Method for detecting genetic mutations using multiplex gene-specific PCR

Abstract

The present disclosure provides methods for the detection of genetic mutations (e.g., Dehejie carrier mutations, beta thalassemia mutations, or alpha thalassemia mutations) using multiplex gene-specific PCR. Kits for use in practices of the methods are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 286,906, filed on December 7, 2021, under 35 U.S.C. §119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure provides methods for detecting genetic mutations (e.g., Ashkenazi Jewish carrier mutations, beta thalassemia mutations, or alpha thalassemia mutations) using multiplexed gene-specific PCR. Also provided are kits for use in practicing the methods. Background Art

[0004] The following background description of the present disclosure is provided only to help the reader understand the present disclosure and is not to be considered as constituting a description of the present disclosure or prior art.

[0005] Thalassemia is an inherited autosomal recessive disease caused by mutations in the alpha globin gene cluster and the beta globin gene cluster, located on chromosome 16 and chromosome 11, respectively. It is characterized by a lack of or reduced synthesis of hemoglobin globin chains and includes two main types: alpha-thalassemia and beta-thalassemia. It is reported that thalassemia is one of the top five major birth defects. Severe thalassemia has brought a huge burden to society and has seriously affected the quality of life of the affected population. A high prevalence of thalassemia has been observed in southern China, Southeast Asia, India, the Middle East, Africa, and the Mediterranean region.

[0006] The Ashkenazi Jewish population, which lives mainly in Central and Eastern Europe, remains genetically isolated, separated from its neighbors by religious and cultural practices as well as by blood ties. The main evidence for this isolation of the Ashkenazi Jewish population is the presence of genetic traits, including a high prevalence of autosomal recessive diseases and a relatively high frequency of alleles that confer risk for common conditions such as breast and ovarian cancer, or variants associated with inflammatory bowel disease or Parkinson's disease.

[0007] There is an urgent need for more robust and sensitive methods that can rapidly detect in a single biological sample whether a subject is a carrier of inherited genetic diseases, including the diseases common among Ashkenazi Jews, beta thalassemia and alpha thalassemia. Summary of the invention

[0008] The present disclosure provides methods for simultaneously identifying hereditary genetic variants (such as Ashkenazi Jewish panel variants, beta thalassemia variants, and alpha thalassemia variants). The present disclosure also provides methods for identifying Ashkenazi Jewish panel variants, beta thalassemia variants, and alpha thalassemia variants in biological samples from subjects. Finally, the present disclosure provides kits for practicing the methods described herein.

[0009] In some embodiments, the method comprises: extracting DNA from a biological sample obtained from a subject; generating a first plurality of amplicons by contacting the biological sample with a first plurality of primer pairs, wherein at least one amplicon corresponds to each gene in 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; 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, wherein the second plurality of primer pairs comprises SEQ ID NOs: 81 and 82, and SEQ ID NOs: 83 and 84; and generating a second 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 The invention relates to a method for preparing a novel nucleic acid sequence for the present invention and a method for preparing the nucleic acid sequence of the first plurality of amplicons, wherein at least one amplicon corresponds to each of HBA1 and HBA2; incorporating barcode sequences into ends of the first plurality of amplicons, the second plurality of amplicons, and the third plurality of amplicons via polymerase chain reaction; and detecting one or more variants in at least one of the first plurality of amplicons, the second plurality of amplicons, and the third plurality of amplicons using high-throughput massively parallel sequencing.

[0010] In some embodiments, the Ashkenazi panel variants include genetic variants (e.g., single nucleotide variants (SNVs), deletions, insertions, or inversions) that are commonly observed in the Ashkenazi population, but may also occur outside the Ashkenazi population, albeit at a lower frequency. In some embodiments, the Ashkenazi panel variants include genetic variants that can cause Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, maple serum urinary system disease, glycogen storage disease I, familial hyperinsulinemia, 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, the Ashkenazi panel variants include the genetic variants listed in Table 1. In some embodiments, the beta thalassemia variants include the genetic variants listed in Table 2. In some embodiments, the beta thalassemia variants include the genetic variants listed in Table 3.

[0011] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only when 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 when a deletion is present.

[0012] In some embodiments, a HEXA gene variant causes Tay-Sachs disease; and a SMPD1 gene variant causes Niemann-Pick disease; and a MCOLN1 gene variant causes mucolipidosis type IV; and a GBA gene variant causes Gaucher disease; and a PANCC gene variant causes Fanconi anemia; and a IKBKAP gene variant causes familial dysautonomia; and a ASPA gene variant causes Canavan disease; and a BLM gene variant causes Bloom syndrome; and a BCKDHB gene variant causes maple serum urinary system disease; and a G6PC gene variant causes variants in the ABCC8 gene cause glycogen storage disease I; and variants in the ABCC8 gene cause familial hyperinsulinemia; and variants in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3); and variants in the NEB gene cause nemaline myopathy; and variants in the PCDH15 gene cause Usher syndrome type IF; and variants in the CLRN1 gene cause Usher syndrome type IIIA; and variants in the TMEM216 gene cause Joubert syndrome 2; and variants in the FKTN gene cause Walker-Warburg syndrome.

[0013] In some embodiments, the Ashkenazi panel gene variants include one or more of the gene variants listed in Table 1. In some embodiments, the HEXA gene variants include R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; and SMPD1 gene variation includes one or more of L302P, fsP330, R496L (R498L) or δR608; and MCOLN1 gene variation includes one or more of IVS3-2A>G or 6.4kb_del; and GBA gene variation includes one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P or R496H; and PANCC gene variation includes one or more of IVS4+4A>T or 322delG; and IKBKAP gene variation includes one or more of R696P or IVS20+6T>C; and ASPA gene variation includes IVS2-2A>G, Y231X, E28 5A or A305E; and BLM gene variations include one or more of 2281del6 / ins7; and BCKDHB gene variations include one or more of R183P, G278S or E372X; and G6PC gene variations include one or more of R83C or Q347X; and ABCC8 gene variations include IVS32-9G>A or F1387del; and DLD gene variations include one or more of Y35* or G229C; and NEB gene variations include R2478_D2512del35; and PCDH15 gene variations include one or more of R245; and CLRN1 gene variations include N48K; and TMEM216 gene variations include R73L; and FKTN gene variations include F390fs.

[0014] In some embodiments, the beta thalassemia gene variants in the HBB gene include one or more gene variants listed in Table 2. In some embodiments, the gene variants include c.*111A>G, c.*110T>C, c.*96T>C, Hb D-Los Angeles (c.364G>C), Hb O-Arabic (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.287288insA, c.251delG, c.230delC, c.216217insA, c.203204delTG, 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.-8 1A>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.

[0015] In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include one or more variants listed in Table 3. In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL; or the single nucleotide variant HBA2 c.427T>C in the HBA2 gene.

[0016] In some embodiments, the first plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two, at least four, at least five, at least eight, or at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, or all primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 4.

[0017] In some embodiments, the second plurality of primer pairs includes at least two, at least four, or at least five primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two, at least four, or at least five primer pairs selected from Table 5.

[0018] In some embodiments, the third plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85% identical to at least two, at least three, or at least four primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 6.

[0019] In some embodiments, next generation sequencing comprises synthesis sequencing (eg, pyrosequencing). In some embodiments, next generation sequencing comprises ligation sequencing.

[0020] In some embodiments, the subject is suspected of being a carrier of at least one disease selected from Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, maple serum urinary disease, glycogen storage disease I, familial hyperinsulinemia, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Ussher syndrome type IF, Ussher syndrome type IIIA, Joubert syndrome 2, Walker-Warburg syndrome, beta thalassemia, or alpha thalassemia.

[0021] In some embodiments, the subject is of Ashkenazi Jewish ancestry or has a family history of beta thalassemia or alpha thalassemia.

[0022] In some embodiments, the biological sample is selected from whole blood, serum, plasma, amniotic fluid, or chorionic villi.

[0023] In some embodiments, the genetic disorder is an autosomal or X-linked recessive genetic disorder.

[0024] In another aspect, the invention provides a kit comprising an oligonucleotide, which may be a primer or a probe for performing amplification as described herein.

[0025] 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.

[0026] The kit of the present technology includes at least (i) a first plurality of primer pairs, which are directed to the amplification region of each of a plurality of genes, wherein the plurality of genes include 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, which are directed to the amplification region of the GBA gene and the HBB gene, wherein the second plurality of primer pairs include SEQ ID NOs:81 and 82 and SEQ ID NOs:83 and 84; and (iii) a third plurality of primer pairs, which are directed to the amplification region of the α-thalassemia gene variation in each of the HBA1 gene and the HBA2 gene, and instructions for use.

[0027] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only when a deletion is present.

[0028] 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.

[0029] In some embodiments, the third plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only when a deletion is present.

[0030] In some embodiments, a HEXA gene variant causes Tay-Sachs disease; and a SMPD1 gene variant causes Niemann-Pick disease; and a MCOLN1 gene variant causes mucolipidosis type IV; and a GBA gene variant causes Gaucher disease; and a PANCC gene variant causes Fanconi anemia; and a IKBKAP gene variant causes familial dysautonomia; and a ASPA gene variant causes Canavan disease; and a BLM gene variant causes Bloom syndrome; and a BCKDHB gene variant causes maple serum urinary system disease; and a G6PC gene variant causes variants in the ABCC8 gene cause glycogen storage disease I; and variants in the ABCC8 gene cause familial hyperinsulinemia; and variants in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3); and variants in the NEB gene cause nemaline myopathy; and variants in the PCDH15 gene cause Usher syndrome type IF; and variants in the CLRN1 gene cause Usher syndrome type IIIA; and variants in the TMEM216 gene cause Joubert syndrome 2; and variants in the FKTN gene cause Walker-Warburg syndrome.

[0031] In some embodiments, the Ashkenazi panel gene variants include one or more of the gene variants listed in Table 1. In some embodiments, the HEXA gene variants include R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; and SMPD1 gene variation includes one or more of L302P, fsP330, R496L (R498L) or δR608; and MCOLN1 gene variation includes one or more of IVS3-2A>G or 6.4kb_del; and GBA gene variation includes one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P or R496H; and PANCC gene variation includes one or more of IVS4+4A>T or 322delG; and IKBKAP gene variation includes one or more of R696P or IVS20+6T>C; and ASPA gene variation includes IVS2-2A>G, Y231X, E2 85A or A305E; and BLM gene variations include one or more of 2281del6 / ins7; and BCKDHB gene variations include one or more of R183P, G278S or E372X; and G6PC gene variations include one or more of R83C or Q347X; and ABCC8 gene variations include IVS32-9G>A or F1387del; and DLD gene variations include one or more of Y35* or G229C; and NEB gene variations include R2478_D2512del35; and PCDH15 gene variations include one or more of R245; and CLRN1 gene variations include N48K; and TMEM216 gene variations include R73L; and FKTN gene variations include F390fs.

[0032] In some embodiments, the beta thalassemia gene variants in the HBB gene include one or more gene variants listed in Table 2. In some embodiments, the gene variants 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.146_147insATCT, c.135del C, 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.-8 1A>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.

[0033] In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include one or more gene variants listed in Table 3. In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include 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 HBA2 c.427T>C in the HBA2 gene.

[0034] In some embodiments, the first plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two, at least four, at least five, at least eight, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or all primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 4.

[0035] In some embodiments, the second plurality of primer pairs includes at least two, at least four, or at least five primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical 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 includes all primer pairs shown in Table 5.

[0036] In some embodiments, the third plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85% identical to at least two, at least four, at least five, at least eight, or at least ten primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 .Sequencing library preparation workflow.

[0038] Figure 2 .Sequencing data analysis workflow. DETAILED DESCRIPTION

[0039] The present disclosure provides a method ("expanded carrier screening") that simultaneously provides hotspot molecular detection of variants for multiple autosomal and X-linked recessive genetic disorders and allows individuals to be tested regardless of their ancestry or geographic origin. Point-of-care carrier screening can easily and quickly identify couples at higher risk of having an affected child to facilitate informed reproductive decision-making.

[0040] The carrier screening panel disclosed in this article 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 hyperinsulinemia, Fanconi anemia type C, Gaucher disease, glycogen storage disease type IA, Joubert syndrome 2, maple serum urinary system 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.

[0041] In addition to the above 19-disorder (20-gene) carrier screening panel, this carrier screening panel includes 24 genes, which also include 3 additional diseases: cystic fibrosis (CFTR), fragile X syndrome (FMR1), and spinal muscular atrophy (SMN1 and SMN2).

[0042] The disclosed carrier screening provides hotspot molecular detection of variants for multiple autosomal and X-linked recessive genetic disorders simultaneously and allows individuals to be tested regardless of their ancestry or geographic origin. The purpose of carrier screening is to identify couples at increased risk of having an affected child to facilitate informed reproductive decision making.

[0043] definition

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this disclosure belongs.

[0045] As used herein, unless otherwise indicated, the singular forms "a", "an", and "the" include plural references. Thus, for example, reference to an "oligonucleotide" includes a plurality of oligonucleotide molecules, and reference to a "nucleic acid" is a reference to one or more nucleic acids. Note also that claims may be written to exclude any optional elements. Therefore, this statement is intended to serve as a pre-conditional basis for the use of such exclusive terms as "solely", "only", etc., or the use of "negative" limitations related to the narration of claim elements.

[0046] As used herein, the term "about" in reference to a number means that the number is generally included within the range of 1%-10% in either direction (greater or less than) of the number, unless otherwise stated or otherwise obvious from the context.

[0047] The term "adapter" refers to a short, chemically synthesized nucleic acid sequence that can be used to connect to the end of a nucleic acid sequence to facilitate attachment to another molecule. An adaptor can be single-stranded or double-stranded. An adaptor can include a short sequence (usually less than 50 base pairs) for PCR amplification or sequencing.

[0048] As used herein, the term "amplify" or "amplification" with respect to a nucleic acid sequence refers to a method of increasing the representativeness of a nucleic acid sequence population in a sample. Copies of a specific target nucleic acid sequence generated in vitro in an amplification reaction are referred to as "amplicons" or "amplification products". Amplification can be exponential or linear. The target nucleic acid can be DNA (such as, for example, genomic DNA and cDNA) or RNA. Although the exemplary methods described below involve amplification using polymerase chain reaction (PCR), many other methods such as isothermal methods, rolling circle methods, etc. are well known to those skilled in the art. The skilled person will appreciate that these other methods can be used in place of, or in conjunction with, the PCR method. See, for example, Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al., eds., Academic Press, San Diego, California, 1990, pp. 13-20; Wharam, et al., Nucleic Acids Res. 29(11): E54-E54 (2001).

[0049] 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, nonionic and ionic detergents (including cationic detergents).

[0050] An "amplification master mix" includes an amplification mixture and primers for amplifying one or more target nucleic acids, but does not contain the sample to be amplified.

[0051] As used herein, the phrase "selective amplification" 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 a selected gene or portion of a gene (e.g., where known / preselected variants occur) is amplified.

[0052] As used herein, the terms "complement", "complementary" or "complementarity" with respect to polynucleotides (i.e., nucleotide sequences, such as oligonucleotides or target nucleic acids) refer to the 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 so that the 5' end of one sequence is paired with the 3' end of another sequence. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Certain bases that are not common 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 (PNA). Complementarity does not have to be perfect; stable duplexes may contain mismatched base pairs, degenerate or mismatched bases. A person skilled in the art of nucleic acid technology can determine duplex stability empirically, taking into account many 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 may also be an RNA sequence complementary to the DNA sequence or its complementary sequence, or may be cDNA.

[0053] As used herein, the term "substantially complementary" refers to two sequences that hybridize under stringent hybridization conditions. It will be appreciated by the skilled artisan that substantially complementary sequences do not need to hybridize along their entire length. In particular, substantially complementary sequences may include a continuous sequence of bases that do not hybridize to a target sequence, which is located 3' or 5' to a continuous sequence of bases that hybridizes to a target sequence under stringent hybridization conditions.

[0054] As used herein, the term "detection" refers to determining the presence of a target nucleic acid in a sample. Detection does not require a method that provides 100% sensitivity and / or 100% specificity.

[0055] As used herein, the term "direct amplification" refers to a nucleic acid amplification reaction in which a target nucleic acid is amplified from a sample without prior purification, extraction, or concentration.

[0056] As used herein, the term "sequencing" refers to a method by which the identity of at least 10 consecutive nucleotides of a polynucleotide is obtained (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides).

[0057] The term "second-generation sequencing" refers to the so-called parallel synthesis sequencing or ligation sequencing platforms currently used by Illumina, Life Technologies and Roche, etc. (e.g., Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' ligation sequencing (SOLiD platform), or Pacific Biosciences' fluorescent base 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 references are incorporated by reference for a general description of the methods and specific steps of the methods, including all starting products, reagents, and final products for each step. Next generation sequencing can generate at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1 M, at least 10 M, at least 100 M, or at least 1 B sequence reads. In many cases, the reads are paired-end reads.

[0058] In some embodiments, the next generation sequencing of the present disclosure comprises the following next generation sequencing system:

[0059] Ion Torrent TM(Life Technologies, Carlsbad, CA) Amplicon sequencing system uses a flow-based method that detects pH changes caused by the release of hydrogen ions during the incorporation of unmodified nucleotides in DNA replication. To use with this system, a sequencing library is first generated by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplification template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is filled with nucleotides one by one, and if the nucleotide is complementary to a DNA molecule in a specific microwell of the chip, it is incorporated. When the polymerase incorporates the nucleotide into the DNA molecule, protons are naturally released, resulting in a detectable local pH change. The pH of the solution then changes in the well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides are incorporated in a single cycle. This results in a corresponding amount of released hydrogen and a proportionally higher electronic signal.

[0060] 454TM GS FLX TM The sequencing system (Roche, Germany) uses a light-based detection method in a massively parallel pyrosequencing system.

[0061] Pyrosequencing uses DNA polymerization to add one nucleotide species at a time and detects and quantifies the number of nucleotides added to a given position by the emission of light from the release of the attached pyrophosphate. TM The PCR system is used together with the PCR-PCR system to fix the adapter-ligated DNA fragments to small DNA capture beads in an oil-in-water emulsion and amplify them by PCR (emulsion PCR). Each DNA-binding bead is placed into a well on a pico-titer plate, and the sequencing reagents are transported across the wells of the plate. During the sequencing run, the four DNA nucleotides are added sequentially across the pico-titer plate device in a fixed order. During the nucleotide flow, millions of DNA copies bound to each bead are sequenced in parallel. When a nucleotide complementary to the template strand is added to the well, the nucleotide is incorporated into the existing DNA strand, generating a light signal that is recorded by the CCD camera in the instrument.

[0062] Reversible dye terminator-based sequencing technology: DNA molecules are first attached to primers on a slide and amplified, forming local clonal colonies. Four types of reversible terminator bases (RT-bases) are added, and unincorporated nucleotides are washed away. Unlike pyrophosphate sequencing, DNA can only be extended one nucleotide at a time. The camera takes an image of the fluorescently labeled nucleotides, and then the dye and terminal 3' blocker are removed from the DNA chemically, allowing the next cycle.

[0063] Helicos single-molecule sequencing uses DNA fragments with added poly-A tail adapters that are attached to the flow cell surface. In each cycle, DNA polymerase and a single species of fluorescently labeled nucleotides are added, resulting in template-dependent extension of the primer-template duplex immobilized on the surface. Reading is performed by the Helioscope sequencer. After acquiring an image of the entire array, chemical cleavage and release of the fluorescent label allow subsequent extension and imaging cycles.

[0064] Synthesis sequencing (SBS), like "old-style" dye-terminator electrophoretic sequencing, relies on DNA polymerase to bind nucleotides to determine base sequence. A DNA library with fixed adapters is denatured into single strands and transplanted into a flow cell, followed by bridge amplification to form a high-density spot array on a glass chip. The reversible terminator method uses a reversible form of dye terminators, adding one nucleotide at a time, and detecting fluorescence at each position by repeatedly removing the blocking group to allow another nucleotide to polymerize. The signal of nucleotide binding can be varied with the use of fluorescently labeled nucleotides, phosphate-driven photoreactions, and hydrogen ion sensing. Examples of SBS platforms include Illumina GA and HiSeq2000. The Personal Sequencing System (Illumina, Inc.) also employs sequencing by synthesis via reversible terminator chemistry.

[0065] In contrast to the sequencing-by-synthesis method, the ligation sequencing method uses DNA ligase to determine the target sequence. This sequencing method relies on the enzymatic ligation of adjacent oligonucleotides on the template DNA strand by local complementarity. The technology employs a partitioning of all possible oligonucleotides of fixed length and labels them according to the sequencing position. The oligonucleotides are annealed and ligated, and preferentially ligated by DNA ligase to matching sequences, generating a dinucleotide-encoded color space signal at that position (by releasing a fluorescently labeled probe corresponding to a known nucleotide at a known position along the oligonucleotide). The method is mainly used by Life Technologies' SOLiD TM Sequencer use. Before sequencing, the 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.

[0066] SMRT TM Sequencing is based on a synthetic sequencing method. DNA is synthesized in a zero-mode waveguide (ZMW), a small well-shaped container in which a capture tool is located 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 well is constructed in such a way that only the fluorescence occurring at the bottom of the well is detected. The fluorescent label is separated from the nucleotides at the point where the nucleotides are incorporated into the DNA chain, leaving the unmodified DNA chain.

[0067] As used herein, the term "barcode sequence" or "molecular barcode" refers to a unique sequence of nucleotides used for: (a) identifying and / or tracking the source of a polynucleotide in a reaction; and / or (b) counting the number of times an initial molecule has been sequenced (e.g., in the case where substantially every molecule in a sample is labeled with a different sequence, and the sample is then amplified). The barcode sequence can be located at the 5' end, 3' end, or in the middle of an oligonucleotide, or at both the 5' end and the 3' end. The size and composition of barcode sequences can vary widely; the following references provide guidance for selecting a barcode sequence set suitable for a particular embodiment: Brenner, U.S. Pat. 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 0799897A1; Wallace, U.S. Pat. No. 5,981,179; etc. In some embodiments, the barcode sequence can have a length ranging from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides.

[0068] 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).

[0069] As used herein, the term "hybridization" 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 to 25 nucleotides) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex by forming hydrogen bonds between complementary base pairs. Hybridization is typically and preferably performed with a probe-length nucleic acid molecule (preferably 15 to 100 nucleotides in length, more preferably 18 to 50 nucleotides in length). Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York. Hybridization and the strength of hybridization (i.e., the strength of binding between nucleic acids) are subject to factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the thermal melting point (T) of the hybrid formed. m) and other factors. Those skilled in the art understand 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 lower complementarity will not. For examples of hybridization conditions and parameters, see, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Plainview, New York; Ausubel, FM et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, New Jersey. In some embodiments, specific hybridization occurs under stringent hybridization conditions. Oligonucleotides or polynucleotides (e.g., probes or primers) specific to a target nucleic acid will "hybridize" with the target nucleic acid under suitable conditions.

[0070] In the context of nucleic acid amplification reactions, "hot start" refers to a protocol in which at least one key reagent is retained from the reaction mixture (or, if present in the reaction mixture, the reagent is kept inactive) until the temperature is raised sufficiently to provide the necessary hybridization specificity of the primer or primers. A "hot start enzyme" is an enzyme, typically a nucleic acid polymerase, that can act as a "holding" or inactivating 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

[0071] As used herein, "Taq Extender" is a PCR additive that increases the efficiency of the Taq DNA polymerase extension reaction during each PCR cycle by reducing mismatch pauses, resulting in a greater percentage of completed extension reactions. In some embodiments, the Taq Extender includes Taq Extender from Agilent or Strategene. TM .

[0072] As used herein, the term "individual", "patient" or "subject" can be an individual organism, a vertebrate, a mammal or a human. In a preferred embodiment, the individual, patient or subject is a human.

[0073] Pseudogenes are defined herein as nucleic acid sequences that do not encode wild-type functional proteins. The term "pseudogene" encompasses nucleic acid sequences that do not encode proteins at all. In addition, the term "pseudogene" encompasses gene alleles including modifications (e.g., insertions or deletions) such that they encode a portion of a homologous protein or protein that is significantly impaired or lost compared to a wild-type protein. For example, such alleles encode truncated proteins as a result of a frameshift caused by the insertion and / or deletion of at least one nucleotide or a frameshift caused by a premature stop codon.

[0074] 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. The multiplex reaction can also include specific probes for each product labeled with different detectable moieties.

[0075] As used herein, "oligonucleotide" refers to a molecule having a nucleic acid base sequence on the main chain, which is mainly composed of the same monomeric units at defined intervals. Bases are arranged on the main chain in this manner: they can be combined with nucleic acids having a base sequence complementary to the bases of the oligonucleotides. The most common oligonucleotides have a main chain of sugar phosphate units. It is possible to distinguish between oligodeoxyribonucleotides without a hydroxyl group at the 2' position and oligoribonucleotides with a hydroxyl group at the 2' position. Oligonucleotides can also include derivatives, in which the hydrogen of the hydroxyl group is replaced by an organic group such as an allyl group. The length of the oligonucleotide serving as a primer or probe is generally at least about 10 to 15 nucleotides or a length of at most about 70, 100, 110, 150 or 200 nucleotides, and more preferably a length of at least about 15 to 25 nucleotides. The oligonucleotides used as primers or probes for specific amplification or specific detection of a specific target nucleic acid can usually be specifically hybridized with the target nucleic acid.

[0076] As used herein, a "positive control nucleic acid" or "internal positive amplification control" is a nucleic acid known to be present in a sample in a certain amount or level. In some embodiments, the positive control nucleic acid does not naturally occur in the sample, and in the disclosed methods it is added to the sample before subjecting the reaction-sample mixture to a real-time polymerase chain reaction.

[0077] As used herein, the term "primer" refers to an oligonucleotide, when placed under conditions where the synthesis of a primer extension product complementary to a target nucleic acid strand is induced, the oligonucleotide can serve as the starting point for nucleic acid sequence synthesis, i.e., in the presence of different nucleotide triphosphates and polymerase, in an appropriate buffer ("buffer" includes pH, ionic strength, cofactors, etc.) and at a suitable temperature. One or more nucleotides of a primer can be modified, for example, by adding a methyl group, biotin or digoxigenin moiety, a fluorescent label, or by using radioactive nucleotides. The primer sequence does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be connected to the 5' end of the primer, and the remainder of the primer sequence is substantially complementary to the chain. As used herein, the term primer includes all forms of primers that can be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, thiophosphate-modified primers, labeled primers, etc. As used herein, the term "forward primer" refers to a primer annealed to the antisense strand of a double-stranded DNA (dsDNA). "Reverse primer" anneals to the sense strand of dsDNA.

[0078] The length of primer is generally at least 10,15,18 or 30 nucleotides, or the length is at most about 100,110,125 or 200 nucleotides.In certain embodiments, the length of primer is preferably between about 15 to about 60 nucleotides, and most preferably the length is between about 25 to about 40 nucleotides.In certain embodiments, the length of primer is 15 to 35 nucleotides.There is no standard length for optimal hybridization or polymerase chain reaction amplification.The optimal length of specific primer application can be described in H.Erlich, PCR Technology, PRINCIPLES AND APPLICATION FOR DNA AMPLIFICATION, (1989) and the mode in easily determines.

[0079] "Primer extension reaction" refers to a synthetic 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 a single complementary nucleotide. In some embodiments, the primer extension reaction is PCR.

[0080] As used herein, the phrase "primer pair" refers to a forward and reverse primer pair (ie, a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.

[0081] As used herein, the phrase "gap primer pair" refers to a primer pair designed to produce a product only when a deletion is present. In some embodiments, large deletions specifically detected using the gap primer pair include: 7.6 Kb deletion in HEXA, 6.4 Kb deletion in MCOLN1, 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 the gap PCR amplicon, rather than on the specific variant referred to.

[0082] As used herein, "probe" refers to a nucleic acid that interacts with a target nucleic acid via hybridization. The probe may be fully complementary or partially complementary to the target nucleic acid sequence. The level of complementarity will generally depend on many factors based on the function of the probe. The probe may be labeled or unlabeled, or modified in any of a variety of ways well known in the art. The probe may specifically hybridize with the target nucleic acid. The probe may be DNA, RNA, or RNA / DNA hybrid. The probe may be an oligonucleotide, an artificial chromosome, a fragmented artificial chromosome, a genomic nucleic acid, a fragmented genomic nucleic acid, RNA, a recombinant nucleic acid, a fragmented recombinant nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid, an oligomer of a cyclic heterocyclic compound, or a conjugate of nucleic acid. The probe may include modified nucleic acid bases, modified sugar moieties, and modified internucleotide bonds. The probe may be used to detect the presence or absence of a methylated target nucleic acid. The length of the probe is generally at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or longer.

[0083] As used herein, "probe element" refers to a stretch of nucleotides that (a) is associated with a primer because it is linked to or located near the primer nucleic acid sequence and (b) specifically hybridizes to a target nucleic acid sequence to be detected under stringent conditions.

[0084] As used herein, "reaction-sample mixture" refers to a mixture containing an amplification master mix and a sample.

[0085] 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 a 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.

[0086] As used herein, the term "sensitivity" used in relation to the methods of the present technology is a measure of the ability of the method to detect preselected sequence variants in a heterogeneous population of sequences. If a given sample is present in which the preselected sequence variant is present in at least F% of the sequence in the sample, the method has a sensitivity of S% to F% variants, and the method can detect the preselected sequence at a preselected confidence of C% and S% times. For example, if a given sample is present in which the preselected sequence variant is present in at least 5% of the sequence in the sample, the method has a sensitivity of 90% for 5% variants, and the method can detect the preselected sequence 9 times out of 10 with a preselected confidence of 99% (F=5%; C=99%; S=90%). Exemplary sensitivities include at least 50%, 60%, 70%, 80%, 90%, 95%, 98% and 99%.

[0087] As used herein, the term "specificity" about oligonucleotide primers refers to that when oligonucleotide and nucleic acid are compared, the nucleotide sequence of the primer has a sequence identity of at least 12 bases with the part of the nucleic acid to be amplified. Oligonucleotide primers specific to nucleic acids are oligonucleotide primers that can hybridize with a target of interest under stringent hybridization or washing conditions and are not substantially hybridized with nucleic acids of no interest. Higher levels of sequence identity include at least 75%, at least 80%, at least 85%, at least 90%, at least 85% to 95%, at least 98%, at least 99% or higher sequence identity. Sequence identity can be determined using commercially available computer programs that have default settings using algorithms well known in the art. As used herein, sequences with "high sequence identity" have 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 aligned nucleotide positions.

[0088] As used herein, "specificity" is a measure of the ability of a method to distinguish true occurring preselected sequence variants (gene variations) from sequencing artifacts or other closely related sequences. It is the ability to avoid false positive detections. False positive detections may be caused by errors introduced into the sequence of interest during sample preparation, sequencing errors, or inadvertent sequencing of closely related sequences (such as pseudogenes or gene family members). If applied to N 总计 When the sample set of the sequence is 真实 The sequence is a true variant, and X 不真实is an untrue variant, then the method has a specificity of X%, and the method selects at least X% of the untrue variants as non-variants. For example, if when applied to a sample set of 1,000 sequences, 500 of which are true variants and 500 are untrue variants, then the method has a specificity of 90%, and the method selects 90% of the 500 untrue variant sequences as non-variants. Exemplary specificities include at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, and 99%.

[0089] As used herein, the term "stringent hybridization conditions" refers to hybridization conditions that are at least as stringent as the following: hybridization in 50% formamide, 5x SSC, 50mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1mg / mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42°C overnight; washing with 2x SSC, 0.1% SDS at 45°C; and washing with 0.2x SSC, 0.1% SDS at 45°C. In another example embodiment, 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.

[0090] 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 a sample to be analyzed. The target nucleic acid can be composed of a chromosome fragment, a complete gene with or without an intergenic sequence, a gene fragment or part with or without an intergenic sequence, or a nucleic acid sequence for designing a probe or primer. The target nucleic acid can include a wild-type sequence, a mutation, a deletion, an insertion or a repetition, a tandem repeat element, a gene of interest, a region of a gene of interest, or any upstream or downstream region thereof. The target nucleic acid can represent an alternative sequence or an allele of a specific gene. The target nucleic acid can be derived from genomic DNA, cDNA, or RNA.

[0091] Methods for detecting gene variants (mutations)

[0092] One aspect of the present disclosure relates to a method for detecting one or more variants for a genetic disorder in a subject suspected of being a carrier of at least one inherited genetic mutation, the method 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, wherein at least one amplicon corresponds to each gene in 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; 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, wherein the second plurality of primer pairs comprises SEQ ID NOs: 81 and 82, and SEQ ID NOs: 83 and 84; and generating a second 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 The invention relates to a method for preparing a novel nucleic acid sequence for the present invention and a method for preparing the nucleic acid sequence of the first plurality of amplicons, wherein at least one amplicon corresponds to each of HBA1 and HBA2; incorporating barcode sequences into ends of the first plurality of amplicons, the second plurality of amplicons, and the third plurality of amplicons via polymerase chain reaction; and detecting one or more variants in at least one of the first plurality of amplicons, the second plurality of amplicons, and the third plurality of amplicons using high-throughput massively parallel sequencing.

[0093] In some embodiments, the Ashkenazi panel variants include genetic variants (e.g., single nucleotide variants (SNVs), deletions, insertions, or inversions) that are commonly observed in the Ashkenazi population, but may also occur outside the Ashkenazi population, albeit at a lower frequency. In some embodiments, the Ashkenazi panel variants include genetic variants that can cause Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, maple serum urinary system disease, glycogen storage disease I, familial hyperinsulinemia, 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, the Ashkenazi panel variants include the genetic variants listed in Table 1. In some embodiments, the beta thalassemia variants include the genetic variants listed in Table 2. In some embodiments, the alpha thalassemia variants include the genetic variants listed in Table 3.

[0094] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only when 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 when a deletion is present.

[0095] In some embodiments, a HEXA gene variant causes Tay-Sachs disease; and a SMPD1 gene variant causes Niemann-Pick disease; and a MCOLN1 gene variant causes mucolipidosis type IV; and a GBA gene variant causes Gaucher disease; and a PANCC gene variant causes Fanconi anemia; and a IKBKAP gene variant causes familial dysautonomia; and a ASPA gene variant causes Canavan disease; and a BLM gene variant causes Bloom syndrome; and a BCKDHB gene variant causes maple serum urinary system disease; and a G6PC gene variant causes variants in the ABCC8 gene cause glycogen storage disease I; and variants in the ABCC8 gene cause familial hyperinsulinemia; and variants in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3); and variants in the NEB gene cause nemaline myopathy; and variants in the PCDH15 gene cause Usher syndrome type IF; and variants in the CLRN1 gene cause Usher syndrome type IIIA; and variants in the TMEM216 gene cause Joubert syndrome 2; and variants in the FKTN gene cause Walker-Warburg syndrome.

[0096] In some embodiments, the Ashkenazi panel gene variants include one or more of the gene variants listed in Table 1. In some embodiments, the HEXA gene variants include R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; and SMPD1 gene variation includes one or more of L302P, fsP330, R496L (R498L) or δR608; and MCOLN1 gene variation includes one or more of IVS3-2A>G or 6.4kb_del; and GBA gene variation includes one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P or R496H; and PANCC gene variation includes one or more of IVS4+4A>T or 322delG; and IKBKAP gene variation includes one or more of R696P or IVS20+6T>C; and ASPA gene variation includes IVS2-2A>G, Y231X, E28 5A or A305E; and BLM gene variations include one or more of 2281del6 / ins7; and BCKDHB gene variations include one or more of R183P, G278S or E372X; and G6PC gene variations include one or more of R83C or Q347X; and ABCC8 gene variations include IVS32-9G>A or F1387del; and DLD gene variations include one or more of Y35* or G229C; and NEB gene variations include R2478_D2512del35; and PCDH15 gene variations include one or more of R245; and CLRN1 gene variations include N48K; and TMEM216 gene variations include R73L; and FKTN gene variations include F390fs.

[0097] In some embodiments, the beta thalassemia gene variants in the HBB gene include one or more gene variants listed in Table 2. In some embodiments, the gene variants 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.146_147insATCT, c.135del C, 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.-8 1A>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.

[0098] In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include one or more variants listed in Table 3. In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include a large deletion selected from variant 3.7, variant 4.2, variant SEA, variant THAI, variant 20.5, variant MED, or variant FIL; or the single nucleotide variant HBA2 c.427T>C in the HBA2 gene.

[0099] In some embodiments, the first plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two, at least four, at least five, at least eight, or at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, or all primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 4.

[0100] In some embodiments, the first plurality of primer pairs 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 two, at least three, at least four, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, or all pairs 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: 27-28), pair 15 (SEQ ID NOs: 29-30), pair 16 (SEQ ID NOs: 31-32), pair 17 (SEQ ID NOs: 33-34), pair 18 (SEQ ID NOs: 35-36), pair 19 (SEQ ID NOs: 37-38), pair 20 (SEQ ID NOs: 39-40), pair 210 (SEQ ID NOs: 31-41), pair 221 (SEQ ID NOs: 32-42), pair 23 NO: 23-24), pair 13 (SEQ ID NO: 25-26), pair 14 (SEQ ID NO: 27-28), pair 15 (SEQ ID NO: 29-30), pair 16 (SEQ ID NO: 31-32), pair 17 (SEQ ID NO: 33-34), pair 18 (SEQ ID NO: 35-36), pair 19 (SEQ ID NO: 37-38), pair 20 (SEQ ID NO: 39-40), pair 21 (SEQ ID NO: 41-42), pair 22 (SEQ ID NO: 43-44), pair 23 (SEQ ID NO: 45-46), pair 24 (SEQ ID NO: 47-48), pair 25 (SEQ ID NO: 49-50), pair 26 (SEQ ID NO: 51-52), pair 27 (SEQ ID NO: 53-54), pair 28 (SEQ ID NO: 55-56), pair 29 (SEQ ID NO: 57-58), pair 30 (SEQ ID NO: 59-60), pair 31 (SEQ ID NO: 61-62), pair 32 (SEQ ID NO: 63-64), pair 33 (SEQ ID NO: 65-66), pair 34 (SEQ ID NO: 67-68), pair 35 (SEQ ID NO: 69-70), pair 36 (SEQ ID NO: 71-72), pair 37 (SEQ ID NO: 73-74), pair 38 (SEQ ID NO: 75-76), pair 39 (SEQ ID NO: 80 NO:57-58), pair 30 (SEQ ID NO:59-60), pair 31 (SEQ ID NO:61-62), pair 32 (SEQ ID NO:63-64), pair 33 (SEQ ID NO:65-66), pair 34 (SEQ ID NO:67-68), pair 35 (SEQ ID NO:69-70), pair 36 (SEQ ID NO:71-72), pair 37 (SEQ ID NO:73-74), pair 37 (SEQ ID NO:75-76), pair 38 (SEQ ID NO:77-78) and pair 40 (SEQ ID NO:79-80).

[0101] In some embodiments, the second plurality of primer pairs includes at least two, at least four, or at least five primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical 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 includes all primer pairs shown in Table 5.

[0102] 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 higher identity with at least two, at least four or at least five 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, SEQID NOs:87 and 88 or SEQ ID NOs:89 and 90.

[0103] In some embodiments, the second plurality of primer pairs includes at least two primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two primer pairs selected from the group consisting of the following items of Table 6: pair 1 (SEQ ID NO:81-82), pair 2 (SEQ ID NO:83-84), pair 3 (SEQ ID NO:85-86), pair 4 (SEQ ID NO:87-88) and pair 5 (SEQ ID NO:89-90).

[0104] In some embodiments, the third plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85% identical to at least two, at least four, at least five, at least eight, or at least ten primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 6.

[0105] 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 higher identity to at least two pairs selected from the group consisting of the following items in Table 6: pair 1 (SEQ ID NO:91-92), pair 2 (SEQ ID NO:93-94), pair 3 (SEQ ID NO:95-96), pair 4 (SEQ ID NO:97-98), pair 5 (SEQ ID NO:99-100), pair 6 (SEQ ID NO:101-102), pair 7 (SEQ ID NO:103-104) and pair 8 (SEQ ID NO:105-106).

[0106] In some embodiments, next generation sequencing comprises synthesis sequencing (eg, pyrosequencing). In some embodiments, next generation sequencing comprises ligation sequencing.

[0107] In some embodiments, next generation sequencing (also known as "high throughput, massively parallel sequencing") employs synthetic sequencing with reversible dye terminators. In other embodiments, sequencing is performed via ligation sequencing. In yet other embodiments, sequencing is single molecule sequencing. Examples of next generation sequencing technologies include, but are not limited to, pyrophosphate sequencing, reversible dye terminator sequencing, SOLiD sequencing, ion semiconductor sequencing, and Heliscope single molecule sequencing.

[0108] In some embodiments, methods of features of the present technology are used in a multiplex, multi-gene assay format, e.g., an assay that incorporates multiple signals from a large number of different genetic alterations in a large number of genes.

[0109] In some embodiments, the subject is of Ashkenazi Jewish ancestry or has a family history of beta thalassemia or alpha thalassemia.

[0110] In some embodiments, the biological sample is selected from whole blood, serum, plasma, amniotic fluid, or chorionic villi.

[0111] In some embodiments, the genetic disorder is an autosomal or X-linked recessive genetic disorder.

[0112] Methods for detecting genetic mutations (e.g., Ashkenazi Jewish population panel variants, beta thalassemia variants, or alpha thalassemia variants) in a single assay

[0113] After preparing the biological sample and next generation sequencing as described above, the present disclosure further provides a method for detecting the presence of a genetic mutation (e.g., an Ashkenazi Jewish population group variation, a β-thalassemia variation, or an α-thalassemia variation) present in a target nucleic acid sequence amplified from the biological sample.

[0114] In some embodiments, the method further comprises determining whether the subject is a carrier of at least one of an Ashkenazi panel variant, a beta thalassemia variant, or an alpha thalassemia variant based on the next generation sequencing results. In some embodiments, the determination is achieved by: demultiplexing the sequencing reads; mapping the sequencing reads to a customized human reference genome, wherein pseudogene sequences within Genome Reference Consortium Human Genome Build 37 (GRCh37) chr1:155184031-155185327, chr1:155188031-155188964, chr16:223705-224240, and chr16:227257-227413 are replaced with nucleotide T; filtering the mapped reads using Phred quality scores, requiring more than 30 reads to be mapped; detecting variants in the filtered reads; and determining whether the subject is a carrier of an Ashkenazi panel variant, a beta thalassemia variant, or an alpha thalassemia variant.

[0115] In some embodiments, the subject is suspected of being a carrier of at least one disease selected from Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, maple serum urinary disease, glycogen storage disease I, familial hyperinsulinemia, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Ussher syndrome type IF, Ussher syndrome type IIIA, Joubert syndrome 2, Walker-Warburg syndrome, beta thalassemia, or alpha thalassemia.

[0116] Reagent test kit

[0117] 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.

[0118] The kit of the present technology comprises at least (i) a first plurality of primer pairs for the amplified region of each gene in a plurality of genes, wherein the plurality of genes comprise hexosaminidase subunit alpha (HEXA), sphingomyelin phosphodiesterase 1 (SMPD1), mucolipid TRP cation channel 1 (MCOLN1), beta glucosylceramidase (GBA), FA complementation group C (FANCC), inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex associated protein (IKBKAP), aspartate acylase (ASPA), Bloom syndrome-like RecQ helicase (BLM), branched-chain ketoacid dehydrogenase E1 subunit beta (BCKDHB), glucose-6-phosphatase catalytic subunit (G6PC), ATP binding cassette subfamily C member 8 (ABCC8), dihydrolipoamide dehydrogenase (DLD), nexin E1 (NEB), protocadherin-related 15 (PCDH15), Clarin 1 (CLRN1), transmembrane protein 216 (TMEM216) and Fukutin (FKTN); (ii) a second plurality of primer pairs for amplifying regions of beta glucosylceramidase (GBA) and hemoglobin beta subunit (HBB) genes, wherein the second plurality of primer pairs include SEQ ID NOs:81 and 82, and SEQ ID NOs:83 and 84; and (iii) a third plurality of primer pairs for amplifying regions of alpha thalassemia gene variation in each of the hemoglobin subunit alpha 1 (HBA1) and hemoglobin subunit alpha 1 (HBA2) genes, and instructions for use.

[0119] In some embodiments, the first plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only when a deletion is present.

[0120] 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.

[0121] In some embodiments, the third plurality of primer pairs includes at least one gap primer pair designed to amplify the target region only when a deletion is present.

[0122] In some embodiments, a HEXA gene variant causes Tay-Sachs disease; and a SMPD1 gene variant causes Niemann-Pick disease; and a MCOLN1 gene variant causes mucolipidosis type IV; and a GBA gene variant causes Gaucher disease; and a PANCC gene variant causes Fanconi anemia; and a IKBKAP gene variant causes familial dysautonomia; and a ASPA gene variant causes Canavan disease; and a BLM gene variant causes Bloom syndrome; and a BCKDHB gene variant causes maple serum urinary system disease; and a G6PC gene variant causes variants in the ABCC8 gene cause glycogen storage disease I; and variants in the ABCC8 gene cause familial hyperinsulinemia; and variants in the DLD gene cause dihydrolipoamide dehydrogenase deficiency (DLD) or lipoamide dehydrogenase deficiency (E3); and variants in the NEB gene cause nemaline myopathy; and variants in the PCDH15 gene cause Usher syndrome type IF; and variants in the CLRN1 gene cause Usher syndrome type IIIA; and variants in the TMEM216 gene cause Joubert syndrome 2; and variants in the FKTN gene cause Walker-Warburg syndrome.

[0123] In some embodiments, the Ashkenazi panel gene variants include one or more of the gene variants listed in Table 1. In some embodiments, the HEXA gene variants include R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; and SMPD1 gene variation includes one or more of L302P, fsP330, R496L (R498L) or δR608; and MCOLN1 gene variation includes one or more of IVS3-2A>G or 6.4kb_del; and GBA gene variation includes one or more of IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P or R496H; and PANCC gene variation includes one or more of IVS4+4A>T or 322delG; and IKBKAP gene variation includes one or more of R696P or IVS20+6T>C; and ASPA gene variation includes IVS2-2A>G, Y231X, E28 5A or A305E; and BLM gene variations include one or more of 2281del6 / ins7; and BCKDHB gene variations include one or more of R183P, G278S or E372X; and G6PC gene variations include one or more of R83C or Q347X; and ABCC8 gene variations include IVS32-9G>A or F1387del; and DLD gene variations include one or more of Y35* or G229C; and NEB gene variations include R2478_D2512del35; and PCDH15 gene variations include one or more of R245; and CLRN1 gene variations include N48K; and TMEM216 gene variations include R73L; and FKTN gene variations include F390fs.

[0124] In some embodiments, the beta thalassemia gene variants in the HBB gene include one or more gene variants listed in Table 2. In some embodiments, the gene variants 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.146_147insATCT, c.135del C, 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.-8 1A>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.

[0125] In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include one or more gene variants listed in Table 3. In some embodiments, the alpha thalassemia variants in the HBA1 gene and the HBA2 gene include 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 HBA2 c.427T>C in the HBA2 gene.

[0126] In some embodiments, the first plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two, at least four, at least five, at least eight, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or all primer pairs selected from Table 4. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 4.

[0127] In some embodiments, the first plurality of primer pairs 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 two, at least three, at least four, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, or all pairs 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: 27-28), pair 15 (SEQ ID NOs: 29-30), pair 16 (SEQ ID NOs: 31-32), pair 17 (SEQ ID NOs: 33-34), pair 18 (SEQ ID NOs: 35-36), pair 19 (SEQ ID NOs: 37-38), pair 20 (SEQ ID NOs: 39-40), pair 210 (SEQ ID NOs: 31-41), pair 221 (SEQ ID NOs: 32-42), pair 23 NO: 23-24), pair 13 (SEQ ID NO: 25-26), pair 14 (SEQ ID NO: 27-28), pair 15 (SEQ ID NO: 29-30), pair 16 (SEQ ID NO: 31-32), pair 17 (SEQ ID NO: 33-34), pair 18 (SEQ ID NO: 35-36), pair 19 (SEQ ID NO: 37-38), pair 20 (SEQ ID NO: 39-40), pair 21 (SEQ ID NO: 41-42), pair 22 (SEQ ID NO: 43-44), pair 23 (SEQ ID NO: 45-46), pair 24 (SEQ ID NO: 47-48), pair 25 (SEQ ID NO: 49-50), pair 26 (SEQ ID NO: 51-52), pair 27 (SEQ ID NO: 53-54), pair 28 (SEQ ID NO: 55-56), pair 29 (SEQ ID NO: 57-58), pair 30 (SEQ ID NO: 59-60), pair 31 (SEQ ID NO: 61-62), pair 32 (SEQ ID NO: 63-64), pair 33 (SEQ ID NO: 65-66), pair 34 (SEQ ID NO: 67-68), pair 35 (SEQ ID NO: 69-70), pair 36 (SEQ ID NO: 71-72), pair 37 (SEQ ID NO: 73-74), pair 38 (SEQ ID NO: 75-76), pair 39 (SEQ ID NO: 80 NO:57-58), pair 30 (SEQ ID NO:59-60), pair 31 (SEQ ID NO:61-62), pair 32 (SEQ ID NO:63-64), pair 33 (SEQ ID NO:65-66), pair 34 (SEQ ID NO:67-68), pair 35 (SEQ ID NO:69-70), pair 36 (SEQ ID NO:71-72), pair 37 (SEQ ID NO:73-74), pair 37 (SEQ ID NO:75-76), pair 38 (SEQ ID NO:77-78) and pair 40 (SEQ ID NO:79-80).

[0128] In some embodiments, the second plurality of primer pairs includes at least two, at least four, or at least five primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical 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 includes all primer pairs shown in Table 5.

[0129] 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 higher identity with at least two, at least four or at least five 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, SEQID NOs:87 and 88 or SEQ ID NOs:89 and 90.

[0130] In some embodiments, the second plurality of primer pairs includes at least two primer pairs that are at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 99% or more identical to at least two primer pairs selected from the group consisting of the following items of Table 6: pair 1 (SEQ ID NO:81-82), pair 2 (SEQ ID NO:83-84), pair 3 (SEQ ID NO:85-86), pair 4 (SEQ ID NO:87-88) and pair 5 (SEQ ID NO:89-90).

[0131] In some embodiments, the third plurality of primer pairs includes at least two, at least four, at least five, at least eight, or at least ten primer pairs that are at least 85% identical to at least two, at least four, at least five, at least eight, or at least ten primer pairs selected from Table 6. In some embodiments, the first plurality of primer pairs includes all primer pairs shown in Table 6.

[0132] 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 higher identity to at least two pairs selected from the group consisting of the following items in Table 6: pair 1 (SEQ ID NO:91-92), pair 2 (SEQ ID NO:93-94), pair 3 (SEQ ID NO:95-96), pair 4 (SEQ ID NO:97-98), pair 5 (SEQ ID NO:99-100), pair 6 (SEQ ID NO:101-102), pair 7 (SEQ ID NO:103-104) and pair 8 (SEQ ID NO:105-106).

[0133] In some embodiments, the kits of the present disclosure further include a buffer, an enzyme having polymerase activity, an enzyme having polymerase activity but lacking 5'→3' exonuclease activity or both 5'→3' and 3'->>5' exonuclease activity, an enzyme cofactor, such as magnesium or manganese, a salt, a chain extending nucleotide, such as a deoxynucleoside triphosphate (dNTP), a modified dNTP, a nuclease-resistant dNTP or a labeled dNTP, which are necessary for performing an assay or reaction, such as amplifying and / or detecting a target nucleic acid sequence corresponding to a specific genetic gene mutation group disclosed herein.

[0134] In some embodiments, the kit of the present disclosure further includes a positive control nucleic acid sequence and a negative control nucleic acid sequence to ensure the integrity of the assay during the experimental run. The kit may also include instructions for use, software for automatic analysis, containers, packaging such as packaging for commercial sale, etc.

[0135] The kit of the present technology may also include other necessary reagents for performing any NGS technology disclosed herein. For example, the kit may further include one or more of the following: adapter sequence, barcode sequence, reaction tube, ligase, ligase buffer, washing buffer and / or reagent, hybridization buffer and / or reagent, labeling buffer and / or reagent and detection tool. Buffer and / or reagent are usually optimized for the specific amplification / detection technology expected by the kit. The kit may also include a scheme for using these buffers and reagents to perform different steps of the procedure.

[0136] In some embodiments, the kits of the present disclosure further comprise a plurality of barcode primers selected from pairs 1-4 of Table 7 or pairs 1-9 of Table 8.

[0137] The kit may further include one or more of the following: washing buffers and / or reagents, hybridization buffers and / or reagents, labeling buffers and / or reagents, and detection means. The buffers and / or reagents are typically optimized for the specific amplification / detection techniques contemplated by the kit. The kit may also include protocols for using these buffers and reagents to perform the different steps of the procedure.

[0138] The kit may also include an assay definition scan card and / or instructions, such as 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.

[0139] Primers, probes and / or primer probes specific for amplification and detection of DNA internal controls can be included in the amplification master mix as target primer pairs to monitor potential PCR inhibition. The reagents required for amplification and detection of targets and internal controls can be formulated as an integrated amplification master mix, which can be provided as single reaction aliquots in the kit.

[0140] Examples

[0141] Example 1: Sequencing library preparation and raw sequencing data generation

[0142] Three multiplex gene-specific PCR reactions (GSP) were set up for each specimen to amplify the target region (see Figure 1 ). Each multiplex PCR reaction consisted of a pool of primer pairs (see Tables 4-6) that amplified various regions of genomic DNA containing the variants to be detected in this assay (see Tables 1-3 for gene variants of interest). A total of fifty-five amplicons were generated by the GSP reactions. After PCR, the GSP product for each patient was diluted 2500-fold with water. The diluted products were amplified using primers that added barcode sequences (BC) and the P5 and P7 adapters required for sequencing on the Illumina MiSeq (Table 7). The barcoded samples were pooled, purified and quantified using Ampure XP beads. The final products were pooled into a single library and sequenced on the MiSeq. The sequencing data were analyzed using an in-house developed pipeline.

[0143] Gene-specific PCR (GSP)

[0144] 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 contained 41 primer pairs, including 3 gap PCR primer pairs, to amplify the target regions of 17 genes. The gap PCR primer pairs were designed to produce products only when a deletion was present. The gene-specific primer pairs were designed to contain Illumina sequencing primer binding sites, R1SP (for forward reads) and R2SP (for reverse and barcode reads).

[0145] Reaction GSP-B contains 2 primer pairs for the GBA gene and 3 primer pairs for the HBB gene. The GBA primers are designed to specifically amplify the target region of the GBA gene while preventing co-amplification of the GBA pseudogene. Reaction GSP-C contains 16 primers for detecting 8 common α-thalassemia variants and 1 amplification control. Gap PCR is used for 7 common α-thalassemia variants, and therefore, products are present only when a deletion is present. A total of 55 amplicons were generated by the GSP reaction.

[0146] Barcode PCR (BCP)

[0147] To facilitate simultaneous incorporation of all specimens into a single library, each individual specimen 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 barcode PCR reactions (BCP-A, -B, and -C). During barcode PCR, the R1SP and R2SP sequences were used as primer sites to add the barcode sequences as well as the P5 and P7 adapters required for sequencing on an Illumina Miseq sequencer.

[0148] Barcoded PCR reaction B (GSP-B – see, Table 5 ) contained 5 primer pairs and reaction C (GSP-C – Table 6 ) contained 16 primers, R1SP and R2SP fusion nested primers, generating 4 GBA-specific nested PCR products and 8 α-thalassemia nested PCR products, respectively.

[0149] A total of 57 amplicons were generated in the BCP reaction.

[0150] Specimen Pool and Ampure Cleaning

[0151] Barcoded samples were pooled into three separate tubes to create pools-A, -B, and -C and purified using AmpureXP beads followed by Qubit quantification. The final products were combined into a single library and sequenced on a MiSeq.

[0152] Sequencing

[0153] The pooled library is denatured with 0.2N NaOH before sequencing. Illumina sequencing chemistry is based on synthesis sequencing. The single-stranded library is loaded into the Miseq sequencing box. In short, the MiSeq workflow is as follows: the instrument starts by flushing the library through the flow cell, where the library is hybridized with 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 done by isothermal bridge amplification. After cluster generation, the clusters are made single-stranded and synthesis sequencing begins. Fluorophore-labeled nucleotide triphosphates are applied to the flow cell. The nucleotides are 3' blocked so that only a single nucleotide is incorporated in each round of synthesis. The fluorophore is then excited by a laser and the emission spectrum is recorded by Miseq. The nucleotide blocker that inhibits further synthesis is cleaved, allowing the next nucleotide triphosphate to be added. In this way, the fragments are sequenced.

[0154] Example 2: Sequencing data analysis

[0155] Alignment, filtering, and variant calling

[0156] The data analysis pipeline process is as follows Figure 2As shown. Demultiplexed FASTQ files were generated using MiSeq analysis software. The raw sequence reads in the FASTQ files were then aligned to the custom reference genome using Burrows-Wheeler Aligner (BWA). The custom reference genome differed from the Genome 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 were replaced with nucleotide T to accurately align GBA and detect α-thalassemia 4.2 deletion variants. Reads were then sorted and indexed using SAMtools, followed by read array alignment using the Picard tool. Local realignment and base quality score recalibration were performed within the target using the Genome Analysis Toolkit (GATK). Mapped reads were further filtered using Phred quality scores prior to downstream analysis, requiring more than 30 reads to be mapped (>99.9% accuracy). The average and minimum coverage depths for each ROI (region of interest) were calculated, and variant calling was performed using GATKUnified and Haploid Genotyper calling programs. A single variant file (*.vcf) was created by merging the variant files from the two variant calling programs. Coverage and variant depth reports were created and loaded into the sequencing database (seqDB). Alamut Batch was used to obtain high-level annotations of the detected variants. Quality control rules for variant detection include the following: 1) the target position must reach a minimum of 40 reads, 2) variants were detected at a frequency of 20% or higher. 3) Detection of large deletions was based on the presence of gap PCR amplicons with a minimum of 100 reads, rather than a specific variant being called.

[0157] Example 3: Detected variants

[0158] Table 1: Ashkenazi Jewish Group

[0159]

[0160]

[0161]

[0162] Table 2: β-thalassemia-associated variants within the HBB gene

[0163]

[0164]

[0165]

[0166] Table 3: α-thalassemia-associated variants

[0167]

[0168]

[0169] Example 4: Primers and other materials

[0170] Table 4: GSP-A Primers

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Table 5: GSP-B Primers

[0178]

[0179] Table 6: GSP-C Primers

[0180]

[0181]

[0182] Table 7: BCB-B Primers

[0183]

[0184]

[0185] Table 8: BCP-C Primers

[0186]

[0187]

[0188] Table 9: GSP-A Primer Pool

[0189]

[0190]

[0191] Table 10: GSP-B Primer Library

[0192]

[0193]

[0194] Table 11: GSP-C Primer Library

[0195]

[0196]

[0197] Table 12: BCP-B Primer Library

[0198]

[0199] Table 13: BCP-C Primer Library

[0200]

[0201] Table 14: Controls used

[0202]

[0203]

[0204] Analytical Measurement Range (AMR)

[0205] Negative (wild type): None of the tested variants were detected. Heterozygous: Positive for a variant with a frequency between ≥20% and <96%. Compound heterozygous: Positive for two variants within a gene (one on each allele) with a frequency between ≥20% and <96%. Homozygous: Positive for the same variant on both alleles with a frequency ≥96%.

[0206] Table 15: Conditions detected by this method and their carrier frequencies.

[0207]

[0208]

[0209] Example 5: Results

[0210] The clinical sensitivity and specificity of the disclosed assay were measured to be 100% and 100%, respectively. The assay also demonstrated 100% inter-assay and intra-assay precision during validation runs.

[0211] Equivalent

[0212] The technology of the present invention is not limited to the specific embodiments described in this application, and these specific embodiments are intended to be separately illustrated as a single aspect of the technology of the present invention. Without departing from the spirit and scope of the present invention, many modifications and changes can be made to the technology of the present invention, which is obvious to those skilled in the art. According to the above description, in addition to those methods and devices listed herein, functionally equivalent methods and devices within the scope of the technology of the present invention will be obvious to those skilled in the art. Such modifications and changes are intended to fall within the scope of the technology of the present invention. It should be understood that the technology of the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course, these specific methods, reagents, compounds, compositions or biological systems can change. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limited.

[0213] Thus, for example, the terms "comprising," "including," "containing," etc. shall be read broadly and not limiting. Additionally, the terms and expressions employed herein have been used as terms of description and not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications may be made within the scope of the present disclosure as claimed.

[0214] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0215] As will be understood by those skilled in the art, for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily considered to fully describe and enable the same range to be decomposed into at least equal halves, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages ​​such as "at most", "at least", "greater than", "less than", etc. include the enumerated numbers and refer to the ranges that can be subsequently decomposed into subranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1-3 cells refers to a group with 1, 2, or 3 cells. Similarly, a group with 1-5 cells refers to a group with 1, 2, 3, 4, or 5 cells, etc.

[0216] All patents, patent applications, provisional applications, and publications mentioned or cited herein are hereby incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1. A method for detecting one or more variants of a genetic disorder in a subject suspected of being a carrier of at least one inherited genetic mutation, the method 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, wherein at least one amplicon corresponds to each gene in 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; 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, wherein the second plurality of primer pairs includes SEQ ID NOs: 81 and 82 and SEQ ID NOs: 83 and 84; and 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 TaqExtender PCR additive, wherein at least one amplicon corresponds to each of HBA1 and HBA2; incorporating barcode sequences into ends of the first plurality of amplicons, the second plurality of amplicons, and the third plurality of amplicons via polymerase chain reaction; as well as One or more variants in at least one of the first plurality of amplicons, the second plurality of amplicons, and the third plurality of amplicons are detected using high throughput massively parallel sequencing. 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 when a deletion is present.

3. The method of claim 1, wherein the GBA primers of the second plurality of primer pairs specifically amplify the GBA gene and do not amplify any GBA pseudogene. 4 . 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 when a deletion is present.

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

6. The method of claim 1, wherein HEXA gene variations include R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; And the SMPD1 gene mutations include L302P, fsP330, R496L (R498L) or δR608; And the MCOLN1 gene mutations include IVS3-2A>G or 6.4kb_del; And the GBA gene mutations include IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P or R496H; And the PANCC gene mutations include IVS4+4A>T or 322delG; And the IKBKAP gene mutations include R696P or IVS20+6T>C; And the ASPA gene mutations included IVS2-2A>G, Y231X, E285A or A305E; And among them, BLM gene mutations include 2281del6 / ins7; And the BCKDHB gene mutations include R183P, G278S or E372X; And the G6PC gene mutations include R83C or Q347X; And the ABCC8 gene mutations include IVS32-9G>A or F1387del; And the DLD gene mutations include Y35* or G229C; And among them, NEB gene variants include R2478_D2512del35; And among them, PCDH15 gene mutations include R245; And among them, CLRN1 gene mutations include N48K; And among them, TMEM216 gene mutations include R73L; And the FKTN gene mutations include F390fs.

7. The method according to claim 1, wherein the β-thalassemia gene variants in the HBB gene 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.146_147insATCT, c.135del C, 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.-8 1A>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.

8. According to the method of claim 1, the α-thalassemia variation in the HBA1 gene and the HBA2 gene includes 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 HBA2 c.427T>C in the HBA2 gene.

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

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

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

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

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

17. The method according to claim 1, wherein the biological sample is whole blood, serum, plasma, Amniotic fluid or chorionic villi.

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

19. The method according to any of the preceding claims, wherein the subject is suspected of being a carrier of at least one disease selected from the group consisting of Tay-Sachs disease, Niemann-Pick disease, mucolipidosis type IV, Gaucher disease, Fanconi anemia, familial dysautonomia, Canavan disease, Bloom syndrome, maple serum urinary disease, glycogen storage disease I, familial hyperinsulinemia, dihydrolipoamide dehydrogenase deficiency (DLD), lipoamide dehydrogenase deficiency (E3), nemaline myopathy, Ussher syndrome type IF, Ussher 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 gene in 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 amplified regions of the GBA gene and the HBB gene, wherein the second plurality of primer pairs comprises SEQ ID NOs: 81 and 82 and SEQ ID NOs: 83 and 84; and (iii) a third plurality of primer pairs directed to amplifying regions of alpha thalassemia gene variation in each of the HBA1 gene and the HBA2 gene, And instructions for use.

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

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

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

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

25. The kit of claim 20, wherein HEXA gene variations include R178H (B1 variant), R247W, R249W, G269S, IVS9+1G>A, 1278_TATC, IVS12+1G>C, or 7.6-kb Del, Ex1; And the SMPD1 gene mutations include L302P, fsP330, R496L (R498L) or δR608; And the MCOLN1 gene mutations include IVS3-2A>G or 6.4kb_del; And the GBA gene mutations include IVS2+1G>A, 84G>GG, N370S, del_55bp, V394L, D409H, L444P or R496H; And the PANCC gene mutations include IVS4+4A>T or 322delG; And the IKBKAP gene mutations include R696P or IVS20+6T>C; And the ASPA gene mutations included IVS2-2A>G, Y231X, E285A or A305E; And among them, BLM gene mutations include 2281del6 / ins7; And the BCKDHB gene mutations include R183P, G278S or E372X; And the G6PC gene mutations include R83C or Q347X; And the ABCC8 gene mutations include IVS32-9G>A or F1387del; And the DLD gene mutations include Y35* or G229C; And among them, NEB gene variants include R2478_D2512del35; And among them, PCDH15 gene mutations include R245; And among them, CLRN1 gene mutations include N48K; And among them, TMEM216 gene mutations include R73L; And the FKTN gene mutations include F390fs.

26. The kit according to claim 20, wherein the β-thalassemia gene variants in the HBB gene 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.146_147insATCT, c.135del C, 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.-8 1A>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.

27. The kit according to claim 20, wherein the α-thalassemia variation in the HBA1 gene and the HBA2 gene 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 HBA2 c.427T>C in the HBA2 gene.

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

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

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

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