Probe composition for screening multiple genetic disease related gene mutation types, kit and preparation method thereof

Abstract

The invention relates to the technical field of gene detection, in particular to a probe composition and a kit for screening various genetic disease related gene mutations and a preparation method of the probe composition and the kit. The hereditary diseases comprise 23 hereditary diseases such as citrullinemia type, hepatolenticular degeneration, 21-hydroxylase deficiency, progressive pseudohypertrophy muscular dystrophy, fragile X syndrome, Friedreich ataxia, glycogen accumulation disease type II, Gaucher disease, autosomal recessive deafness type 1A, Fabry disease, alpha-thalassemia, beta-thalassemia and the like. The probe composition disclosed by the invention can be used for simultaneously screening the specific mutation types of the various genetic disease related genes, and is more accurate, comprehensive, convenient, high in detection efficiency, simple and economical compared with the traditional screening.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a probe composition, a kit and a preparation method thereof for screening for multiple genetic disease-related gene mutation types. Background Art

[0002] Monogenic diseases are hereditary diseases caused by mutations in a single gene. These diseases include but are not limited to thalassemia, hemophilia, 21-hydroxylase deficiency, etc. At present, the diagnosis of these diseases mainly relies on traditional short-read sequencing technology, such as NGS sequencing. However, due to the diverse types of gene mutations in monogenic diseases, including point mutations, small fragment insertions or deletions, large fragment duplications or deletions, gene fusions with high homology or pseudogenes, dynamic mutations, etc., traditional gene mutation detection methods are often difficult to fully cover these mutation types, and often require multiple methods for combined detection, such as next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), linkage disequilibrium (LD), polymerase chain reaction (PCR) and other methods for combined detection. The operation process is complicated, requires multiple equipment, and has a long detection cycle.

[0003] As an emerging genomics tool, long-read sequencing (LRS) provides a possible solution to the above problems with its ability to read thousands or even tens of thousands of base pairs at a time. Compared with traditional short-read sequencing, long-read sequencing technology has obvious advantages in detecting large-fragment structural variations, complex genomic regions, and highly repetitive sequences. In addition, the development of long-read sequencing technology has also promoted in-depth research and understanding of gene mutation types related to single-gene genetic diseases.

[0004] Although long-read sequencing technology has shown great potential in the detection of gene mutations in single-gene genetic diseases, there are still some challenges and limitations. First, the cost of long-read sequencing is relatively high, which limits its application in large-scale screening and clinical diagnosis. Second, the analysis and interpretation of long-read sequencing data require complex bioinformatics tools and expertise. In addition, for the detection of gene mutations for specific diseases, it is necessary to develop probe sets and kits with strong specificity and high sensitivity.

[0005] In view of the above-mentioned deficiencies of the prior art, it is necessary to provide a screening method for genetic disease-related gene mutation types that has strong specificity, high sensitivity and is applicable to a variety of diseases. Summary of the invention

[0006] In view of the technical problems existing in the background technology, one or more embodiments of the present application provide a probe composition, a kit and a preparation method thereof for screening multiple genetic disease-related gene mutation types, which are used to simultaneously screen for specific mutations in multiple genetic disease-related genes.

[0007] In a first aspect, an embodiment of the present application provides a probe composition, wherein the probe composition specifically captures pathogenic genes of genetic diseases, wherein the genetic diseases include citrullinemia, hepatolenticular degeneration, 21-hydroxylase deficiency, pseudohypertrophic muscular dystrophy, fragile X syndrome, Friedreich ataxia, glycogen storage disease type II, Gaucher disease, autosomal recessive deafness type 1A, Fabry disease, a-thalassemia, β-thalassemia, mucopolysaccharidosis type II, methylmalonic aciduria and homocystinuria cbIC type, methylmalonic aciduria mut(0) type, phenylketonuria, BH4-deficient hyperphenylalaninemia type A, primary carnitine deficiency, citrullinemia type II, Pendred syndrome, autosomal recessive deafness with dilatation of the vestibular aqueduct type 4, spinal muscular atrophy, and X-linked ichthyosis;

[0008] The probe composition includes 866 probes, wherein the capture starting site of each probe is shown in Table 1;

[0009] Table 1 Capture start and end sites of each probe among 866 probes

[0010]

[0011]

[0012]

[0013]

[0014]

[0015] At present, screening methods or detection kits for pathogenic genes of various genetic diseases cannot detect all mutation types simultaneously in a single experimental system. In order to comprehensively assess the genetic risk of an individual, it is usually necessary to combine multiple detection technologies, such as NGS combined with multiplex ligation probe amplification (MLPA), long-distance PCR (LD-PCR), etc., which not only increases the complexity of the experiment, but also increases the cost and difficulty of operation.

[0016] In the technical solution of the embodiment of the present application, the probe composition can simultaneously screen for specific mutation types of multiple genetic disease-related genes, which is more accurate, comprehensive, convenient, efficient, simple and economical than traditional screening.

[0017] In a second aspect, the present application provides a method for preparing a probe composition, comprising: selecting respective target capture regions according to the pathogenic genes of each genetic disease, wherein the target capture regions include each pathogenic gene and its upstream and downstream extending 3 kb regions and its CNV coordinate regions;

[0018] The probe is designed according to the target capture region to obtain the probe composition.

[0019] Currently, screening methods or detection kits for pathogenic genes of multiple genetic diseases are unable to detect all mutation types simultaneously in a single experimental system. Specific probes need to be designed for each disease, which not only increases costs but also limits the flexibility and efficiency of detection.

[0020] The embodiments of the present application can conveniently and quickly design capture probes based on the selected target capture region, thereby saving probe preparation costs and improving detection efficiency.

[0021] In one embodiment of the present application, the probe composition satisfies at least one of the following conditions:

[0022] (i) the length of each probe in the probe composition is 100 nt;

[0023] (ii) the error tolerance of each probe in the probe composition is less than 5%; and,

[0024] (iii) The coverage density of each probe in the probe composition is 0.2268×.

[0025] In a third aspect, an embodiment of the present application provides a kit, which contains the probe composition as described above, or the probe composition prepared by the preparation method as described above.

[0026] In a fourth aspect, an embodiment of the present application provides a method for detecting a target region, comprising: using the probe composition described above, or the kit described above, to capture the target region.

[0027] Furthermore, the detection method comprises the following steps:

[0028] The genomic DNA of the sample to be tested is fragmented and adapter-added to obtain DNA fragments with adapters added to the ends;

[0029] After the DNA fragment with a connector added to the end is amplified, it is subjected to hybridization capture treatment using the probe composition or the kit to capture the target region.

[0030] Furthermore, the steps of taking genomic DNA of the sample to be tested, fragmenting and adding adapters to obtain DNA fragments with adapters added to the ends include:

[0031] The genomic DNA of the sample to be tested, the transposase and the transposase buffer are mixed at a first temperature for a first time to obtain a DNA fragment with a linker added to the end;

[0032] The amount of the transposase used is 2.5 μL to 3.5 μL: 1 μg relative to the genomic DNA, the first temperature is 50° C. to 60° C., and the first time is 8 min to 12 min.

[0033] Furthermore, the amplification enzyme used in the amplification process includes at least one of Phanta Flash Super-Fidelity DNA Polymerase, 2×KeyPo SE Master Mix (Dye Plus) and KOD FX Neo.

[0034] Furthermore, the amount of DNA fragments with adapters added to the ends after amplification treatment used in the hybridization capture treatment is 750 ng to 1000 ng.

[0035] The target region detection method of the present application can be used to screen carriers of various variant types of various monogenic disease genes in phenotypically normal people, identify specific variant types, and can achieve simultaneous detection of multiple variant types such as point mutations, deletions and duplications, gene fusions, large-segment gene conversions, short tandem repeat mutations, and gene inversion mutations in the same reaction system. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 This is a comparison chart of the coverage of the DMD region by the gene library finally constructed by treating genomic DNA under different shearing / amplification conditions in Example 1 of the present application;

[0038] Figure 2 This is the IGV spectrum detected from the positive sample in Example 1 of the present application. DETAILED DESCRIPTION

[0039] The present application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the claims attached to the present application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0041] the term

[0042] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0043] The term "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0044] Herein, "preferred", "preferable", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0045] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0046] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0047] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0048] In a first aspect of the present application, a probe composition is provided, wherein the probe composition is used to specifically capture pathogenic genes of genetic diseases, and the genetic diseases include citrullinemia, hepatolenticular degeneration, 21-hydroxylase deficiency, pseudohypertrophic muscular dystrophy, fragile X syndrome, Friedreich ataxia, glycogen storage disease type II, Gaucher disease, autosomal recessive deafness type 1A, Fabry disease, a-thalassemia, β-thalassemia, mucopolysaccharidosis type II, methylmalonic aciduria and homocystinuria cbIC type, methylmalonic aciduria mut(0) type, phenylketonuria, BH4-deficient hyperphenylalaninemia type A, primary carnitine deficiency, citrullinemia type II, Pendred syndrome, autosomal recessive deafness with dilatation of the vestibular aqueduct type 4, spinal muscular atrophy and X-linked ichthyosis;

[0049] The probe composition includes 866 probes, and the capture start and end sites of each probe are shown in Table 1 above.

[0050] Furthermore, the pathogenic genes of the above genetic diseases include:

[0051] (1) ASSI, a pathogenic gene associated with citrullinemia;

[0052] (2) ATPTB, a pathogenic gene associated with Wilson's disease;

[0053] (3) CYP21A2, a pathogenic gene associated with 21-hydroxylase deficiency;

[0054] (4) Duchenne muscular dystrophy (DMD)

[0055] (5) Fragile X syndrome-related pathogenic gene FMR1;

[0056] (6) FXN, a pathogenic gene associated with Friedreich's ataxia;

[0057] (7) GAA, a pathogenic gene associated with glycogen storage disease type II;

[0058] (8) Gaucher disease-related pathogenic gene GBA1;

[0059] (9) GJB2, a pathogenic gene associated with autosomal recessive deafness type 1A;

[0060] (10) Fabry disease-related pathogenic gene GLA;

[0061] (11) a-thalassemia-related pathogenic genes HBA1 / HBA2;

[0062] (12) β-thalassemia-related pathogenic gene HBB;

[0063] (13) IDS, a pathogenic gene associated with mucopolysaccharidosis type II;

[0064] (14) MMACHC, the pathogenic gene associated with methylmalonic aciduria and homocystinuria cbIC type;

[0065] (15) MMUT, the pathogenic gene associated with methylmalonic aciduria mut(0);

[0066] (16) Phenylketonuria-related pathogenic gene PAH;

[0067] (17) PTS, a pathogenic gene associated with BH4-deficient hyperphenylalaninemia type A;

[0068] (18) Primary carnitine deficiency-related pathogenic gene SLC22A5;

[0069] (19) SLC25A13, a pathogenic gene associated with citrullinemia type II;

[0070] (20) Pendred syndrome-related pathogenic gene SLC26A4;

[0071] (21) SLC26A4, a pathogenic gene associated with autosomal recessive deafness with vestibular aqueduct dilatation type 4;

[0072] (22) SMNI, a disease-causing gene associated with spinal muscular atrophy; and

[0073] (23) X-linked ichthyosis-related pathogenic gene STS.

[0074] The second aspect of the present application provides a method for preparing a probe composition, which can efficiently construct a capture probe group for different pathogenic genes of various diseases, effectively saving costs and improving detection efficiency.

[0075] In one embodiment, the method for preparing the probe composition includes selecting respective target capture regions according to each pathogenic gene, wherein the target capture region includes each pathogenic gene and its upstream and downstream extending 3 kb regions and its CNV coordinate region;

[0076] The probes are designed according to the target capture region to obtain a probe composition.

[0077] Furthermore, the probe composition satisfies at least one of the following conditions:

[0078] (i) The length of each probe in the probe composition is 100 nt;

[0079] (ii) the error tolerance of each probe in the probe composition is less than 5%; and,

[0080] (iii) The coverage density of each probe in the probe composition is 0.2268×.

[0081] The method for preparing the probe composition can obtain the probe composition of the first aspect of the present application.

[0082] In one embodiment, the method for preparing the probe composition satisfies at least one of the following conditions:

[0083] (i) After designing the probes, each probe is synthesized separately;

[0084] (ii) After synthesizing each probe individually, quality control is performed individually;

[0085] (vii) After designing the probe, synthesizing the probe at equal concentrations;

[0086] (iv) synthesizing the probes at equal concentrations and mixing them in equal amounts;

[0087] (v) The error tolerance of each probe is less than 5%;

[0088] (vi) the coverage density of each probe is 0.2268×;

[0089] (vii) genetic diseases including citrullinemia, hepatolenticular degeneration, 21-hydroxylase deficiency, pseudohypertrophic muscular dystrophy, fragile X syndrome, Friedreich's ataxia, glycogen storage disease type II, Gaucher disease, autosomal recessive deafness type 1A, Fabry disease, α-thalassemia, β-thalassemia, mucopolysaccharidosis type II, methylmalonic aciduria and homocystinuria cbIC type, methylmalonic aciduria mut(0) type, phenylketonuria, BH4-deficient hyperphenylalaninemia type A, primary carnitine deficiency, citrullinemia type II, Pendred syndrome, autosomal recessive deafness with vestibular aqueduct dilatation type 4, spinal muscular atrophy and X-linked ichthyosis;

[0090] (viii) The target capture region of the probe composition includes the following regions:

[0091] (1) ASSI, a pathogenic gene associated with citrullinemia;

[0092] (2) ATPTB, a pathogenic gene associated with Wilson's disease;

[0093] (3) CYP21A2, a pathogenic gene associated with 21-hydroxylase deficiency;

[0094] (4) Duchenne muscular dystrophy (DMD)

[0095] (5) Fragile X syndrome-related pathogenic gene FMR1;

[0096] (6) FXN, a pathogenic gene associated with Friedreich's ataxia;

[0097] (7) GAA, a pathogenic gene associated with glycogen storage disease type II;

[0098] (8) Gaucher disease-related pathogenic gene GBA1;

[0099] (9) GJB2, a pathogenic gene associated with autosomal recessive deafness type 1A;

[0100] (10) Fabry disease-related pathogenic gene GLA;

[0101] (11) a-thalassemia-related pathogenic genes HBA1 / HBA2;

[0102] (12) β-thalassemia-related pathogenic gene HBB;

[0103] (13) IDS, a pathogenic gene associated with mucopolysaccharidosis type II;

[0104] (14) MMACHC, the pathogenic gene associated with methylmalonic aciduria and homocystinuria cbIC type;

[0105] (15) MMUT, the pathogenic gene associated with methylmalonic aciduria mut(0);

[0106] (16) Phenylketonuria-related pathogenic gene PAH;

[0107] (17) PTS, a pathogenic gene associated with BH4-deficient hyperphenylalaninemia type A;

[0108] (18) Primary carnitine deficiency-related pathogenic gene SLC22A5;

[0109] (19) SLC25A13, a pathogenic gene associated with citrullinemia type II;

[0110] (20) Pendred syndrome-related pathogenic gene SLC26A4;

[0111] (21) SLC26A4, a pathogenic gene associated with autosomal recessive deafness with vestibular aqueduct dilatation type 4;

[0112] (22) SMNI, a pathogenic gene associated with spinal muscular atrophy;

[0113] (23) X-linked ichthyosis-related pathogenic gene STS.

[0114] The third aspect of the present application provides a kit, which contains the probe composition of the first aspect of the present application, or the probe composition prepared by the preparation method of the second aspect of the present application. The kit can be used to simultaneously screen for specific mutation types of genes related to multiple genetic diseases, and has the advantages of high specificity and high sensitivity.

[0115] In a fourth aspect, an embodiment of the present application provides a method for detecting a target region, comprising: using the probe composition of the first aspect of the present application, or the kit of the third aspect of the present application, to capture the target region.

[0116] The target region detection method of the present application can be used to perform carrier screening for multiple variant types of multiple monogenic disease genes in phenotypically normal people, identify specific variant types, and can achieve simultaneous detection of multiple variant types such as point mutations, deletion duplications, gene fusions, large-segment gene conversions, short tandem repeat mutations, and gene inversion mutations in the same reaction system.

[0117] In one embodiment, the method for detecting the target area comprises the following steps:

[0118] The genomic DNA of the sample to be tested is fragmented and adapter-added to obtain DNA fragments with adapters added to the ends;

[0119] After the DNA fragment with the connector added to the end is amplified, it is subjected to hybridization capture treatment using the above-mentioned probe composition or kit to capture the target area.

[0120] The target region of the present application corresponds to the genetic disease-causing gene region mentioned above. Furthermore, the target region refers to the full length of each pathogenic gene and its region extending 3kb upstream and downstream, as well as its CNV coordinate region.

[0121] Furthermore, the steps of taking genomic DNA of the sample to be tested, fragmenting and adding adapters to obtain DNA fragments with adapters added to the ends include:

[0122] The genomic DNA of the sample to be tested, the transposase and the transposase buffer are mixed at a first temperature for a first time to obtain a DNA fragment with a linker added to the end;

[0123] The amount of transposase used is 2.5 μL to 3.5 μL: 1 μg relative to the genomic DNA, the first temperature is 50° C. to 60° C., and the first time is 8 min to 12 min.

[0124] Optionally, the amount of transposase used is 2.5 μL to 3.5 μL: 1 μg relative to genomic DNA, and the first time is 8 min to 12 min, so that a higher 5000-15000 bp fragment ratio and fragment distribution peak can be obtained, which is beneficial to improve the yield of the product screened before capture.

[0125] Furthermore, the amplification enzyme used in the amplification process includes at least one of Phanta Flash Super-Fidelity DNA Polymerase, 2×KeyPo SE Master Mix (Dye Plus) and KOD FX Neo.

[0126] The average length of the DNA fragments with adapters added to the ends is 6kb to 7kb, and some of the DNA fragments have high GC / AT content.

[0127] Optionally, the amplification enzyme 2×KeyPo SE Master Mix (Dye Plus) is used to amplify the DNA fragments with connectors added to the ends. This can be applied to DNA fragments with an average length of 6kb to 7kb and DNA fragments with high GC / AT content. 2×KeyPo SE Master Mix (Dye Plus) has the advantages of good extension performance, high fidelity, and good amplification uniformity when applied to the above fragments.

[0128] Furthermore, after the DNA fragment with a connector added to the end is amplified, it is subjected to hybridization capture treatment with a probe composition to capture the target region.

[0129] In one embodiment, the amount of DNA fragments with connectors added to the ends after amplification treatment in the hybrid capture process is 750ng to 1000ng. This feed ratio is conducive to improving the capture efficiency, obtaining a better library yield, and obtaining a higher proportion of 5000bp to 15000bp fragments and a peak value of fragment distribution, which is conducive to improving the capture rate. Increasing the proportion of 5000bp to 15000bp fragment length is conducive to the analysis of chromosome structural variation data, improving the accuracy of analysis, and avoiding false negatives.

[0130] In some of the embodiments, after hybridization capture is completed, the captured product is purified and thermally washed.

[0131] Optionally, the number of thermal washes is 4 times, so increasing the number of thermal washes can reduce non-specific binding and improve capture efficiency.

[0132] The above-mentioned optimization of some conditions in the detection method, such as optimization of the probe composition, optimization of the transposase reaction conditions, optimization of the hybridization capture conditions, optimization of the amplification conditions, etc., the optimized parameter characteristics can be arbitrarily combined with each other.

[0133] The following are some specific embodiments.

[0134] For experimental parameters not specified in the following specific embodiments, reference is made to the instructions given in the present application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers.

[0135] The raw materials and reagents involved in the following specific examples can be obtained from commercial sources, or can be prepared by those skilled in the art according to known methods.

[0136] Example 1

[0137] This example optimizes the main steps of the detection method and screens the conditions, as follows:

[0138] 1. Optimization of transposase reaction conditions

[0139] The optimal conditions for fragmentation length of 6 kb to 7 kb were screened out by gradient testing of transposase interruption time (3 min, 5 min, 10 min) and transposase dosage (1 μL, 2 μL, 3 μL, 4 μL). The test results are shown in Table 2.

[0140] Table 2 Distribution of fragments after interruption under different conditions

[0141]

[0142] According to the data in Table 2, the interruption time of 10 min and the amount of transposase of 3 μL were determined as the final conditions for fragmentation, where “ / ” means that the “fragment ratio of 5000 bp to 15000 bp” did not meet expectations, so no subsequent testing was performed.

[0143] 2. Optimization of reaction conditions for the first amplification (amplification enzyme)

[0144] In this part, the amplification enzymes KOD FX Neo, Phanta Flash Super-Fidelity DNA Polymerase, and 2×KeyPo SE Master Mix (Dye Plus) are denoted as KOD, P521, and PK512, respectively.

[0145] Using the physical shearing method g-tube (excluding fragmentation preference) combined with the amplification enzyme KOD (g-tube+KOD) as the standard, the concentration of total DNA fragments obtained by combining the transposase Tn5 with the amplification enzymes P521, PK512 and KOD (Tn5+P521, Tn5+PK512, Tn5+KOD) were compared. The comparison results are shown in Tables 3-4. Figure 1 .

[0146] According to the data in Tables 3 and 4: a) Tn5+P521 has a more obvious preference than Tn5+PK512; b) Tn5+PK512 has a similar regional coverage to the standard condition g-tube+KOD, with the average coverage of the former being slightly lower than the standard condition. Based on the above analysis, the transposase Tn5+amplifier PK512 was determined as the final condition.

[0147] Table 3 Statistical results of DNA fragment concentration at different stages after treating genomic DNA with different shearing / amplification conditions (including two stages before hybridization capture and after hybridization capture).

[0148]

[0149] Table 4 Statistical results of sequencing data after treating genomic DNA with different shearing / amplification conditions

[0150]

[0151]

[0152] 3. Optimization of hybridization capture conditions

[0153] The amount of the first amplification product used for hybridization capture was mainly investigated. The input amounts were set to 1000 ng, 2000 ng, and 3000 ng, and the capture yield and fragment distribution were compared. The results are shown in Table 5.

[0154] According to Table 5, after inputting 1000 ng, the capture yield is > 1200 ng, which meets the requirements for the subsequent three-generation library construction.

[0155] Table 5 Sequencing analysis results of different capture input amounts

[0156]

[0157] 4. Thermal cleaning times

[0158] The thermal washing times were set to 3 times / 4 times, and the capture efficiency of different washing times was compared. The results are shown in Table 6.

[0159] According to Table 6, the capture efficiency of 4 washes was slightly improved compared with 3 washes.

[0160] Table 6

[0161] condition Number of HiFi reads Number of reads covering the target region Capture efficiency % 4 cleanings 211590.00 125792.00 59.45 4 times cleaning 335323.00 164958.00 49.19 3 times cleaning 145560.00 73681.00 50.62 3 times cleaning 172057.00 72531.00 42.16

[0162] In summary, this embodiment determines that the genomic DNA of the sample to be tested is fragmented and treated with linkers using transposase Tn5, the genomic DNA of the sample to be tested is 1 μg, the amount of transposase is 3 μL; the transposase treatment time is 10 min; the amplification enzyme 2×KeyPo SE Master Mix (Dye Plus) is used for amplification; 1000 ng is input for hybridization capture; and the number of thermal washings is 4 times.

[0163] Example 2

[0164] This embodiment provides a test kit of the present application and the prior art for the same 29 positive samples and the results. The 29 positive samples were first tested using the kit of the present application, and each sample was tested using an appropriate prior art technology based on its known variant type: Samples 1-2 used LR-PCR+NGS capture sequencing RFLP to accurately detect 21-enhanced enzyme deficiency-related genes CYP21A2. Samples 3-7 used qPCR to detect SMN1 / SMN2 copy number; samples 8-14 used Gap-PCR combined with PCR-reverse dot hybridization to detect thalassemia-related genes. Samples 12-20 used the patented technology CNVplex TM To achieve gene detection of fragment deletion and duplication. Samples 21-23 were tested for citrullinemia-related genes using whole exon sequencing technology. Samples 24-29 were tested for point mutations using Sanger sequencing technology.

[0165] (I) Mutation detection process based on the kit of this application

[0166] 1.1 Pre-capture library construction

[0167] 1.1.1 Tn5 transposase labeling

[0168] 1.1.1.1 Prepare the system on ice according to Table 7, flick or pipette slowly to mix, and centrifuge briefly.

[0169] Table 7

[0170] Reagents Volume (μL) 1 μg DNA X Nuclease-free water 37-X Labeling buffer 10 Labeling enzyme 3 Total volume 50

[0171] 1.1.1.2 Place the PCR tube in the PCR instrument and react according to Table 8.

[0172] Table 8

[0173]

[0174]

[0175] 1.1.2 Transposase release

[0176] 1.1.2.1 After the reaction is completed, immediately add 10 μL of stop buffer to the reaction system, vortex to mix, and centrifuge instantly.

[0177] 1.1.2.2 Place the PCR tube in the PCR instrument and react according to Table 9.

[0178] Table 9

[0179] temperature Time (heat cover 105℃) 55℃ 5min 4℃ Keep

[0180] 1.1.3 Purification

[0181] 1.1.3.1 Add 0.7× purified magnetic beads (i.e. 42 μL) to the above labeled product. Mix by flicking gently and react at room temperature for 10 minutes.

[0182] 1.1.3.2 Insert the PCR tube into the tube hole of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, aspirate the solution, being careful not to aspirate the magnetic beads (about 5 μL of solution can be left). Leave the PCR tube on the magnetic rack.

[0183] 1.1.3.3 Pipette 200 μL of 80% ethanol into the PCR tube and let it stand for 30 seconds. Aspirate the solution, being careful not to absorb the magnetic beads. 80% ethanol should be prepared on the same day.

[0184] 1.1.3.4 Repeat the previous step once.

[0185] 1.1.3.5 Remove the PCR tube and centrifuge for 5 seconds. Insert the PCR tube into the tube hole of the magnetic rack again. After the magnetic beads are completely adsorbed, use a pipette to remove the residual solution. Be careful not to leave any residue on the tube wall. Open the PCR tube cap and dry it. Be careful not to crack the magnetic beads.

[0186] 1.1.3.6 Pipette 24 μL of nuclease-free water into the PCR tube, flick to mix, and react at room temperature for 5 minutes.

[0187] 1.1.3.7 Centrifuge briefly and insert the PCR tube into the tube hole of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, use a pipette to transfer 22 μL into a new PCR tube with the corresponding number. Be careful not to absorb the magnetic beads.

[0188] 1.1.4 PCR amplification

[0189] 1.1.4.1 Prepare the system on ice according to Table 10, mix by flicking or pipetting with a wide-mouthed pipette tip, and centrifuge briefly.

[0190] Table 10

[0191]

[0192] 1.1.4.2 Place the PCR tube on the PCR instrument and react according to Table 11.

[0193] Table 11

[0194]

[0195] 1.1.5 PCR product purification

[0196] 1.1.5.1 Add 0.8× purified magnetic beads to the PCR product, i.e. 40 μL. Mix by flicking gently and react at room temperature for 10 minutes.

[0197] 1.1.5.2 Insert the PCR tube into the tube hole of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, aspirate the solution, being careful not to aspirate the magnetic beads (about 5 μL of solution can be left). Leave the PCR tube on the magnetic rack.

[0198] 1.1.5.3 Pipette 200 μL of 80% ethanol into the PCR tube and let it stand for 30 seconds. Aspirate the solution, being careful not to aspirate the magnetic beads. 80% ethanol should be prepared on the same day.

[0199] 1.1.5.4 Repeat the previous step once.

[0200] 1.1.5.5 Remove the PCR tube and centrifuge for 5 seconds. Insert the PCR tube into the tube hole of the magnetic rack again. After the magnetic beads are completely adsorbed, use a pipette to remove the residual solution. Be careful not to leave any residue on the tube wall. Open the PCR tube cap and dry it. Be careful not to overdry it.

[0201] 1.1.5.6 Pipette 52 μL of nuclease-free water into the PCR tube, flick to mix, and react at room temperature for 5 minutes.

[0202] 1.1.5.7 Centrifuge briefly and insert the PCR tube into the tube hole of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, use a pipette to transfer 50 μL into a new PCR tube with the corresponding number. Be careful not to absorb the magnetic beads.

[0203] 1.1.5.8 Take 1 μL and determine the concentration.

[0204] 1.1.6 Product screening

[0205] 1.1.6.1 Use elution buffer to dilute AMPure PB screening beads to 35% (v / v) screening beads for later use (the appropriate amount can be prepared according to the actual number of samples). The diluted screening beads can be stored in a 4°C refrigerator for 1 month.

[0206] 1.1.6.2 Take 2500 ng of the purified product from the previous step, add nuclease-free water to make up the volume to 50 μL, and add 155 μL of 35% screening magnetic beads.

[0207] 1.1.6.3 Mix by gently inverting, centrifuge at low speed for 1 second, and incubate at room temperature for 20 minutes.

[0208] 1.1.6.4 Place the PCR tube on the magnetic rack and let it stand for 2 minutes until the liquid becomes clear. Use a pipette to carefully aspirate and discard the supernatant.

[0209] 1.1.6.5 Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0210] 1.1.6.6Repeat the previous step once.

[0211] 1.1.6.7 Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing outward. Centrifuge at low speed for 1 second, then put it back on the magnetic rack. After standing for 5 seconds, aspirate and discard the supernatant and let it dry slightly.

[0212] 1.1.6.8 Remove the PCR tube from the magnetic rack, add 32 μL of nuclease-free water, and use a wide-mouth pipette tip to slowly pipette 10 times or flick until completely mixed.

[0213] 1.1.6.9 Incubate at room temperature for 5 minutes.

[0214] 1.1.6.10 Centrifuge briefly, place the PCR tube on a magnetic rack, let it stand for 2 minutes until the liquid is clear, slowly pipette 30 μL of supernatant into a new PCR tube, making sure there are no magnetic beads left in the supernatant. Pipet 1 μL and measure the concentration.

[0215] 1.2 Hybridization capture

[0216] 1.2.1 Magnetic bead concentration and probe hybridization

[0217] 1.2.1.1 Take 1000 ng (single hybrid) pre-capture library from each sample and add it to a PCR tube, and make up to 30 μL with nuclease-free water.

[0218] 1.2.1.2 Add 1× purified magnetic beads, i.e. 30 μL, flick to mix, and incubate at room temperature for 10 min.

[0219] 1.2.1.3 Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid becomes clear. Use a pipette to carefully aspirate and discard the supernatant.

[0220] 1.2.1.4 Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0221] 1.2.1.5 Repeat the previous step once.

[0222] 1.2.1.6 Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing outward. Centrifuge at low speed for 1 second, then put it back on the magnetic rack. After standing for 5 seconds, aspirate and discard the supernatant, dry it, and ensure that there is no ethanol residue.

[0223] 1.2.1.7 Remove the PCR tube from the magnetic rack, add 28 μL of hybridization reaction solution, pipette slowly 10 times or flick gently to mix thoroughly, and prepare the hybridization reaction solution according to Table 12.

[0224] Table 12

[0225]

[0226] 1.2.1.8 Add the newly prepared reaction solution to 1.2.1.6 for elution (make sure that the ethanol is completely evaporated before adding), and incubate at room temperature for 5 minutes.

[0227] 1.2.1.9 Pipette 28 μL of eluted supernatant into a new PCR tube, and then add 2 μL of Target Probes.

[0228] 1.2.1.10 Place the PCR tube in the PCR instrument and perform the reaction according to Table 13.

[0229] Table 13

[0230]

[0231]

[0232] The recommended hybridization time is 12h to 18h, and preparations for the capture experiment should be carried out before the end of the hybridization.

[0233] 1.2.2 Experimental preparation before capture

[0234] 1.2.2.1 Take the Cap Beads out of the 4℃ refrigerator in advance, mix thoroughly and equilibrate at room temperature for 30 minutes.

[0235] 1.2.2.2 Prepare 80% ethanol with anhydrous ethanol and Nuclease Free Water in advance and store it at room temperature for use in the purification step.

[0236] 1.2.2.3 TargetSeq Take out Wash Buffer 1 from Hybridization and Wash Kit v2.0-Box2. If there is precipitation, heat Wash Buffer 1 in a 37℃ water bath and use it after the precipitation is completely dissolved.

[0237] 1.2.2.4 TargetSeq TargetSeq in Hybridization and Wash Kit v2.0-Box2 Take out Wash Buffer 2v2 and preheat it in a 50℃ water bath.

[0238] 1.2.2.5 Take 50 μL of Cap Beads and add them to a new PCR tube. Place it on a magnetic rack for 1 min. Wait for the solution to become clear and discard the supernatant.

[0239] 1.2.2.6 Remove the PCR tube from the magnetic stand and add 180 μL TargetSeq Mix the Binding Buffer in the Hybridization and Wash Kit v2.0-Box2 by pipetting or vortexing to resuspend the magnetic beads.

[0240] 1.2.2.7 After instant centrifugation, place the PCR tube on a magnetic rack for 1 minute, wait for the solution to become clear, and discard the supernatant.

[0241] 1.2.2.8 Repeat steps 1.2.2.6-1.2.2.7 twice, washing the magnetic beads three times with Binding Buffer.

[0242] 1.2.2.9 Remove the PCR tube from the magnetic stand, add 180 μL of Binding Buffer, pipette or vortex to mix, and proceed to the next step immediately.

[0243] 1.2.3 DNA capture in target region

[0244] 1.2.3.1 Keep the hybridization product of 1.2.1 on the PCR instrument, add 180 μL of Cap Beads prepared in 1.2.2 to the hybridization product, and mix by pipetting.

[0245] 1.2.3.2 Cover the tube cap, remove the PCR tube from the PCR instrument, place it on a vertical rotary mixer, rotate at a speed not exceeding 10 rpm, and combine at room temperature for 30 minutes (if the laboratory does not have a vertical rotary mixer, you can combine at room temperature for 30 minutes, during which time you can mix by turning it upside down several times every 5 minutes).

[0246] 1.2.3.3 Remove the PCR tube, centrifuge it instantly, place it on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear.

[0247] 1.2.3.4 Remove the PCR tube from the magnetic rack, add 150 μL of Wash Buffer 1 into the PCR tube, gently pipette and mix to resuspend the magnetic beads, replace the tube cap, and then place it on a vertical rotating mixer for washing at room temperature for 15 minutes, with a speed not exceeding 10 rpm.

[0248] 1.2.3.5 Remove the PCR tube, centrifuge it instantly, place it on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear.

[0249] 1.2.3.6 Remove the PCR tube from the magnetic stand and add 150 μL of 50℃ preheated TargetSeq WashBuffer 2v2, gently pipette to mix, centrifuge briefly, place on a constant temperature oscillating mixer or metal bath, and incubate at 50℃ for 10min.

[0250] 1.2.3.7 Remove the PCR tube, centrifuge it instantly, place it on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear.

[0251] 1.2.3.8 Repeat steps 1.2.3.6-1.2.3.7 three times, using TargetSeq Wash the beads four times with Wash Buffer 2v2.

[0252] 1.2.3.9 Keep the PCR tube on the magnetic rack, add 200μL 80% ethanol into the PCR tube, let it stand for 30s and then completely discard the ethanol solution (you can use a 10μL pipette to discard the residual ethanol), and dry the magnetic beads at room temperature to allow the residual ethanol to evaporate completely.

[0253] 1.2.3.10 Add 23 μL Nuclease Free Water to the PCR tube, remove the PCR tube from the magnetic stand, vortex briefly to resuspend the magnetic beads, and proceed to the next amplification reaction.

[0254] 1.2.4. Post-capture PCR amplification

[0255] 1.2.4.1 Prepare the reaction system according to Table 14, and then perform the amplification reaction according to Table 15.

[0256] Table 14

[0257] Components Volume (μL) Ligation of purified product 23 2×KeyPo SE Master Mix(Dye Plus) 25 Post PCR Primer 2 Total volume 50

[0258] Table 15

[0259]

[0260] 1.2.5 Post-amplification purification

[0261] 1.2.5.1 Add 0.8× purified magnetic beads to the PCR product, i.e. 40 μL. Mix by flicking gently and react at room temperature for 10 minutes.

[0262] 1.2.5.2 Insert the PCR tube into the tube hole of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, aspirate the solution, being careful not to aspirate the magnetic beads (about 5 μL of solution can be left). Leave the PCR tube on the magnetic rack.

[0263] 1.2.5.3 Pipette 200 μL of 80% ethanol into a PCR tube, let it stand for 30 seconds, and then aspirate the solution. Be careful not to aspirate the magnetic beads. 80% ethanol should be prepared on the same day.

[0264] 1.2.5.4 Repeat the previous step once.

[0265] 1.2.5.5 Remove the PCR tube and centrifuge for 5 seconds. Insert the PCR tube into the tube hole of the magnetic rack again. After the magnetic beads are completely adsorbed, use a pipette to remove the residual solution. Be careful not to leave any residue on the tube wall. Open the PCR tube cap and let it dry.

[0266] 1.2.5.6 Pipette 32 μL of nuclease-free water into the PCR tube, flick to mix, and react at room temperature for 5 minutes.

[0267] 1.2.5.7 Insert the PCR tube into the tube hole of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, use a pipette to transfer 30 μL into a new PCR tube with the corresponding number. Be careful not to absorb the magnetic beads.

[0268] 1.2.5.8 Take 1 μL of the captured product and measure the concentration. The concentration should be > 5 ng / μL.

[0269] 1.3PacBio amplicon library construction

[0270] 1.3.1 Mixing samples: Take equal masses of each captured library and mix them to a total mass of 800-1000ng.

[0271] If the mixed sample volume is greater than 46 μL, use 1× purified magnetic beads to concentrate and elute with 46 μL of nuclease-free water; if the mixed sample volume is less than 46 μL, use nuclease-free water to make up to 46 μL.

[0272] 1.3.2 Damage Repair & End Repair & Adding A

[0273] 1.3.2.1 Prepare the system on ice according to Table 16.

[0274] Table 16

[0275] Components Volume (μL) Post-capture pooled libraries 46 Repair buffer 8 End repair mix 4 DNA repair mix 2 Total volume 60

[0276] 1.3.2.2 Mix by flicking gently and centrifuge briefly.

[0277] 1.3.2.3 Place the reaction tube on the PCR instrument and run the PCR instrument according to Table 17 (cover temperature 75°C, volume 60 μL).

[0278] Table 17

[0279] temperature time 37℃ 30min 65℃ 5min 4℃ Keep

[0280] 1.3.3 Adapter ligation and purification

[0281] 1.3.3.1 Add 4 μL of SMRTbell adapter 3.0 with barcode to the reaction product of the previous step;

[0282] 1.3.3.2 Prepare the system on ice according to Table 18.

[0283] Table 18

[0284] Components Volume (μL) Repair product + connector 64 Ligation mix 30 Ligation enhancer 1 Total volume 95

[0285] 1.3.3.3 Tap gently to mix.

[0286] 1.3.3.4 Place the PCR tube on the PCR instrument and run the PCR instrument according to Table 19 (lid temperature OFF, volume 95 μL).

[0287] Table 19

[0288] temperature time 20℃ 30min 4℃ Keep

[0289] 1.3.3.5 After the reaction is completed, add 124 μL SMRTbell cleanup beads to the ligation product, gently flip and mix, and centrifuge at low speed for 1 second.

[0290] 1.3.3.6 Incubate at room temperature for 10 minutes.

[0291] 1.3.3.7 Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid becomes clear. Use a pipette to carefully aspirate and discard the supernatant.

[0292] 1.3.3.8 Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0293] 1.3.3.9 Repeat the previous step once.

[0294] 1.3.3.10 Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing outward. Centrifuge at low speed for 1 second, then put it back on the magnetic rack. After standing for 5 seconds, aspirate and discard the supernatant.

[0295] 1.3.3.11 Remove the PCR tube from the magnetic rack, immediately add 40 μL of elution buffer, and use a wide-mouth pipette tip to slowly pipette 10 times to mix thoroughly.

[0296] 1.3.3.12 Incubate at room temperature for 5 minutes.

[0297] 1.3.3.13 Centrifuge the PCR tube briefly and place it on a magnetic rack. Let it stand for 2 minutes until the liquid becomes clear. Slowly transfer 40 μL of supernatant to a new PCR tube to ensure that there are no magnetic beads remaining in the supernatant.

[0298] 1.3.4 Enzymatic digestion and product purification

[0299] 1.3.4.1 Prepare the system on ice according to Table 20 and flick to mix.

[0300] Table 20

[0301] Components Volume (μL) Connect products 40 Nuclease buffer 5 Nuclease mix 5 Total volume 50

[0302] 1.3.4.2 Place the PCR tube on the PCR instrument and run the PCR instrument according to Table 21 (lid temperature 75°C, volume 50 μL).

[0303] Table 21

[0304] temperature time 37℃ 15min 4℃ Keep

[0305] 1.3.4.3 Add 65 μL SMRTbell cleanup beads to the digestion product, gently flick and invert to mix, and centrifuge at low speed for 1 second.

[0306] 1.3.4.4 Incubate at room temperature for 10 minutes.

[0307] 1.3.4.5 Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid becomes clear. Use a pipette to carefully aspirate and discard the supernatant.

[0308] 1.3.4.6 Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0309] 1.3.4.7 Repeat the previous step once.

[0310] 1.3.4.8 Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing outward. Centrifuge at low speed for 1 second, then put it back on the magnetic rack. After standing for 5 seconds, aspirate and discard the supernatant.

[0311] 1.3.4.9 Remove the PCR tube from the magnetic rack and immediately add 15 μL of elution buffer and flick to mix.

[0312] 1.3.4.10 Incubate at room temperature for 5 minutes.

[0313] 1.3.4.11 Centrifuge the PCR tube briefly and place it on a magnetic rack. Let it stand for 2 minutes until the liquid becomes clear. Transfer 36 μL of the supernatant to a new PCR tube, making sure there are no magnetic beads left in the supernatant.

[0314] Transfer the eluted library to a new 1.5mL EP tube and take 1μL to measure the concentration. The total amount of the library is not less than 300ng, which is qualified.

[0315] 1.4 Detection of positive samples

[0316] 29 positive samples were sent out for testing and tested using the library construction kit of the present invention. The results are shown in Table 22. According to Table 22, it can be found that the overall performance is in line with expectations.

[0317] Table 22

[0318]

[0319]

[0320] Example 3

[0321] This example is the carrier screening process and results of 31 normal people using the kit of the present application. The detection process refers to the steps of Example 1, and the results are as follows:

[0322] In the samples tested, the target regions of 23 diseases were uniformly covered with a capture efficiency of 45%-50%. The probes had good capture uniformity and no GC preference.

[0323] Among the 31 normal samples, one case was found to carry a thalassemia pathogenic mutation, one case was found to carry an SMA-related pathogenic mutation, and one case was found to carry a Wilson disease pathogenic mutation. If the traditional method is used to detect the above three diseases, it is necessary to combine Gap PCR+Sanger sequencing+qPCR, which is cumbersome and expensive. However, this process can detect them all at once, simplifying the process and reducing costs.

[0324] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0325] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0326] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A probe composition, It is characterized in that The probe composition is used to screen for a variety of genetic disease-related gene mutation types; the probe composition can specifically capture pathogenic genes of genetic diseases, and the genetic diseases include citrullinemia, hepatolenticular degeneration, 21-hydroxylase deficiency, pseudohypertrophic muscular dystrophy, fragile X syndrome, Friedreich ataxia, glycogen storage disease type II, Gaucher disease, autosomal recessive deafness type 1A, Fabry disease, a-thalassemia, β-thalassemia, mucopolysaccharidosis type II, methylmalonic aciduria and homocystinuria cbIC type, methylmalonic aciduria mut(0) type, phenylketonuria, BH4-deficient hyperphenylalaninemia type A, primary carnitine deficiency, citrullinemia type II, Pendred syndrome, autosomal recessive deafness with vestibular aqueduct dilatation type 4, spinal muscular atrophy and X-linked ichthyosis; The probe composition includes 866 probes, wherein the capture start and end sites of each probe are respectively as follows:

2. The probe composition according to claim 1, It is characterized in that The causative genes of the genetic disease include: (1) ASSI, a pathogenic gene associated with citrullinemia; (2) ATPTB, a pathogenic gene associated with Wilson's disease; (3) CYP21A2, a pathogenic gene associated with 21-hydroxylase deficiency; (4) Duchenne muscular dystrophy (DMD) (5) Fragile X syndrome-related pathogenic gene FMR1; (6) FXN, a pathogenic gene associated with Friedreich's ataxia; (7) GAA, a pathogenic gene associated with glycogen storage disease type II; (8) Gaucher disease-related pathogenic gene GBA1; (9) GJB2, a pathogenic gene associated with autosomal recessive deafness type 1A; (10) Fabry disease-related pathogenic gene GLA; (11) a-thalassemia-related pathogenic genes HBA1 / HBA2; (12) β-thalassemia-related pathogenic gene HBB; (13) IDS, a pathogenic gene associated with mucopolysaccharidosis type II; (14) MMACHC, the pathogenic gene associated with methylmalonic aciduria and homocystinuria cbIC type; (15) MMUT, the pathogenic gene associated with methylmalonic aciduria mut(0); (16) Phenylketonuria-related pathogenic gene PAH; (17) PTS, a pathogenic gene associated with BH4-deficient hyperphenylalaninemia type A; (18) Primary carnitine deficiency-related pathogenic gene SLC22A5; (19) SLC25A13, a pathogenic gene associated with citrullinemia type II; (20) Pendred syndrome-related pathogenic gene SLC26A4; (21) SLC26A4, a pathogenic gene associated with autosomal recessive deafness with vestibular aqueduct dilatation type 4; (22) SMNI, a disease-causing gene associated with spinal muscular atrophy; and (23) X-linked ichthyosis-related pathogenic gene STS.

3. A method for preparing the probe composition according to claim 1 or 2, It is characterized in that include: Selecting respective target capture regions according to the pathogenic genes of the genetic diseases, wherein the target capture regions include the pathogenic genes and the regions extending 3 kb upstream and downstream thereof and the CNV coordinate regions thereof; The probe is designed according to the target capture region to obtain the probe composition.

4. The preparation method according to claim 3, It is characterized in that The probe composition satisfies at least one of the following conditions: (i) the length of each probe in the probe composition is 100 nt; (ii) the error tolerance of each probe in the probe composition is less than 5%; and, (iii) The coverage density of each probe in the probe composition is 0.2268×.

5. The preparation method according to claim 3, It is characterized in that At least one of the following conditions is met: (i) after designing the probes, synthesizing each probe separately; (ii) synthesizing each of the probes individually and performing individual quality control; (vii) after designing the probe, synthesizing the probe at equal concentrations; (iv) synthesizing the probes at equal concentrations and mixing them in equal amounts; (v) the error tolerance of each of the probes is less than 5%; (vi) the coverage density of each of the probes is 0.2268×; (vii) the genetic diseases include citrullinemia, hepatolenticular degeneration, 21-hydroxylase deficiency, pseudohypertrophic muscular dystrophy, fragile X syndrome, Friedreich's ataxia, glycogen storage disease type II, Gaucher disease, autosomal recessive deafness type 1A, Fabry disease, α-thalassemia, β-thalassemia, mucopolysaccharidosis type II, methylmalonic aciduria and homocystinuria cbIC type, methylmalonic aciduria mut(0) type, phenylketonuria, BH4-deficient hyperphenylalaninemia type A, primary carnitine deficiency, citrullinemia type II, Pendred syndrome, autosomal recessive deafness with vestibular aqueduct dilatation type 4, spinal muscular atrophy and X-linked ichthyosis; (viii) The target capture region of the probe composition includes the following regions: (1) ASSI, a pathogenic gene associated with citrullinemia; (2) ATPTB, a pathogenic gene associated with Wilson's disease; (3) CYP21A2, a pathogenic gene associated with 21-hydroxylase deficiency; (4) Duchenne muscular dystrophy (DMD) (5) Fragile X syndrome-related pathogenic gene FMR1; (6) FXN, a pathogenic gene associated with Friedreich's ataxia; (7) GAA, a pathogenic gene associated with glycogen storage disease type II; (8) Gaucher disease-related pathogenic gene GBA1; (9) GJB2, a pathogenic gene associated with autosomal recessive deafness type 1A; (10) Fabry disease-related pathogenic gene GLA; (11) a-thalassemia-related pathogenic genes HBA1 / HBA2; (12) β-thalassemia-related pathogenic gene HBB; (13) IDS, a pathogenic gene associated with mucopolysaccharidosis type II; (14) MMACHC, the pathogenic gene associated with methylmalonic aciduria and homocystinuria cbIC type; (15) MMUT, the pathogenic gene associated with methylmalonic aciduria mut(0); (16) Phenylketonuria-related pathogenic gene PAH; (17) PTS, a pathogenic gene associated with BH4-deficient hyperphenylalaninemia type A; (18) Primary carnitine deficiency-related pathogenic gene SLC22A5; (19) SLC25A13, a pathogenic gene associated with citrullinemia type II; (20) Pendred syndrome-related pathogenic gene SLC26A4; (21) SLC26A4, a pathogenic gene associated with autosomal recessive deafness with vestibular aqueduct dilatation type 4; (22) SMNI, a pathogenic gene associated with spinal muscular atrophy; (23) X-linked ichthyosis-related pathogenic gene STS.

6. A kit, It is characterized in that The kit contains the probe composition according to any one of claims 1 to 2, or the probe composition prepared by the preparation method according to any one of claims 3 to 5.

7. A method for detecting a target region, It is characterized in that include: The target region is captured using the probe composition according to any one of claims 1 to 2 or the kit according to claim 6.

8. The detection method according to claim 7, It is characterized in that The steps include: The genomic DNA of the sample to be tested is fragmented and adapter-added to obtain DNA fragments with adapters added to the ends; After the DNA fragment with a connector added to the end is amplified, it is subjected to hybridization capture treatment using the probe composition or the kit to capture the target region.

9. The detection method according to claim 8, It is characterized in that The steps of taking genomic DNA of the sample to be tested, fragmenting and adding adapters to obtain DNA fragments with adapters added to the ends include: The genomic DNA of the sample to be tested, the transposase and the transposase buffer are mixed at a first temperature for a first time to obtain a DNA fragment with a linker added to the end; Optionally, the amount of the transposase used is 2.5 μL to 3.5 μL: 1 μg relative to the genomic DNA; Optionally, the first temperature is 50°C to 60°C, and the first time is 8 minutes to 12 minutes.

10. The detection method according to claim 8, It is characterized in that The amplification enzyme used in the amplification process includes at least one of Phanta Flash Super-Fidelity DNA Polymerase, 2×KeyPo SE Master Mix (Dye Plus) and KOD FX Neo.

11. The detection method according to claim 8, It is characterized in that The amount of DNA fragments with adapters added to the ends after amplification treatment put into the hybridization capture treatment is 750 ng to 1000 ng.

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