)may be identified by the analyzer subsystem 210 as a deletion or duplication. Thus, the relative probe ratio, or fold change, of a sample with no deletions or duplications relative to a reference sample is ~1, and a decrease or increase in the probe ratio beyond a copy numbercalling thresholdmaybe interpretedasadeletionorduplication, respectively, of the target sequence.Theanalyzer subsystem 210 may generate and export an initial results file including the relative probe ratio, or fold change, of each sample 225.

[0036] At block 280, the relative probe ratio data calculated on the analyzer subsystem 210 is analyzed by the HBA genotyping subsystem 215 for aberrant probe ratio patterns based on deletion(s) and / or duplications(s) to identify genotypes targeted for testing. More specifically, the HBA genotyping subsystem 215 is configured to serve several purposes: a) to check the data quality; b) to select samples for creating a synthetic referencewith a negative selector tool; and c) to sequentially parse the relative probe ratio data calculated by the analyzer subsystem210 through a decision tree matrix for genotype calling. Tomaximize the number of tests that can be run in a batch and since ~85‑90% of the samples for carrier screening will be negative for an HBA deletion or duplication, the negative selector tool of the HBA genotyping subsystem 215 was developed to provide a technical advantage to identify three to six best quality samples that have the best quality metrics and are negative for any copy number variation (no deletions / duplications) for the control sample selection application in the analyzer subsystem 210. The three to six best quality samples are then communicated and / or selected by the control sample selection application in the analyzer subsystem 210, and the analyzer subsystem 210 combines the three to six best quality samples to create a synthetic reference sample. The relative probe ratio, or fold change for each sample 225 are then recalculated by the analyzer subsystem 210 using this synthetic reference sample. The recalculation takes intoaccount samples fromall parts of theplate, so inaddition tomaximizing thenumberof samples that can be run per plate, it the synthetic reference sample normalizes fold-change ratios across the plate, making it less likely that samples will fail. The analyzer subsystem 210 may generate and export a new results file including the new calculated relative probe ratio, or fold change of each sample 225. The HBA genotyping subsystem 215 uses these new calculated relative probe ratios, or fold changes and the decision treematrix to classify and / or identify the HBAgenotypes in each sample 225.

[0037] In various embodiments, after checking the new calculated relative probe ratios, or fold changes for data quality, the HBA genotyping subsystem 215 uses copy number calling thresholds to identify regions of copy number losses or 7 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 gainswith probeswhich are involved inmultiple abnormal genotypes and sorts each sample into a normal, polymorphic or copy number variation (CNV) category. The CNV samples may then be classified as either large targeted deletions, duplications or other. Samples in the large targeted deletion group are determined for zygosity and further classified as SEA, FIL / THAI, MEDorα‑20.5. Samples that are heterozygous are further checked for theα3.7 and α4.2 deletions. Those in the ’other’ category may also be checked for the α3.7 and α4.2 deletions, and if either are detected, for zygosity. At this point, all samples should have been classified as either a targeted genotype or ’other’. The HBA genotyping subsystem 215 then compares the combination of probes against a table that has each possible scenario to identify compound heterozygotes for the targeted deletions and duplications. All samples may also be analyzed by the HBA genotyping subsystem 215 for the HS‑40 deletion and the Hb Constant Spring point mutation, and any positive results are concatenated onto the previously determined genotype. Samples that are still classified as ’other’ are flagged for review by a healthcare professional such as a clinical director, thereby ensuring that the HBA genotyping subsystem 215 will not miss any clinically important results, targeted or not.

[0038] TheHBAgenotype of each sample and an optional risk result for each sample are output by the analyzer system 205 as a final result 285. In some instances, all threshold and QC parameters and the decision tree matrix used by the analyzer subsystem 205 and the HBA genotyping subsystem 215 are maintained in one or more separate configuration files and can be used across any number of HBA PCR assays. III. HBA Genotyping Techniques

[0039] FIG.4 illustratesaprocess400 forHBAgenotypingusingaHBAassayplatformandgenotyping techniques (e.g., theHBAassayplatform100describedwith respect toFIG.1).Process400beginsat block405,where rawdata isobtained from aHBAassay performed on a plurality of samples. In some instances, the HBAassay is performed using a plurality of probes (e.g., ligated probes) capable of detection of copy number losses or gains (e.g., deletions, duplications and Hb ConstantSpringPointMutation) in theα-globin genecluster regionof each sampleof theplurality of samples. Theplurality of probesmay include oneormore control probes andoneormore test probes. In some instances, the rawdata comprises HBA copy number data for the plurality of probes resolved by capillary electrophoresis for each sample of the plurality of samples.At block410,a referencesample is selected from theplurality of samples.The referencesamplemaybeselected to obtain a reference sample that has the least variability among the plurality of probes andno copy number variation (e.g., no deletion(s) and / or duplication(s)) based on the raw data for the plurality of probes.

[0040] In some instances, prior to selectionof a referenceprobe, thequality of the rawdata is checked.Thequality check of the raw data may include confirming quality of the raw data using one or more parameters listed in Table 1. In some instances, one ormore of the following quality checks are performed and the quality of the raw data is assessed based on the result(s) of the one or more quality checks: (i) a total probe number being equal to a predetermined number of total probessuchas45, (ii) a test (normal) probenumberbeingequal toapredeterminednumberof test probessuchas33, (iii) a control probe number being equal to a predetermined number of control probes such as 11, (iv) peak signals being greater than or equal to a predetermined peak height threshold (e.g., greater than or equal to 200RFUafter normalization), (v) the Q-frgaments (single oligonucleotides (not ligatedprobes) thatwill preferentially amplifywhen theDNAamount is too lowor ligation fails) represent ≤ a predetermined threshold, e.g., 33% of signal for 92 nt benchmark fragment, and (vi) the D- fragments (single oligonucleotides (not ligated probes) that will preferentially amplify when the denaturation reaction is incomplete) represent ≤ a predetermined threshold, e.g., 50%of signal for 92 nt benchmark fragment.When the quality of the raw data fails, then the processmay stop and optionally request the HBA assay be performed again to obtain new raw data. When the quality of the raw data passes, then the process may continue. Table 1: Quality Metrics and Thresholds used in this Study Parameter Description / Purpose Threshold Total Probe Number All (normal and control) probes in the assay; used to iden- tify normal samples (0 CNV probes) Must equal 45 Normal Probe Number Normalized probes that target the α-globin gene cluster region and surrounding sequences Must equal 33 Control Probe Number Control probes that target stable (0 CNV) sequences on autosomal chromosomes besides chr 16; used to normal- ize signals within a sample and assess normalized probe data between a sample and reference Must equal 11 Peak Height Threshold Minimum RFU threshold of fluorescent signal peak height required for detection ≥200 RFU after normalization 8 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Parameter Description / Purpose Threshold MLPA Lane Score Signal-to-noise quality of the normalized traces >10 Q-fragments Single oligonucleotides (not ligated probes) that will pre- ferentially amplify when the DNA amount is too low or liga- tion fails ≤ 33% of signal for 92 nt benchmark fragment D-fragments Single oligonucleotides (not ligated probes) that will pre- ferentially amplify when denaturation reaction is incomplete ≤ 50% of signal for 92 nt benchmark fragment Copy Number Calling Threshold Relative signal between normalized sample probe and corresponding reference probe; used to detect deletions and duplications; also referred to as relative probe ratio or fold change v. reference in this document Deletion<0.75<Normal<1.30<Dupli cation Copy Number Calling Threshold for Duplicated Probes Same as copy number calling threshold except 4 copies detected in a normal sample due to duplicated probes re- cognizing homologous sequences in HBA1 and HBA2 Deletion<0.87<Normal<1.12<Dupli cation Control Probe Standard De- viation Measure of control probe variability in the reference sample when compared to the corresponding control probe in the test sample < 0.125 Hb Constant Spring detec- tion A signle probe is used to detect presence of the mutation; does not determine zygosity Constant Spring mutant probe peak height ≥10% of mean peak height of all control probes in a sample

[0041] At block 415, a first set of probe ratios are calculated for each sample of the plurality of samples (in some instances, the first set of probe ratios is not calculated for the reference sample). In some instances, the calculating the first set of probe ratios comprises: (i) comparing the control probe peak heights, or signals, in each sample to corresponding control probe peak heights in the selected reference sample, (ii) calculating variability in the signals between the control probe peak heights, or signals, in each sample and the control probes of the reference sample as the control probe standard deviation, (iii) determining a sample of the plurality of samples fails when the control probe standard deviation is greater than or equal to a predetermined threshold (e.g., ≥ 0.125 threshold), (iv) determining a sample of the plurality samples does not fail when the control probe standard deviation is less than a predetermined threshold (e.g., < 0.125 threshold), and (v) for each sample that does not fail, comparing the test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the selected reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights in the selected reference sample.

[0042] At block 420, an initial results file is generated and output that includes the first set of probe ratios calculated for each sample. Thefirst set of probe ratios includes the relative probe ratio, or fold change, of each sample calculated in step 415 that did not fail (and optionally is not the reference sample). At block 425, the initial results file is accessed and the first set of probe ratios for the samples is parsed to identify a predetermined number (e.g., between three and six) of reference samples to be combined as a synthetic reference sample for the plurality of samples. In certain instances, the reference samples are identified based on qualitymetrics and copy number variation. For example, the identified reference samples should be negative for any copy number variation (e.g., have no probes out of normal range based on the copy number calling thresholds, and thusnodeletionsor duplications), passall probenumbermetrics, andhave the lowest control probe standard deviation metrics on the plate of samples. A list may be generated that comprises the identified reference samples and a synthetic reference sample file is generated and output that includes the list of the identified samples. At block 430, the synthetic reference sample file is accessed or uploaded to a computing system, and a synthetic reference sample is generated based on the identified reference samples within the list. In some instances, the synthetic reference sample is createdasa functional concatenationof peakheights, or signals, for eachof the identified sampleswithin the list. For example, the synthetic reference samplemay be amean,median, or mode of the peak heights, or signals, for each of the identified samples within the list.

[0043] At block 435, a second set of probe ratios are calculated for each sample of the plurality of samples. In some instances, the calculating the second set of probe ratios comprises: (i) comparing the control probe peak heights, or 9 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 signals, in each sample to corresponding control probe peak heights in the synthetic reference sample, (ii) calculating variability in the signals between the control probe peak heights, or signals, in each sample and the control probes of the synthetic reference sample as the control probe standard deviation, (iii) determining a sample of the plurality of samples fails when any variability metrics are greater than or equal to a predetermined threshold (e.g., ≥ 0.125 threshold), (iv) determining a sample of the plurality of samples does not fail when none of the variabilitymetrics are greater than or equal to a predetermined threshold (e.g., ≥ 0.125 threshold), and (v) for each sample that does not fail, comparing the test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the synthetic reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights in the synthetic reference sample.

[0044] At block 440, a new results file is generated and output that includes new relative probe ratio data for each sample. The new relative probe ratio data includes the relative probe ratio, or fold change, of each sample calculated in step435 that did not fail. At block 445, thenew results file is accessedand thenew relativeprobe ratio data for eachsample is iteratively input into a decision tree matrix to: (i) determine aberrant probe ratio patterns for each sample based on deletion(s) and / or duplications(s), and (ii) identify genotypes targeted for testing based on the aberrant probe ratio patterns. The determination of the aberrant probe ratio patterns and identification of genotypes targeted for testing may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletion(s) and / or duplications(s)) based on the new relative probe ratio data for the plurality of probes and normal ranges or the copy number calling thresholds for the sample probe / reference probe ratios associated with each probe. For example, any changes to the sample probe / reference probe ratios that are outside a normal range (e.g., the copy number calling threshold) are identifiedasadeletionor duplicationandmaybeused to identify oneormore regionsof copynumber losses or gains. The determination of the aberrant probe ratio patterns may further comprise classifying the sample as normal, having a copy number variation (CNV), or as polymorphic based on the identified one or more regions of copy number losses or gains. For example, if a sample has no probes out of normal range, then the samplemay be classified as normal. If a samplehasoneormoreprobesoutof normal range, then thesamplemaybeclassifiedasCNV.For sampleswithoneor more probes out of normal range, if none of the probes out of normal range are within a subset of probes indicative of deletions (e.g., a subset of deletion probes identified as being important for calling a CNV), then the sample may be classified as polymorphic.

[0045] The determination of the aberrant probe ratio patterns and identification of genotypes targeted for testing may further comprise sub classifying the sample classified as having a CNVas large targeted deletions, duplications or ’other’ basedon the identifiedoneormore regionsof copynumber lossesor gains. For example, if a sample classifiedashavinga CNV has one or more probes indicative of large targeted deletions that are out of normal range, then the sample may be further classified as having large targeted deletions. If a sample classified as having a CNV has one or more probes indicative of duplications that are out of normal range, then the samplemaybe further classifiedas havingduplications. If a sample classified as having aCNVdoes not have one ormore probes indicative of large targeted deletions or duplications that are out of normal range, then the sample may be further classified as ’other’.

[0046] The determination of the aberrant probe ratio patterns and identification of genotypes targeted for testing may further comprise determining deletion status and zygosity and duplication status for samples in the CNV group (e.g., homozygous or heterozygous deletion) based on one or more probes indicative of zygosity, and further classifying the samples for one of the following deletions, or a duplication: α3.7 and α4.2 based on one or more probes indicative of deletions α3.7 and α4.2 that are out of normal range. Large deletions defined as deletions that encompass both theHBA1 and HBA2 genes. Samples in the large targeted deletion group may be determined for zygosity (e.g., homozygous or heterozygous large deletions) based on one or more probes indicative of zygosity, and further classified for one of the following deletions: SEA, FIL / THAI, MED or α20.5, or the α3.7A / α4.2C deletion of indeterminate phasing based on one or moreprobes indicativeofdeletionsSEA,FIL / THAI,MED,α20.5α3.7A / α4.2C thatareout of normal range.Samplesclassified as heterozygous large deletions may be further classified for the smaller α3.7 and α4.2 deletions based on one or more probes indicative of deletionsα3.7 andα4.2 that are out of normal range. At this point, a sample should havebeen classified aseither a targetedgenotypeor ’other’. Examplesof targetedgenotypesa result of this process, the result implication, and possible clinical interpretation are provided in Table. 2. Table 2: Possible MLPA Results and Their Implications for Mutations Targeted in the Test Result* Genotype Result Implication Clinical Interpretation No deletions or CS point muta- tion (Normal) αα / αα 2 copies ofHBA1 andHBA2 present and expressed normally NEG Polymorphism αα / αα 2 copies ofHBA1 andHBA2 present and expressed normally NEG 10 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Result* Genotype Result Implication Clinical Interpretation Additional copy of α4.2 or α3.7 ααα / αα 3 copies of HBA1 and 2 copies of HBA2 present OR 3 copies of HBA2 and 2 co- pies of HBA1 present NEG HET, α3.7 deletion or α4.2 dele- tion -α / αα 1 copy of either HBA1 or HBA2 Silent carrier HS‑40 promoter deletion αα / αα 2 copies each of HBA1 and HBA2 pre- sent but low / no expression forHBA1 and HBA2 Carrier withα-thal trait HOM of either α3.7 deletion or α4.2 deletion -α / -α 0 copies of either HEA1 or HBA2 Carrier withα-thal trait HET α‑3.7 deletion in trans α4.2 deletion -α / -α 1 copy each of HBA1 and HBA2 in trans Carrier withα-thal trait HET SEA, FIL, THAI, MED or α20.5 deletion -‑ / αα 1 copy each of HBA1 and HBA2 in cis Carrier withα-thal trait HET CS SNV ααCS / αα Low expression of HBA2 on one chro- mosome Carrier withα-thal trait HOM CS SNV ααCS / ααCS No or very low expression of HBA2 Carrier withα-thal trait HET α3.7 or α4.2 deletion AND HET SEA, FIL, MED, THAI, or α20.5 -‑ / ‑ α 1 copy of HEA1 or HBA2 Affected with HbH disease SEA, FIL, THAI, MED or α20.5 deletion in trans with Constant Spring SNV -‑ / αCSα 1 copy of HBA1 and CS point mutation in cis Affected with HbH disease HOM SEA, FIL, THAI, MED or α20.5 deletion -‑ / -- 0 copy of HEA1 and HBA2 Affected with Hb Bart’s hydrops fetalis HET SEA, FIL, THAI, MED or α20.5 deletion in any combination -‑ / -- 0 copy of HEA1 and HBA2 Affected with Hb Bart’s hydrops fetalis HET SEA, FIL, THAI, MED or α20.5 deletion in trans with HET HS‑40 deletion -‑ / αα 1 copy each of HBA1 and HBA2 in cis expressed at low or no levels Affected with Hb Bart’s hydrops fetalis *Note: The MLPA technology cannot detect copy number neutral inversions or translocations. This table lists sce- narios only for the targeted mutations tested and does not include situations that would trigger review by a health- care provider such as a clinical director. HET, heterozygous; HOM, homozygous; CS SNV, Constant Spring single nucleotide variant

[0047] The determination of the aberrant probe ratio patterns and identification of genotypes targeted for testing may further comprise comparing the new relative probe ratio data against a table that has every possible scenario to identify non-target genotypes such as compound heterozygotes for the targeted deletions and duplications. Samples that are still classified as ’other’ after the table comparison may be flagged for review by a healthcare provider such as a clinical director, thereby ensuring that the genotyping technique will not miss any clinically important results, targeted or not. Moreover, all samples (those classified as normal, CNV, and polymorphic) may also be analyzed for the HS‑40 deletion and the Hb Constant Spring point mutation based on one or more probes indicative the HS‑40 deletion and the Hb Constant Spring point mutation, that are out of normal range, and any positive results may be concatenated onto the previously determined targeted or non-targeted genotype.

[0048] Optionally at block 450, the genotype of each sample determined in block 445 may be used to determine a risk score(s) for asubject associatedwith thesample.The riskscore(s)may identify: (i) a riskof thesubject beingacarrier of the α3.7, α4.2, SEA, MED, THAI, FIL, α20.5 and HS‑40 deletions and Hb Constant Spring point mutation, (ii) a risk for couples 11 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 identifiedasbeingacarrier of theα3.7,α4.2, SEA,MED,THAI, FIL,α20.5 andHS‑40deletionsandHbConstantSpringpoint mutation, and / or (iii) a risk of a fetus inheriting the α3.7, α4.2, SEA, MED, THAI, FIL, α20.5 and HS‑40 deletions and Hb Constant Spring point mutation. At block 455, the genotype of each sample determined in block 445 and optional risk score(s) determined in block 450maybeoutput. Theoutput of the genotypeof each sample andoptional risk score(s)may comprise providing an end user with the output and / or recording the output in a storage device (e.g., displaying the output on a user interface and / or storing the output in a results file of a database). At block 460, when the genotype of the sample determined in block 445 indicates one or more of the α3.7, α4.2, SEA, MED, THAI, FIL, α20.5 and HS‑40 deletions and Hb Constant Spring point mutation are assigned to the sample, confirmation testing may be performed on the sample. Confirmation testing may be performed by multiplex assaymethodology and gel electrophoresis or Sanger sequencing. When the genotype of the sample is undetermined in block 445,manual reviewmay be performed for the sample. At block 465, when the genotype of the sample determined in block 445 indicates a normal classification is assigned to the sample, processing of a sample is stopped. As such, the non- linear classificationof thedecision treematrixmaybeused togatewhetherornot confirmation testingshouldbeperformed onasample,andconsequently savesoncostsof erroneouslyperformingconfirmation testingand increases robustnessof the overall screening HBA assay.

[0049] FIG.5 shows a decision tree matrix 500 illustrating genotyping techniques that may be implemented to perform HBA genotyping of one or more samples. At block 502, a new results file is accessed and raw data including HBA copy numberdata for eachsampleof aplurality of samples is iteratively input intoadecision treematrix to: (i) determineaberrant probe ratio patterns for each sample based on deletion(s) and / or duplications(s), and (ii) identify genotypes targeted for testing based on the aberrant probe ratio patterns. The raw data includes new relative probe ratio data (as discussed with respect to steps 435, 440, and 445 in FIG. 4) for each sample obtained based on a synthetic reference sample (as discussedwith respect to steps425and430 inFIG.4).Thenewrelativeprobe ratiodata includes the relativeprobe ratio, or fold change, of each probe of a plurality of probes used to analyze the α-globin gene cluster region of each sample of the plurality of samples. In some instances, the new relative probe ratio data is obtained fromanHBAassayperformedusing a plurality of probes (e.g., ligated probes), which are capable of detection of deletions, duplications andHbConstant Spring Point Mutation in the α-globin gene cluster region of each sample of the plurality of samples, as discussed with respect to FIGS. 1 and 4. Table 3 provides an exemplary list of a plurality of probes that may be used to perform the HBA assay and genotyping. The plurality of probes include one ormore control probes (e.g., Ctrl_5q31) and one ormore test probes (e.g., HBA_HBA1). Table. 3: List of Probes - Total of 45 probes including 11 control probes and 34 test probes Manufacturer Probe Number Probe Name HG19 coordinates Probe Number 11435-L12163 *Ctrl_1q41 chr1:215813987‑215814050 C05 15318-L17117 *Ctrl_2q33 chr2:200188562‑200188633 C11 03272-L02709 *Ctrl_3q29 chr3:193412522‑193412595 C09 03075-L19996 *Ctrl_5p15 chr5:9437899‑9437965 C06 00797-L13645 *Ctrl_5q31 chr5:132009710‑132009772 C01 07641-L07326 *Ctrl_8p23 chr8:11612591‑11612664 C02 05846-L11214 *Ctrl_10q26 chr10:131557466‑131557538 C03 00547-L00116 *Ctrl_11q22 chr11:102220951‑102221012 C08 11331-L12056 *Ctrl_12p13 chr12:6101020‑6101086 C04 03250-L02687 *Ctrl_13q14 chr13:52511732‑52511800 C07 07607-L07292 *Ctrl_15q26 chr15:99482536‑99482612 C10 19236-L25316 POLR3K_TeloHBA chr16:97132‑97217 1 04799-L04797 HS‑40_HS‑40 chr16:163528‑163595 2 04800-L04175 HS‑40_HS‑40_2 chr16:163685‑163749 3 04926-L23886 HBA_HBZ_2 chr16:193637‑193702 4 04622-L04001 HBA_HBZ chr16:199336‑199407 5 17214-SP0457-L20489 HBA_HBZ_3 chr16:202592‑202698 6 12 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Manufacturer Probe Number Probe Name HG19 coordinates Probe Number 04624-L04004 HBA_HBZHBZP1 chr16:209467‑209540 7 04637-L04018 HBA_HBAP2HBAP1 chr16:217274‑217344 8 18097-L22521 HBA_HBA2_4 chr16:219798‑219876 9 18090-L08415 HBA_HBA2_3 chr16:220313‑220396 10 18098-L22522 HBA_HBA2 chr16:221951‑222031 11 18092-L22516 HBA_HBA2_2 chr16:222190‑222260 12 18099-L22524 HBA1HBA2 ex1 2 chr16:222841‑222896 13 18881-L06288 HBA1HBA2 ex1 chr16:222921‑222988 14 08498-L08422 HBA2_intron2 chr16:223362‑223420 15 04633-L23748 HBA2_intron2_2 chr16:223427‑223490 16 15857-L21812 HBA1HBA2 ex3 chr16:223484‑223544 17 S0585-SP0043-L09493 HBA2_mutation chr16:223569‑223657 18 18096-L22520 HBA_HBA1_2 chr16:224084‑224160 19 18880-L24428 HBA_HBA1_3 chr16:224602‑224690 20 08494-L08417 HBA_HBA1_5 chr16:225160‑225221 21 14855-L23604 HBA_HBA1_8 chr16:225728‑225789 22 18093-L22517 HBA_HBA1_4 chr16:226018‑226093 23 18099-L22524 HBA1HBA2 ex1 2 chr16:226644‑226700 13 18881-L06288 HBA1HBA2 ex1 chr16:226725‑226792 14 08498-L21607 HBA1_intron2 chr16:227166‑227223 26 04633-L23600 HBA1_intron2_2 chr16:227237‑227301 27 15857-L21812 HBA1HBA2 ex3 chr16:227295‑227353 17 08499-L23594 HBA_HBA1 chr16:227572‑227638 29 04638-L23602 HBA_HBA1_6 chr16:227890‑227962 30 04639-L04020 HBA_HBA1_7 chr16:229744‑229814 31 19233-L25313 HBQ1_ex3 chr16:231170‑231236 32 15859-L21960 LUC7L_CentroHBA chr16:256304‑256385 33 17227-L20554 ITFG3_CentroHBA chr16:289853‑289926 34 18102-L20488 RGS11_CentroHBA chr16:321757‑321830 35 17212-L13393 AXIN1_CentroHBA chr16:338106‑338166 36 17613-L23601 DECR2_CentroHBA chr16:457523‑457589 37 probes that map specifically to HBA1 or HBA2, potentially indicating a gene conversion probes that map to both HBA1 and HBA2

[0050] At block 504, a quality checkmay be performed by the decision treematrix to confirm that the raw data is valid for genotyping analysis. The quality check of the raw data may include the decision tree matrix confirming quality of the raw data using one or more parameters listed in Table 1. In some instances, one or more of the following quality checks are performed and the quality of the raw data is assessed based on the result(s) of the one or more quality checks: (i) a total probe number being equal to a predetermined number of total probes such as 45, (ii) a test (normal) probe number being equal to a predetermined number of test probes such as 33, (iii) a control probe number being equal to a predetermined 13 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 number of control probes such as 11, (iv) peak signals being greater than or equal to a predetermined peak height threshold (e.g., greater than or equal to 200 RFU after normalization), (v) the Q-fragments (single oligonucleotides (not ligatedprobes) thatwill preferentially amplifywhen theDNAamount is too lowor ligation fails) represent≤apredetermined threshold, e.g., 33% of signal for 92 nt benchmark fragment, (vi) the D-fragments (single oligonucleotides (not ligated probes) that will preferentially amplify when the denaturation reaction is incomplete) represent ≤ a predetermined threshold, e.g., 50% of signal for 92 nt benchmark fragment, and (vii) any deviations in the control probe standard deviationgreater thanapredetermined threshold (e.g., 0.125 threshold).When thequalityof the rawdata fails, theprocess stops at block 506, and optionally a request is issued to perform the HBA assay again to obtain new raw data. When the quality of the raw data, the process continues at block 508.

[0051] At block 508, a classification of the sample being normal or having a CNV is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associatedwith aCNV. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a CNV based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for all probes listed in Table. 3, (ii) the copy number calling threshold is ≥ 0.75and≤1.3 forpredicting thesampleasnormal, and (iii) thecopynumbercalling threshold is<0.75or≥1.3 for predicting the sample as having a CNV. If a result of the analysis is the sample is predicted as being normal, then the sample is classified as normal. If a result of the analysis is the sample is predicted as having a CNV, then the sample is classified as having aCNV.When the sample is classified as normal, the process continues at block 510.When the sample is classified as having a CNV, the process continues at block 512.

[0052] At block 510, for any sample having a "normal" or "polymorphism" classification check 13<0.85, 14<0.85, 17<0.85. If true, thenaberrant probesaredetectedand the sample is re-classifiedasbeingHaberrant,whereH=3. This is essentially a check on repeated probes 13, 14, 17 to determine whether repeated probes 13, 14, 17 are lower than expected. This could indicate a deletion not detected by probes 11, 21, or 22 due to subpar performance. If any of the probes have a probe ratio >0.85, the sample is classified as normal. When the sample is classified as normal or classified as H=3 as the genotype, the process continues at block 540.

[0053] At block 512, a classification of the sample having a large deletion is determined based on sample probe / r- eference probe ratios indicative of an aberrant probe ratio pattern associated with a large deletion. A large deletion encompassesboth theHBA1andHBA2genes,whereasa small deletion contains onlyHBA1orHBA2. Thedetermination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a large deletion based on the new relative probe ratiodataandnormal ranges (copynumbercalling thresholds) for thesampleprobe / referenceprobe ratiosassociatedwith one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for theanalysis include: (i) the relativeprobe ratiodata forafirst set of probes includingprobes8,10, 11,13, 14,and17 listed in Table. 3, (ii) the copy number calling threshold for probe 10 is <0.75 for predicting the sample as having a large deletion, and (iii) the copy number calling threshold for probes 8 and 11 is <0.75 and for probes 13, 14, and 17 is <0.63 for predicting the sample as having a large deletion. If a result of the analysis predicts the sample as having a large deletion, then sample is classified as having a large deletion. If a result of the analysis fails to predict the sample as having a large deletion, then the sample is classified as having aCNVwithout a large deletion.When the sample is classified as having a large deletion, the process continues at block 528.When the sample is classified ashavingaCNVwithout a large deletion, the process continues at block 516.

[0054] At block 516, a classification of the sample having a polymorphism is determined based on sample probe / r- eference probe ratios indicative of an aberrant probe ratio pattern associated with a polymorphism. The determination of theclassificationmaycomprise identifying, by thedecision treematrix, oneormore regionsof copynumber lossesor gains (e.g., deletions, duplications, or point mutations) associated with a polymorphism based on the new relative probe ratio data andnormal ranges (copynumber calling thresholds) for the sample probe / referenceprobe ratios associatedwith one ormoreprobes. In certain instances, thesampleprobe / referenceprobe ratiosandcopynumbercalling thresholdsused for the analysis include: (i) the relative probe ratio data for a second set of probes including probes 4, 8, 11, 21, and 22 listed in Table. 3, and (ii) the copy number calling threshold for probes 4, 8, 11, 21, and 22 is ≥ 0.75 and ≤ 1.3. for predicting the sample as being polymorphic. If a result of the analysis predicts the sample as having a polymorphism, then sample is classified as having a polymorphism. If a result of the analysis fails to predict the sample as having a polymorphism, then the sample is classifiedashavingaCNVwithout a polymorphism.When the sample is classifiedasbeingpolymorphic, the process continues at block 540. When the sample is classified as having a CNV without polymorphism, the process continues at block 520.

[0055] At block 520, a classification of the sample having a α3.7 deletion and zygosity is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associatedwith aα3.7 deletion and zygosity. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy 14 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 number losses or gains (e.g., deletions, duplications, or point mutations) associated with a α3.7 deletion and zygosity based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a third set of probes including probes 21, 22, and 8 listed in Table. 3 for α3.7 homozygous, (ii) the copy number calling threshold checks if 21<0.1, 22<0.1, 8>0.75 for predicting the sample as having a α3.7 homozygous deletion, (iii) the relative probe ratio data for a fourth set of probes including probes 21, 22, and 8 listed in Table. 3 forα3.7 heterozygous, and (iv) the copy number calling threshold checks if 21<0.75, 22<0.75, 8>0.75 for predicting the sample as having a α3.7 heterozygous deletion. If a result of the analysis predicts the sample as having a α3.7 homozygous deletion, then the sample is classified as having a α3.7 homozygous deletion. If a result of the analysis predicts the sample as having a α3.7 heterozygous deletion, then the sample is classified as having a α3.7 heterozygous deletion.

[0056] Additionally at block 520, in conjunctionwith, prior to, or subsequent to determining a classification of the sample having a α3.7 deletion and zygosity, a classification of the sample having a α4.2 deletion and zygosity is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with a α4.2 deletion and zygosity. The determination of the classificationmay comprise identifying, by the decision treematrix, one ormore regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a α3.7 deletion and zygosity based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associatedwith one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a fourth set of probes including probes 11, 21, and 22 listed in Table. 3 for α4.2 heterozygous and homozygous, (ii) the copy number calling threshold checks if 11<0.1, 21>0.75, 22>0.75 for predicting the sample as having a α4.2 homozygous deletion, and (iii) the copy number calling threshold checks if 11<0.75, 21>0.75, 22>0.75 for predicting the sample as having a α4.2 heterozygous deletion. If a result of the analysis predicts the sample as having a α4.2 homozygous deletion, then the sample is classified as having a α4.2 homozygous deletion. If a result of the analysis predicts the sample as having a α4.2 heterozygous deletion, then the sample is classified as having a α4.2 heterozygous deletion. If a result of the analysis fails topredict thesampleashavingaα4.2 deletion; however, the result of theα3.7 deletionanalysis concludes thesamplesdoes have a α3.7 heterozygous or homozygous deletion, then the sample is classified as having a α3.7 heterozygous or homozygous deletion. If a result of the analysis fails to predict the sample as having a α4.2 deletion or a α3.7 deletion, then the sample is classified as having a CNV without a α4.2 heterozygous or homozygous deletion or a α3.7 heterozygous or homozygous deletion. When the sample is classified as having a α3.7 heterozygous or homozygous deletion or α4.2 heterozygousorhomozygousdeletion, theprocesscontinuesatblock522.When thesample is classifiedashavingaCNV without aα3.7 heterozygous or homozygous deletion orα4.2 heterozygous or homozygous deletion, the process continues at block 524.

[0057] At block 522, a classification of the sample having a α4.2 heterozygous or homozygous deletion, and / or having a α3.7 duplication is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with a α3.7 duplication. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a α3.7 duplication based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a fifth set of probes including probes 8, 11, 21, and 22 listed in Table. 3 for α3.7 duplication when a α4.2 heterozygous or homozygous deletion is present, and (ii) the copy number calling threshold checks if 8<1.3,>0.75, 11<0.75,21>1.3,22>1.3 for predicting the sample as having a α3.7 duplication. If a result of the analysis predicts the sample, classified as having a α4.2 heterozygous or homozygous deletion, as also having a α3.7 duplication, then the sample is classified ashaving aα4.2 heterozygous or homozygous deletion andaα3.7 duplication. If a result of the analysis predicts the sample, classified as having a α4.2 heterozygous or homozygous deletion, does not have a α3.7 duplication, then the sample is left with the classification of having a α4.2 heterozygous or homozygous deletion. When the sample is classified as having a α4.2 heterozygous or homozygous deletion and a α3.7 duplication, the process continues at block 540. When the sample is classified as having a α3.7 heterozygous or homozygous deletion or a α4.2 heterozygous or homozygous deletion, the process continues at block 540.

[0058] At block 524, a classification of the sample having compound heterozygotes including an α3.7 deletion and a α4.2 deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associatedwithbothaα3.7 deletionandaα4.2 deletion.Thedeterminationof theclassificationmaycomprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations)associatedwithaα3.7 deletionandzygositybasedon thenewrelativeprobe ratiodataandnormal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a sixth set of probes including probes 8, 29, 1111, 21, 22, 16, and 19 listed in Table. 3 forα3.7 15 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 deletion and a α4.2 deletion, and (ii) the copy number calling threshold checks if 8<1.3,>0.75, 29<1.3,>0.75,11<0.75,21<0.75, 22<0.75, either 16<0.1, or 19<0.1 for predicting the sample as having compound heterozygotes including an α3.7 deletion and a α4.2 deletion. If a result of the analysis predicts the sample as having a compound heterozygotes including an α3.7 deletion and a α4.2 deletion, then the sample is classified as having a compound heterozygotes including an α3.7 deletion and a α4.2 deletion. When the sample is classified as having a compound heterozygotes including an α3.7 deletion and a α4.2 deletion, the process continues at block 540.

[0059] Additionally at block 524, in conjunctionwith, prior to, or subsequent to determining a classification of the sample having a compound heterozygotes including an α3.7 deletion and a α4.2 deletion, a classification of the sample having a duplication is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associatedwith a duplication. Thedetermination of the classificationmaycomprise identifying, by thedecision treematrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a duplication based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associatedwith one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a seventh set of probes including probes 8, 21, and 22 listed in Table. 3 for duplication, and (ii) the copy number calling threshold checks if 8>0.75, 21>1.3, 22>1.3 for predicting the sample as having a duplication. If a result of the analysis predicts the sample as having a duplication, then the sample is classified as having a duplication. When the sample is classified as having a duplication, the process continues at block 540. If a result of the analysis fails to predict the sample as having a compound heterozygotes including an α3.7 deletion and a α4.2 deletion and / or a duplication, then the sample is classified as having a CNV without a α4.2 heterozygous or homozygous deletion, a α3.7 heterozygous or homozygous deletion, and / or a duplication. When the sample is classified as having a CNV without a α4.2 heterozygous or homozygous deletion, a α3.7 heterozygous or homozygous deletion, and a duplication, the process continues at block 526.

[0060] At block 526, a classification of the sample being a CNV that is H aberrant is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with a CNV that is H aberrant. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number lossesorgains (e.g., deletions, duplications, or pointmutations)associatedwithaCNV that isHaberrant basedon the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for aneighth set of probes including probes 1, 4‑6, 8, 11, 21, 22, 29‑37 listed in Table. 3 for a CNV that is H aberrant, and (ii) the copy number calling threshold checks if 8 <0.75 or >1.3 for probes 1, 4‑6, 8, 11, 21, 22, 29‑37 (determines how many important deletion probes are aberrant, and provides a count H of the those probes that are aberrant) for predicting the sample as having aCNV that is H aberrant. If a result of theanalysis predicts the sampleashavingaCNV that isHaberrant, then the sample is classifiedasa CNV that is H aberrant (where H represents how many important deletion probes are aberrant). When the sample is classified as having a CNV that is H aberrant, the process continues at block 540.

[0061] At block 528, a classification of the sample having a large homozygous deletion or a large heterozygous deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with largedeletion zygosity. Thedeterminationof the classificationmaycomprise identifying, by thedecision treematrix, oneor more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with large deletion zygosity based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / r- eferenceprobe ratiosandcopynumber calling thresholdsused for theanalysis include: (i) the relativeprobe ratio data for a ninth set of probes including probes 8, 10, 13, 14, and 17 listed in Table. 3 for large homozygous deletion, (ii) the copy number calling threshold checks if probes 8, 10, or 13, 14, 17 are <0.1 for predicting the sample as having a large homozygous deletion, (iii) the relative probe ratio data for a tenth set of probes including probes 8, 11, 13, 14, and 17 listed inTable. 3 for largeheterozygousdeletion, and (iv) thecopynumbercalling thresholdchecks if probes8and11<0.75or13, 14, 17 <0.63 for predicting the sample as having a large heterozygous deletion. If a result of the analysis predicts the sample as having a large homozygous deletion, then the sample is classified as having a large homozygous deletion. If a result of theanalysis predicts the sampleashavinga largeheterozygousdeletion, then the sample is classifiedashavinga largeheterozygousdeletion.When thesample is classifiedashavinga largehomozygousdeletion, the process continues at block 530. When the sample is classified as having a large heterozygous deletion, the process continues at block 532.

[0062] At block 530, a classification of the sample having a specific large homozygous deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with specification deletions includingSEA,MED1,MED2,THAI,FIL, andα20.5. Thedeterminationof theclassificationmaycomprise identifying,by the decision treematrix, one ormore regions of copy number losses or gains (e.g., deletions, duplications, or pointmutations) associatedwithaspecificationdeletionbasedon thenewrelativeprobe ratiodataandnormal ranges (copynumber calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative 16 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 probe ratio data for a eleventh set of probes including probes 8, 31, 32, 5, 6, 33, and 34 listed in Table. 3 for large SEA homozygous deletion, (ii) the copy number calling threshold checks if 8<0.1, 31<0.1, 32<0.1, 5>0.75, 6>0.75, 33>0.75, 34>0.75 for predicting the sampleashavinga largeSEAhomozygousdeletion, (iii) the relativeprobe ratiodata fora twelfth setof probes includingprobes8,30, 31,5, 6, 32,and33 listed inTable. 3 for largeMED1homozygousdeletion, (iv) thecopy number calling threshold checks if 8<0.1, 30<0.1, 31<0.1, 5>0.75, 6>0.75, 32>0.75, 33>0.75 for predicting the sample as havinga largeMED1homozygousdeletion, (v) the relativeprobe ratiodata fora thirteenthsetof probes includingprobes5, 6, 30, 4, 31, and 32 listed in Table. 3 for largeMED2homozygous deletion, (vi) the copy number calling threshold checks if 5<0.1, 6<0.1, 30<0.1, 4>0.75, 31>0.75, 32>0.75 for predicting the sample as having a largeMED2 homozygous deletion, (vii) the relative probe ratio data for a fourteenth set of probes including probes 8, 6, 29, and 30 listed in Table. 3 for large α20.5 homozygous deletion, (viii) the copy number calling threshold checks if 8<0.1, 6>0.75, 29>0.75, 30>0.75 for predicting the sample as having a large α20.5 homozygous deletion, (ix) the relative probe ratio data for a fifteenth set of probes including probes 6, 8, 31, 32, 5, 33, and 34 listed in Table. 3 for large FIL / THAI homozygous deletion, (x) the copy number calling threshold checks if 6<0.1, 8<0.1, 31<0.1, 32<0.1, 5>0.75, 33>0.75, 34>0.75 for predicting the sample as having a large FIL / THAI homozygous deletion, (xi) the relative probe ratio data for a sixteenth set of probes including probes 9, 22, 6, 8, 29, and 30 listed in Table. 3 for large α3.7A / α4.2C homozygous deletion, (xii) the copy number calling threshold checks if 9<0.1, 22<0.1, 6>0.75, 8>0.75, 29>0.75, 30>0.75 for predicting the sample as having a large α3.7A / α4.2C homozygous deletion, (xiii) the relative probe ratio data for a seventeenth set of probes including probes 11, 21, and 22 listed in Table. 3 for α4.2 homozygous deletion, (xiv) the copy number calling threshold checks if 11<0.1, 21>0.75, 22>0.75 for predicting the sample as having a α4.2 homozygous deletion, (xv) the relative probe ratio data for a eighteenth set of probes including probes 8, 21, 22, and 11 listed in Table. 3 for large α4.2 heterozygous deletion, and (xvi) the copy number calling threshold checks if 8<0.75, >0.25, 21<0.75, >0.25, 22<0.75, >0.25, 11<0.1 for predicting the sample as having a large α4.2 heterozygous deletion.

[0063] If a result of the analysis predicts the sample as having a large SEA homozygous deletion, then the sample is classifiedashavinga largeSEAhomozygousdeletion. If a result of theanalysis predicts thesampleashavinga largeMED 1 homozygous deletion, then the sample is classified as having a large MED1 homozygous deletion. If a result of the analysis predicts the sample ashavinga largeMED2homozygous deletion, then the sample is classified ashavinga large MED2 homozygous deletion. If a result of the analysis predicts the sample as having a large α20.5 homozygous deletion, then the sample is classified as having a largeα20.5 homozygous deletion. If a result of the analysis predicts the sample as having a large FIL / THAI homozygous deletion, then the sample is classified as having a large FIL / THAI homozygous deletion. If a result of the analysis predicts the sample as having a largeα3.7A / α4.2C homozygous deletion, then the sample is classified as having a large α3.7A / α4.2C homozygous deletion. If a result of the analysis predicts the sample as having a α4.2 homozygous deletion, then the sample is classified as having a α4.2 homozygous deletion. If a result of the analysis predicts the sample as having a large α4.2 heterozygous deletion, then the sample is classified as having a large α4.2 heterozygous deletion. When the sample is classified as having a large homozygous deletion, the process continues at block 530.When the sample is classified as having a large SEA,MED1,MED2,α20.5, FIL / THAI, orα3.7A / α4.2C deletion, or α4.2 homozygous deletion, or large α4.2 heterozygous deletion, the process continues at block 540.

[0064] At this point, if the sample remains as a nonspecific classified homozygous deletion (not a large SEA, MED1, MED2, α20.5, FIL / THAI, or α3.7A / α4.2C deletion, or α4.2 homozygous deletion, or large α4.2 heterozygous deletion), the processcontinuesatblock530withdeterminingaclassificationof thesamplehavingaspecific largehomozygousdeletion based on sample probe / reference probe ratios indicative of other aberrant probe ratio patterns associated with specifica- tion deletions including α3.7, α4.2, SEA, MED1, MED2, THAI, FIL, and α20.5. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a specification deletion based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a nineteenth set of probes including probes 8, 13, 14, 17, 32, 6, and 33 listed in Table. 3 for largeSEA / MED1homozygous deletion, (ii) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 32<0.75,>0.25, 6>0.75, 33>0.75 for predicting the sample as having a large SEA / MED1 homozygous deletion, (iii) the relative probe ratio data for a twentieth set of probes including probes 8, 13, 14, 17, 30, 31, 5, 6, and 33 listed in Table. 3 for large SEA / (MED2 / DUTCH) homozygous deletion, (iv) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 30<0.1, 31<0.75,>0.25, 5<0.75,>0.25, 6>0.75, 33>0.75 for predicting the sample as having a large SEA / (MED2 / DUTCH) homozygous deletion, (v) the relative probe ratio data for a twenty-first set of probes including probes 8, 13, 14, 29, 30, 31, 32, 8, and33 listed in Table. 3 for largeSEA / α20.5 homozygousdeletion, (vi) the copy numbercalling thresholdchecks if 8<0.1,13<0.1, 14<0.1, 29<0.75,>0.25,30<0.75,>0.25, 31<0.75,>0.25, 32<0.75,>0.25, 6>0.75, 33>0.75 for predicting the sample as having a large SEA / α20.5 homozygous deletion, (vii) the relative probe ratio data for a twenty-second set of probes including probes 8, 13, 14, 17, 32, 8, 6, and 33 listed in Table. 3 for large SEA / (FIL / THAI) homozygous deletion, (viii) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 32<0.1, 8<0.75,>0.25, 6>0.75, 33>0.75 for predicting the sampleashavinga largeSEA / (FIL / THAI) homozygousdeletion, 17 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 (ix) the relative probe ratio data for a twenty-third set of probes including probes 8, 13, 14, 17, 30, 31, 5, 6, 4, and 32 listed in Table. 3 for large MED 1 / (MED2 / DUTCH) homozygous deletion, (x) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 30<0.1, 31<0.75,>0.25, 5<0.75,>0.25, 6<0.75,>0.25, 4>0.75,32>0.75 for predicting the sample as having a largeMED1 / (MED2 / DUTCH) homozygous deletion, (xi) the relative probe ratio data for a twenty-fourth set of probes including probes 8, 13, 14, 17, 31, 6, and 32 listed in Table. 3 for large MED1 / α20.5 homozygous deletion, (xii) the copynumber calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 31<0.75,>0.25, 6>0.75, 32>0.75 for predicting the sample as having a large MED1 / α20.5 homozygous deletion, (xiii) the relative probe ratio data for a twenty-fifth set of probes including probes 8, 13, 14, 17, 31, 32, 6, 5, and 33 listed in Table. 3 for large MED1 / (FIL / THAI) homozygous deletion, (xiv) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 31<0.1, 32<0.75,>0.25, 6<0.75,>0.25, 5>0.75, 33>0.75 for predicting the sample as having a large MED1 / (FIL / THAI) homozygous deletion, (xiii) the relativeprobe ratiodata for a twenty-sixth set of probes includingprobes13, 14, 17, 5, 6, 29, 30,31, and4 listed inTable. 3 for large (MED2 / DUTCH) / α20.5 homozygous deletion, (xiv) the copy number calling threshold checks if 13<0.1, 14<0.1, 17<0.1,5<0.75,>0.25, 6<0.75,>0.25, 29<0.75,>0.25, 30<0.75,>0.25, 31>0.75, 4>0.75 for predicting thesampleashaving a large (MED2 / DUTCH) / α20.5 homozygous deletion, (xv) the relative probe ratio data for a twenty-seventh set of probes includingprobes8, 13, 14,17, 6, 31, 5, and33 listed inTable. 3 for large (MED2 / DUTCH) / (FIL / THAI) homozygousdeletion, (xvi) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 6<0.75,>0.25, 31<0.75,>0.25, 5>0.75, 33>0.75 for predicting the sample as having a large (MED2 / DUTCH) / (FIL / THAI) homozygous deletion, (xvii) the relative probe ratio data for a twenty-eighth set of probes including probes 8, 13, 14, 17, 6, 29, 5, and 33 listed in Table. 3 for large α20.5 / (FIL / THAI) homozygous deletion, and (xviii) the copy number calling threshold checks if 8<0.1, 13<0.1, 14<0.1, 17<0.1, 6<0.75,>0.25, 29<0.75,>0.25, 5>0.75, 33>0.75 for predicting the sample as having a large α20.5 / (FIL / THAI) homozygous deletion.

[0065] If a result of theanalysis predicts thesampleashavinga largeSEA / MED1homozygousdeletion, then thesample is classified as having a large SEA / MED1 homozygous deletion. If a result of the analysis predicts the sample as having a large SEA / (MED2 / DUTCH) homozygous deletion, then the sample is classified as having a large SEA / (MED2 / DUTCH) homozygous deletion. If a result of the analysis predicts the sample as having a large SEA / (MED2 / DUTCH) homozygous deletion, then the sample is classified as having a large SEA / (MED2 / DUTCH) homozygous deletion. If a result of the analysis predicts the sample as having a large SEA / α20.5 homozygous deletion, then the sample is classified as having a large SEA / α20.5 homozygous deletion. If a result of the analysis predicts the sample as having a large SEA / (FIL / THAI) homozygous deletion, then the sample is classified as having a large SEA / (FIL / THAI) homozygous deletion. If a result of the analysis predicts the sample as having a large MED 1 / (MED2 / DUTCH) homozygous deletion, then the sample is classifiedashavinga largeMED1 / (MED2 / DUTCH)homozygousdeletion. If a result of the analysis predicts the sampleas havinga largeMED1 / α20.5 homozygousdeletion, then the sample is classified ashavinga largeMED1 / α20.5 homozygous deletion. If a result of the analysis predicts the sample as having a largeMED1 / (FIL / THAI) homozygous deletion, then the sample is classified as having a large MED1 / (FIL / THAI) homozygous deletion. If a result of the analysis predicts the sample as having a large (MED2 / DUTCH) / α20.5 homozygous deletion, then the sample is classified as having a large (MED2 / DUTCH) / α20.5 homozygous deletion. If a result of the analysis predicts the sample as having a large (MED2 / - DUTCH) / (FIL / THAI) homozygous deletion, then the sample is classified as having a large (MED2 / DUTCH) / (FIL / THAI) homozygous deletion. If a result of the analysis predicts the sample as having a large α20.5 / (FIL / THAI) homozygous deletion, then the sample is classified as having a large α20.5 / (FIL / THAI) homozygous deletion. At this point, when the sample remains as a nonspecific classified homozygous deletion (not a large SEA, MED1, MED2, α20.5, FIL / THAI, or α3.7A / α4.2C deletion, or α4.2 homozygous deletion, or large α4.2 heterozygous deletion), the process continues at block 540. When the sample is classified as having a large SEA, MED1, MED2, α20.5, FIL / THAI, or α3.7A / α4.2C deletion, or α4.2 homozygous deletion, or large α4.2 heterozygous deletion, the process continues at block 540.

[0066] At block 532, a classification of the sample having a specific large heterozygous deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with specification deletions includingSEA,MED1,MED2,THAI,FIL, andα20.5. Thedeterminationof theclassificationmaycomprise identifying,by the decision treematrix, one ormore regions of copy number losses or gains (e.g., deletions, duplications, or pointmutations) associatedwithaspecificationdeletionbasedon thenewrelativeprobe ratiodataandnormal ranges (copynumber calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a twenty ninth set of probes including probes 8, 31, 32, 5, 6, 33, and 34 listed in Table. 3 for large SEA heterozygous deletion, (ii) the copy number calling threshold checks if 8<0.75,31<0.75,32<0.75,5>0.75,6>0.75,33>0.75,345>0.75 for predicting the sample as having a large SEA hetero- zygousdeletion, (iii) the relativeprobe ratio data for a thirtieth set of probes includingprobes8, 30, 31, 5, 6, 32, and33 listed in Table. 3 for large MED1 heterozygous deletion, (iv) the copy number calling threshold checks if 8<0.75, 30<0.75, 31<0.75, 5>0.75, 6>0.75, 32>0.75, 33>0.75 for predicting the sample as having a largeMED1 heterozygous deletion, (v) the relative probe ratio data for a thirty-first set of probes including probes 5, 6, 30, 4, 31, and 32 listed in Table. 3 for large MED2heterozygous deletion, (vi) the copy number calling threshold checks if 5<0.75, 6<0.75, 30<0.75, 4>0.75, 31>0.75, 18 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 32>0.75 for predicting the sample as having a large MED2 heterozygous deletion, (vii) the relative probe ratio data for a thirty-second set of probes includingprobes 8, 6, 29, and30 listed in Table. 3 for largeα20.5 heterozygousdeletion, (viii) the copynumbercalling thresholdchecks if 8<0.75, 6>0.75, 29>0.75,30>0.75 forpredicting thesampleashavinga largeα20.5 heterozygous deletion, (ix) the relative probe ratio data for a thirty-third set of probes including probes 6, 8, 31, 32, 5, 33, and 34 listed in Table. 3 for large FIL / THAI heterozygous deletion, (x) the copy number calling threshold checks if 6<0.75, 85<0.75, 31<0.75, 32<0.75, 5>0.75, 33>0.75, 34>0.75 for predicting the sample ashavinga largeFIL / THAI heterozygous deletion, (xi) the relative probe ratio data for a thirty-fourth set of probes including probes 9, 22, 6, 8, 29, and 30 listed in Table. 3 for large α3.7A / α4.2C heterozygous deletion, (xii) the copy number calling threshold checks if 9<0.75, 22<0.75, 6>0.75, 8>0.75, 29>0.75, 30>0.75 for predicting the sample as having a large α3.7A / α4.2C heterozygous deletion, (xiii) the relative probe ratio data for a thirty-fifth set of probes including probes 11, 21, and 22 listed in Table. 3 forα4.2 homozygous deletion, (xiv) the copy number calling threshold checks if 11<0.1, 21>0.75, 22>0.75 for predicting the sample as having a α4.2 homozygous deletion, (xv) the relative probe ratio data for a thirty-sixth set of probes including probes 21, 22, and 11 listed in Table. 3 for large α3.7 heterozygous deletion, (xvi) the copy number calling threshold checks if 21<0.15, 22<0.15, 11<0.75,>0.25 for predicting the sample ashavinga largeα3.7 heterozygous deletion, (xvii) the relative probe ratio data for a thirty-seventh set of probes includingprobes21, 22, and11 listed inTable. 3 forα3.7 homozygousdeletion, (xviii) the copy number calling threshold checks if 21<0.15, 22< 0.15, 11<0.75,>0.25 for predicting the sample as having a α3.7 homozygous deletion, (xix) the relative probe ratio data for a thirty-eighth set of probes including probes 8, 21, 22, and 11 listed in Table. 3 for large α4.2 heterozygous deletion, and (xviii) the copy number calling threshold checks if 8<0.75,>0.25, 21<0.75,>0.25, 22<0.75,>0.25, 11<0.1 for predicting the sample as having a large α4.2 heterozygous deletion.

[0067] If a result of the analysis predicts the sample as having a large SEA heterozygous deletion, then the sample is classified as having a large SEA heterozygous deletion. If a result of the analysis predicts the sample as having a large MED1heterozygousdeletion, then the sample is classifiedashavinga largeMED1heterozygousdeletion. If a result of the analysispredicts thesampleashavinga largeMED2heterozygousdeletion, then thesample is classifiedashavinga large MED2heterozygous deletion. If a result of the analysis predicts the sample as having a largeα20.5 heterozygous deletion, then the sample is classifiedashavinga largeα20.5 heterozygousdeletion. If a result of the analysis predicts the sampleas having a large FIL / THAI heterozygous deletion, then the sample is classified as having a large FIL / THAI heterozygous deletion. If a result of theanalysis predicts thesampleashavinga largeα3.7A / α4.2Cheterozygousdeletion, then thesample is classified as having a largeα3.7A / α4.2C heterozygous deletion. If a result of the analysis predicts the sample as having a α4.2 homozygous deletion, then the sample is classified as having a α4.2 homozygous deletion. If a result of the analysis predicts the sample as having a large α3.7 heterozygous deletion, then the sample is classified as having a large α3.7 heterozygous deletion. If a result of the analysis predicts the sample as having a α3.7 homozygous deletion, then the sample is classified as having a α3.7 homozygous deletion. If a result of the analysis predicts the sample as having a large α4.2 heterozygous deletion, then the sample is classified as having a largeα4.2 heterozygous deletion.When the sample is classified as having a large heterozygous deletion, the process continues at block 534. When the sample is classified as having a large SEA, MED1, MED2, α20.5, FIL / THAI, or α3.7A / α4.2C, or α4.2 homozygous deletion, or large α3.7 hetero- zygous deletion, or α3.7 homozygous deletion, or large α4.2 heterozygous deletion, the process continues at block 536.

[0068] At block 534, a classification of the sample having a compound heterozygotic genotype including an unspecified large deletion and a α3.7 deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with both a large deletion and α3.7 deletion. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or pointmutations)associatedwithaα3.7 deletionandzygosity basedon thenewrelativeprobe ratiodataand normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a thirty-ninth set of probes including probes 8, 29, 11, 21, 22, 16, and 19 listed in Table. 3 for α3.7 deletion, and (ii) the copy number calling threshold checks if 8<0.75, 29<0.75,11<0.75,21<0.1, 22<0.1, either 16<0.1, or 19<0.1 for predicting the sample as having compound heterozygotes including an unspecified large deletion and an α3.7 deletion. If a result of the analysis predicts the sample as having a compound heterozygotes including a α3.7 deletion, then the sample is classified as being a large heterozygous deletion having a compound heterozygotes including an α3.7 deletion . If a result of the analysis predicts the sample as not having a compound heterozygotes including a α3.7 deletion, then the sample is classified as being a large heterozygous deletion.

[0069] Additionally at block 534, a classification of the sample having a compound heterozygotic genotype including an unspecified large deletion andaα4.2 deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associatedwith both a large deletion andα4.2 deletion . The determination of the classification may comprise identifying, by the decision treematrix, one ormore regions of copy number losses or gains (e.g., deletions, duplications, or pointmutations)associatedwithaα4.2 deletionandzygosity basedon thenewrelativeprobe ratiodataand normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the 19 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 analysis include: (i) the relative probe ratio data for a fortieth set of probes including probes 8, 29, 11, 21, 22, 16, and 19 listed in Table. 3 for α4.2 deletion, and (ii) the copy number calling threshold checks if 8<0.75, 29<0.75,11<0.1,21<0.75, 22<0.75, for predicting the sample as having compound heterozygotes including an unspecified large deletion and anα4.2 deletion . If a result of the analysis predicts the sample as having a compoundheterozygotes including aα4.2 deletion, then the sample is classified as being a large heterozygous deletion having a compound heterozygotes including an α4.2 deletion . If a result of the analysis predicts the sample as not having a compound heterozygotes including a α4.2 deletion, then the sample is classified as being a large heterozygous deletion.

[0070] Additionally at block 534, in conjunctionwith, prior to, or subsequent to determining a classification of the sample having a compound heterozygotes including an α3.7 deletion and a α4.2 deletion, a classification of the sample having a duplication is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associatedwith a duplication. Thedetermination of the classificationmaycomprise identifying, by thedecision treematrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a duplication based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associatedwith one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a forty-first set of probes including probes 8, 21, and 22 listed in Table. 3 for duplication, and (ii) the copy number calling threshold checks if 8<0.75, 21>0.75, 22>0.75 for predicting the sample as having a duplication.

[0071] If a result of theanalysis predicts the sample, havingaunspecified largeheterozygousdeletion combinedwith an α3.7 deletion or, then the sample is classified as being a large unspecified heterozygous deletion combined with an α3.7 deletion . When the sample is classified as being a large unspecified heterozygous deletion having a compound heterozygotes including a α4.2 deletion, the process continues at block 540. When the sample is classified as being a large unspecified heterozygous deletion combined with a duplication, the sample is classified as having a large, unspecified deletion and a duplication, and the process continues at block 540. If a result of the analysis predicts the sample, having a large heterozygous deletion, as not having a duplication, then the sample is classified as being a large heterozygous deletion. When the sample is classified as being a large heterozygous deletion, the process continues at block 540.

[0072] At block 536, a classification of the sample having compound heterozygotes including an specific targeted large deletion and a α3.7 deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with both a specific targeted large deletion α3.7 deletion. The determination of the classification may comprise identifying, by the decision treematrix, one ormore regions of copy number losses or gains (e.g., deletions, duplications, or pointmutations)associatedwithaα3.7 deletionandzygosity basedon thenewrelativeprobe ratiodataand normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one ormore probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a forty-second set of probes including probes 8, 29, 11, 21, 22, 16, and 19 listed in Table. 3 for α3.7 deletion, and (ii) the copy number calling threshold checks if 8<0.75, 29<0.75,11<0.75,21<0.1, 22<0.1, either 16<0.1, or 19<0.1 forpredicting thesampleashavingcompoundheterozygotes includingaα3.7 deletion. If a result of the analysis predicts the sample as having a compoundheterozygotes including anα3.7 deletion, then the sample is classified as being a specific large heterozygous deletion (e.g., large SEA, MED1, MED2, α20.5, FIL / THAI, or α3.7A / α4.2C,) having a compound heterozygotes including an α3.7 deletion. If a result of the analysis predicts the sample as not containing a α3.7 deletion then the sample is classified as being a specific large heterozygous deletion (e.g., large SEA, MED1, MED2, α20.5 , FIL / THAI, or α3.7A / α4.2C.

[0073] Additionally at block 536, a classification of the sample having compound heterozygotes including a specific targeted large deletion and a α4.2 deletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratiopatternassociatedwithbothaspecific targeted largedeletionandaα4.2 deletion.Thedeterminationof theclassificationmaycomprise identifying, by thedecision treematrix, oneormore regionsof copynumber lossesor gains (e.g., deletions, duplications, or point mutations) associated with a α4.2 deletion and zygosity based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a forty-third set of probes including probes 8, 29, 11, 21, 22, 16, and 19 listed in Table. 3 for α3.7 deletion, and (ii) the copy number calling threshold checks if 8<0.75, 29<0.75, 11<0.1, 21<0.75, 22<0.75, for predicting the sample ashaving compoundheterozygotes includingaα4.2 deletion. If a result of theanalysis predicts thesampleashavingacompoundheterozygotes includinganα4.2 deletion, then the sample is classified asbeing a specific large heterozygous deletion (e.g., largeSEA,MED1,MED2,α20.5, FIL / THAI, or α3.7A / α4.2C,) having a compoundheterozygotes including anα4.2 deletion. If a result of the analysis predicts the sample as not containing aα3.7 deletion then the sample is classifiedasbeinga specific largeheterozygousdeletion (e.g., largeSEA, MED1, MED2, α20.5 , FIL / THAI, or α3.7A / α4.2C.

[0074] Additionally at block 536, in conjunctionwith, prior to, or subsequent to determining a classification of the sample having a compound heterozygous deletions including a specific large deletion and a α3.7 deletion or a α4.2 deletion, a 20 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 classification of the sample having a specific large deletion and a duplication is determined based on sample probe / r- eference probe ratios indicative of an aberrant probe ratio pattern associated with a duplication. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with a duplication based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a forty-fourth set of probes including probes 8, 21, and 22 listed in Table. 3 for duplication, and (ii) the copy number calling threshold checks if 8>0.1, 21>0.75, 22>0.75 for predicting the sample as having a specific large deletion and a duplication.

[0075] If a result of the analysis predicts the sample, having a specific large deletion and a compound heterozygote including a α3.7 deletion, then the sample is classified as being a specific large heterozygous deletion having a compound heterozygotes including anα3.7 deletion. If a result of the analysis predicts the sample, having a specific large deletion and a compound heterozygote including a duplication, then the sample is classified as being a specific large heterozygous deletion having a compound heterozygotes including a duplication. If a result of the analysis predicts the sample, having a specific large deletion and a compound heterozygote including a α4.2 duplication, then the sample is classified as being a specific large heterozygous deletion having a compound heterozygotes including a duplication. When the sample is classified as being a specific large heterozygous deletion having a compound heterozygotes including a α3.7 deletion, a α4.2 deletion or duplication, the process continues at block 540. If a result of the analysis predicts the sample, having a specific large heterozygous deletion, as not having a α3.7 deletion, a α4.2 deletion, or a duplication, then the sample is classified as being a specific large heterozygous deletion. When the sample is classified as being a specific large heterozygous deletion, the process continues at block 540.

[0076] Atblock540,aclassificationof thesamplehavingaHS40homozygousdeletionoraHS40heterozygousdeletion is determined based on sample probe / reference probe ratios indicative of an aberrant probe ratio pattern associated with HS40 deletion zygosity. The determination of the classification may comprise identifying, by the decision tree matrix, one or more regions of copy number losses or gains (e.g., deletions, duplications, or point mutations) associated with HS40 deletion zygosity based on the new relative probe ratio data and normal ranges (copy number calling thresholds) for the sample probe / reference probe ratios associated with one or more probes. In certain instances, the sample probe / r- eferenceprobe ratiosandcopynumber calling thresholdsused for theanalysis include: (i) the relativeprobe ratio data for a forty-fifth set of probes including probes 1, 4, 2, and 3 listed inTable. 3 for HS40homozygous deletion, (ii) the copy number calling threshold checks if 1>0.75,<1.3, 4>0.75,<1.3, 2<0.1, 3<0.1 for predicting the sample as having a HS40 homo- zygousdeletion, (iii) the relativeprobe ratio data for a forty-fifth set of probes includingprobes1, 4, 2, and3 listed in Table. 3 for HS40 heterozygous deletion, and (iv) the copy number calling threshold checks if probes 8 and 1>0.75,<1.3, 4>0.75,<1.3, 2<0.75,3<0.75 for predicting the sample as having a large heterozygous deletion. Additionally, in instances in which the sample is classified as having a CNV that is H aberrant, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the relative probe ratio data for a forty-second set of probes including probes 2 and 3 listed in Table. 3 for additional aberrant probes, (ii) the copy number calling threshold checks if 2>0.75, 3<0.75 or 2>0.75, 3<0.75 for predicting the sample as having an additional aberrant probe and the number H of probes that are aberrant is updated (if either is true, add 1 to any number H (1‑17)).

[0077] Additionally at block 540, for any sample having a "normal" or "polymorphism" classification check 13<0.85, 14<0.85, 17<0.85. If true, thenaberrant probesaredetectedand thesample is re-classifiedasbeingHaberrant,whereH= 3. For any sample having a " CNV that is H aberrant " classification, the algorithm checks whether probes 13<0.85, 14<0.85, 17<0.85. If true, then additional aberrant probes are detected and the number H of probes that are aberrant is updated (add 3 to any number H (1‑17)).

[0078] Additionally at block 540, all samples are checked for a classification of the sample having the Hb Constant Spring single nucleotide variant (SNV). The presence or absence of the SNV is determined by the presence or absence of the Constant Spring SNV probe. In certain instances, the sample probe / reference probe ratios and copy number calling thresholds used for the analysis include: (i) the positive probe signal data for probe18 listed in Table. 3 for HS40 homozygous deletion, (ii) the probe signal is >0.

[0079] If the result of theanalysis predicts the sampleashavingaHS40homozygousdeletion, thenprior classificationof the sample is concatenated with HS40 homozygous deletion. If the result of the analysis predicts the sample as having a HS40heterozygous deletion, then prior classification of the sample is concatenatedwithHS40 heterozygous deletion. If a result of the analysis predicts the sample as not having a HS40 homozygous or heterozygous deletion, then the previous classification of the sample is unchanged. If a result of the analysis predicts the sample as having an additional aberrant probe, then thenumberHof probes that are aberrant is updatedand the classification remains asaCNV that isHaberrant. If the result of the analysis predicts the sample as having a Hb Constant Spring mutation, then prior classification of the sample is concatenated with with Hb Constant Spring. If a result of the analysis predicts the sample as not having a Hb Constant Spring, then the previous classification of the sample is unchanged.

[0080] The results of this step 540 are a final classification. At this point all samples should be classified as normal, 21 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 contain only polymorphisms, have one or more confirmable mutations (e.g., α3.7, α4.2, SEA, MED1, MED2, THAI, FIL, and / or α20.5), a CNV with H aberrant probes, or an unclassifiable large deletion. If the sample has a CNV with H aberrant probes or an unclassifiable large deletion the sample should be manually reviewed.

[0081] At block 545, the HBA genotype for each sample is determined based on the final classification for each sample. For example, a sample that hasaHETα3.7 orα4.2 deletionANDHETSEA,FIL,MED,THAI, orα20.5 classificationwouldbe genotypedas -‑ / ‑α (see, e.g., Table2).At optional block550, a risk score(s)maybecomputedbasedon theHBAgenotype and result implication determined for each sample, as shown in Table 2. In some instances, the risk score(s) may identify: (i) a risk of the subject being a carrier of the α3.7, α4.2, SEA, MED, THAI, FIL, α20.5 and HS‑40 deletions and Hb Constant Spring point mutation, (ii) a risk for couples identified as being a carrier of the α3.7, α4.2, SEA, MED, THAI, FIL, α20.5 and HS‑40 deletions and Hb Constant Spring point mutation, and / or (iii) a risk of a fetus inheriting the α3.7, α4.2, SEA, MED, THAI, FIL, α20.5 and HS‑40 deletions and Hb Constant Spring point mutation.

[0082] Atblock555, theHBAgenotypedetermined foreachalleleandoptional risk score(s)maybeoutput.Theoutput of theHBAgenotypedetermined foreachalleleandoptional risk score(s)maycompriseprovidinganenduserwith theoutput and / or recording the output in a storage device (e.g., displaying the output on a user interface and / or storing the output in a results file of a database).

[0083] FIG. 6 illustrates an example computing device 600 suitable for use with systems and methods for HBA genotyping using aHBA assay platform and genotyping techniques according to this disclosure. The example computing device 600 includes a processor 605 which is in communication with the memory 610 and other components of the computing device 600 using one or more communications buses 615. The processor 605 is configured to execute processor-executable instructions stored in thememory610 toperformoneormoremethods for searching and identifying HBA peaks that are present within the raw data, determining an HBA genotype of a specimen, and / or determining a risk score of a patient according to different examples, such as part or all of the example process 400 or 500 described above with respect to FIGS. 4 and5. In this example, thememory 610 stores processor-executable instructions that provideHBA peak analysis 620 and HBA genotype determination 625, as discussed above with respect to FIGS. 1, 2, 4, and 5.

[0084] The computing device 600, in this example, also includes one or more user input devices 630, such as a keyboard,mouse, touchscreen,microphone, etc., to accept user input. The computing device 600 also includes a display 635 to provide visual output to a user such as a user interface. The computing device 600 also includes a communications interface 640. In some examples, the communications interface 640 may enable communications using one or more networks, including a local area network ("LAN"); wide area network ("WAN"), such as the Internet; metropolitan area network ("MAN"); point-to-point or peer-to-peer connection; etc. Communicationwith other devicesmaybeaccomplished using any suitable networking protocol. For example, one suitable networking protocol may include the Internet Protocol ("IP"), TransmissionControl Protocol ("TCP"), User DatagramProtocol ("UDP"), or combinations thereof, such as TCP / IP or UDP / IP. IV. Examples

[0085] The systems and methods implemented in various embodiments may be better understood by referring to the following examples. Example 1: The HBA assay and the HBA Genotyping Decision Tree Matrix Specimens, Data Collection, and HBA Assay Analysis

[0086] Genomic DNA was extracted from 224 bloods, 41 prenatal specimens and 2 cell lines (Coriell NA03433, NA10797)wereused in this exemplary study.Of theblood specimens, 49wereeither freshor archived specimens.Prior to de-identification, anyα-thalassemia genotype information, if available, was retained for each sample. All specimenswere anonymized prior to their use in this exemplary study. Raw data were collected on an ABI 3730XL genetic analyzer using theABI FoundationDataCollection software v3.0 and uploaded into aGeneMarker software v2.7.0 for analysis of sample quality, generation of the synthetic reference and normalization of sample signal to that of the synthetic reference to detect deletions and duplications. The quality metrics for analysis are listed in Table 1. The manufacturer’s recommended thresholds for relative probe ratios (fold changes v. reference) when no deletions or duplications are present were used in this study. The threshold for the control probe standard deviation was determined empirically. Analytical Sensitivity and Specificity

[0087] To establish analytical sensitivity and specificity, 69 specimens and cell lines of known genotypes (39 positives and 30 negatives; Table 4) were tested in the HBA MLPA assay as described with respect to FIGS. 1‑3. Genotype calls were made with both the HBA Genotyping decision tree matrix (discussed with respect to FIGS. 4 and 5) and by manual 22 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 review. All samples were quantified using a SpectraMax M2 Fluorometer, and inputs ranged from 12.5 ng to 100 ng. The genotypes were blinded to the operator prior to their use. Table 4. List of Deletion and Duplication Genotypes Genotype Number of Samples α3.7 deletion, HET 6 α3.7 deletion, HOM 8 α4.2 deletion, HET 1 α3.7 deletion / α4.2 deletion, cpd HET 2 α20.5 deletion, HET 1 SEA deletion, HET 4 SEA deletion / α3.7 deletion, cpd HET 1 SEA deletion / FIL deletion, cpd HET 1 FIL deletion, HET 2 MED deletion, HET 1 THAI deletion, HET 1 HS40 deletion, HET 1 BRIT deletion, HET 2 α3.7 duplication, HET 4 α4.2 duplication, HET 1 -‑ / α 1 -‑ / -- 2 Normal and Polymorphisms 30 Quality Metrics

[0088] The overall average control probe standard deviation was 0.048± 0.030. Of the 69 samples tested, six did not initially pass the sample qualitymetrics (6 / 69; 8.7%; Table 5) andwere repeatedwith the samealiquots of DNA. The inputs for three of these samples were 15 ng,16 ng and 29 ng, which are below the lower limit that is recommended by the manufacturer (50‑100 ng). The other three samples had been archived frozen for at least 15 years, and no information on the extraction method used was available. Upon retesting, all 6 samples passed quality metrics and were deemed acceptable for genotype calling (Table 5). Table 5. Quality Metrics for Sensitivity / Specificity Sample Set Number of Samples Average of Control probe std dev Std dev of control probe std dev No. of samples failing QC - 1st pass No. of samples failing QC 2nd pass Positive: 39 0.052 0.031 3 0 Negative: 30 0.043 0.028 3 0 Total: 69 0.048 0.030 6 0 Genotype Calls

[0089] All sampleswereanalyzedusing thequality check thresholds inTable1.Witheither theHBAgenotypingdecision tree matrix (discussed with respect to FIGS. 4 and 5) or by manual analysis, no false negatives were detected, and all 30 negative samples were called correctly, resulting in 100% specificity. Of the 39 positive samples, 35 and 37 sampleswere called fora targetedgenotypewith theHBAgenotypingdecision treematrix andbymanualanalysis, respectively (Table6). For the samples inwhich the variationswere detected but the genotypes not specifically called, twowere duplications and 23 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 were not targeted variants.

[0090] For the two duplication samples, manual analysis correctly identified one as a heterozygous α3.7 duplication (sample ZZ‑59) and the other as a heterozygous α4.2 duplication (sample ZZ‑48). The HBA genotyping decision tree matrix alsodetected theduplicationsbut couldnot assignoneof the targetedgenotypes to themand instead, flagged these samples for manual review. That is, for ZZ‑59, all but one probe for theα3.7 regionwas above the 1.30 threshold to call the duplication and rather than call this as a normal, the HBA genotyping decision tree matrix flagged it for manual review. As for ZZ‑48 with the α4.2 duplication, the HBA genotyping decision tree matrix was not programmed to call this particular genotype. Importantly, for either sample, theHBAgenotyping decision treematrix did notmiss the call but rather defaulted to manual review.

[0091] Although not one of the intended targeted mutations for this assay, two samples that were heterozygous for a BRIT deletion were included in this study to test the HBA genotyping decision tree matrix. With both the HBA genotyping decision tree matrix and by manual analysis, the samples were called as heterozygous SEA deletions since the same MLPA probes will detect either a SEA or BRIT deletion. Multiplex PCR did not confirm the SEA deletion in either sample. The SEA deletion call by HBAMLPA for these samples should not be considered false positive since the BRIT deletion is not a targeted mutation in the assay and both calling methods did detect a large deletion that was about the same size as theBRITdeletion.Since theHBAMLPAassaycandetect anynumberof largedeletionsbesides those targeted for this test that could have clinical significance, the HBA genotyping decision tree matrix was designed to ensure that any copy number changes detected by multiple probes were not missed and instead went to manual review. Thus, all targeted mutationswere identified in thepositive samples, resulting inanassay sensitivity of 100%usingeither theHBAgenotyping decision tree matrix or manual method. Table 6. Summary of Sensitivity / Specificity Genotype Calls Sample Number of Samples Decision Tree Matrix Manual Correct Genotype Call CNV Detected and Correct Genotype Call after Manual Review CNV Detected Correct Genotype Call CNV Detected Positive 39 35 2 2* 37 2* Negative 30 30 0 0 30 0 *BRIT Deletions Reproducibility

[0092] For intra-assay reproducibility, 3 samples were tested in triplicate on the same assay run. These same samples were also used for inter-assay reproducibility with a second lot of reagents, different operator and on different days. Data analysiswas carried out by both amanual analysis and theHBAgenotyping decision treematrix. For both intra‑assayand inter-assay reproducibility, all but one sample (G05-intra1) passedQCmetrics for control probe standard deviation (Table 7) although replicates of this sample passed suggesting that sample quality was not the issue. The MLPA Lane Score or quality control fragments did not detect issues with the MLPA reaction and no evaporation was visibly detected for this sample. With passing samples, the fold change v. reference for each probe was reproducible (see, FIG. 7 for example - Single probes (#15,16,19‑23) consistently detected the deletion at ~0.5 fold change and with duplicated probes (#13,14,17) at ~0.75. Polymorphic probe #12 also detected a deletion at ~0.75 fold change, indicating the likely span of this particularα3.7 deletion), and genotype callswith the algorithmwere 100%concordantwith the callsmademanually. Based on this data, the assay was reproducible from run to run. Table 7. Intra- and Inter- Assay Reproducibility Results Sample QC Metrics Decision Tree Matrix Matrix / Manual CallControl Probe Number of Control Probes Number* of Normal Probes Number* of Del / Dup Probes B09-intra1 0.097 11 23 10 α3.7 het B09-intra2 0.06 11 23 10 α3.7 het B09-intra3 / inter3 0.071 11 23 10 α3.7 het 24 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Sample QC Metrics Decision Tree Matrix Matrix / Manual CallControl Probe Number of Control Probes Number* of Normal Probes Number* of Del / Dup Probes B09-inter1 0.049 11 23 10 α3.7 het B09-inter2 0.064 11 23 10 α3.7 het C01-intra1 0.029 11 33 0 Normal C01-intra2 0.068 11 33 0 Normal C01-intra3 / inter 3 0.07 11 33 0 Normal C01-inter1 0.043 11 33 0 Normal C01-inter2 0.066 11 33 0 Normal G05-intra1 0.147 11 27 6 Fail G05-intra2 0.055 11 28 5 Polymorphism G05-intra3 / inter3 0.093 11 27 6 Polymorphism G05-inter1 0.043 11 27 6 Polymorphism G05-inter2 0.094 11 27 6 polymorphism *Total Normal Probe Number = number of normal probes + number of del / dup probes HBA genotyping decision tree matrix performance

[0093] The performance of theHBAgenotyping decision treematrix was further evaluatedwith 267 blinded specimens. Genotype calls were compared to that by manual review and / or results from previous testing by another laboratory. Overall, the HBA genotyping decision tree matrix and manual method / previous testing results agreed for 261 samples (261 / 267; 97.8%; Table 8) at first pass through the HBAMLPA assay. Two samples with minimal genotyping information, one labeled as ’hydrops’ and the other as ’Hemo. H’, were genotyped by the HBA genotyping decision tree matrix as a homozygousSEAdeletion andaSEAdeletion in transwithα3.7 deletion, respectively, and confirmedbymultiplexPCR. In addition, as aforementioned, two samples with BRIT deletions that are indistinguishable from the SEA deletions by the HBA MLPA assay were not confirmed as SEA or any of the other targeted deletions in the multiplex PCR assay. These samples would be repeated in the MLPA assay for confirmation testing in the clinical laboratory. Table 8. Performance of Genotyping Algorithm Compared to Manual Analysis Sample No. Sample Genotype Truth Correct Genotype Called Incorrect Genotype Called Matrix Manual Matrix Manual 1 Normal 164 161* 164 0 0 2 Polymorphism 28 28 28 0 0 3 HS40 del 1 1 1 0 0 4 α3.7 or α4.2 deletion 35 35 35 0 0 6 SEA, FIL, THAI, MED, α20.5 deletion 9 9 9 2** 2* * 7 combination of 4&6 in trans 19 19 19 0 0 8 α3.7 duplication 7 6* 7 0 0 9 α4.2 duplication 2 0* 2 0 0 25 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Sample No. Sample Genotype Truth Correct Genotype Called Incorrect Genotype Called Matrix Manual Matrix Manual Total: 267 265 265 2 2 *Samples that were not specifically called for expected genotype by the HBA genotyping decision tree matrix but instead indicated for Director review ** Two samples with BRIT deletions that are indistinguishable from the SEA deletions by the HBA MLPA assay

[0094] The HBA genotyping decision tree matrix is trained to detect fold change patterns in normalized sample probes whether or not it can assign one of the targeted genotypes, and as shown in Table 9, all six samples that the algorithm marked for clinical director reviewweredue to inconsistencies in theconfigurationof the relativeprobe ratios for a region. In addition, the HBA genotyping decision treematrix is designed to error on the side of sensitivity (i.e., false positives) rather thanmiss any calls (i.e., false negatives). Thus, although the HBA genotyping decision treematrix did not specifically call the α3.7 duplication, it did detect an anomaly with one probe and rather than miss the call, flagged the sample for manual review. Likewise, with the three normal samples, the HBA genotyping decision treematrix identified irregularities with the relative probe ratio for multiple probes and designated these for manual review instead of miscalling the genotypes. Table 9. Discrepancy in Calls Between the Genotyping Algorithm and Manual Method Truth Number of Samples Matrix Call Reason for Discrepancy α3.7 dup 1 Manual Review One probe slightly below 1.30 fold change dup calling threshold; probe pattern recognized as abnormal -> marked for review α4.2 dup 2 Manual Review α4.2 genotype is not included in the algorithm; probe pattern recognized as abnormal -> marked for review Normal 3 Manual Review Multiple specific probes slightly above / below normal calling thresholds; probe pattern recognized as abnormal -desig- nated for review Additional Considerations

[0095] Specific details are given in the above description to provide a thorough understanding of the embodiments. However, it is understood that the embodiments can be practiced without these specific details. For example, circuits can beshown inblockdiagrams inorder not to obscure theembodiments in unnecessarydetail. In other instances,well-known circuits, processes, algorithms, structures, and techniques can be shown without unnecessary detail in order to avoid obscuring the embodiments.

[0096] Implementation of the techniques, blocks, steps and means described above can be done in various ways. For example, these techniques, blocks, steps and means can be implemented in hardware, software, or a combination thereof. For a hardware implementation, the processing units can be implementedwithin one ormore application specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field programmable gate arrays (FPGAs), processors, controllers, micro-controllers, microproces- sors, other electronic units designed to perform the functions described above, and / or a combination thereof.

[0097] Also, it is noted that the embodiments can be described as a process which is depicted as a flowchart, a flow diagram, adata flowdiagram,a structurediagram, or ablockdiagram.Althoughaflowchart candescribe theoperationsas a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations canbe rearranged.A process is terminatedwhen its operations are completed, but could haveadditional steps not included in the figure.Aprocess can correspond to amethod, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a function, its termination corresponds to a return of the function to the calling function or the main function.

[0098] Furthermore, embodiments can be implemented by hardware, software, scripting languages, firmware, mid- dleware, microcode, hardware description languages, and / or any combination thereof. When implemented in software, firmware, middleware, scripting language, and / or microcode, the program code or code segments to perform the necessary tasks can be stored in a machine readable medium such as a storage medium. A code segment or 26 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 machine-executable instructioncan representaprocedure,a function, asubprogram,aprogram,a routine, asubroutine, a module, a software package, a script, a class, or any combination of instructions, data structures, and / or program statements. A code segment can be coupled to another code segment or a hardware circuit by passing and / or receiving information, data, arguments, parameters, and / or memory contents. Information, arguments, parameters, data, etc. can be passed, forwarded, or transmitted via any suitablemeans includingmemory sharing,message passing, ticket passing, network transmission, etc.

[0099] For a firmware and / or software implementation, the methodologies can be implemented with modules (e.g., procedures, functions, and so on) that perform the functions described herein. Any machine-readable medium tangibly embodying instructions can be used in implementing the methodologies described herein. For example, software codes can be stored in amemory.Memory can be implementedwithin the processor or external to the processor. As used herein the term "memory" refers to any type of long term, short term, volatile, nonvolatile, or other storagemediumand is not to be limited to any particular type of memory or number of memories, or type of media upon which memory is stored.

[0100] Moreover, as disclosed herein, the term "storage medium", "storage" or "memory" can represent one or more memories for storing data, including read only memory (ROM), random access memory (RAM), magnetic RAM, core memory,magnetic disk storagemediums, optical storagemediums, flashmemorydevicesand / or othermachine readable mediums for storing information. The term "machine-readable medium" includes, but is not limited to portable or fixed storagedevices, optical storage devices,wireless channels, and / or various other storagemediums capable of storing that contain or carry instruction(s) and / or data.

[0101] While the principles of the disclosure have been described above in connection with specific apparatuses and methods, it is to be clearly understood that this description is made only by way of example and not as limitation on the scope of the disclosure.

[0102] The invention will now be defined by reference to the following clauses 1. A method comprising: obtaining rawdata fromaHemoglobinA (HBA)assayperformedonaplurality of samples,wherein theHBAassay is performed using a plurality of probes capable of detection of copy number losses or gains in a 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 resolved by capillary electrophoresis for each sample of the plurality of samples; selecting a reference sample from the plurality of samples; calculatingafirst set ofprobe ratios foreachsampleof thepluralityof samplesbasedon the rawdata from theHBA assay and the reference sample; identifying 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 the synthetic reference sample based on the predetermined number of reference samples; calculating a second set of probe ratios for each sample of the plurality of samples based on the rawdata from the HBA assay and the synthetic reference sample; iteratively inputting the second set of probe ratios for each sample into a decision tree matrix; determining, by thedecision treematrix, aHBAgenotype for each samplebasedon the secondset 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; and providing the HBA genotype for each sample. 2. Themethod of clause 1, wherein the calculating the first set of probe ratios comprises: (i) comparing control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals, in the reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metrics are greater than a predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variabilitymetrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probepeakheights, or signals, in the sample to corresponding test probepeakheights in the reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the reference sample. 3. The method of clause 1 or 2, wherein the calculating the second set of probe ratios comprises: (i) comparing the control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the synthetic reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals in the synthetic 27 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metrics are greater than the predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the synthetic reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the synthetic reference sample. 4. The method of clause 1, 2, or 3, wherein the determining the HBA genotype for each sample comprises: (i) determining aberrant probe ratio patterns for each sample based on the second set of probe ratios for each sample and the copy number calling thresholds for the sample probe / referenceprobe ratios associatedwith eachprobeof the plurality of probes, and (ii) identify the HBA genotype for each sample based on the aberrant probe ratio patterns. 5. The method of clause 4, wherein the determining the aberrant probe ratio patterns and the identifying the HBA genotype for each sample comprises: classifying each sample as normal, having a copy number variation (CNV), or as polymorphic based on the aberrant probe ratio patterns; and sub classifying any sample classified as having theCNVas a large targeted deletion, duplication or ’other’ based on the aberrant probe ratio patterns. 6. The method of clause 5, wherein the determining the aberrant probe ratio patterns and the identifying the HBA genotype for each sample further comprises: sub classifying any sample classified as having the large targeted deletion as a large heterozygous deletion or a homozygous deletion; sub classifying any sample classified as the large heterozygous deletion or the homozygous deletion for one or more of the following deletions: SEA, FIL / THAI, MED or α20.5 based on the aberrant probe ratio patterns; and sub classifying any sample classified as having the large heterozygous deletion for one or more of the following: α3.7 deletions, α4.2 deletions and / or α3.7 duplications based on the aberrant probe ratio patterns. 7. The method of clause 5, wherein the determining the aberrant probe ratio patterns and the identifying the HBA genotype for each sample further comprises: sub classifying any sample classified as ’other’ as havingα3.7 deletions,α4.2 deletions, and / orα3.7 duplications based on the aberrant probe ratio patterns. 8. The method of any one of clauses 1‑7, further comprising triggering performance of a confirmatory test on each sample of the plurality of samples that has the HBA genotype that is not normal or does not require manual review. 9. A system comprising: one or more data processors; and a non-transitory computer readable storagemedium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform actions including: obtaining raw data from a Hemoglobin A (HBA) assay performed on a plurality of samples, wherein the HBA assay is performed using a plurality of probes capable of detection of copy number losses or gains in a 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 resolvedby capillary electrophoresis for each sample of the plurality of samples; selecting a reference sample from the plurality of samples; calculating a first set of probe ratios for each sampleof the plurality of samplesbasedon the rawdata from the HBA assay and the reference sample; identifyingapredeterminednumberof referencesamples tobecombinedasasynthetic referencesample for the plurality of samples based on the first set of probe ratios; generating the synthetic reference sample based on the predetermined number of reference samples; calculatinga secondset of probe ratios for eachsampleof theplurality of samplesbasedon the rawdata from the HBA assay and the synthetic reference sample; 28 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 iteratively inputting the second set of probe ratios for each sample into a decision tree matrix; determining, by the decision treematrix, a 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; and providing the HBA genotype for each sample. 10. The system of clause 9, wherein the calculating the first set of probe ratios comprises: (i) comparing control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals, in the reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metrics are greater than a predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variabilitymetricsare greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probepeakheights, or signals, in the sample to corresponding test probepeakheights in the reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the reference sample. 11. The system of clause 9 or 10, wherein the calculating the second set of probe ratios comprises: (i) comparing the control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the synthetic reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals in the synthetic reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metricsare greater than the predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metricsare greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the synthetic reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the synthetic reference sample. 12. The system of clause 9, 10, or 11, wherein the determining the HBA genotype for each sample comprises: (i) determining aberrant probe ratio patterns for each sample based on the second set of probe ratios for each sample and the copy number calling thresholds for the sample probe / referenceprobe ratios associatedwith eachprobeof the plurality of probes, and (ii) identify the HBA genotype for each sample based on the aberrant probe ratio patterns. 13. The system of clause 12, wherein the determining the aberrant probe ratio patterns and the identifying the HBA genotype for each sample comprises: classifying each sample as normal, having a copy number variation (CNV), or as polymorphic based on the aberrant probe ratio patterns; and sub classifying any sample classified as having theCNVas a large targeted deletion, duplication or ’other’ based on the aberrant probe ratio patterns. 14. The system of clause 12, wherein the determining the aberrant probe ratio patterns and the identifying the HBA genotype for each sample further comprises: sub classifying any sample classified as having the large targeted deletion as a large heterozygous deletion or a homozygous deletion; sub classifying any sample classified as the large heterozygous deletion or the homozygous deletion for one or more of the following deletions: SEA, FIL / THAI, MED or α20.5 based on the aberrant probe ratio patterns; and sub classifying any sample classified as having the large heterozygous deletion for one or more of the following deletions: α3.7 and α4.2 deletions based on the aberrant probe ratio patterns. 15. The system of any one of clauses 9‑14, wherein the actions further include triggering performance of a confirmatory test on each sample of the plurality of samples that has the HBA genotype that is not normal or does not require manual review. 16. A computer-programproduct tangibly embodied in a non-transitorymachine-readable storagemedium, including instructions configured to cause one or more data processors to perform actions including: 29 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 obtaining rawdata fromaHemoglobinA (HBA)assayperformedonaplurality of samples,wherein theHBAassay is performed using a plurality of probes capable of detection of copy number losses or gains in a 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 resolved by capillary electrophoresis for each sample of the plurality of samples; selecting a reference sample from the plurality of samples; calculatingafirst set ofprobe ratios foreachsampleof thepluralityof samplesbasedon the rawdata from theHBA assay and the reference sample; identifying 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 the synthetic reference sample based on the predetermined number of reference samples; calculating a second set of probe ratios for each sample of the plurality of samples based on the rawdata from the HBA assay and the synthetic reference sample; iteratively inputting the second set of probe ratios for each sample into a decision tree matrix; determining, by thedecision treematrix, aHBAgenotype for each samplebasedon the secondset 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; and providing the HBA genotype for each sample. 17. The computer-program product of clause 16, wherein the calculating the first set of probe ratios comprises: (i) comparing control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the reference sample, (ii) calculating variability in signals between the control probe peakheights, or signals, in eachsampleand thecorrespondingcontrol probepeakheights, or signals, in the reference sample as a control probe standard deviation, (iii) determining a sample of the plurality samples fails when any variability metricsare greater than a predetermined threshold, (iv) determining a sample of the plurality of samples doesnot fail whennoneof the variabilitymetricsare greater than thepredetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the reference sample. 18. The computer-program product of clause 17 or 17, wherein the calculating the second set of probe ratios comprises: (i) comparing the control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the synthetic reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals in the synthetic reference sample as a control probe standard deviation, (iii) determining a sample of theplurality samples failswhenanyvariabilitymetricsaregreater than thepredetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metricsare greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in thesample to corresponding test probepeakheights in thesynthetic referencesample, andcalculatingaprobe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the synthetic reference sample. 19. Thecomputer-programproduct of clause16, 17, or 18,wherein theactions further includedetermininga risk score for a subject associatedwith the samplebasedon theAGGgenotypegenerated for the first allele, the secondallele, or both the first allele and the second allele, and wherein the risk score identifies a risk of the subject developing late- onset neurodegenerative disease fragile X-associated tremor / ataxia syndrome (FXTAS) or fragile X-associated primaryovarian insufficiency (FXPOI) or transmittinga fullmutationallele to their offspringor any combination thereof. 20. The computer-program product of clause 16, 17, or 18, wherein the determining the HBA genotype for each sample comprises: (i) determining aberrant probe ratio patterns for each sample based on the second set of probe ratios for eachsampleand the copynumber calling thresholds for the sampleprobe / referenceprobe ratiosassociated with each probe of the plurality of probes, and (ii) identify the HBA genotype for each sample based on the aberrant probe ratio patterns. 21. The computer-program product of any one of clauses 16‑20, wherein the actions further include triggering performance of a confirmatory test on each sample of the plurality of samples that has the HBA genotype that is not normal or does not require manual review. 30 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 Claims 1. A method comprising: performing a Hemoglobin A (HBA) assay on a plurality of samples, wherein the performing the HBA assay comprises: obtaining a pool of oligonucleotides, wherein each oligonucleotide of the pool of oligonucleotides targets a specific region within an α-globin gene cluster region and / or its surrounding regions; hybridizing nucleic acid obtained from theplurality of sampleswith thepool of oligonucleotides or a derivative thereof to allow binding of a plurality of probes to sequences corresponding to the specific regions in each sample of the plurality of samples; and detecting signals for each probe of the plurality of probes to generate raw data, wherein the raw data comprises HBA copy number data for the plurality of probes; determininga first set of probe ratios for eachsampleof theplurality of samplesbasedon (i) theHBAcopynumber data associated with each sample of the plurality of samples and (ii) HBA copy number data associated with a reference sample; combining a subset of samples from the plurality of samples to create a synthetic reference sample, wherein the subset of samples is negative for copy number variation as determined based on the first set of probe ratios; determining a second set of probe ratios for each sample of the plurality of samples based on (i) the HBA copy number data associated with each sample of the plurality of samples and (ii) HBA copy number data for the synthetic reference sample; determining, using a decision treematrix, an HBA genotype for each sample of the plurality of samples based on aberrant probe ratiopatterns foreachsampledeterminedbasedon thesecondsetof probe ratios for eachsample and copy number calling thresholds for probe ratios associated with each probe of the plurality of probes; and outputting the HBA genotype for each sample of the plurality of samples. 2. The method of claim 1, wherein the performing the HBA assay further comprises obtaining at least one control oligonucleotide targeting at least one locus outside the α-globin gene cluster region and / or its surrounding regions, wherein the nucleic acid obtained from the plurality of samples is hybridizedwith (i) the pool of oligonucleotides or the derivative thereof and (ii) the at least one control oligonucleotide. 3. Themethodof claim2, further comprisingnormalizing the first set of probe ratios for each samplebasedon the signals or probe ratios corresponding to the at least one control oligonucleotide,wherein the subset of samples is negative for copy number variation as determined based on the normalized first set of probe ratios. 4. The method of claim 2, wherein at least 10 control oligonucleotides are obtained. 5. Themethod of claim 2, wherein the at least one control oligonucleotide targets the locus outside of Chromosome 16. 6. The method of claim 1, wherein the pool of oligonucleotides comprises at least 30 oligonucleotides. 7. Themethod of claim 1, wherein the decision treematrix is configured to identify compound heterozygotes involving at least two distinct deletions or duplications in the α-globin gene cluster region. 8. The method of claim 1, wherein: thedetermining thefirst setof probe ratioscomprises: (i) comparingcontrol probepeakheights, or signals, ineach sample of the plurality of samples to corresponding control probe peak heights, or signals, in the reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probepeakheights, or signals, in the referencesample asa control probestandard deviation, (iii) determining a sample of the plurality of samples fails when any variabilitymetrics are greater than a predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the referencesample, andcalculatingaprobe ratiobetween the test probepeakheights, or signals, in thesampleand the corresponding test probe peak heights, or signals, in the reference sample; and / or 31 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 thedetermining thesecondset of probe ratios comprises: (i) comparing thecontrol probepeakheights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the synthetic reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals in the synthetic reference sample as a control probe standard deviation, (iii) determining a sample of the plurality of samples fails when any variability metrics are greater than the predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variabilitymetrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the synthetic reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the synthetic reference sample. 9. The method of claim 1, further comprising: classifying each sample as normal, having a copy number variation (CNV), or as polymorphic based on the aberrant probe ratio patterns; and sub classifying any sample classified as having theCNVas a large targeted deletion, duplication or ’other’ based on the aberrant probe ratio patterns. 10. The method of claim 9, further comprising: sub classifying any sample classified as having the large targeted deletion as a large heterozygous deletion or a homozygous deletion; sub classifying any sample classified as the large heterozygous deletion or the homozygous deletion for one or more of the following deletions: SEA, FIL / THAI, MED or α20.5 based on the aberrant probe ratio patterns; sub classifying any sample classified as having the large heterozygous deletion for one or more of the following: α3.7 deletions, α4.2 deletions and / or α3.7 duplications based on the aberrant probe ratio patterns; or sub classifying any sample classified as ’other’ as having α3.7 deletions, α4.2 deletions, and / or α3.7 duplications based on the aberrant probe ratio patterns. 11. Themethod of claim 1, wherein the performing theHBAassay further comprises resolvingHBA copy number data for the plurality of probes by capillary electrophoresis to generate the raw data. 12. The method of claim 1, wherein the performing the HBA assay further comprises: hybridizing a primer of each oligonucleotide of the pool of oligonucleotides to the nucleic acid from the plurality of samples; ligating the hybridized oligonucleotides to the nucleic acid to obtain the plurality of probes; amplifying at least some of the ligated probes using PCR primers to generate multiplex assay products; and generating, by capillary electrophoresis, the HBA copy number data for the multiplex assay products. 13. Themethod of claim 1, wherein the copy number calling thresholds are between 0.75 and 1.3 for predicting a sample as normal, and the copy number calling thresholds are greater than 1.3 or less than 0.75 for predicting a sample as having a copy number variation. 14. A system comprising: one or more data processors; and a non-transitory computer readable storagemedium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform the method in any one of claims 1‑13. 15. A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform the method in any one of claims 1‑13. 32 EP 4 693 299 A2 5 10 15 20 25 30 35 40 45 50 55 33 EP 4 693 299 A2 34 EP 4 693 299 A2 35 EP 4 693 299 A2 36 EP 4 693 299 A2 37 EP 4 693 299 A2 38 EP 4 693 299 A2 39 EP 4 693 299 A2 40 EP 4 693 299 A2 41 EP 4 693 299 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • US 62986152

[0001] 摘要 本公開涉及一種血紅蛋白 A（HBA）檢測方法和一種用於臨床檢測的 HBA基因分型決 策樹矩陣。 具體而言，本文旨在從對多個樣本進行的 HBA 檢測中獲取原始數據，基 於原始數據計算每個樣本的第一組探針比率，基於第一組探針比率確定要組合成合成參 考樣本的若干參考樣本，基於原始數據和合成參考樣本計算多個樣本中每個樣本的第二 組探針比率，並通過決策樹矩陣，基於每個樣本的第二組探針比率以及與多個探針中每 個探針相關的樣本探針 / 參考探針比率的拷貝數調用閾值，確定每個樣本的 HBA 基因 型。

Claims

1. A method comprising: performing a Hemoglobin A (HBA) assay on a plurality of samples, wherein the performing the HBA assay comprises: obtaining a pool of oligonucleotides, wherein each oligonucleotide of the pool of oligonucleotides targets a specific region within an α-globin gene cluster region and / or its surrounding regions; hybridizing nucleic acid obtained from the plurality of samples with the pool of oligonucleotides or a derivative thereof to allow binding of a plurality of probes to sequences corresponding to the specific regions in each sample of the plurality of samples; and detecting signals for each probe of the plurality of probes to generate raw data, wherein the raw data comprises HBA copy number data for the plurality of probes; determining a first set of probe ratios for each sample of the plurality of samples based on (i) the HBA copy number data associated with each sample of the plurality of samples and (ii) HBA copy number data associated with a reference sample; combining a subset of samples from the plurality of samples to create a synthetic reference sample, wherein the subset of samples is negative for copy number variation as determined based on the first set of probe ratios; determining a second set of probe ratios for each sample of the plurality of samples based on (i) the HBA copy number data associated with each sample of the plurality of samples and (ii) HBA copy number data for the synthetic reference sample; determining, using a decision tree matrix, an HBA genotype for each sample of the plurality of samples based on aberrant probe ratio patterns for each sample determined based on the second set of probe ratios for each sample and copy number calling thresholds for probe ratios associated with each probe of the plurality of probes; and outputting the HBA genotype for each sample of the plurality of samples.

2. The method of claim 1, wherein the performing the HBA assay further comprises obtaining at least one control oligonucleotide targeting at least one locus outside the α-globin gene cluster region and / or its surrounding regions, wherein the nucleic acid obtained from the plurality of samples is hybridized with (i) the pool of oligonucleotides or the derivative thereof and (ii) the at least one control oligonucleotide.

3. The method of claim 2, further comprising normalizing the first set of probe ratios for each sample based on the signals or probe ratios corresponding to the at least one control oligonucleotide, wherein the subset of samples is negative for copy number variation as determined based on the normalized first set of probe ratios.

4. The method of claim 2, wherein at least 10 control oligonucleotides are obtained.

5. The method of claim 2, wherein the at least one control oligonucleotide targets the locus outside of Chromosome 16.

6. The method of claim 1, wherein the pool of oligonucleotides comprises at least 30 oligonucleotides.

7. The method of claim 1, wherein the decision tree matrix is configured to identify compound heterozygotes involving at least two distinct deletions or duplications in the α-globin gene cluster region.

8. The method of claim 1, wherein: the determining the first set of probe ratios comprises: (i) comparing control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals, in the reference sample as a control probe standard deviation, (iii) determining a sample of the plurality of samples fails when any variability metrics are greater than a predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the reference sample; and / or the determining the second set of probe ratios comprises: (i) comparing the control probe peak heights, or signals, in each sample of the plurality of samples to corresponding control probe peak heights, or signals, in the synthetic reference sample, (ii) calculating variability in signals between the control probe peak heights, or signals, in each sample and the corresponding control probe peak heights, or signals in the synthetic reference sample as a control probe standard deviation, (iii) determining a sample of the plurality of samples fails when any variability metrics are greater than the predetermined threshold, (iv) determining a sample of the plurality of samples does not fail when none of the variability metrics are greater than the predetermined threshold, and (v) for each sample that does not fail, comparing test probe peak heights, or signals, in the sample to corresponding test probe peak heights in the synthetic reference sample, and calculating a probe ratio between the test probe peak heights, or signals, in the sample and the corresponding test probe peak heights, or signals, in the synthetic reference sample.

9. The method of claim 1, further comprising: classifying each sample as normal, having a copy number variation (CNV), or as polymorphic based on the aberrant probe ratio patterns; and sub classifying any sample classified as having the CNV as a large targeted deletion, duplication or 'other' based on the aberrant probe ratio patterns.

10. The method of claim 9, further comprising: sub classifying any sample classified as having the large targeted deletion as a large heterozygous deletion or a homozygous deletion; sub classifying any sample classified as the large heterozygous deletion or the homozygous deletion for one or more of the following deletions: SEA, FIL / THAI, MED or α20.5 based on the aberrant probe ratio patterns; sub classifying any sample classified as having the large heterozygous deletion for one or more of the following: α3.7 deletions, α4.2 deletions and / or α3.7 duplications based on the aberrant probe ratio patterns; or sub classifying any sample classified as 'other' as having α3.7 deletions, α4.2 deletions, and / or α3.7 duplications based on the aberrant probe ratio patterns.

11. The method of claim 1, wherein the performing the HBA assay further comprises resolving HBA copy number data for the plurality of probes by capillary electrophoresis to generate the raw data.

12. The method of claim 1, wherein the performing the HBA assay further comprises: hybridizing a primer of each oligonucleotide of the pool of oligonucleotides to the nucleic acid from the plurality of samples; ligating the hybridized oligonucleotides to the nucleic acid to obtain the plurality of probes; amplifying at least some of the ligated probes using PCR primers to generate multiplex assay products; and generating, by capillary electrophoresis, the HBA copy number data for the multiplex assay products.

13. The method of claim 1, wherein the copy number calling thresholds are between 0.75 and 1.3 for predicting a sample as normal, and the copy number calling thresholds are greater than 1.3 or less than 0.75 for predicting a sample as having a copy number variation.

14. A system comprising: one or more data processors; and a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform the method in any one of claims 1-13.

15. A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform the method in any one of claims 1-13.

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