Library establishment kit, library hybridization capture method, library establishment method and application of X-linked dyspepsia related genes

Abstract

According to the library building kit, the library hybridization capture method, the library building method and the application of the X-linked dyspepsia-related genes, the library building kit comprises a probe pool containing a capture probe, and the capture probe is used for hybridizing and capturing the X-linked dyspepsia-related genes. According to the kit, firstly, a DNA library is constructed, the initial quantity of a template can be as low as 10 ng, enzyme digestion breaking, terminal filling and A tail adding are completed in one system, the consumed time is short, and the efficiency is high; secondly, as many as 384 pairs of UDI probes are arranged in the library building kit, and 384 libraries can be simultaneously built and can be loaded on a machine for sequencing together; in addition, according to a rapid hybridization system of the library building kit, the hybridization time can be as short as 15 min, and the efficiency of common overnight hybridization can be achieved. The size of the finally obtained library fragment is about 300-500bp, and the library fragment can be compatible with various sequencing platforms of illumina for sequencing.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a library construction kit for X-linked intellectual disability related genes, a library hybridization capture method, a library construction method and an application. Background Art

[0002] Intellectual disability (ID), i.e., low intelligence and decreased social adaptability, is the main cause of childhood disability and genetic counseling, accounting for about 1-3% of the normal population. The genetic factors of ID etiology are mainly abnormal chromosome number or structure, copy number variation and single gene variation. X-linked intellectual disability (XLID) accounts for about 5-10% of all ID patients, among which fragile X chromosome syndrome is more common. XLID has genetic heterogeneity, and most of them are sporadic cases. There is a lack of large and systematic pedigree studies, making clinical diagnosis and genetic counseling of XLID relatively difficult. XLID has a high degree of clinical phenotypic diversity, usually manifested as mental retardation, global developmental delay, autism and epileptic seizures, with or without microcephaly, eye and ear malformations, cardiovascular system and other mental and behavioral abnormalities.

[0003] MECP2 (Methyl-CpG-binding Protein 2), also known as methylated CpG binding protein, is located on X chromosome Xq28 and is the main pathogenic gene of Rett syndrome. About 90% of Rett syndrome patients have MECP2 gene mutations. Due to the X-linked inheritance of the MECP2 gene, Rett syndrome is mainly seen in females and is the main cause of female mental retardation. Typical Rett syndrome is a progressive neurodevelopmental disorder that mainly affects girls. The main manifestations are that mental development seems normal within 6 to 18 months, then enters a short period of developmental stagnation, followed by rapid decline in language and motor development, and then enters a longer period of stability. During the rapid decline period, the hands repeat stereotyped movements. Other manifestations include paroxysmal screaming and crying, panic-like aggressive behavior, teeth grinding, occasional apnea or gasping, ataxic gait, akinesia, tremor, epileptic seizures, and acquired microcephaly. Unfortunately, the current examination of genes related to X-linked intellectual disability is usually carried out by direct sequencing, but this method is limited in clinical application due to its cumbersome operation, long time consumption, low throughput and low hybridization efficiency.

[0004] Therefore, there is an urgent need for a kit and a detection method that can quickly detect genes related to X-linked intellectual disability. Summary of the invention

[0005] In view of this, the present invention proposes a library construction kit for X-linked intellectual disability-related genes, a library hybridization capture method, a library construction method and an application, which are used to solve the problems of cumbersome operation, time-consuming and low throughput of the current X-linked intellectual disability-related gene detection methods.

[0006] In order to solve the above problems, the technical solution adopted by the present invention is:

[0007] The present invention provides a library construction kit for X-linked intellectual disability-related genes, characterized in that the library construction kit comprises a probe pool containing capture probes, and the capture probes are used for hybridization capture of X-linked intellectual disability-related genes.

[0008] As a preferred embodiment of the present application, the X-linked intellectual disability-associated gene comprises a molecule or a fragment thereof selected from the following group: ARX, ATRX, BCOR, CASK, CUL4B, HUWE1, IQSEC2, KDM5C, L1CAM, MECP2, PORCN, PQBP1, RPS6KA3, SLC16A2, and SL C9A6.

[0009] As a preferred embodiment of the present application, the X-linked intellectual disability-related gene comprises a molecule or a fragment thereof selected from the following group: CASK, CUL4B, MECP2, and HUWE1.

[0010] As a preferred embodiment of the present application, the probe pool includes at least one of the capture probes having base sequences of SEQ ID NOs 1 to 720 as shown in Table 7.

[0011] As a preferred embodiment of the present application, the probe pool includes at least 300 capture probes having base sequences of SEQ ID NOs 1 to 720 as shown in Table 7.

[0012] As a preferred embodiment of the present application, the probe pool includes all of the capture probes having base sequences of SEQ ID NOs 1 to 720 as shown in Table 7.

[0013] As a preferred embodiment of the present application, the probe pool includes at least 40 capture probes having base sequences of SEQ ID NOs 1 to 720 as shown in Table 7.

[0014] As a preferred embodiment of the present application, the probe pool includes at least one of the capture probes having base sequences of SEQ ID NOs 1 to 40 as shown in Table 7.

[0015] As a preferred embodiment of the present application, the probe pool includes all of the capture probes having base sequences of SEQ ID NOs 1 to 40 as shown in Table 7.

[0016] The present invention also provides a hybridization capture method, characterized in that it comprises using the library construction kit to perform hybridization capture on X-linked intellectual disability-related genes in a targeted sequencing library.

[0017] As a preferred embodiment of the present application, before the capture kit is used to capture the library to be captured, the hybridization capture method further includes the step of constructing a targeted sequencing library.

[0018] The present invention also provides a library construction method, characterized in that the library construction method comprises:

[0019] obtaining a DNA sample containing an X-linked intellectual disability-associated gene, and isolating the X-linked intellectual disability-associated gene DNA from the DNA sample;

[0020] Construct a DNA sample library containing genes related to X-linked intellectual disability;

[0021] Mixing multiple DNA sample libraries to establish a library pool containing multiple different DNA sample libraries;

[0022] The library construction kit or the hybridization capture method for X-linked intellectual disability-related genes is used to perform hybridization capture on the X-linked intellectual disability-related genes in the library pool, and the X-linked intellectual disability-related genes in the library pool are hybridized with the capture probe to generate a target complex;

[0023] After hybridization, the target complex is amplified by PCR;

[0024] The PCR amplification products of the target complex were purified to obtain a targeted sequencing library with an average fragment length of 300 to 500 bp.

[0025] As a preferred embodiment of the present application, a DNA sample library containing genes associated with X-linked intellectual disability is constructed, including:

[0026] The X-linked intellectual disability-related gene DNA is fragmented and phosphorylated to obtain the X-linked intellectual disability-related gene;

[0027] Connectors are added to both ends of the X-linked intellectual disability-related gene, and the connection products obtained by connecting the X-linked intellectual disability-related gene and the connector are PCR amplified and purified to obtain a DNA sample library containing the X-linked intellectual disability-related gene.

[0028] As a preferred embodiment of the present application, the genomic DNA fragmentation is obtained by using an enzyme digestion method to interrupt the X-linked intellectual disability-related gene DNA in the whole blood sample.

[0029] As a preferred embodiment of the present application, the dosage of each DNA sample library in the DNA sample library is calculated according to the DNA mass concentration in each DNA sample library to ensure that the total amount of DNA in the library pool is 1.5-4 μg.

[0030] As a preferred embodiment of the present application, the step of adding linkers at both ends of the hereditary deafness-related gene comprises:

[0031] Adding Universal adapters to both ends of the gene associated with hereditary deafness; and

[0032] A step of adding UDI adapters at both ends of the gene associated with hereditary deafness.

[0033] As a preferred embodiment of the present application, after adding Universal adapters to both ends of the hereditary deafness-related gene, the ligation product is PCR amplified and purified.

[0034] As a preferred embodiment of the present application, after adding UDI connectors to both ends of the hereditary deafness-related gene, the ligation product is PCR amplified and purified.

[0035] As a preferred embodiment of the present application, the purification method for the PCR amplification product of the target complex is a magnetic bead enrichment method.

[0036] The present invention also provides the library construction kit, the hybridization capture method or the use of the library construction method in preparing a kit for detecting genes related to X-linked intellectual disability.

[0037] This patent constructs a library construction kit and hybrid capture method that can quickly detect 15 X-linked intellectual disability-related genes. Using the library construction kit or hybrid capture method of this application, only a trace amount of genomic DNA is needed. The library pool is constructed by enzyme cutting, end modification, and adding adapters, and then the X-linked intellectual disability-related genes in the library pool are hybridized and captured and enriched using 720 customized capture probes to quickly complete the construction of the targeted library. Through a simple and easy-to-use NGS sequencing solution, all coding regions of 15 X-linked intellectual disability-related genes can be detected. The variation results of 15 X-linked intellectual disability-related genes can be quickly obtained in one test. It is suitable for screening and diagnosis related to complex intellectual disabilities, and can discover new pathogenic variations with high specificity and sensitivity.

[0038] Compared with the prior art, the present invention has the following technical effects:

[0039] The present application discloses a highly efficient targeted library preparation kit: first, the kit constructs a DNA library, and the template starting amount can be as low as 10ng, and enzyme cutting, end filling and A tailing are completed in one system, which is time-saving and efficient; secondly, the UDI probes of this library construction kit are as many as 384 pairs, which can realize the simultaneous construction and sequencing of 384 libraries; in addition, the rapid hybridization system of this library construction kit can achieve the efficiency of general overnight hybridization with a hybridization time as short as 15min. The final library fragment size is about 300-500bp, which is compatible with various sequencing platforms of Illumina for sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a flow chart of the library construction method of Example 1 of the present application.

[0041] Figure 2 This is the fragment analysis result of the targeted sequencing library constructed in Example 1 of the present application. DETAILED DESCRIPTION

[0042] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0043] The present invention will be further described below in conjunction with the embodiments.

[0044] Implementation 1

[0045] S1 Construction of a DNA sample library containing genes related to X-linked intellectual disability

[0046] 1.0 DNA was sheared by enzyme digestion to shear the DNA of X-linked intellectual disability-related genes in the whole blood sample, and the fragmented DNA of X-linked intellectual disability-related genes was repaired and A-tailed (amplified region) was added. The specific operation is as follows:

[0047] 1.0.1 Turn on the PCR instrument and set the temperature of the PCR cover to 70°C. The starting amount of DNA library construction is 10-100 ng. Mix thoroughly, centrifuge, and place on ice for later use.

[0048] 1.0.2 Go to the reagent preparation area, take a new 1.5mL centrifuge tube, and prepare the enzyme digestion reaction mixture. After melting 10xFragmentation Buffer and 5xFragmentation Enzyme (Twist Bioscience) on ice, prepare the mixture according to Table 1, mix and centrifuge, divide the enzyme digestion reaction mixture into eight tubes, and then transfer it to the amplification area through the transfer window. (At the same time, the DNA purification magnetic bead solution used for the next step of purification can be taken out of the refrigerator, shaken and mixed thoroughly, and then transferred to the amplification area together to equilibrate at room temperature for at least 30 minutes);

[0049] Table 1

[0050] Reagents Single reaction volume 10x Fragmentation Buffer 5μL 5x Fragmentation Enzyme 10μL Total volume 40μL

[0051] 1.0.3 Take 35 μL of each DNA sample to be tested and add them to the eight-tube strip containing the enzyme digestion reaction mixture. Gently shake and mix. Centrifuge for a moment and then quickly put it into the PCR instrument. Make sure the temperature of the PCR instrument's hot cover is 70°C, and then amplify using the program shown in Table 2:

[0052] Table 2

[0053] temperature time 32℃ 22min 65℃ 30min 4℃ HOLD

[0054] 1.1 Connect to Universal adapter

[0055] 1.1.1 After the above steps are completed, add 5 μL of Universal adapter (Twist Bioscience) to each reaction system, shake gently to mix, centrifuge and place on ice for later use.

[0056] 1.1.2 Go to the reagent preparation area and prepare the ligation reaction mixture. After melting DNA Ligation Buffer and DNA Ligation Mix (Twist Bioscience) on ice, prepare the mixture according to Table 3, mix well, centrifuge, place on ice, and transfer to the amplification area for use.

[0057] Table 3

[0058] Reagents Single reaction volume water 15μL DNA Ligation Buffer 20μL DNA Ligation Mix 10μL Total volume 45μL

[0059] 1.1.3 Add 45 μL of the ligation reaction mixture prepared in step 1.1.2 to each sample prepared in step 1.0.3. Gently shake and mix, centrifuge and quickly put into the PCR instrument, connect at 20℃ for 15 minutes (heat cover closed). After the end, take out and immediately start magnetic bead purification.

[0060] 1.2 First step magnetic bead purification (amplification area)

[0061] 1.2.1 Library purification magnetic beads (KAPA) should be taken out from 4°C before use, vortexed and mixed thoroughly, and equilibrated at room temperature for at least 30 minutes.

[0062] 1.2.2 Vortex the purified magnetic bead suspension (KAPA) equilibrated at room temperature for 1 min and dispense into 1.5 mL centrifuge tubes at a rate of 80 μL (0.8 times the sample volume) per sample.

[0063] 1.2.3 Transfer all the ligation products to the magnetic beads in the corresponding centrifuge tube and gently shake to mix to avoid bubbles.

[0064] 1.2.4 After leaving the centrifuge tube at room temperature for 5 minutes, place it on a magnetic rack for 5 minutes until the supernatant becomes clear. Carefully remove the supernatant.

[0065] 1.2.5 Keep the centrifuge tubes on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to each tube, let stand at room temperature for 1 min, and discard the supernatant.

[0066] 1.2.6 Repeat step 1.2.5. After a brief centrifugation, place the centrifuge tube back into the magnetic rack and use a 10 μL pipette to remove as much residual ethanol solution as possible from the bottom of the tube.

[0067] 1.2.7 Leave the centrifuge tube on the magnetic rack with the lid open for 5 minutes at room temperature to dry it so that all the ethanol evaporates. Avoid over-drying.

[0068] 1.2.8 Add 17 μL of enzyme-free water to each tube and mix up and down 10 times using a pipette. Incubate at room temperature for 5 min.

[0069] 1.2.9 Place each tube on a magnetic rack and let it stand for 5 minutes until the supernatant becomes clear.

[0070] 1.3UDI adapter primer PCR amplification

[0071] 1.3.1 During the rest period in step 1.2.9, go to the reagent preparation area and prepare eight tube strips on the ice box according to the number of samples. Take 25 μL KAPA HiFi HotStart ReadyMix (KAPA) and dispense it into each well of the eight tube strips.

[0072] 1.3.2 Add 10 μL of pre-set UDI primers with different indexes (Twist Bioscience) to each well of the eight-tube strip (UDI is a pair of primers, a total of 384 pairs, the sequences of these primers contain 8 bases (we call it index) which are different for distinguishing, except for these 8 bases, the other sequences of the 384 pairs of UDI are the same). Then transfer to the amplification area I through the transfer window.

[0073] 1.3.3 According to the set position order, pipette 15 μL of purified supernatant obtained in step 1.2.9 and add it to each well of the PCR reaction mixture that has been dispensed, cover the tube, shake gently, and centrifuge briefly to collect the liquid.

[0074] 1.3.4 Transfer the prepared PCR reaction tube to the amplification area through the transfer window for PCR reaction. Heat the cover at 105℃ and set the reaction program as shown in Table 4.

[0075] Table 4

[0076]

[0077] 1.4 The second step of magnetic bead purification (amplification area)

[0078] 1.4.1 Library purification magnetic beads (same as KAPA) should be taken out from 4°C before use, vortexed and mixed thoroughly, and equilibrated at room temperature for at least 30 minutes.

[0079] 1.4.2 Vortex the purified magnetic bead suspension equilibrated at room temperature for 1 min and dispense 50 μL (equal volume of sample) / sample into 1.5 mL centrifuge tubes.

[0080] 1.4.3 Take out the PCR reaction product, centrifuge for 30 seconds, transfer all the products to the magnetic beads in the corresponding centrifuge tube, and shake gently to avoid generating bubbles.

[0081] 1.4.4 After standing at room temperature for 5 minutes, place on a magnetic separation rack for 5 minutes until the supernatant is clear. Carefully remove the supernatant.

[0082] 1.4.5 Place on a magnetic separation stand, add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant.

[0083] 1.4.6 Repeat step 1.4.5 and try to remove all the residual ethanol solution.

[0084] 1.4.7 Leave the centrifuge tube on the magnetic rack with the lid open for 5 minutes at room temperature to dry, ensuring that all the ethanol evaporates.

[0085] 1.4.8 Add 22 μL of enzyme-free water, shake gently, and incubate at room temperature for 5 min.

[0086] 1.4.9 Place on the magnetic stand for 5 minutes, take 20 μL of supernatant (library) for the next step of library enrichment. Take 1 μL and dilute 10 times, use Qubit and fragment analyzer to measure library concentration and fragment size respectively. The average fragment length of the test library should be around 300-500 bp.

[0087] S2 mixes multiple DNA sample libraries to establish a library pool containing multiple different DNA sample libraries;

[0088] 2.0.1 Calculate the amount of different DNA sample libraries and determine the mixing scheme of library pool DNA: The library pool often contains multiple different DNA sample libraries. Calculate the amount of each DNA sample library pool according to the concentration of the DNA sample library, and mix to obtain the library pool. The total amount of DNA in the library pool is 1.5-4μg. It is recommended to establish a library pool with samples of the same mass. Refer to Table 5 for the amount of DNA library:

[0089] Table 5

[0090] Number of mixed samples The amount of each library Total library volume per reaction 1 1000ng 1000ng 2 1000ng 2000ng 3 1000ng 3000ng 4 800ng 3200ng 8 400ng 3200ng

[0091] 2.0.2 Take samples according to the calculated amount of different DNA sample libraries, mix each DNA sample library in a 1.5mL centrifuge tube, shake gently, and centrifuge for standby use.

[0092] S3: using the library construction kit or the X-linked intellectual disability-related gene hybridization capture method to perform hybridization capture on the X-linked intellectual disability-related gene in the library pool, wherein the X-linked intellectual disability-related gene in the library pool is hybridized with the capture probe to generate a target complex, and after the hybridization is completed, the target complex is amplified by PCR;

[0093] 3.0.1 Design and synthesize capture probes, design probe sequences according to target genes, remove regions with multiple alignments, and adjust the number of probe layers according to GC content to construct a probe pool.

[0094] 3.0.2 Add the following pre-hybridization reagents to the mixed sample, mix well, centrifuge briefly, and try not to generate bubbles. The pre-hybridization reagents are configured according to Table 6. Capture probes, also known as XLID Panel probes, are personalized DNA probes designed to capture X-linked intellectual disability-related genes for hybridization capture. The base sequence is at least one of SEQ ID NOs 1 to 720 shown in Table 1.

[0095] Table 6

[0096] Reagents volume Capture probe (base sequence is SEQ ID NO 1~720) 4μL Universal Blockers(Twist Bioscience) 8μL Blocking Solution(Twist Bioscience) 5μL

[0097] Table 7

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] 3.0.3 Open the cap of the pre-hybridization reagent tube, place the tube in a vacuum concentrator, balance it, and dry it at 45℃ until the solution in the tube is completely dry. The drying process takes about 30 minutes to 1 hour.

[0119] 3.0.4 When drying is almost finished, incubate Fast Hybridization Mix (Twist Bioscience) at 65°C for 10 min or until all precipitates are dissolved, and then set the PCR instrument program according to Table 8, with the heated cover at 85°C:

[0120] Table 8

[0121] temperature time 95℃ 5min 60℃ 15min~4h

[0122] 3.0.5 After drying, remove the centrifuge tube from the dryer, tear off the sealing film, quickly vortex Fast Hybridization Mix (Twist Bioscience) and add 20 μL to the tube to resuspend the sample (please do not let the hybridization solution return to room temperature), flick with your fingertips to mix, avoid bubbles, and let stand at room temperature for 5 minutes. Use a pipette to pipette up and down 10 times. After the DNA is completely dissolved, transfer all the solution in the tube to a new PCR tube as much as possible to avoid bubbles.

[0123] 3.0.6 Add 30 μL of paraffin oil to the tube to cover the surface of the reagent inside, and then centrifuge it instantly to remove bubbles.

[0124] 3.0.7 Place the PCR tube in the preheated PCR instrument for hybridization and incubate for 15 minutes to 2 hours. (During this period, you can go to the reagent preparation area to take out the Streptavidin Binding Beads (Twist Bioscience) used for the next capture from the refrigerator, shake and mix thoroughly, and then equilibrate to room temperature for at least 30 minutes).

[0125] S4 Purification of PCR amplification products of target complex

[0126] 4.0.1 Go to the reagent preparation area and dispense the enrichment reagent (reagent amount for a single enrichment reaction) into 1.5 mL centrifuge tubes according to Table 9, and then transfer each component and the balanced magnetic bead component (Streptavidin Binding Beads) to the amplification area through the transfer window.

[0127] Table 9

[0128]

[0129]

[0130] 4.0.2 Enter the amplification area and preheat 450μL Fast Wash Buffer 1 at 70℃ and 700μL Wash Buffer 2 at 48℃ in a thermostatic metal bath.

[0131] 4.0.3 Shake the pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, and transfer 100 μL of magnetic beads to a 1.5 mL centrifuge tube;

[0132] 4.0.4 Add 200 μL of Fast Binding Buffer to the centrifuge tube and mix well by pipetting with a pipette;

[0133] 4.0.5 Place the centrifuge tube on the magnetic rack for 1 minute or until the solution is clear, discard the supernatant, and remove the centrifuge tube;

[0134] 4.0.6 Repeat the washing steps 4.0.4 to 4.0.5 twice, for a total of 3 times;

[0135] 4.0.7 After the third wash, add 200 μL of Last Binding Buffer and resuspend by vortexing to mix thoroughly.

[0136] 4.0.8 After the hybridization in step 3.0.6 is completed, open the cover of the PCR instrument and quickly transfer all the hybridization solution to the balanced magnetic bead solution. Note: Rapidly transferring the reagents in the PCR instrument to the magnetic beads is a key step. The sample must be loaded directly on the PCR instrument. Do not remove the hybridization tube to prevent the temperature of the hybridization solution from dropping.

[0137] 4.0.9 Close the cap of the centrifuge tube and seal the tube mouth with sealing film, then place it on the Biocate oscillator at 1200 rpm and mix thoroughly at room temperature for 30 minutes.

[0138] 4.0.10 Remove the centrifuge tube from the mixer, centrifuge quickly and place it on the magnetic rack for 1 minute, remove the supernatant, remove the tube, add 200μL preheated Fast Wash Buffer 1 (do not remove it from the metal bath), and gently shake to mix;

[0139] 4.0.11 Rapidly incubate the centrifuge tube at 66°C for 5 minutes;

[0140] 4.0.12 Centrifuge the tube and place it on a magnetic rack for 1 minute. Remove the supernatant, remove the tube, add 200 μL preheated Fast Wash Buffer 1 again, and gently shake to mix;

[0141] 4.0.13 Rapidly incubate the centrifuge tube at 66°C for 5 minutes;

[0142] 4.0.14 Centrifuge the centrifuge tube and transfer all the liquid in it to a new 1.5mL centrifuge tube; place it on the magnetic rack for 1 minute and remove the supernatant;

[0143] 4.0.15 Remove the tube, add 200 μL of preheated Wash Buffer 2 (do not remove it from the metal bath), and gently shake to mix;

[0144] 4.0.16 Rapidly incubate the centrifuge tube at 48°C for 5 minutes;

[0145] 4.0.17 After centrifugation, place the centrifuge tube on a magnetic rack for 1 minute, remove the supernatant and take out the tube;

[0146] 4.0.18 Repeat (steps 4.0.15 to 4.0.17) and wash twice, three times in total;

[0147] 4.0.19 After centrifugation, place the centrifuge tube on a magnetic rack and use a 10μL pipette tip to suck out the wash solution;

[0148] 4.0.20 Remove the centrifuge tube from the magnetic rack, add 45 μL of nuclease-free water, gently shake to mix, and incubate the solution on ice. If PCR amplification is not performed for the time being, the magnetic bead mixture can be stored at 20°C.

[0149] S5 PCR amplification of the target complex

[0150] 5.0.1 Enter the reagent preparation area, take a PCR tube, prepare the mixed solution, add (25μL KAPAHiFiHotStartReadyMix(KAPA)+2.5μL Amplification Primers(Twist Bioscience)=22.5μL) to the tube respectively (this Amplification Primer is unique, just like the Universal Adapter mentioned above, there is only one), and then transfer the PCR tube to the amplification area I through the transfer window. (Note: At this time, you can take out the DNA PurificationBeads(KAPA) from the refrigerator, shake and mix thoroughly, and transfer it to the amplification area together with the PCR tube to equilibrate at room temperature for at least 30 minutes)

[0151] 5.0.2 Take 22.5 μL of magnetic bead suspension obtained in step 5.0.1 and add it to the PCR tube, shake gently to mix and centrifuge instantly. The remaining 22.5 μL can be stored at –20℃ for later use.

[0152] 5.0.3 Place the PCR tube on the PCR instrument, heat the cover to 105°C, and amplify according to the reaction program in Table 10:

[0153] Table 10

[0154]

[0155] S6 uses magnetic beads to purify the PCR amplification products (amplification regions) of the target complex to obtain a targeted sequencing library with an average fragment length of 300 to 500 bp

[0156] 6.0.1 Vortex and thoroughly mix the pre-equilibrated DNA purification magnetic beads (same as KAPA above), take a clean 1.5mL centrifuge tube, add 90μL (1.8*) DNA purification magnetic beads, and after the PCR is completed, centrifuge the PCR tube instantly, then transfer all the solutions in the PCR tube to a 1.5mL centrifuge tube and vortex to mix thoroughly;

[0157] 6.0.2 Incubate the centrifuge tube at room temperature for 5 minutes;

[0158] 6.0.3 Place the centrifuge tube on the magnetic rack for 1 minute and remove the supernatant after the solution becomes clear;

[0159] 6.0.4 Keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the 80% ethanol wash once (2 times in total);

[0160] 6.0.5 After instant centrifugation, place the centrifuge tube on a magnetic rack and carefully remove the remaining ethanol with a 10μL pipette tip. Leave at room temperature for 5-10 minutes or until the magnetic beads are dry, but avoid cracking.

[0161] 6.0.6 Remove the tube from the magnetic rack and add 32 μL of nuclease-free water, gently shake to mix, and incubate at room temperature for 5 min;

[0162] 6.0.7 Place the centrifuge tube on the magnetic rack for 3 minutes or until the solution becomes clear;

[0163] 6.0.8 Transfer 30 μL of supernatant to a clean 1.5 mL centrifuge tube, label the library name and library construction date on the tube cap, and then transfer to the library quality inspection area. Note: If the enriched library cannot be sequenced immediately after purification, it can be stored below -20°C for 30 days.

[0164] 6.0.9 Take another 1 μL for use The library concentration was determined by dsDNA HS Assay Kit (KAPA), which should be greater than 0.5 ng / μL. The 2100 fragment analyzer (Agilent) was also used to detect the length distribution of the library fragments, and the average fragment length should be between 300 and 500 bp.

[0165] Example 2

[0166] The difference between this embodiment and embodiment 1 is: 3.0.2 Add the following pre-hybridization reagents to the mixed sample respectively, mix well, centrifuge instantaneously, and try not to generate bubbles. The pre-hybridization reagents are configured according to Table 6. The capture probe, also known as the XLID Panel probe, is a personalized DNA probe designed for capturing X-linked intellectual disability-related genes for hybridization capture. The base sequence is shown in Table 1. All of the capture probes in SEQ ID NO 1 to 40. The rest of the detection conditions and methods are the same.

[0167] Example 3 Clinical sample detection

[0168] Take 2 clinical whole blood samples with known positive gene mutation results, and detect the mutation of X-linked intellectual disability related genes in these 2 samples. First, extract the genomic DNA of the 2 whole blood samples, and then build the library according to the methods in Example 1 and Example 2, and successfully build the targeted sequencing library. The fragment analysis results of the constructed targeted sequencing library in Example 1 are shown in Figure 2 .

[0169] The targeted sequencing library of Example 1 was sequenced on an Illumina high-throughput sequencer, with sequencing parameters PE, 2×150. After quality control of the data off the machine, the data was analyzed using a self-built bioinformatics analysis process to obtain the gene mutations associated with X-linked intellectual disability in each sample (Table 11 is the test results of patient 1; Table 12 is the test results of patient 2), and each site was verified by first-generation sequencing, with a compliance rate of 100%.

[0170] Table 11 Test results of patient 1:

[0171]

[0172] Table 12 Test results of patient 2:

[0173]

[0174] The targeted sequencing library of Example 2 was sequenced on an Illumina high-throughput sequencer, with sequencing parameters PE, 2×150. After quality control of the data off the machine, the data was analyzed using a self-built bioinformatics analysis process to obtain the gene mutations associated with X-linked intellectual disability in each sample (Table 13 is the test results of patient 1; Table 14 is the test results of patient 2), and then each site was verified by first-generation sequencing, with a compliance rate of 100%.

[0175] Table 13 Test results of patient 1:

[0176]

[0177]

[0178] Table 14 Patient 2 test results:

[0179]

[0180] It can be seen from the test results that the kit of the present invention can detect X-linked intellectual disability-related gene mutations and can accurately detect X-linked intellectual disability. The XLID Panel hybridization capture probe of Example 1 of the present invention can accurately capture 15 X-linked intellectual disability-related genes, and the XLID Panel hybridization capture probe of Example 2 can accurately capture 4 X-linked intellectual disability-related genes. The detection of conventional specimens shows that the probes, methods and kits of the present invention can detect X-linked intellectual disability-related gene mutations. The above examples are intended to illustrate the embodiments disclosed in the present invention and cannot be construed as limitations on the present invention. In addition, the various modifications listed herein and the changes in the methods and compositions of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with a variety of specific preferred embodiments of the present invention, it should be understood that the present invention is not limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims

1. A library construction kit for X-linked intellectual disability-related genes, characterized in that: The library construction kit comprises a probe pool containing capture probes, and the capture probes are used for hybridization and capture of X-linked intellectual disability-related genes.

2. The library construction kit for X-linked intellectual disability-related genes according to claim 1, characterized in that: The X-linked intellectual disability-associated gene comprises a molecule or a fragment thereof selected from the following group: ARX, ATRX, BCOR, CASK, CUL4B, HUWE1, IQSEC2, KDM5C, L1CAM, MECP2, PORCN, PQBP 1, RPS6KA3, SLC16A2, and SLC9A6.

3. The library construction kit for X-linked intellectual disability-related genes according to claim 1, characterized in that: The probe pool includes at least one of the capture probes having base sequences of SEQ ID NOs 1 to 720 as shown in the following table:

4. A hybrid capture method, characterized in that: The method comprises using the library construction kit described in claim 1 to perform hybridization capture on X-linked intellectual disability-related genes in a targeted sequencing library.

5. The hybridization capture method according to claim 4, characterized in that: Before using the capture kit to capture the library to be captured, the hybridization capture method also includes the step of constructing a targeted sequencing library.

6. A library construction method, characterized in that: The library construction method comprises: obtaining a DNA sample containing an X-linked intellectual disability-associated gene, and isolating the X-linked intellectual disability-associated gene DNA from the DNA sample; Construct a DNA sample library containing genes related to X-linked intellectual disability; Mixing multiple DNA sample libraries to establish a library pool containing multiple different DNA sample libraries; Using the library construction kit according to any one of claims 1 to 3, or the X-linked intellectual disability-related gene hybridization capture method according to any one of claims 4 to 5, the X-linked intellectual disability-related gene in the library pool is hybridized and captured, and the X-linked intellectual disability-related gene in the library pool is hybridized with the capture probe to generate a target complex; After hybridization, the target complex is amplified by PCR; The PCR amplification products of the target complex were purified to obtain a targeted sequencing library with an average fragment length of 300 to 500 bp.

7. The library construction method according to claim 6, characterized in that: Construction of a DNA sample library containing genes related to X-linked intellectual disability, including: The X-linked intellectual disability-related gene DNA is fragmented and phosphorylated to obtain the X-linked intellectual disability-related gene; Connectors are added to both ends of the X-linked intellectual disability-related gene, and the connection products obtained by connecting the X-linked intellectual disability-related gene and the connector are PCR amplified and purified to obtain a DNA sample library containing the X-linked intellectual disability-related gene.

8. The library construction method according to claim 7, characterized in that: The genomic DNA fragmentation is obtained by using enzyme digestion to interrupt the X-linked intellectual disability-related gene DNA in the whole blood sample.

9. The library construction method according to claim 6, characterized in that: The amount of each DNA sample library in the DNA sample library is calculated according to the DNA mass concentration in each DNA sample library to ensure that the total amount of DNA in the library pool is 1.5-4 μg.

10. Use of the library construction kit according to any one of claims 1 to 3, the hybridization capture method according to claim 4, or the library construction method according to claim 6 in preparing a kit for detecting genes associated with X-linked intellectual disability.

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