Methods for detecting inherited mutations using multiplex gene-specific PCR.

Abstract

The present disclosure provides methods for detecting inherited mutations (e.g., Ashkenazi Jewish carrier mutations, beta thalassemia mutations, or alpha thalassemia mutations) using multiplex gene-specific PCR. Kits for use in practicing the methods are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 286,906, filed December 7, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure provides methods for detecting inherited mutations (e.g., Ashkenazi Jewish carrier mutations, beta thalassemia mutations, or alpha thalassemia mutations) using multiplex gene-specific PCR. Kits for use in practicing the methods are also provided. [Background technology]

[0003] The following description of the background of the present disclosure is provided merely to aid the reader in understanding the present disclosure and is not admitted to describe or constitute prior art to the present disclosure.

[0004] Thalassemia is an inherited autosomal recessive disorder resulting from mutations in the α- and β-globin gene clusters on chromosome 16 and chromosome 11, respectively. It is characterized by the absence or reduced synthesis of the globin chains of hemoglobin and includes two main types, α- and β-thalassemia. Thalassemia is reported to be one of the top five major congenital defects. Thalassemia major imposes a great burden on society and severely impacts the quality of life of affected populations. High prevalence of thalassemia has been observed in Southern China, Southeast Asia, India, the Middle East, Africa, and the Mediterranean region.

[0005] The Ashkenazi Jewish population, which lived primarily in Central and Eastern Europe, maintained a genetic isolation from its neighbors, separated by religious and cultural practices, as well as by kinship ties. The primary evidence for this isolation of the Ashkenazi Jewish population is the presence of genetic characteristics, including a high prevalence of autosomal recessive diseases, and a relatively high frequency of alleles that confer risk for common diseases such as breast and ovarian cancer, or variants associated with inflammatory bowel disease or Parkinson's disease.

[0006] There is a substantial need for more robust and sensitive methods that can rapidly detect whether a subject is a carrier for inherited genetic diseases, including beta-thalassemia and alpha-thalassemia, diseases that are frequent in the Ashkenazi Jewish population, in a single biological sample. Summary of the Invention

[0007] The present disclosure provides methods for simultaneous identification of inherited genetic variants, such as Ashkenazi Jewish panel variations, beta thalassemia variations, and alpha thalassemia variations. The present disclosure also provides methods for identifying Ashkenazi Jewish panel variations, beta thalassemia variations, and alpha thalassemia variations in a biological sample from a subject. Finally, the present disclosure provides kits for carrying out the methods described herein.

[0008] In some embodiments, the method includes extracting DNA from a biological sample obtained from the subject; contacting the biological sample with a first plurality of primer pairs to generate a first plurality of amplicons, where at least one amplicon corresponds to each of a plurality of genes, the plurality of genes including HEXA, SMPD1, MCOLN1, GBA, FANCC, IKBKAP, ASPA, BLM, BCKDHB, G6PC, ABCC8, DLD, NEB, PCDH15, CLRN1, TMEM216, and FKTN; and contacting the biological sample with a second plurality of primer pairs to generate a second plurality of amplicons, where at least one amplicon corresponds to each of GBA and HBB, the second plurality of primer pairs including SEQ ID NOs:81 and 82, and SEQ ID NOs:83 and 84; and contacting the biological sample with a third plurality of primer pairs, Hot Start Taq generating a third plurality of amplicons by contacting with a DNA polymerase and a Taq extender PCR additive, at least one amplicon corresponding to each of HBA1 and HBA2; incorporating barcode sequences onto ends of the first, second, and third plurality of amplicons via polymerase chain reaction; and detecting one or more variants in at least one of the first, second, and third plurality of amplicons using high throughput massively parallel sequencing.

[0009] In some embodiments, Ashkenazi Jewish panel variations include genetic variations (e.g., single nucleotide variations (SNVs), deletions, insertions, or inversions) that are commonly observed in Ashkenazi Jewish populations, but may also occur, albeit at a lower frequency, outside of Ashkenazi Jewish populations. In some embodiments, Ashkenazi Jewish panel variations include genetic variations that may cause Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, Maple serum uria, glycogen storage disease I, familial hyperinsulinism, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Usher syndrome type IF, Usher syndrome, type IIIA, Joubert syndrome 2, or Walker-Warburg syndrome. In some embodiments, Ashkenazi Jewish panel variations include genetic variations listed in Table 1. In some embodiments, the beta thalassemia variations include the genetic variations listed in Table 2. In some embodiments, the beta thalassemia variations include the genetic variations listed in Table 3.

[0010] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present. In some embodiments, the GBA primers of the second plurality of primer pairs specifically amplify the GBA gene and do not amplify any GBA pseudogenes. In some embodiments, the third plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present.

[0011] In some embodiments, variations in the HEXA gene cause Tay-Sachs disease, variations in the SMPD1 gene cause Niemann-Pick disease, variations in the MCOLN1 gene cause mucolipidosis type IV, variations in the GBA gene cause Gaucher disease, variations in the PANCC gene cause Fanconi anemia, variations in the IKBKAP gene cause familial dysautonomia, variations in the ASPA gene cause Canavan disease, variations in the BLM gene cause Bloom syndrome, variations in the BCKDHB gene cause Maple serum uria, variations in the G6PC gene cause G6PC disease, Variations in the ABCC8 gene cause familial hyperinsulinism, variations in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3), variations in the NEB gene cause nemaline myopathy, variations in the PCDH15 gene cause Usher syndrome, type IF, variations in the CLRN1 gene cause Usher syndrome, type IIIA, variations in the TMEM216 gene cause Joubert syndrome 2, and variations in the FKTN gene cause Walker-Warburg syndrome.

[0012] In some embodiments, the Ashkenazi Jewish panel genetic variations include variations in one or more genes listed in Table 1. In some embodiments, the HEXA gene variation is R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or the 7.6-kb Del, Ex1, SMPD1 gene variations include one or more of L302P, fsP330, R496L (R498L), or deltaR608, MCOLN1 gene variations include one or more of IVS3-2A>G or 6.4kb_del, GBA gene variations include one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P, or R496H, PANCC gene variations include one or more of IVS4+4A>T or 322delG, IKBKAP gene variations include one or more of R696P or IVS20+6T>C, ASPA gene variations include IVS2-2A>G, Y231X, E285A, or A 305E, BLM gene variations include one or more of 2281del6 / ins7, BCKDHB gene variations include one or more of R183P, G278S, or E372X, G6PC gene variations include one or more of R83C or Q347X, ABCC8 gene variations include IVS32-9G>A or F1387del, DLD gene variations include one or more of Y35* or G229C, NEB gene variations include R2478_D2512del35, PCDH15 gene variations include one or more of R245, CLRN1 gene variations include N48K, TMEM216 gene variations include R73L, and FKTN gene variations include F390fs.

[0013] In some embodiments, the beta thalassemia genetic variation in the HBB gene comprises one or more genetic variations listed in Table 2. In some embodiments, the genetic variations include c.*111A>G, c.*110T>C, c.*96T>C, Hb D-Los Angeles (c.364G>C), Hb O-Arab(c.364G>A), c.321_322insG, c.316-2A>C, c.316-2A>G, c.316-3C>A, c.316-106C>G, c.316-125A>G, c.316-146T>G, c.316-197C>T, c.315+1G>A, c.287_288insA, c.251delG, c.230delC, c.216_217insA, c.203_204delTG, c.143_144insA, c.1 46_147insATCT, c.135delC, c.130G>T, c.126_129delCTTT, c.124_127delTTCT, c.118C>T, c.114G>A, c.112delT, c.93-1G>C , c.93-1G>A, c.93-21G>A, c.92+6T>C, c.92+5G>A, c.92+5G>C, c.92+5G>T, c.92+2T>A, c.92+2T>C, c.92+1G>A, c.92+1G>T, Hb Monroe(c.92G>C), c.92G>A, c.84_85insC, c.79G>T, HB E(c.79G>A), c.75T>A, c.59A>G, c.52A>T(LYS17*), c.51delC, c.48G>A, c.47G>A( Trp15), c.46delT, c.36delT, c.33C>A, c.27_28insG, c.25_26delAA, c.20delA, HB S(c.20A>T), HB C(c.19G>A), c.17_18delCT, c.2T>C, c.2T>G, c.1A>G, c.-78A>C, c.-78A>G, c.-79A>G, c.-80T>A, c.-81A >G, c.-136C>G, c.-137C>A, c.-137C>G, c.-137C>T, c.-138C>T, c.-138C>A, c.-140C>T, or c.-151C>T.

[0014] In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise one or more variations listed in Table 3. In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL, or a single nucleotide variant in the HBA2 gene HBA2 c.427T>C.

[0015] In some embodiments, the first plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least 2, at least 4, at least 5, at least 8, or at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or all of the primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs set forth in Table 4.

[0016] In some embodiments, the second plurality of primer pairs comprises at least two, at least four, or at least five primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two, at least four, or at least five primer pairs selected from Table 5.

[0017] In some embodiments, the third plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85% identity to at least 2, at least 3, or at least 4 primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 6.

[0018] In some embodiments, next generation sequencing comprises sequencing by synthesis (e.g., pyrosequencing). In some embodiments, next generation sequencing comprises sequencing by ligation.

[0019] In some embodiments, the subject is suspected of being a carrier for at least one disease selected from Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom's syndrome, Maple serum urine, glycogen storage disease I, familial hyperinsulinism, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Usher syndrome type IF, Usher syndrome, type IIIA, Joubert syndrome 2, Walker-Warburg syndrome, beta thalassemia, or alpha thalassemia.

[0020] In some embodiments, the subject is of Ashkenazi Jewish descent or has a family history of beta or alpha thalassemia.

[0021] In some embodiments, the biological sample is selected from whole blood, serum, plasma, amniotic fluid, or chorionic villi.

[0022] In some embodiments, the genetic disorder is an autosomal disorder or an X-linked recessive disorder.

[0023] In another aspect, the disclosure provides kits that include oligonucleotides, which can be primers or probes for carrying out the amplifications described herein.

[0024] The present disclosure also provides kits for detecting the presence of an Ashkenazi Jewish panel variation, a beta thalassemia variation, or an alpha thalassemia variation in a biological sample from a subject.

[0025] The kit of the present technology includes at least: (i) a first plurality of primer pairs directed to an amplified region of each of a plurality of genes, the plurality of genes including HEXA, SMPD1, MCOLN1, GBA, FANCC, IKBKAP, ASPA, BLM, BCKDHB, G6PC, ABCC8, DLD, NEB, PCDH15, CLRN1, TMEM216, and FKTN; (ii) a second plurality of primer pairs directed to an amplified region of GBA and HBB genes, the second plurality of primer pairs including SEQ ID NOs: 81 and 82, and SEQ ID NOs: 83 and 84; and (iii) a third plurality of primer pairs directed to an amplified region of an alpha thalassemia genetic variation in each of HBA1 and HBA2 genes, and instructions for use.

[0026] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present.

[0027] In some embodiments, the GBA primers of the second plurality of primer pairs specifically amplify the GBA gene and do not amplify any GBA pseudo-genes.

[0028] In some embodiments, the third plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present.

[0029] In some embodiments, variations in the HEXA gene cause Tay-Sachs disease, variations in the SMPD1 gene cause Niemann-Pick disease, variations in the MCOLN1 gene cause mucolipidosis type IV, variations in the GBA gene cause Gaucher disease, variations in the PANCC gene cause Fanconi anemia, variations in the IKBKAP gene cause familial dysautonomia, variations in the ASPA gene cause Canavan disease, variations in the BLM gene cause Bloom syndrome, variations in the BCKDHB gene cause Maple serum uria, variations in the G6PC gene cause G6PC disease, Variations in the ABCC8 gene cause familial hyperinsulinism, variations in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3), variations in the NEB gene cause nemaline myopathy, variations in the PCDH15 gene cause Usher syndrome, type IF, variations in the CLRN1 gene cause Usher syndrome, type IIIA, variations in the TMEM216 gene cause Joubert syndrome 2, and variations in the FKTN gene cause Walker-Warburg syndrome.

[0030] In some embodiments, the Ashkenazi Jewish panel genetic variations include variations in one or more genes listed in Table 1. In some embodiments, the HEXA gene variation is R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or the 7.6-kb Del, Ex1, SMPD1 gene variations include one or more of L302P, fsP330, R496L (R498L), or deltaR608, MCOLN1 gene variations include one or more of IVS3-2A>G or 6.4kb_del, GBA gene variations include one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P, or R496H, PANCC gene variations include one or more of IVS4+4A>T or 322delG, IKBKAP gene variations include one or more of R696P or IVS20+6T>C, ASPA gene variations include IVS2-2A>G, Y231X, E285A, or A 305E, BLM gene variations include one or more of 2281del6 / ins7, BCKDHB gene variations include one or more of R183P, G278S, or E372X, G6PC gene variations include one or more of R83C or Q347X, ABCC8 gene variations include IVS32-9G>A or F1387del, DLD gene variations include one or more of Y35* or G229C, NEB gene variations include R2478_D2512del35, PCDH15 gene variations include one or more of R245, CLRN1 gene variations include N48K, TMEM216 gene variations include R73L, and FKTN gene variations include F390fs.

[0031] In some embodiments, the beta thalassemia genetic variation in the HBB gene comprises one or more genetic variations listed in Table 2. In some embodiments, the genetic variations include c.*111A>G, c.*110T>C, c.*96T>C, Hb D-Los Angeles (c.364G>C), Hb O-Arab(c.364G>A), c.321_322insG, c.316-2A>C, c.316-2A>G, c.316-3C>A, c.316-106C>G, c.316-125A>G, c.316-146T>G, c.316-197C>T, c.315+1G>A, c.287_288insA, c.251delG, c.230delC, c.216_217insA, c.203_204delTG, c.143_144insA, c.1 46_147insATCT, c.135delC, c.130G>T, c.126_129delCTTT, c.124_127delTTCT, c.118C>T, c.114G>A, c.112delT, c.93-1G>C , c.93-1G>A, c.93-21G>A, c.92+6T>C, c.92+5G>A, c.92+5G>C, c.92+5G>T, c.92+2T>A, c.92+2T>C, c.92+1G>A, c.92+1G>T, Hb Monroe(c.92G>C), c.92G>A, c.84_85insC, c.79G>T, HB E(c.79G>A), c.75T>A, c.59A>G, c.52A>T(LYS17*), c.51delC, c.48G>A, c.47G>A( Trp15), c.46delT, c.36delT, c.33C>A, c.27_28insG, c.25_26delAA, c.20delA, HB S(c.20A>T), HB C(c.19G>A), c.17_18delCT, c.2T>C, c.2T>G, c.1A>G, c.-78A>C, c.-78A>G, c.-79A>G, c.-80T>A, c.-81A >G, c.-136C>G, c.-137C>A, c.-137C>G, c.-137C>T, c.-138C>T, c.-138C>A, c.-140C>T, or c.-151C>T.

[0032] In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise variations in one or more genes listed in Table 3. In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL, or a single nucleotide variant in the HBA2 gene HBA2 c.427T>C.

[0033] In some embodiments, the first plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least 2, at least 4, at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or all of the primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs set forth in Table 4.

[0034] In some embodiments, the second plurality of primer pairs comprises at least two, at least four, or at least five primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two, at least four, or at least five primer pairs selected from Table 5. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 5.

[0035] In some embodiments, the third plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85% identity to at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 6. [Brief description of the drawings]

[0036] [Figure 1] 1 shows the sequencing library preparation workflow. [Diagram 2] 1 shows the sequencing data analysis workflow. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure provides methods that simultaneously provide hotspot molecular detection of variants for multiple autosomal and X-linked recessive disorders, allowing testing of individuals regardless of ancestry or geographic origin ("extended carrier screening"). This carrier screening easily and quickly identifies couples at high risk of having affected children to facilitate informed reproductive decisions.

[0038] The carrier screening panel disclosed herein includes 20 genes associated with 19 diseases, including: alpha thalassemia, beta thalassemia (including sickle cell anemia), Bloom's syndrome, Canavan disease, dihydrolipoamide dehydrogenase deficiency, familial dysautonomia, familial hyperinsulinism, Fanconi anemia type C, Gaucher disease, glycogen storage disease type IA, Joubert syndrome 2, maple syrup urine disease, mucolipidosis IV, nemaline myopathy, Niemann-Pick disease type A, Tay-Sachs disease, Usher syndrome type IF, Usher syndrome type III, and Walker-Warburg syndrome.

[0039] This carrier screening panel contains 24 genes by including three additional diseases, Cystic Fibrosis (CFTR), Fragile X Syndrome (FMR1), and Spinal Muscular Atrophy (SMN1 and SMN2), in addition to the previously described carrier screening panel (20 genes) for 19 disorders.

[0040] The disclosed carrier screening provides hotspot molecular detection of variants for multiple autosomal and X-linked recessive disorders simultaneously, allowing testing of individuals regardless of ancestry or geographic origin. Carrier screening aims to identify couples at high risk of having affected children to facilitate informed reproductive decisions.

[0041] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0042] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise specified. Thus, for example, a reference to an "oligonucleotide" includes a plurality of oligonucleotide molecules, and a reference to a "nucleic acid" is a reference to one or more nucleic acids. It is further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0043] As used herein, the term "about" in reference to numerical values ​​is generally interpreted as including numerical values ​​that fall within a range of 1% to 10% in either direction (greater or lesser) of the numerical value, unless otherwise specified or apparent from the context.

[0044] The term "adapter" refers to a short, chemically synthesized nucleic acid sequence that can be used to ligate to the end of a nucleic acid sequence to facilitate attachment to another molecule. Adapters can be single-stranded or double-stranded. Adapters can incorporate short (typically less than 50 base pairs) sequences useful for PCR amplification or sequencing.

[0045] As used herein, the term "amplify" or "amplification" with respect to a nucleic acid sequence refers to a method of increasing the representation of a population of nucleic acid sequences in a sample. The copies of a particular target nucleic acid sequence generated in vitro in an amplification reaction are called "amplicons" or "amplification products". Amplification can be exponential or linear. The target nucleic acid can be DNA (e.g., genomic DNA and cDNA, etc.) or RNA. The exemplary method described below relates to amplification using polymerase chain reaction (PCR), although many other methods, such as isothermal methods, rolling circle methods, etc., are well known to those skilled in the art. Those skilled in the art will understand that these other methods can either be used instead of or together with the PCR method. See, e.g., Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, CA 1990, pp 13-20; Wharam, et al., Nucleic Acids Res. 29(11):E54-E54 (2001).

[0046] As used herein, an "amplification mixture" is a mixture of reagents used in a nucleic acid amplification reaction, but does not contain primers or samples. The amplification mixture includes a buffer, dNTPs, and a DNA polymerase. The amplification mixture may further include at least one of MgCl2, KCl, non-ionic and ionic detergents (including cationic detergents).

[0047] An "amplification master mix" contains an amplification mixture and primers for amplifying one or more target nucleic acids, but does not contain the sample to be amplified.

[0048] As used herein, the phrase "selectively amplify" refers to an amplification reaction (e.g., a PCR reaction) in which only selected sequences are amplified. The phrase "gene-specific amplification" refers to an amplification reaction (e.g., a PCR reaction) in which only selected genes or portions of genes (e.g., where a known / preselected variant occurs) are amplified.

[0049] The terms "complement," "complementary," or "complementarity" as used herein with respect to polynucleotides (i.e., sequences of nucleotides such as oligonucleotides or target nucleic acids) refer to Watson / Crick base pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide that is in "antiparallel association" when aligned with a nucleic acid sequence such that the 5' end of one sequence pairs with the 3' end of the other. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Certain bases not commonly found in naturally occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7-deazaguanine, locked nucleic acids (LNA), and peptide nucleic acids (PNAs). Complementarity need not be perfect, and stable duplexes may contain mismatched base pairs, degenerates, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically, taking into account several variables including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs. The complementary sequence can be a DNA sequence or an RNA sequence complementary to the complementary sequence, or it can be a cDNA.

[0050] The term "substantially complementary" as used herein means that two sequences hybridize under stringent hybridization conditions. Those skilled in the art will understand that substantially complementary sequences do not have to hybridize along their entire length. In particular, a substantially complementary sequence may include a contiguous sequence of bases that do not hybridize to the target sequence, located 3' or 5' to a contiguous sequence of bases that hybridize to the target sequence under stringent hybridization conditions.

[0051] As used herein, the term "detecting" refers to determining the presence of a target nucleic acid in a sample. Detection does not require a method to provide 100% sensitivity and / or 100% specificity.

[0052] As used herein, the term "direct amplification" refers to a nucleic acid amplification reaction in which the target nucleic acid is amplified from a sample without prior purification, extraction, or concentration.

[0053] The term "sequencing," as used herein, refers to a method in which the identity of at least 10 consecutive nucleotides of a polynucleotide (e.g., the identity of at least 20, at least 50, at least 100, or at least 200, or more consecutive nucleotides) is obtained.

[0054] The term "next-generation sequencing" refers to so-called parallelized sequencing-by-synthesis or sequencing-by-ligation platforms currently used by Illumina, Life Technologies, and Roche, etc. (e.g., Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' sequencing-by-ligation (SOLiD platform), or Pacific Biosciences' fluorescence-based cleavage method). Next-generation sequencing methods can also include nanopore sequencing methods, or electronic detection-based methods such as the Ion Torrent technology commercialized by Life Technologies. Examples of such methods are described in the following references: Margulies et al (Nature 2005 437:376-80), Ronaghi et al (Analytical Biochemistry 1996 242:84-9), Shendure (Science 2005 309:1728), Imelfort et al (Brief Bioinform. 2009 10:609-18), Fox et al (Methods Mol Biol. 2009;553:79-108), Appleby et al (Methods Mol Biol. 2009;513:19-39), English (PLoS One. 2012 7:e47768), and Morozova (Genomics. 2008 92:255-64), which are incorporated by reference for a general description of the method and specific steps of the method, including all starting products, reagents, and end products for each of the steps. Next generation sequencing can produce at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1M at least 10M at least 100M, or at least 1B sequence reads. Often the reads are paired end reads.

[0055] In some embodiments, the next generation sequencing of the present disclosure comprises the following next generation sequencing systems:

[0056] The Ion Torrent™ (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during the incorporation of unmodified nucleotides in DNA replication. For use with this system, a sequencing library is first produced by generating DNA fragments flanked by sequencing adaptors. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified templates are then placed into a silicon semiconductor sequencing chip. During replication, the chip is poured with one nucleotide after another, and if the nucleotide complements a DNA molecule in a specific microwell of the chip, it will be incorporated. When a nucleotide is incorporated into a DNA molecule by a polymerase, a proton is spontaneously released, resulting in a detectable local change in pH. The pH of the solution is then changed in that well and detected by an ion sensor. If homopolymeric repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

[0057] The 454TM GS FLX™ Sequencing System (Roche, Germany) employs a light-based detection methodology in a massively parallel pyrosequencing system.

[0058] Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time, and detecting and quantifying the number of nucleotides added at a given position through light emitted by the release of attached pyrophosphate. For use with the 454™ system, adapter-ligated DNA fragments are immobilized on small DNA capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR). Each DNA-bound bead is placed into a well on a picotiter plate, and sequencing reagents are delivered across the wells of the plate. During a sequencing run, the four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device. During the flow of nucleotides, millions of copies of the DNA bound to each of the beads are sequenced in parallel. When a nucleotide complementary to the template strand is added to the well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument.

[0059] A sequencing technique based on reversible dye terminators: DNA molecules are first attached to a primer on a slide and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT bases) are added and unincorporated nucleotides are washed away. Unlike pyrosequencing, DNA can only be extended one nucleotide at a time. A camera takes an image of the fluorescently labeled nucleotides, and then the dye, along with the terminal 3' blocker, is chemically removed from the DNA to allow for the next cycle.

[0060] Helicos single molecule sequencing uses DNA fragments with polyA tail adapters attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplex. Reads are performed by the Helioscope sequencer. After acquisition of images tiling the complete array, chemical cleavage and release of the fluorescent label allows for subsequent cycles of extension and imaging.

[0061] Sequencing by synthesis (SBS), like "old-style" dye-termination electrophoretic sequencing, relies on the incorporation of nucleotides by DNA polymerase to determine base sequence. A DNA library with immobilized adapters is denatured into single strands, grafted to a flow cell, and subsequently bridge-amplified to form a high-density array of spots on a glass chip. The reversible terminator method uses a reversible version of the dye terminator, adding one nucleotide at a time and detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide. The signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate-driven photoreactions, and hydrogen ion sensing, all of which are used. Examples of SBS platforms include the Illumina GA and HiSeq 2000. The MiSeq® Personal Sequencing System (Illumina, Inc.) also employs sequencing by synthesis using reversible terminator chemistry.

[0062] In contrast to sequencing by synthesis, sequencing by ligation uses DNA ligase to determine the target sequence. This sequencing method relies on the enzymatic ligation of adjacent oligonucleotides via local complementarity on the template DNA strand. This technology employs partitions of all possible oligonucleotides of fixed length, labeled according to the sequenced position. Preferential ligation by DNA ligase to anneal and ligate oligonucleotides to match sequences results in a dinucleotide-coded color space signal at that position (through the release of a fluorescently labeled probe corresponding to a known nucleotide at a known position along the oligo). This method is primarily used by Life Technologies' SOLiD™ sequencers. Prior to sequencing, DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.

[0063] SMRT™ sequencing is based on a sequencing-by-synthesis approach. DNA is synthesized in a zero-mode waveguide (ZMW), which is a small well-like container with a capture tool placed at the bottom of the well. Sequencing is performed using an unmodified polymerase (attached to the bottom of the ZMW) and fluorescently labeled nucleotides that flow freely in solution. The wells are constructed so that only the fluorescence generated at the bottom of the well is detected. The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unmodified DNA strand.

[0064] The term "barcode sequence" or "molecular barcode" as used herein refers to a unique sequence of nucleotides that is used to (a) identify and / or track the source of a polynucleotide in a reaction and / or (b) count the number of times an initial molecule is sequenced (e.g., when substantially all molecules in a sample are tagged with a different sequence and the sample is then amplified). The barcode sequence can be at the 5' end, 3' end, or in the middle, or at both the 5' and 3' ends of the oligonucleotide. Barcode sequences can vary widely in size and composition. The following references provide guidance for selecting an appropriate set of barcode sequences for a particular embodiment. Brenner, U.S. Patent No. 5,635,400; Brenner et al, Proc. Natl. Acad. Sci., 97:1665-1670 (2000); Shoemaker et al, Nature Genetics, 14:450-456 (1996); Morris et al, European Patent Publication No. 0799897A1; Wallace, U.S. Patent No. 5,981,179, etc. In some embodiments, the barcode sequence can have a length ranging from 4 to 36 nucleotides, or 6 to 30 nucleotides, or 8 to 20 nucleotides.

[0065] As used herein, the term "extraction" refers to any action taken to remove nucleic acids from other (non-nucleic acid) materials present in a sample. The term extraction includes mechanical or chemical lysis, addition of detergents or proteases, or precipitation and removal of non-nucleic acids such as proteins.

[0066] The term "hybridizing" as used herein refers to the process by which two substantially complementary nucleic acid strands (at least about 65% complementary, at least about 75%, or at least about 90% complementary over a stretch of at least 14-25 nucleotides) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through the formation of hydrogen bonds between complementary base pairs. Hybridization is typically and preferably performed with probe-length nucleic acid molecules, preferably 15-100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) depend on the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the thermal melting temperature (T m) and other factors. Those skilled in the art know how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the desired level of complementarity will stably hybridize, while sequences with less complementarity will not hybridize. For examples of hybridization conditions and parameters, see, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, NJ. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or primer) that is specific for a target nucleic acid will "hybridize" to the target nucleic acid under suitable conditions.

[0067] "Hot start" in the context of nucleic acid amplification reactions refers to a protocol in which at least one key reagent is withheld from the reaction mixture (or the reagent remains inactive, if present in the reaction mixture) until the temperature is raised sufficiently to provide the required hybridization specificity of the primer or primers. A "hot start enzyme" is an enzyme, typically a nucleic acid polymerase, that can act as a "withheld" or inactive reagent in a hot start protocol. For example, some hot start enzymes can be obtained by chemically modifying the enzyme. Examples of hot start enzymes include AZ05-Gold polymerase, KAPA HiFi, and AmpliTaq Gold®.

[0068] As used herein, a "Taq extender" is a PCR additive that increases the efficiency of the Taq DNA polymerase extension reaction during each cycle of PCR by reducing mismatch pausing, resulting in a greater percentage of completed extension reactions. In some embodiments, the Taq extender comprises Taq Extender™ from Agilent or Strategene.

[0069] As used herein, the terms "individual," "patient," or "subject" may refer to an individual organism, a vertebrate, a mammal, or a human. In preferred embodiments, the individual, patient, or subject is a human.

[0070] A pseudogene is defined herein as a wild-type, functional, non-protein-encoding nucleic acid sequence. The term "pseudogene" encompasses nucleic acid sequences that do not encode any protein. Additionally, the term "pseudogene" encompasses gene alleles that contain modifications, e.g., insertions or deletions, such that they encode proteins or portions of proteins with significantly impaired or lost function compared to the wild-type protein of the same type. Such alleles encode truncated proteins, e.g., as a result of a frameshift caused by the insertion and / or deletion of at least one nucleotide or caused by a premature stop codon.

[0071] As used herein, the term "multiplex PCR" refers to the simultaneous generation of two or more PCR products or amplicons in the same reaction vessel. Each PCR product is primed using a different primer pair. A multiplex reaction may further include specific probes for each product that are labeled with different detectable moieties.

[0072] As used herein, "oligonucleotide" refers to a molecule having a sequence of nucleic acid bases on a backbone composed primarily of identical monomer units at defined intervals. The bases are arranged on the backbone so that it can bind to a nucleic acid having a sequence of bases that is complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction can be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides can also include derivatives in which the hydrogen of the hydroxyl group is replaced with an organic group, e.g., an allyl group. Oligonucleotides that function as primers or probes are generally at least about 10-15 nucleotides in length, or up to about 70, 100, 110, 150, or 200 nucleotides in length, and more preferably at least about 15-25 nucleotides in length. Oligonucleotides used as primers or probes to specifically amplify or specifically detect a particular target nucleic acid are generally capable of specific hybridization to the target nucleic acid.

[0073] As used herein, a "positive control nucleic acid" or "internal positive amplification control" is a nucleic acid known to be present in a sample at a certain amount or level. In some embodiments, the positive control nucleic acid is not naturally present in the sample and is added to the sample in the disclosed methods prior to subjecting the reaction sample mixture to real-time polymerase chain reaction.

[0074] As used herein, the term "primer" refers to an oligonucleotide that can act as an initiation point for nucleic acid sequence synthesis when placed under conditions that induce the synthesis of a primer extension product that is complementary to a target nucleic acid strand, i.e., in an appropriate buffer ("buffer" includes pH, ionic strength, cofactors, etc.) and in the presence of different nucleotide triphosphates and a polymerase at a suitable temperature. One or more of the nucleotides of the primer can be modified, for example, by the addition of a methyl group, a biotin, or a digoxigenin moiety, a fluorescent tag, or by using a radioactive nucleotide. The primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. The term primer as used herein includes all forms of primers that can be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, etc. As used herein, the term "forward primer" refers to a primer that anneals to the antisense strand of double-stranded DNA (dsDNA). A "reverse primer" anneals to the sense strand of dsDNA.

[0075] Primers are typically at least 10, 15, 18, or 30 nucleotides in length, or up to about 100, 110, 125, or 200 nucleotides in length. In some embodiments, primers are preferably about 15 to about 60 nucleotides in length, and most preferably about 25 to about 40 nucleotides in length. In some embodiments, primers are 15 to 35 nucleotides in length. There is no standard length for optimal hybridization or polymerase chain reaction amplification. The optimal length for a particular primer application can be readily determined in the manner described in H. Erlich, PCR Technology, Principles and Application for DNA Amplification, (1989).

[0076] "Primer extension reaction" refers to a synthesis reaction in which an oligonucleotide primer hybridizes to a target nucleic acid and a complementary copy of the target nucleic acid is produced by polymerase-dependent 3' addition of individual complementary nucleotides. In some embodiments, the primer extension reaction is PCR.

[0077] As used herein, the phrase "primer pair" refers to a forward and reverse primer pair (i.e., a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.

[0078] As used herein, the phrase "gap primer pair" refers to a primer pair designed to produce a product only if a deletion is present. In some embodiments, large deletions specifically detected using gap primer pairs include a 7.6 Kb deletion in HEXA, a 6.4 Kb deletion in MCOLN1, a 2.5 Kb deletion in NEB, and the following alpha thalassemia variants: 3.7, 4.2, SEA, THAI, 20.5, MED, and FIL. In some embodiments, detection of any of the 10 large deletions is based on the presence of a gap PCR amplicon and not on the specific variant called.

[0079] A "probe" as used herein refers to a nucleic acid that interacts with a target nucleic acid through hybridization. A probe may be fully or partially complementary to a target nucleic acid sequence. The level of complementarity will generally depend on a number of factors based on the function of the probe. A probe can be labeled, unlabeled, or modified in any of several ways well known in the art. A probe can specifically hybridize to a target nucleic acid. A probe can be DNA, RNA, or an RNA / DNA hybrid. A probe can be an oligonucleotide, an artificial chromosome, a fragmented artificial chromosome, a genomic nucleic acid, a fragmented genomic nucleic acid, an RNA, a recombinant nucleic acid, a fragmented recombinant nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid, an oligomer of a cyclic heterocycle, or a conjugate of a nucleic acid. A probe can include modified nucleobases, modified sugar moieties, and modified internucleotide linkages. A probe can be used to detect the presence or absence of a methylated target nucleic acid. Probes are typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, or more nucleotides in length.

[0080] As used herein, a "probe element" refers to a stretch of nucleotides that (a) is associated with a primer in that it is connected to or located adjacent to a primer nucleic acid sequence, and (b) specifically hybridizes to a target nucleic acid sequence to be detected under stringent conditions.

[0081] As used herein, a "reaction sample mixture" refers to a mixture containing an amplification master mix and a sample.

[0082] As used herein, the term "sample" refers to a clinical sample obtained from a patient or an isolated microorganism. In a preferred embodiment, the sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, body fluid, or microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum (processed or unprocessed), bronchoalveolar lavage fluid (BAL), bronchial washings (BW), blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material). Preferred sample sources include nasopharyngeal and / or throat swabs or nasal washes.

[0083] The term "sensitivity" as used herein with respect to the method of the present technology is a measure of the ability of the method to detect preselected sequence variants in a heterogeneous population of sequences. Considering a sample in which a preselected sequence variant is present as at least F% of the sequences in the sample, if the method can detect the preselected sequence with a preselected C% confidence, S% of the time, the method has a sensitivity of S% to the F% variant. As an example, considering a sample in which a preselected variant sequence is present as at least 5% of the sequences in the sample, if the method can detect the preselected sequence with a preselected 99% confidence, 9 times out of 10 (F=5%, C=99%, S=90%), the method has a sensitivity of 90% to the 5% variant. Exemplary sensitivities include at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, and 99%.

[0084] The term "specific" as used herein with respect to an oligonucleotide primer means that the nucleotide sequence of the primer has at least 12 bases of sequence identity with a portion of the nucleic acid to be amplified when the oligonucleotide and the nucleic acid are aligned. An oligonucleotide primer that is specific for a nucleic acid is one that can hybridize to a target of interest under stringent hybridization or washing conditions and does not substantially hybridize to nucleic acids that are not of interest. Higher levels of sequence identity include at least 75%, at least 80%, at least 85%, at least 90%, at least 85-95%, at least 98%, at least 99%, or more sequence identity. Sequence identity can be determined using commercially available computer programs with default settings that use algorithms well known in the art. As used herein, sequences with "high sequence identity" have identical nucleotides at at least about at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more of the aligned nucleotide positions.

[0085] "Specificity" as used herein is a measure of the ability of a method to distinguish truly existing preselected sequence variants (genetic variations) from sequencing artifacts or other closely related sequences. This is the ability to avoid false positive detection. False positive detection can result from errors introduced into the sequence of interest during sample preparation, sequencing errors, or inadvertent sequencing of closely related sequences such as spurious genes or members of a gene family. True If the sequence is truly variant and X Not true is not truly variant N TotalWhen applied to a sample set of sequences, if the method selects at least X% of the truly non-variant sequences as not variants, the method has a specificity of X%. For example, when applied to a sample set of 1,000 sequences, of which 500 sequences are truly variants and 500 are not truly variants, if the method selects 90% of the 500 truly non-variant sequences as not variants, the method has a specificity of 90%. Exemplary specificities include at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, and 99%.

[0086] The term "stringent hybridization conditions" as used herein refers to hybridization conditions that are at least as stringent as the following: overnight hybridization in 50% formamide, 5xSSC, 50mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1mg / mL sonicated salmon sperm DNA, and 5xDenhart's solution at 42°C; washing with 2xSSC, 0.1% SDS at 45°C; and washing with 0.2xSSC, 0.1% SDS at 45°C. In another example, stringent hybridization conditions should not allow hybridization of two nucleic acids that differ by more than two bases over a stretch of 20 consecutive nucleotides.

[0087] The term "target nucleic acid" or "target sequence" as used herein refers to a nucleic acid sequence of interest to be detected and / or quantified in an analyzed sample. A target nucleic acid may consist of a segment of a chromosome, a complete gene with or without intergenic sequences, a segment or portion of a gene with or without intergenic sequences, or a sequence of a nucleic acid for which a probe or primer is designed. A target nucleic acid may include a wild-type sequence(s), a mutation, a deletion, an insertion or duplication, a tandem repeat element, a gene of interest, a region of a gene of interest, or any upstream or downstream region thereof. A target nucleic acid may represent an alternative sequence or allele of a particular gene. A target nucleic acid may be derived from genomic DNA, cDNA, or RNA.

[0088] Methods for detecting genetic variants An embodiment of the disclosure is a method for detecting one or more variants for a genetic disorder in a subject suspected of being a carrier for at least one inherited genetic variation, comprising: extracting DNA from a biological sample obtained from the subject; and contacting the biological sample with a first plurality of primer pairs to generate a first plurality of amplicons, at least one amplicon corresponding to each of a plurality of genes, the plurality of genes including HEXA, SMPD1, MCOLN1, GBA, FANCC, IKBKAP, generating a second plurality of amplicons by contacting the biological sample with a second plurality of primer pairs, wherein at least one amplicon corresponds to each of GBA and HBB, the second plurality of primer pairs comprising SEQ ID NOs:81 and 82, and SEQ ID NOs:83 and 84; and contacting the biological sample with a third plurality of primer pairs, Hot Start Taq generating a third plurality of amplicons by contacting with a DNA polymerase and a Taq extender PCR additive, where at least one amplicon corresponds to each of HBA1 and HBA2; incorporating barcode sequences at the ends of the first, second, and third plurality of amplicons via polymerase chain reaction; and detecting one or more variants in at least one of the first, second, and third plurality of amplicons using high throughput massively parallel sequencing.

[0089] In some embodiments, Ashkenazi Jewish panel variations include genetic variations (e.g., single nucleotide variations (SNVs), deletions, insertions, or inversions) that are commonly observed in Ashkenazi Jewish populations, but may also occur, albeit at a lower frequency, outside of Ashkenazi Jewish populations. In some embodiments, Ashkenazi Jewish panel variations include genetic variations that may cause Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, Maple serum uria, glycogen storage disease I, familial hyperinsulinism, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Usher syndrome type IF, Usher syndrome, type IIIA, Joubert syndrome 2, or Walker-Warburg syndrome. In some embodiments, Ashkenazi Jewish panel variations include genetic variations listed in Table 1. In some embodiments, the beta thalassemia variations include the genetic variations listed in Table 2. In some embodiments, the alpha thalassemia variations include the genetic variations listed in Table 3.

[0090] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present. In some embodiments, the GBA primers of the second plurality of primer pairs specifically amplify the GBA gene and do not amplify any GBA pseudogenes. In some embodiments, the third plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present.

[0091] In some embodiments, variations in the HEXA gene cause Tay-Sachs disease, variations in the SMPD1 gene cause Niemann-Pick disease, variations in the MCOLN1 gene cause mucolipidosis type IV, variations in the GBA gene cause Gaucher disease, variations in the PANCC gene cause Fanconi anemia, variations in the IKBKAP gene cause familial dysautonomia, variations in the ASPA gene cause Canavan disease, variations in the BLM gene cause Bloom syndrome, variations in the BCKDHB gene cause Maple serum uria, variations in the G6PC gene cause G6PC disease, Variations in the ABCC8 gene cause familial hyperinsulinism, variations in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3), variations in the NEB gene cause nemaline myopathy, variations in the PCDH15 gene cause Usher syndrome, type IF, variations in the CLRN1 gene cause Usher syndrome, type IIIA, variations in the TMEM216 gene cause Joubert syndrome 2, and variations in the FKTN gene cause Walker-Warburg syndrome.

[0092] In some embodiments, the Ashkenazi Jewish panel genetic variations include variations in one or more genes listed in Table 1. In some embodiments, the HEXA gene variation is R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or the 7.6-kb Del, Ex1, SMPD1 gene variations include one or more of L302P, fsP330, R496L (R498L), or deltaR608, MCOLN1 gene variations include one or more of IVS3-2A>G or 6.4kb_del, GBA gene variations include one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P, or R496H, PANCC gene variations include one or more of IVS4+4A>T or 322delG, IKBKAP gene variations include one or more of R696P or IVS20+6T>C, ASPA gene variations include IVS2-2A>G, Y231X, E285A, or A 305E, BLM gene variations include one or more of 2281del6 / ins7, BCKDHB gene variations include one or more of R183P, G278S, or E372X, G6PC gene variations include one or more of R83C or Q347X, ABCC8 gene variations include IVS32-9G>A or F1387del, DLD gene variations include one or more of Y35* or G229C, NEB gene variations include R2478_D2512del35, PCDH15 gene variations include one or more of R245, CLRN1 gene variations include N48K, TMEM216 gene variations include R73L, and FKTN gene variations include F390fs.

[0093] In some embodiments, the beta thalassemia genetic variation in the HBB gene comprises one or more genetic variations listed in Table 2. In some embodiments, the genetic variations include c.*111A>G, c.*110T>C, c.*96T>C, Hb D-Los Angeles (c.364G>C), Hb O-Arab(c.364G>A), c.321_322insG, c.316-2A>C, c.316-2A>G, c.316-3C>A, c.316-106C>G, c.316-125A>G, c.316-146T>G, c.316-197C>T, c.315+1G>A, c.287_288insA, c.251delG, c.230delC, c.216_217insA, c.203_204delTG, c.143_144insA, c.1 46_147insATCT, c.135delC, c.130G>T, c.126_129delCTTT, c.124_127delTTCT, c.118C>T, c.114G>A, c.112delT, c.93-1G>C , c.93-1G>A, c.93-21G>A, c.92+6T>C, c.92+5G>A, c.92+5G>C, c.92+5G>T, c.92+2T>A, c.92+2T>C, c.92+1G>A, c.92+1G>T, Hb Monroe(c.92G>C), c.92G>A, c.84_85insC, c.79G>T, HB E(c.79G>A), c.75T>A, c.59A>G, c.52A>T(LYS17*), c.51delC, c.48G>A, c.47G>A( Trp15), c.46delT, c.36delT, c.33C>A, c.27_28insG, c.25_26delAA, c.20delA, HB S(c.20A>T), HB C(c.19G>A), c.17_18delCT, c.2T>C, c.2T>G, c.1A>G, c.-78A>C, c.-78A>G, c.-79A>G, c.-80T>A, c.-81A >G, c.-136C>G, c.-137C>A, c.-137C>G, c.-137C>T, c.-138C>T, c.-138C>A, c.-140C>T, or c.-151C>T.

[0094] In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise one or more variations listed in Table 3. In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL, or a single nucleotide variant in the HBA2 gene HBA2 c.427T>C.

[0095] In some embodiments, the first plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least 2, at least 4, at least 5, at least 8, or at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or all of the primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs set forth in Table 4.

[0096] In some embodiments, the first plurality of primer pairs are selected from the group consisting of pair 1 (SEQ ID NOs: 1-2), pair 2 (SEQ ID NOs: 3-4), pair 3 (SEQ ID NOs: 5-6), pair 4 (SEQ ID NOs: 7-8), pair 5 (SEQ ID NOs: 9-10), pair 6 (SEQ ID NOs: 11-12), pair 7 (SEQ ID NOs: 13-14), pair 8 (SEQ ID NOs: 15-16), pair 9 (SEQ ID NOs: 17-18), pair 10 (SEQ ID NOs: 19-20), pair 11 (SEQ ID NOs: 21-22), pair 12 (SEQ ID NOs: 23-24), pair 13 (SEQ ID NOs: 25-26), pair 14 (SEQ ID NOs: 26-27), pair 15 (SEQ ID NOs: 27-28), pair 16 (SEQ ID NOs: 28-29), pair 17 (SEQ ID NOs: 29-30), pair 18 (SEQ ID NOs: 30-31), pair 19 (SEQ ID NOs: 31-32), pair 20 (SEQ ID NOs: 32-33), pair 21 (SEQ ID NOs: 33-34), pair 22 (SEQ ID NOs: 34-35), pair 23 (SEQ ID NOs: 35-36), pair 24 (SEQ ID NOs: 36-37), pair 25 (SEQ ID NOs: 37-38), pair 26 (SEQ ID NOs: 38-39), pair 27 (SEQ ID NOs: 39-40), pair 28 (SEQ ID NOs: 41-42), pair 29 (SEQ ID 25 to 26), pair 14 (SEQ ID NOs: 27 to 28), pair 15 (SEQ ID NOs: 29 to 30), pair 16 (SEQ ID NOs: 31 to 32), pair 17 (SEQ ID NOs: 33 to 34), pair 18 (SEQ ID NOs: 35 to 36), pair 19 (SEQ ID NOs: 37 to 38), pair 20 (SEQ ID NOs: 39 to 40), pair 21 (SEQ ID NOs: 41 to 42), pair 22 (SEQ ID NOs: 43 to 44), pair 23 (SEQ ID NOs: 45 to 46), pair 24 (SEQ ID NOs: 47 to 48), pair 25 (SEQ ID NOs: 49 to 50), pair 26 (SEQ ID NOs: 51 to 52) ), pair 27 (SEQ ID NOs: 53 to 54), pair 28 (SEQ ID NOs: 55 to 56), pair 29 (SEQ ID NOs: 57 to 58), pair 30 (SEQ ID NOs: 59 to 60), pair 31 (SEQ ID NOs: 61 to 62), pair 32 (SEQ ID NOs: 63 to 64), pair 33 (SEQ ID NOs: 65 to 66), pair 34 (SEQ ID NOs: 67 to 68), pair 35 (SEQ ID NOs: 69 to 70), pair 36 (SEQ ID NOs: 71 to 72), pair 37 (SEQ ID NOs: 73 to 74), pair 37 (SEQ ID NOs: 75 to 76), pair 38 (SEQ ID NOs: 77 to 78), and The present invention includes at least two primer pairs having at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identity to at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or all of the pairs selected from the group consisting of 40 pairs (SEQ ID NOs: 79 to 80).

[0097] In some embodiments, the second plurality of primer pairs comprises at least two, at least four, or at least five primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two, at least four, or at least five primer pairs selected from Table 5. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 5.

[0098] In some embodiments, the second plurality of primer pairs comprises at least two primer pairs having at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to primer pairs of at least two, at least four, or at least five primers selected from SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:85 and 86, SEQ ID NOs:87 and 88, or SEQ ID NOs:89 and 90.

[0099] In some embodiments, the second plurality of primer pairs includes at least two primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two pairs selected from the group consisting of pair 1 (SEQ ID NOs:81-82), pair 2 (SEQ ID NOs:83-84), pair 3 (SEQ ID NOs:85-86), pair 4 (SEQ ID NOs:87-88), and pair 5 (SEQ ID NOs:89-90) in Table 6.

[0100] In some embodiments, the third plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85% identity to at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 6.

[0101] In some embodiments, the third plurality of primer pairs includes at least two primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two pairs selected from the group consisting of pair 1 (SEQ ID NOs:91-92), pair 2 (SEQ ID NOs:93-94), pair 3 (SEQ ID NOs:95-96), pair 4 (SEQ ID NOs:97-98), pair 5 (SEQ ID NOs:99-100), pair 6 (SEQ ID NOs:101-102), pair 7 (SEQ ID NOs:103-104), and pair 8 (SEQ ID NOs:105-106) in Table 6.

[0102] In some embodiments, next generation sequencing comprises sequencing by synthesis (e.g., pyrosequencing). In some embodiments, next generation sequencing comprises sequencing by ligation.

[0103] In some embodiments, next generation sequencing (also known as "high throughput, massively parallel sequencing") employs sequencing by synthesis using reversible dye terminators. In other embodiments, sequencing is performed via sequencing by ligation. In yet other embodiments, sequencing is single molecule sequencing. Examples of next generation sequencing techniques include, but are not limited to, pyrosequencing, reversible dye terminator sequencing, SOLiD sequencing, ion semiconductor sequencing, and Helioscope single molecule sequencing.

[0104] In some embodiments, the methods featured in the technology are used in multiplex, multi-gene assay formats, e.g., assays that incorporate multiple signals from multiple diverse genetic alterations in multiple genes.

[0105] In some embodiments, the subject is of Ashkenazi Jewish descent or has a family history of beta or alpha thalassemia.

[0106] In some embodiments, the biological sample is selected from whole blood, serum, plasma, amniotic fluid, or chorionic villi.

[0107] In some embodiments, the genetic disorder is an autosomal disorder or an X-linked recessive disorder.

[0108] Methods for detecting inherited mutations (eg, Ashkenazi Jewish panel variations, beta thalassemia variations, or alpha thalassemia variations) in a single assay Upon preparing a biological sample and next generation sequencing as described above, the present disclosure further provides a method for detecting the presence of an inherited variation (e.g., an Ashkenazi Jewish panel variation, a beta thalassemia variation, or an alpha thalassemia variation) present in a target nucleic acid sequence amplified from the biological sample.

[0109] In some embodiments, the method further comprises determining whether the subject is a carrier for at least one of an Ashkenazi Jewish panel variation, a beta thalassemia variation, or an alpha thalassemia variation based on the next generation sequencing results. In some embodiments, the determining is accomplished by demultiplexing the sequencing reads, mapping the sequencing reads to a custom human reference genome, where pseudogene sequences in Reference Consortium Human Genome Build 37 (GRCh37) chr1:155184031-155185327, chr1:155188031-155188964, chr16:223705-224240, and chr16:227257-227413 have been replaced with the nucleotide T, filtering the mapped reads using a Phred Quality score requiring read mappings of greater than 30, detecting variants in the filtered reads, and determining whether the subject is a carrier for an Ashkenazi Jewish panel variation, a beta thalassemia variation, or an alpha thalassemia variation.

[0110] In some embodiments, the subject is suspected of being a carrier for at least one disease selected from Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom's syndrome, Maple serum urine, glycogen storage disease I, familial hyperinsulinism, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Usher syndrome type IF, Usher syndrome, type IIIA, Joubert syndrome 2, Walker-Warburg syndrome, beta thalassemia, or alpha thalassemia.

[0111] kit The present disclosure also provides kits for detecting the presence of an Ashkenazi Jewish panel variation, a beta thalassemia variation, or an alpha thalassemia variation in a biological sample from a subject.

[0112] The kit of the present technology includes at least (i) a first plurality of primer pairs directed to an amplified region of each of a plurality of genes, the plurality of genes being selected from the group consisting of hexosaminidase subunit alpha (HEXA), sphingomyelin phosphodiesterase 1 (SMPD1), mucolipin TRP cation channel 1 (MCOLN1), glucosylceramidase beta (GBA), FA complementation group C (FANCC), inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex associated protein (IKBKAP), aspartoacylase (ASPA), Bloom's syndrome RecQ-like helicase (BLM), branched-chain ketoacid dehydrogenase E1 subunit beta (BCKDHB), glucose-6-phosphatase catalytic subunit (G6PC), ATP-binding cassette subfamily C member (ATP-binding cassette C member (ATP-binding cassette D ... (ii) a first plurality of primer pairs directed to amplified regions of glucosylceramidase beta (GBA) and hemoglobin subunit beta (HBB) genes, the second plurality of primer pairs comprising SEQ ID NOs: 81 and 82, and SEQ ID NOs: 83 and 84; and (iii) a third plurality of primer pairs directed to amplified regions of alpha thalassemia genetic variations in each of the hemoglobin subunit alpha 1 (HBA1) and hemoglobin subunit alpha 1 (HBA2) genes, and instructions for use.

[0113] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present.

[0114] In some embodiments, the GBA primers of the second plurality of primer pairs specifically amplify the GBA gene and do not amplify any GBA pseudo-genes.

[0115] In some embodiments, the third plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only if a deletion is present.

[0116] In some embodiments, variations in the HEXA gene cause Tay-Sachs disease, variations in the SMPD1 gene cause Niemann-Pick disease, variations in the MCOLN1 gene cause mucolipidosis type IV, variations in the GBA gene cause Gaucher disease, variations in the PANCC gene cause Fanconi anemia, variations in the IKBKAP gene cause familial dysautonomia, variations in the ASPA gene cause Canavan disease, variations in the BLM gene cause Bloom syndrome, variations in the BCKDHB gene cause Maple serum uria, variations in the G6PC gene cause G6PC disease, Variations in the ABCC8 gene cause familial hyperinsulinism, variations in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3), variations in the NEB gene cause nemaline myopathy, variations in the PCDH15 gene cause Usher syndrome, type IF, variations in the CLRN1 gene cause Usher syndrome, type IIIA, variations in the TMEM216 gene cause Joubert syndrome 2, and variations in the FKTN gene cause Walker-Warburg syndrome.

[0117] In some embodiments, the Ashkenazi Jewish panel genetic variations include variations in one or more genes listed in Table 1. In some embodiments, the HEXA gene variation is R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or the 7.6-kb Del, Ex1, SMPD1 gene variations include one or more of L302P, fsP330, R496L (R498L), or deltaR608, MCOLN1 gene variations include one or more of IVS3-2A>G or 6.4kb_del, GBA gene variations include one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P, or R496H, PANCC gene variations include one or more of IVS4+4A>T or 322delG, IKBKAP gene variations include one or more of R696P or IVS20+6T>C, ASPA gene variations include IVS2-2A>G, Y231X, E285A, or A 305E, BLM gene variations include one or more of 2281del6 / ins7, BCKDHB gene variations include one or more of R183P, G278S, or E372X, G6PC gene variations include one or more of R83C or Q347X, ABCC8 gene variations include IVS32-9G>A or F1387del, DLD gene variations include one or more of Y35* or G229C, NEB gene variations include R2478_D2512del35, PCDH15 gene variations include one or more of R245, CLRN1 gene variations include N48K, TMEM216 gene variations include R73L, and FKTN gene variations include F390fs.

[0118] In some embodiments, the beta thalassemia genetic variation in the HBB gene comprises one or more genetic variations listed in Table 2. In some embodiments, the genetic variations include c.*111A>G, c.*110T>C, c.*96T>C, Hb D-Los Angeles (c.364G>C), Hb O-Arab(c.364G>A), c.321_322insG, c.316-2A>C, c.316-2A>G, c.316-3C>A, c.316-106C>G, c.316-125A>G, c.316-146T>G, c.316-197C>T, c.315+1G>A, c.287_288insA, c.251delG, c.230delC, c.216_217insA, c.203_204delTG, c.143_144insA, c.1 46_147insATCT, c.135delC, c.130G>T, c.126_129delCTTT, c.124_127delTTCT, c.118C>T, c.114G>A, c.112delT, c.93-1G>C , c.93-1G>A, c.93-21G>A, c.92+6T>C, c.92+5G>A, c.92+5G>C, c.92+5G>T, c.92+2T>A, c.92+2T>C, c.92+1G>A, c.92+1G>T, Hb Monroe(c.92G>C), c.92G>A, c.84_85insC, c.79G>T, HB E(c.79G>A), c.75T>A, c.59A>G, c.52A>T(LYS17*), c.51delC, c.48G>A, c.47G>A( Trp15), c.46delT, c.36delT, c.33C>A, c.27_28insG, c.25_26delAA, c.20delA, HB S(c.20A>T), HB C(c.19G>A), c.17_18delCT, c.2T>C, c.2T>G, c.1A>G, c.-78A>C, c.-78A>G, c.-79A>G, c.-80T>A, c.-81A >G, c.-136C>G, c.-137C>A, c.-137C>G, c.-137C>T, c.-138C>T, c.-138C>A, c.-140C>T, or c.-151C>T.

[0119] In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise variations in one or more genes listed in Table 3. In some embodiments, the alpha thalassemia variations in the HBA1 and HBA2 genes comprise a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL, or a single nucleotide variant in the HBA2 gene HBA2 c.427T>C.

[0120] In some embodiments, the first plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least 2, at least 4, at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or all of the primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs set forth in Table 4.

[0121] In some embodiments, the first plurality of primer pairs are selected from the group consisting of pair 1 (SEQ ID NOs: 1-2), pair 2 (SEQ ID NOs: 3-4), pair 3 (SEQ ID NOs: 5-6), pair 4 (SEQ ID NOs: 7-8), pair 5 (SEQ ID NOs: 9-10), pair 6 (SEQ ID NOs: 11-12), pair 7 (SEQ ID NOs: 13-14), pair 8 (SEQ ID NOs: 15-16), pair 9 (SEQ ID NOs: 17-18), pair 10 (SEQ ID NOs: 19-20), pair 11 (SEQ ID NOs: 21-22), pair 12 (SEQ ID NOs: 23-24), pair 13 (SEQ ID NOs: 25-26), pair 14 (SEQ ID NOs: 26-27), pair 15 (SEQ ID NOs: 27-28), pair 16 (SEQ ID NOs: 28-29), pair 17 (SEQ ID NOs: 29-30), pair 18 (SEQ ID NOs: 30-31), pair 19 (SEQ ID NOs: 31-32), pair 20 (SEQ ID NOs: 32-33), pair 21 (SEQ ID NOs: 33-34), pair 22 (SEQ ID NOs: 34-35), pair 23 (SEQ ID NOs: 35-36), pair 24 (SEQ ID NOs: 36-37), pair 25 (SEQ ID NOs: 37-38), pair 26 (SEQ ID NOs: 38-39), pair 27 (SEQ ID NOs: 39-40), pair 28 (SEQ ID NOs: 41-42), pair 29 (SEQ ID 25 to 26), pair 14 (SEQ ID NOs: 27 to 28), pair 15 (SEQ ID NOs: 29 to 30), pair 16 (SEQ ID NOs: 31 to 32), pair 17 (SEQ ID NOs: 33 to 34), pair 18 (SEQ ID NOs: 35 to 36), pair 19 (SEQ ID NOs: 37 to 38), pair 20 (SEQ ID NOs: 39 to 40), pair 21 (SEQ ID NOs: 41 to 42), pair 22 (SEQ ID NOs: 43 to 44), pair 23 (SEQ ID NOs: 45 to 46), pair 24 (SEQ ID NOs: 47 to 48), pair 25 (SEQ ID NOs: 49 to 50), pair 26 (SEQ ID NOs: 51 to 52) ), pair 27 (SEQ ID NOs: 53 to 54), pair 28 (SEQ ID NOs: 55 to 56), pair 29 (SEQ ID NOs: 57 to 58), pair 30 (SEQ ID NOs: 59 to 60), pair 31 (SEQ ID NOs: 61 to 62), pair 32 (SEQ ID NOs: 63 to 64), pair 33 (SEQ ID NOs: 65 to 66), pair 34 (SEQ ID NOs: 67 to 68), pair 35 (SEQ ID NOs: 69 to 70), pair 36 (SEQ ID NOs: 71 to 72), pair 37 (SEQ ID NOs: 73 to 74), pair 37 (SEQ ID NOs: 75 to 76), pair 38 (SEQ ID NOs: 77 to 78), and The present invention includes at least two primer pairs having at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identity to at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or all of the pairs selected from the group consisting of 40 pairs (SEQ ID NOs: 79 to 80).

[0122] In some embodiments, the second plurality of primer pairs comprises at least two, at least four, or at least five primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two, at least four, or at least five primer pairs selected from Table 5. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 5.

[0123] In some embodiments, the second plurality of primer pairs comprises at least two primer pairs having at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to primer pairs of at least two, at least four, or at least five primers selected from SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:85 and 86, SEQ ID NOs:87 and 88, or SEQ ID NOs:89 and 90.

[0124] In some embodiments, the second plurality of primer pairs includes at least two primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two pairs selected from the group consisting of pair 1 (SEQ ID NOs:81-82), pair 2 (SEQ ID NOs:83-84), pair 3 (SEQ ID NOs:85-86), pair 4 (SEQ ID NOs:87-88), and pair 5 (SEQ ID NOs:89-90) in Table 6.

[0125] In some embodiments, the third plurality of primer pairs comprises at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs that have at least 85% identity to at least 2, at least 4, at least 5, at least 8, or at least 10 primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs comprises all of the primer pairs shown in Table 6.

[0126] In some embodiments, the third plurality of primer pairs includes at least two primer pairs that have at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99%, or more identity to at least two pairs selected from the group consisting of pair 1 (SEQ ID NOs:91-92), pair 2 (SEQ ID NOs:93-94), pair 3 (SEQ ID NOs:95-96), pair 4 (SEQ ID NOs:97-98), pair 5 (SEQ ID NOs:99-100), pair 6 (SEQ ID NOs:101-102), pair 7 (SEQ ID NOs:103-104), and pair 8 (SEQ ID NOs:105-106) in Table 6.

[0127] In some embodiments, the kits of the present disclosure further comprise buffers, enzymes having polymerase activity, enzymes having polymerase activity and lacking 5'→3' exonuclease activity or both 5'→3' and 3'->5' exonuclease activity, enzyme cofactors, e.g., magnesium or manganese, salts, chain elongation nucleotides, e.g., deoxynucleoside triphosphates (dNTPs), modified dNTPs, nuclease resistant dNTPs, or labeled dNTPs, necessary to perform an assay or reaction, such as amplification and / or detection of alterations in a target nucleic acid sequence corresponding to a particular set of inherited genetic variations disclosed herein.

[0128] In some embodiments, the kits of the present disclosure further comprise positive and negative control nucleic acid sequences to ensure the integrity of the assay during experimental run-off. The kits may also include instructions for use, software for automated analysis, containers, packages, e.g., packaging intended for commercial sale, etc.

[0129] The kit of the present technology may also include other necessary reagents for carrying out any of the NGS techniques disclosed herein. For example, the kit may further include one or more of adapter sequences, barcode sequences, reaction tubes, ligase, ligase buffer, washing buffer and / or reagents, hybridization buffer and / or reagents, labeling buffer and / or reagents, and detection means. The buffers and / or reagents are usually optimized for the particular amplification / detection technique for which the kit is intended. Protocols for using these buffers and reagents to carry out the different steps of the procedure may also be included in the kit.

[0130] In some embodiments, a kit of the present disclosure further comprises a plurality of barcoded primers selected from pairs 1-4 of Table 7 or pairs 1-9 of Table 8.

[0131] The kit may further comprise one or more of washing buffers and / or reagents, hybridization buffers and / or reagents, labeling buffers and / or reagents, and detection means. The buffers and / or reagents are usually optimized for the particular amplification / detection technique for which the kit is intended. Protocols for using these buffers and reagents to perform the different steps of the procedure may also be included in the kit.

[0132] The kit may additionally include instructions, such as an assay definition scan card and / or printed or electronic instructions for using the oligonucleotides in the assay. In some embodiments, the kit includes an amplification reaction mixture or an amplification master mix. The reagents included in the kit may be contained in one or more containers, such as vials.

[0133] Primers, probes, and / or primer-probes specific for amplification and detection of the DNA internal control may be included in the amplification master mix as a target primer pair to monitor potential PCR inhibition. The reagents required for amplification and detection of the target and internal control may be formulated as an all-in-one amplification master mix, which may be provided as a single reaction aliquot in the kit. EXAMPLES

[0134] Example 1: Preparation of sequencing libraries and generation of raw sequencing data Three multiplex gene-specific PCR reactions (GSP) were set up for each specimen to amplify the target regions (Figure 1). Each multiplex PCR reaction consisted of a pool of primer pairs (see Tables 4-6), amplifying different regions of genomic DNA containing the variations detected by this assay (see Tables 1-3 for variations in genes of interest). In total, 55 amplicons were generated from the GSP reactions. After PCR, the GSP products for each patient were diluted 2500-fold with water. The diluted products were amplified with primers that add the barcode sequence (BC), as well as the P5 and P7 adapters (Table 7) required for sequencing on the Illumina MiSeq. The barcoded samples were pooled, purified with Ampure XP beads, and quantified. The final products were pooled into a single library and subjected to sequencing on the MiSeq. The sequencing data were analyzed by an in-house developed pipeline.

[0135] Gene-specific PCR (GSP) Three multiplex gene-specific PCR reactions (GSP-A, -B, and -C) were set up for each specimen to amplify the target regions. Reaction GSP-A contains 41 primer pairs, including three gap PCR primer pairs, to amplify the target regions of 17 genes. The gap PCR primer pairs were designed to produce product only if a deletion was present. The gene-specific primer pairs were designed to contain Illumina sequencing primer binding sites, R1SP for the forward read, and R2SP for the reverse and barcode reads.

[0136] Reaction GSP-B contains two primer pairs for the GBA gene and three primer pairs for the HBB gene. The GBA primers were designed to specifically amplify the GBA gene target region while preventing co-amplification of the GBA pseudogene. Reaction GSP-C contains 16 primers for detection of eight alpha thalassemia common variations and one amplification control. Gap PCR was used for seven alpha thalassemia common variations so that product is only present if a deletion is present. In total, 55 amplicons were generated from the GSP reactions.

[0137] Barcoded PCR (BCP) To facilitate the simultaneous combination of all samples into a single library, each individual sample was spiked with one of 96 unique barcode sequences. After gene-specific PCR, the three GSP reactions for each patient were diluted 2500-fold with water and subjected to three barcoded PCR reactions (BCP-A, -B, and -C). During barcoded PCR, the R1SP and R2SP sequences were used as priming sites to add the barcode sequence, as well as the P5 and P7 adapters required for sequencing on an Illumina Miseq sequencer.

[0138] Barcoded PCR reaction B (GSP-B - see Table 5) contains 5 primer pairs and reaction C (GSP-C - Table 6) contains 16 primers of R1SP and R2SP fusion nested primers to generate 4 GBA-specific nested PCR products and 8 alpha-thalassemia nested PCR products, respectively.

[0139] In total, 57 amplicons were generated from the BCP reactions.

[0140] Sample Pooling and Ampure Cleanup Barcoded samples were pooled into three separate tubes to create Pool A, Pool B, and Pool C, and purified with Ampure XP beads followed by Qubit quantification. The final products were combined into a single library and subjected to sequencing on a MiSeq.

[0141] Sequencing The combined library was denatured before sequencing with 0.2 N NaOH. Illumina sequencing chemistry is based on sequencing by synthesis. The single-stranded library was loaded onto a Miseq sequencing cartridge. In summary, the MiSeq workflow is as follows: the instrument starts by flushing the library through a flow cell, where it hybridizes to antisense P5 and P7 oligonucleotides that are complementary to the adapters on the library. The library is diluted so that amplification produces well-separated clusters of identical products from a single DNA molecule (clonal amplification). This is achieved by isothermal bridge amplification. After cluster generation, the clusters are made single-stranded and sequencing by synthesis begins. Fluorophore-labeled nucleotide triphosphates are applied to the flow cell. The nucleotides are 3'-blocked so that only a single nucleotide is incorporated into each round of synthesis. The fluorophore is then excited by a laser and the emission spectrum is recorded by the Miseq. The nucleotide blockers that were inhibiting further synthesis are cleaved to allow the addition of the next nucleotide triphosphate. In this way, the fragments are sequenced.

[0142] Example 2: Sequencing Data Analysis Alignment, filtering, and variant calling The data analysis pipeline workflow is shown in Figure 2. MiSeq analysis software was used to generate non-multiplexed FASTQ files. The raw sequence reads in the FASTQ files were then aligned to a custom reference genome using Burrows-Wheeler Aligner (BWA). The custom reference genome differs from the Genomic Reference Consortium Human Genome Build 37 (GRCh37) in that highly homologous pseudogene sequences within chr1:155184031-155185327, chr1:155188031-155188964, chr16:223705-224240, and chr16:227257-227413 have been replaced with the nucleotide T for accurate alignment of GBA and detection of the alpha thalassemia 4.2 deletion variant. Reads were then sorted and indexed using SAMtools, followed by arrangement of read groups using Picard Tools. Local realignment and base quality score recalibration were performed within targets using the Genome Analysis Toolkit (GATK). Mapped reads were further filtered using a Phred Quality score (>99.9% accuracy) that required more than 30 reads mapping before downstream analysis. The average and minimum depth of coverage for each ROI (region of interest) was calculated and variant calling was performed using the GATK Unified and Haploid Genotyper callers. A single variant file (*.vcf) was created by merging variant files from both variant callers. Coverage and variant depth reports were generated and loaded into a sequencing database (seqDB). Alamut batch was used to obtain high-level annotation for detected variants. Quality control rules for variant detection included: 1) a minimum of 40 reads must be achieved at the target position(s) and 2) variants are detected at a frequency of 20% or higher. 3) Detection of large deletions was based on the presence of gapped PCR amplicons with a minimum of 100 reads and was not based on specific variants being called.

[0143] Example 3: Detected variants [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3]

[0144] Example 4: Primers and other materials [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9-1] [Table 9-2] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0145] Analytical Measurement Range (AMR) Negative (wild type): None of the variants tested are detected. Heterozygous: A variant with a frequency of 20% or more but less than 96% is positive. Compound heterozygous: Two variations, one on each allele, within a gene with a frequency of 20% or more but less than 96% are positive. Homozygous: The same variant on both alleles with a frequency of 96% or more is positive. [Table 15-1] [Table 15-2]

[0146] Example 5: Results The clinical sensitivity and specificity of the disclosed assay were measured to be 100% and 100%, respectively. The assay also demonstrated 100% inter- and intra-assay precision during validation runs.

[0147] Equivalent The present technology is not limited with respect to the specific embodiment described in this application, which is intended as a single illustration of each aspect of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present technology. It should be understood that the present technology is not limited to specific methods, reagents, compound compositions, or biological systems, which, of course, can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0148] Terms such as "comprising," "including," "containing," and the like, are to be read broadly and without limitation. Additionally, the terms and expressions used herein are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the shown and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure as claimed.

[0149] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0150] As would be understood by one of ordinary skill in the art, for any or all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. Also, as would be understood by one of ordinary skill in the art, all language, such as "up to," "at least," "greater than," "less than," etc., refers to a range that includes the recited numbers and can then be broken down into subranges as discussed above. Finally, as would be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so on.

[0151] All patents, patent applications, provisional applications, and publications mentioned or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent they do not contradict the explicit teachings of this specification.

Claims

1. 1. A method for detecting one or more variants for a genetic disorder in a subject suspected of being a carrier for at least one inherited genetic variation, comprising: extracting DNA from a biological sample obtained from the subject; generating a first plurality of amplicons by contacting the biological sample with a first plurality of primer pairs, at least one amplicon corresponding to each of a plurality of genes, the plurality of genes including HEXA, SMPD1, MCOLN1, GBA, FANCC, IKBKAP, ASPA, BLM, BCKDHB, G6PC, ABCC8, DLD, NEB, PCDH15, CLRN1, TMEM216, and FKTN; generating a second plurality of amplicons by contacting the biological sample with a second plurality of primer pairs, wherein at least one amplicon corresponds to each of GBA and HBB, and wherein the second plurality of primer pairs includes SEQ ID NOs:81 and 82, and SEQ ID NOs:83 and 84; generating a third plurality of amplicons by contacting the biological sample with a third plurality of primer pairs, a hot start Taq DNA polymerase, and a Taq extender PCR additive, wherein at least one amplicon corresponds to each of HBA1 and HBA2; incorporating barcode sequences onto ends of the first, second, and third plurality of amplicons via polymerase chain reaction; detecting one or more variants in at least one of said first, second, and third plurality of amplicons using high throughput massively parallel sequencing.

2. 2. The method of claim 1, wherein the first plurality of primer pairs comprises at least one gap primer pair designed to amplify a target region only if a deletion is present.

3. 2. The method of claim 1, wherein the GBA primers of the second plurality of primer pairs specifically amplify GBA genes and do not amplify any GBA pseudogenes.

4. 2. The method of claim 1, wherein the third plurality of primer pairs comprises at least one gap primer pair designed to amplify a target region only if a deletion is present.

5. Mutations in the HEXA gene cause Tay-Sachs disease, Mutations in the SMPD1 gene cause Niemann-Pick disease, A variation in the MCOLN1 gene causes type IV mucolipidosis; Mutations in the GBA gene cause Gaucher disease, a mutation in the PANCC gene causes Fanconi anemia; A variation in the IKBKAP gene causes familial dysautonomia, A mutation in the ASPA gene causes Canavan disease, Variation in the BLM gene causes Bloom syndrome, A mutation in the BCKDHB gene causes maple serum urinary syndrome, A mutation in the G6PC gene causes glycogen storage disease I; A variation in the ABCC8 gene causes familial hyperinsulinism, The DLD gene mutation causes dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3); Mutations in the NEB gene cause nemaline myopathy, Mutations in the PCDH15 gene cause Usher syndrome, IF type; Mutations in the CLRN1 gene cause Usher syndrome, type IIIA; Mutations in the TMEM216 gene cause Joubert syndrome 2; The method of claim 1, wherein the FKTN gene mutation causes Walker-Warburg syndrome.

6. the HEXA gene variation comprises R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; The SMPD1 gene variation comprises L302P, fsP330, R496L (R498L), or deltaR608; the MCOLN1 gene variation comprises IVS3-2A>G or 6.4kb_del; the GBA gene variation comprises IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P, or R496H; the PANCC gene variation comprises IVS4+4A>T or 322delG; the variation of the IKBKAP gene comprises R696P or IVS20+6T>C; the ASPA gene variation comprises IVS2-2A>G, Y231X, E285A, or A305E; the BLM gene variation comprises 2281del6 / ins7, the variation in the BCKDHB gene comprises R183P, G278S, or E372X; the G6PC gene variation comprises R83C or Q347X; the variation in the ABCC8 gene comprises IVS32-9G>A or F1387del; the DLD gene variation comprises Y35* or G229C; the NEB gene variation comprises R2478_D2512del35; the PCDH15 gene variation comprises R245; the CLRN1 gene variation comprises N48K; the variation of the TMEM216 gene comprises R73L; 2. The method of claim 1, wherein the FKTN gene variation comprises F390fs.

7. Preface: Changes in the HBB legacy, c. *111A>G、c. *110T>C、c. *96T>C、Hb D-Los Angeles(c.364G>C)、Hb O-Arab(c.364G>A)、c. 321_322insG、c. 316-2A>C、c. 316-2A>G、c. 316-3C>A、c. 316-106C>G、c. 316-125A>G、c. 316-146T>G、c. 316-197C>T、c. 315+1G>A, c. 287_288insA, c. 251delG, c. 230delC, c. 216_217insA, c. 203_204delTG, c. 143_144insA, c. 146_147insATCT, c. 135delC, c. 130G>T, c. 126_129delCTTT, c. 124_127delTTCT, c. 118C>T, c. 114G>A, c. 112delT, c. 93-1G>C, c. 93-1G>A, c. 93-21G>A, c. 92+6T>C, c. 92+5G>A, c. 92+5G>C, c. 92+5G>T, c. 92+2T>A, c. 92+2T>C, c. 92+1G>A, c. 92+1G>T, Hb Monroe (c. 92G>C), c. 92G>A, c. 84_85insC, c. 79G>T, HB E (c. 79G>A), c. 75T>A, c. 59A>G, c. 52A>T (LYS17*), c. 51delC, c. 48G>A, c. 47G>A (Trp15), c. 46delT, c. 36delT, c. 33C>A, c. 27_28insG, c. 25_26delAA,c. 20delA, HB S (c.20A>T), HB C (c.19G>A), c. 17_18delCT, c. 2T>C, c. 2T>G, c. 1A>G, c. -78A>C, c. -78A>G, c. -79A>G, c. -80T>A, c. -81A>G, c. -136C>G, c. -137C>A, c. -137C>G, c. -137C>T, c. -138C>T, c. -138C>A, c. -140C>T、also はc. -151C>T includes the method described in request item 1. 【Request 8】 2. The method of claim 1, wherein the alpha thalassemia variation in the HBA1 and HBA2 genes comprises a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL, or a single nucleotide variant in the HBA2 gene HBA2 c.427T>C.

9. 2. The method of claim 1, wherein the first plurality of primer pairs comprises at least two primer pairs having at least 85% identity to at least two primer pairs selected from Table 4.

10. 10. The method of claim 9, wherein the first plurality of primer pairs comprises all of the primer pairs shown in Table 4.

11. 2. The method of claim 1, wherein the second plurality of primer pairs comprises at least two primer pairs having at least 85% identity to at least two primer pairs selected from SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:85 and 86, SEQ ID NOs:87 and 88, or SEQ ID NOs:89 and 90.

12. 2. The method of claim 1 , wherein the second plurality of primer pairs comprises all of the primer pairs shown in Table 5.

13. 10. The method of claim 1 , wherein the third plurality of primer pairs comprises at least two primer pairs having at least 85% identity to at least two primer pairs selected from Table 6.

14. 14. The method of claim 13, wherein the first plurality of primer pairs comprises all of the primer pairs shown in Table 6.

15. 2. The method of claim 1, wherein next generation sequencing comprises sequencing by synthesis or sequencing by ligation.

16. 10. The method of claim 1, wherein the subject is of Ashkenazi Jewish descent or has a family history of beta or alpha thalassemia.

17. 10. The method of claim 1, wherein the biological sample is whole blood, serum, plasma, amniotic fluid, or chorionic villi.

18. 2. The method of claim 1, wherein the disorder is an autosomal disorder or an X-linked recessive disorder.

19. 19. The method of any one of claims 1 to 18, wherein the subject is suspected of being a carrier for at least one disease selected from Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom's syndrome, Maple serum urine, glycogen storage disease I, familial hyperinsulinism, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Usher syndrome type IF, Usher syndrome type IIIA, Joubert syndrome 2, Walker-Warburg syndrome, beta thalassemia, or alpha thalassemia.

20. A kit comprising: (i) a first plurality of primer pairs directed to an amplified region of each of a plurality of genes, the plurality of genes comprising HEXA, SMPD1, MCOLN1, GBA, FANCC, IKBKAP, ASPA, BLM, BCKDHB, G6PC, ABCC8, DLD, NEB, PCDH15, CLRN1, TMEM216, and FKTN; (ii) a second plurality of primer pairs directed to the amplified regions of the GBA and HBB genes, the second plurality of primer pairs comprising SEQ ID NOs:81 and 82, and SEQ ID NOs:83 and 84; (iii) a third plurality of primer pairs directed to the amplified region of the alpha thalassemia genetic variation in each of the HBA1 and HBA2 genes; and A kit including instructions for use.

21. 21. The kit of claim 20, wherein the first plurality of primer pairs comprises at least one gap primer pair designed to amplify a target region only if a deletion is present.

22. 21. The kit of claim 20, wherein the GBA primers of the second plurality of primer pairs specifically amplify GBA genes and do not amplify any GBA pseudogenes.

23. 21. The kit of claim 20, wherein the third plurality of primer pairs comprises at least one gap primer pair designed to amplify a target region only if a deletion is present.

24. Mutations in the HEXA gene cause Tay-Sachs disease, Mutations in the SMPD1 gene cause Niemann-Pick disease, A variation in the MCOLN1 gene causes type IV mucolipidosis; Mutations in the GBA gene cause Gaucher disease, a mutation in the PANCC gene causes Fanconi anemia; A variation in the IKBKAP gene causes familial dysautonomia, A mutation in the ASPA gene causes Canavan disease, Variation in the BLM gene causes Bloom syndrome, A mutation in the BCKDHB gene causes maple serum urinary syndrome, A mutation in the G6PC gene causes glycogen storage disease I; A variation in the ABCC8 gene causes familial hyperinsulinism, The DLD gene mutation causes dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3); Mutations in the NEB gene cause nemaline myopathy, Mutations in the PCDH15 gene cause Usher syndrome, IF type; Mutations in the CLRN1 gene cause Usher syndrome, type IIIA; Mutations in the TMEM216 gene cause Joubert syndrome 2; The kit of claim 20, wherein the FKTN gene mutation causes Walker-Warburg syndrome.

25. the HEXA gene variation comprises R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; The SMPD1 gene variation comprises L302P, fsP330, R496L (R498L), or deltaR608; the MCOLN1 gene variation comprises IVS3-2A>G or 6.4kb_del; the GBA gene variation comprises IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P, or R496H; the PANCC gene variation comprises IVS4+4A>T or 322delG; the variation of the IKBKAP gene comprises R696P or IVS20+6T>C; the ASPA gene variation comprises IVS2-2A>G, Y231X, E285A, or A305E; the BLM gene variation comprises 2281del6 / ins7, the variation in the BCKDHB gene comprises R183P, G278S, or E372X; the G6PC gene variation comprises R83C or Q347X; the variation in the ABCC8 gene comprises IVS32-9G>A or F1387del; the DLD gene variation comprises Y35* or G229C; the NEB gene variation comprises R2478_D2512del35; the PCDH15 gene variation comprises R245; the CLRN1 gene variation comprises N48K; the variation of the TMEM216 gene comprises R73L; 21. The kit of claim 20, wherein the FKTN gene variation comprises F390fs.

26. Preface: Changes in the HBB legacy, c. *111A>G、c. *110T>C、c. *96T>C、Hb D-Los Angeles(c.364G>C)、Hb O-Arab(c.364G>A)、c. 321_322insG、c. 316-2A>C、c. 316-2A>G、c. 316-3C>A、c. 316-106C>G、c. 316-125A>G、c. 316-146T>G、c. 316-197C>T、c. 315+1G>A, c. 287_288insA, c. 251delG, c. 230delC, c. 216_217insA, c. 203_204delTG, c. 143_144insA, c. 146_147insATCT, c. 135delC, c. 130G>T, c. 126_129delCTTT, c. 124_127delTTCT, c. 118C>T, c. 114G>A, c. 112delT, c. 93-1G>C, c. 93-1G>A, c. 93-21G>A, c. 92+6T>C, c. 92+5G>A, c. 92+5G>C, c. 92+5G>T, c. 92+2T>A, c. 92+2T>C, c. 92+1G>A, c. 92+1G>T, Hb Monroe (c. 92G>C), c. 92G>A, c. 84_85insC, c. 79G>T, HB E (c. 79G>A), c. 75T>A, c. 59A>G, c. 52A>T (LYS17*), c. 51delC, c. 48G>A, c. 47G>A (Trp15), c. 46delT, c. 36delT, c. 33C>A, c. 27_28insG, c. 25_26delAA,c. 20delA, HB S (c.20A>T), HB C (c.19G>A), c. 17_18delCT, c. 2T>C, c. 2T>G, c. 1A>G, c. -78A>C, c. -78A>G, c. -79A>G, c. -80T>A, c. -81A>G, c. -136C>G, c. -137C>A, c. -137C>G, c. -137C>T, c. -138C>T, c. -138C>A, c. -140C>T、also はc. -151C>Tをincludes, request item 20にrecordsのキット. 【Request 27】 21. The kit of claim 20, wherein the alpha thalassemia variation in the HBA1 and HBA2 genes comprises a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL, or a single nucleotide variant in the HBA2 gene HBA2 c.427T>C.

28. 21. The kit of claim 20, wherein the first plurality of primer pairs comprises at least two primer pairs having at least 85% identity to at least two primer pairs selected from Table 4.

29. 30. The kit of claim 28, wherein the first plurality of primer pairs comprises all of the primer pairs shown in Table 4.

30. 21. The kit of claim 20, wherein the second plurality of primer pairs comprises at least two primer pairs having at least 85% identity to at least two primer pairs selected from SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:85 and 86, SEQ ID NOs:87 and 88, or SEQ ID NOs:89 and 90.

31. 31. The kit of claim 30, wherein the first plurality of primer pairs comprises all of the primer pairs shown in Table 5.

32. 21. The kit of claim 20, wherein the third plurality of primer pairs comprises at least two primer pairs having at least 85% identity to at least two primer pairs selected from Table 6.

33. 33. The kit of claim 32, wherein the first plurality of primer pairs comprises all of the primer pairs shown in Table 6.

Images