Rigosity-adjustable probe hybridization capture method

Abstract

The invention discloses a preciseness-adjustable probe hybridization capture method which is characterized in that a hybridization resolution enhancing probe is added into a system of a probe hybridization capture target area, a potential non-specific capture area is competitively combined, and the hybridization capture resolution of the probe to the target area is improved. The binding strength of the hybridization resolution enhanced probe and a potential non-specific capture region is higher than that of the probe and the potential non-specific capture region, and the binding strength of the hybridization resolution enhanced probe and a target region is far lower than that of the probe and the target region, so that the detection sensitivity of the target region is not influenced. The method realizes local fine control of hybridization capture preciseness of the nucleic acid target area, and simultaneously achieves high-sensitivity and high-specificity detection of the nucleic acid target area compared with a traditional method.

Description

Technical Field

[0001] This invention belongs to the field of high-throughput sequencing, specifically relating to a probe hybridization capture method with adjustable rigor. Background Technology

[0002] With advancements in technology, gene sequencing has developed into a crucial life science technology, playing a key role in genomics research, disease diagnosis and prevention, drug development and personalized treatment, biotechnology innovation, and agriculture. The widespread adoption of gene sequencing across industries has led to the rapid development of next-generation sequencing (NGS) technology, significantly reducing the cost of DNA sequencing. However, the cost of whole-genome sequencing remains high, and the massive amounts of data present challenges for subsequent analysis and applications.

[0003] Hybrid capture sequencing (HMRS) is a technique that uses designed probes to enrich target regions of interest based on the principle of complementary base pairing before sequencing. This technique significantly reduces sequencing data volume, lowers sequencing costs, increases sequencing depth of target regions, and improves the sensitivity and accuracy of target region detection, leading to its widespread application in scientific research and medical fields. However, traditional HMRS techniques still suffer from low capture efficiency and an inability to simultaneously achieve high specificity and high sensitivity. Common adjustment strategies to improve the enrichment effect of HMRS sequencing on target regions include adjusting hybridization temperature, hybridization time, and buffer solutions. However, all these strategies affect all target regions simultaneously, making it impossible to achieve high capture efficiency, high specificity, and high sensitivity for all target regions. Furthermore, low-complexity target sequences can severely negatively impact probe capture specificity and sensitivity.

[0004] Therefore, there is an urgent need in this field for a rigorous and adjustable target region sequence capture method that can flexibly and uncoupledly control the capture effect of the target region, while taking into account the capture specificity and sensitivity of all target regions. Summary of the Invention

[0005] To address the problems of low capture efficiency, large sequencing data volume, high sequencing cost, and inability to balance capture specificity and sensitivity for all target regions in conventional capture methods, this application proposes a rigorously adjustable probe hybridization capture method.

[0006] The method includes adding a hybridization resolution-enhancing probe to a system in which the probe hybridizes to capture the target region, competitively binding to potential non-specific capture regions, and independently and uncouplingly improving the hybridization capture resolution of the probe for both the target region and non-specific capture regions.

[0007] The hybridization resolution-enhancing probe binds to the potential nonspecific capture region with greater strength than the probe binds to the potential nonspecific capture region in the system.

[0008] The binding strength between the probe and the target capture region is higher than that between the hybridization resolution enhancement probe and the target capture region.

[0009] The probes and hybridization resolution-enhancing probes include nucleic acids or nucleic acid analogs.

[0010] The stringency adjustment includes adjusting the overall stringency of probe capture and / or adjusting the stringency of specific capture areas of the probe locally.

[0011] The method specifically includes the following steps: 1) extracting genomic DNA from the sample and constructing a whole-genome library; 2) hybridizing the whole-genome library obtained in step 1) with a probe of the target region sequence and a hybridization resolution enhancement probe at a first temperature; 3) incubating the reaction system of step 2) with a solid-phase carrier to obtain a solid-phase carrier with hybridization products; 4) washing the solid-phase carrier with hybridization products obtained in step 3) in a washing solution at a second temperature to remove non-target libraries; 5) performing an amplification reaction on the hybridization products obtained in step 4); 6) purification.

[0012] Preferably, the solid support comprises a modified solid support having reactive sites and sufficient connection points.

[0013] The probe contains a nucleic acid or nucleic acid analogue that can bind to the solid-phase carrier, while the hybridization resolution enhancement probe does not contain a marker that can bind to the solid-phase carrier.

[0014] The marker includes one of biotin, digoxigenin, isotopes, or antibodies.

[0015] In another aspect of this application, a hybridization resolution-enhanced probe is provided, wherein the binding strength of the hybridization resolution-enhanced probe to a potential target region is much lower than the binding strength of the probe to the target region.

[0016] The preparation method of the hybridization resolution enhancement probe includes: (1) Calculation: Calculate the binding strength of the probe to the specific capture region and the probe to the potential non-specific capture region, respectively denoted as T. m1 T m4 The specific capture regions of the hybridization resolution enhancement probe and its corresponding probe are calculated, and the binding strengths of the potential non-specific capture regions of the hybridization resolution enhancement probe and its corresponding probe are denoted as T. m2 T m3(2) Design: Based on the calculation results, a hybridization resolution-enhancing probe is designed such that its binding strength with the probe's specific capture region is lower than the binding strength between the probe and the probe's specific capture region; and the binding strength of the hybridization resolution-enhancing probe with the probe's potential non-specific capture region is higher than the binding strength between the probe and the probe's potential non-specific capture region. That is, the design conforms to T m1 >T m2 T m3 >T m4 (3) Synthesis of hybridization resolution enhancement probes.

[0017] The calculation method includes methods commonly used in the art, which are not limited in this application. The calculation method based on base stacking force and base proximity model is used as an example.

[0018] The calculation process includes calculating the effects of base mismatch, base mutation type, base mutation position, and incorporation of base analogs on binding force in the optimal hybridization system.

[0019] The synthesis steps include methods commonly used in the art for synthesizing nucleic acids or nucleic acid analogs, and this application does not limit them.

[0020] The hybridization resolution-enhancing probe comprises a nucleic acid or nucleic acid analog fragment with a length greater than 5 nt.

[0021] Preferably, the hybridization resolution-enhancing probe comprises a nucleic acid or nucleic acid analog fragment with a length between 50 nt and 200 nt.

[0022] The beneficial effects of the present invention include: (1) allowing independent and uncoupling adjustment of the capture efficiency, specificity and sensitivity of the probe target region; (2) achieving high capture efficiency of the target region, reducing the amount of sequencing data and saving sequencing costs. Attached Figure Description

[0024] Figure 1 The diagrams show a comparison between the conventional capture method and the probe hybridization capture method with adjustable rigor. (a) is a schematic diagram of the conventional capture method, and (b) is a schematic diagram of the probe hybridization capture method with adjustable rigor.

[0025] The labels in the figure mean: P represents a probe, T represents a target sequence, T' represents a non-target sequence, P' represents a hybridization resolution enhancement probe; T m1 Indicates the binding ability of PT products; T m2 Indicates the binding affinity of P'T products; T m3 Indicates the binding affinity of P'T' products; T m4 This indicates the binding ability of PT' products. Detailed Implementation

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0027] In this article, the term "probe" refers to a single-stranded oligonucleotide that is typically used to detect a target sequence complementary to the probe's sequence, and also encompasses nucleic acids with different backbone compositions, such as, but not limited to, peptide nucleic acids (PNA), locked nucleic acids (LNA), diol nucleic acids (GNA), and threonine nucleic acids (TNA).

[0028] In this article, the term "solid support" includes, but is not limited to, supports made of materials such as silica gel, polystyrene, and polyacrylamide.

[0029] In this document, the term "hybridization resolution enhancement probe" refers to a substance that can bind to nucleic acid sequences. This includes, but is not limited to, linear polynucleotide fragments composed of nucleotide residues linked by phosphodiester bonds. Hybridization resolution enhancement probes may contain one or more non-standard nucleotides, nucleotide analogs and / or modified nucleotides, as well as other substances that can bind to nucleic acid sequences.

[0030] On one hand, the present invention provides a method for target region capture with adjustable rigor. The method includes adding a hybridization resolution-enhancing probe to a system in which probes hybridize to capture the target region, competitively binding to potential non-specific capture regions, and independently and uncouplingly improving the hybridization capture resolution of the probe for both the target region and non-specific capture regions.

[0031] The method is characterized in that the binding strength between the hybridization resolution-enhancing probe and the potential nonspecific capture region is higher than the binding strength between the probe and the potential nonspecific capture region in the system.

[0032] The binding strength between the probe and the target capture region is higher than that between the hybridization resolution enhancement probe and the target capture region.

[0033] The probes and hybridization resolution-enhancing probes include nucleic acids or nucleic acid analogs.

[0034] The stringency adjustment includes adjusting the overall stringency of probe capture and / or adjusting the stringency of specific capture areas of the probe locally.

[0035] The method specifically includes the following steps: 1) extracting genomic DNA from the sample and constructing a whole-genome library; 2) hybridizing the whole-genome library obtained in step 1) with a probe of the target region sequence and adding a hybridization resolution enhancement probe at a first temperature; 3) incubating the reaction system of step 2) with a solid-phase carrier to obtain a solid-phase carrier with hybridization products; 4) washing the solid-phase carrier with hybridization products obtained in step 3) in a washing solution at a second temperature to remove non-target libraries; 5) performing an amplification reaction on the hybridization products obtained in step 4); 6) purifying to obtain the target region sequence.

[0036] The solid-phase carrier for capture includes carriers commonly used in the art, and this application does not limit this to any particular carrier; magnetic beads are used as an example.

[0037] Preferably, the solid support comprises a modified solid support having reactive sites and sufficient connection points.

[0038] The probe includes nucleic acids or nucleic acid analogs containing markers that can bind to a solid-phase carrier.

[0039] The hybridization resolution-enhancing probe does not contain nucleic acids or nucleic acid analogs that can bind to a solid-phase carrier.

[0040] The marker includes one of biotin, digoxigenin, isotopes, or antibodies.

[0041] The target region sequence refers to the genome sequence that needs to be detected in scenarios such as genomics research, disease diagnosis and prevention, and genetic research. For example, in the clinical treatment of patients with congenital achondroplasia, it is necessary to detect the mutation status of the FGFR3 gene to guide medication. In this case, the exon region of the FGFR3 gene is the target region sequence.

[0042] The attached diagrams compare and contrast conventional capture methods with controllable and rigorous probe hybridization capture methods. Here, P represents the probe, T represents the target sequence, T' represents the non-target sequence bound by the probe, and P' represents a hybridization resolution-enhancing probe. In the conventional capture method, the probe binds to the target sequence to form PT, and the probe binds to the non-target sequence to form PT'. x is the probe number, and there are a total of x probe sequences. The diagrams also show the average capture efficiency C1 and the capture efficiency Cs of a single probe in the conventional capture method. x The calculation formula is as follows:

[0043] (1), (2),

[0044] Among them PT, PT x PT`, PT` x This indicates the capture depth of the hybrid product.

[0045] A controllable and rigorous probe hybridization capture method adds hybridization resolution-enhancing probes to competitively bind potential non-target sequences, reducing hybridization of non-target sequences and improving probe capture efficiency, thereby increasing the average capture efficiency of the probes. Here, y represents the number of the hybridization resolution-enhancing probe. Adding hybridization resolution-enhancing probes produces four products: PT product (complete match, denoted by Tm1); P'T product (Tm2); P'T' product (Tm3); and PT' product (Tm4). The binding abilities of the four products are Tm1 > Tm2, ​​and Tm3 > Tm4. The system rigor of the controllable and rigorous target region capture method lies between Tm1 and Tm3 and between Tm2 and Tm4; therefore, the capture products in the region with added hybridization resolution-enhancing probes are mainly PT and P'T', while PT' and P'T are negligible. The capture efficiency C of adding hybridization resolution-enhancing probes... y The calculation formula is as follows:

[0046] (3),

[0047] Among them PT x PT` x PT represents the capture depth of hybridization products in a conventional hybridization capture system. y P'T y P'T' y This indicates a probe hybridization capture method with adjustable rigor, where the capture depth of hybridization products is enhanced by adding hybridization resolution.

[0048] Hybridization resolution-enhanced probes can adjust the capture efficiency of the entire target region or a portion of the target region. Based on whether it is modulated by the hybridization resolution-enhanced probe, the probe is divided into two parts: the modulated part is y, and the unmodulated part is z. These two parts together constitute the entire probe x. The probe capture efficiency of the rigorously adjustable probe hybridization capture method is C², calculated using the following formula:

[0049] (4),

[0050] Among them PT x PT` x P`T` represents the capture depth of the probe. y P'T' represents the capture depth of the hybridization resolution-enhancing probe. yz This indicates the capture depth of the probe affected by the hybridization resolution enhancement probe.

[0051] When C y > C x，C2 > C1. A hybridization resolution-enhancing probe is added to adjust the probe's capture efficiency through uncoupling. By improving the overall or partial probe capture efficiency, the average capture efficiency of the probe can be increased, achieving high capture efficiency while preserving the entire target region.

[0052] On the other hand, the present invention provides a method for preparing the hybridization resolution-enhancing probe.

[0053] The preparation method of the hybridization resolution enhancement probe includes: (1) Calculation: Calculate the binding strength of the probe to the specific capture region and the probe to the potential non-specific capture region, respectively denoted as T. m1 T m4 The specific capture regions of the hybridization resolution enhancement probe and its corresponding probe are calculated, and the binding strengths of the potential non-specific capture regions of the hybridization resolution enhancement probe and its corresponding probe are denoted as T. m2 T m3 (2) Design: Design hybridization resolution-enhancing probes such that the binding strength between the hybridization resolution-enhancing probe and the probe's specific capture region is lower than the binding strength between the probe and the probe's specific capture region; and the binding strength between the hybridization resolution-enhancing probe and the probe's potential non-specific capture region is higher than the binding strength between the probe and the probe's potential non-specific capture region. That is, design hybridization resolution-enhancing probes that meet the conditions Tm1>Tm2, Tm3>Tm4; (3) Synthesis.

[0054] This application does not specifically limit the calculation methods and synthesis methods involved in the preparation method. In practice, the calculation method based on base stacking force and base proximity model and the in situ synthesis method of oligonucleotides are used as examples.

[0055] The capture effectiveness of the capture protocol was evaluated by sequencing the captured library and analyzing the data using the same data analysis methods, comparing the capture efficiency and the sequencing depth of the target and non-target regions.

[0056] In this invention, all reagents used in the capture experiments of the embodiments are from iGeneTech. There are no special restrictions on the source of the purified magnetic beads and the library quantification kit; any quantification kit well-known to those skilled in the art can be used.

[0057] Example 1: Hybridization Resolution Enhancement Probe Design

[0058] 1. Calculation

[0059] Twenty human genomic loci were selected as target regions. Based on the total nucleic acid pool sequence and the target region sequence to be captured, the binding affinity of the probe to specific capture regions and the probe to potential non-specific capture regions was calculated using the Tbinding-proximity model, based on base stacking force and base proximity. mThe ability of a probe to bind to a target region is defined as T. m1x The ability of a probe to bind to a non-target region is defined as T. m4x The binding product of probe (P) with the target region (T) is PT, and the binding product of probe (P) with the non-target region (T') is PT'.

[0060] Based on the binding affinity T between the probe and the target region and potential non-target regions in the total nucleic acid pool m1 and T m4 Calculate the binding force T between the desired hybridization resolution-enhancing probe (P') and the potential nonspecific capture region (T') product P'T'. m3 And the binding force T between the hybridization resolution-enhancing probe (P') and the target region (T) product P'T. m2 The range of bonding forces.

[0061] Based on base stacking forces and base proximity models, the effects of base mismatch, base mutation type, base mutation location, and incorporation of base analogs (such as 5-bromouracil, 2-aminopurine, 6-bromouracil, and peptide nucleic acids) on binding force in the optimal hybridization system are calculated and used as parameters for designing hybridization resolution-enhancing probes. A binding force T is constrained. m1 Greater than T m2 T m3 Greater than T m4 Based on the parameters calculated above, the optimal hybridization resolution enhanced probe is designed. The length of the optimal hybridization resolution enhanced probe can be 5 nt-500 nt or even higher. The binding ability of the optimal hybridization resolution enhanced probe to the potential nonspecific capture region (T') is 2-50℃ higher or even higher than the binding ability of the probe to T' to achieve the best competitive effect. At the same time, the binding force of the hybridization resolution enhanced probe to the target region (T) is the weakest, thus completing the design of the hybridization resolution enhanced probe.

[0062] Using the hybridization resolution enhancement probe design method described in this invention, the target region sequences and potential non-target regions of 20 probes (Seq ID No. 1 to Seq ID No. 20) were predicted. Based on the design principles of the hybridization resolution enhancement probes described above, 20 corresponding hybridization resolution enhancement probes (Seq ID No. 21 to Seq ID No. 40) were designed. The binding strength of the probe to the target region sequence hybridization product Probe-Target is higher than that of the hybridization resolution enhancement probe to the target region sequence hybridization product Eprobe-Target. The binding strength of the hybridization resolution enhancement probe to the potential non-target region hybridization product Eprobe-unTarget is higher than that of the probe to the potential non-target region hybridization product Probe-unTarget (see Table 1).

[0063] Table 1 Results of hybridization resolution enhancement probe design

[0064]

[0065] Example 2 Synthesis of Hybridization Resolution Enhancement Probes

[0066] The specific experimental steps for probe synthesis are as follows:

[0067] 1) Targeting the coding sequence of the gene, starting from the first base, synthesize probes from 5' to 3' in the direction of reverse sequence complementation, based on the designed hybridization resolution enhancement probe sequence;

[0068] 2) Add primers with the sequence Seq ID No. 41 and Seq ID No. 42 to the 5' and 3' ends of each hybridization resolution enhancement probe sequence, respectively, to form a list of hybridization resolution enhancement probe sets with the same sequence at both ends;

[0069] 3) Oligonucleotides were synthesized on a large scale on a chip using in situ oligonucleotide synthesis technology, using sequences from the hybridization resolution enhancement probe sequence list mentioned above;

[0070] 4) Elute the oligonucleotides from the chip to form an oligonucleotide mixture;

[0071] 5) Using an oligonucleotide mixture as a template, and primers with the sequence Seq ID No. 41 and the sequence Seq ID No. 42 as primers, a polymerase chain reaction was performed using Taq polymerase to amplify the double-stranded DNA pool.

[0072] 6) Use a PCR purification kit to purify the PCR product according to its instructions to obtain a purified double-stranded DNA pool.

[0073] 7) Using 1 µL of purified double-stranded DNA pool as a template, and using primers with the sequences Seq ID No 43 and Seq ID No 44, polymerase chain reaction (PCR) amplification was performed using Taq polymerase to form a double-stranded DNA pool with the T7 sequence.

[0074] 8) The PCR product from the previous step was separated by gel electrophoresis to remove non-specific bands, and the 170-180 bp region fragment was recovered and purified using a gel extraction kit.

[0075] 9) Using the T7 large-scale RNA transcription kit and dNTPs as substrates, the purified product from the previous gel extraction step was transcribed in vitro to prepare a ribonucleic acid hybridization resolution-enhanced probe set. The reagents used are shown in Table 2.

[0076] Table 2. Reagents for synthesizing hybridization resolution-enhancing probes

[0077] Reagent Name Volume (µL) ATP (10 mmol / L) 2 CTP (10 mmol / L) 2 GTP (10 mmol / L) 2 UTP (10 mmol / L) 2 Reaction buffer (10×) 2 Gel recovery and purification products with T7 sequence 10

[0078] Incubate at 37℃ for 8-12 hours to obtain the highest yield of ribonucleic acid hybridization resolution-enhanced probe set, and store in a -80℃ freezer.

[0079] Example 3: Construction of a genomic library

[0080] Human whole genome libraries were constructed by loading 200 ng of human genomic DNA samples.

[0081] Example 4: Conventional Capture Experiment

[0082] Step 1. Hybridization reaction

[0083] The human whole genome library constructed in Example 3, the probes for the target region obtained, and the optimal hybridization reaction solution were mixed to prepare a hybridization reaction system. Hybridization reaction conditions: 5 min at 80°C, followed by 4 h at 50°C.

[0084] Step 2: Combining the capture magnetic bead with the probe

[0085] (1) The capture magnetic beads modified with streptavidin were taken out in advance and placed at room temperature for 30 min to equilibrate. The supernatant was then removed by placing them on a magnetic rack.

[0086] (2) Wash the magnetic beads with a binding reagent three times. Resuspend the magnetic beads with the binding reagent to complete the preparation for capturing the magnetic beads.

[0087] (3) Add the prepared capture magnetic beads to the hybridization product obtained from the above steps and mix thoroughly. Rotate slowly at room temperature for 30 min to fully combine.

[0088] Step 3: Wash the captured magnetic beads.

[0089] (1) Place the magnetic bead binding product obtained above on a magnetic rack for 3 min to bind, and then discard the supernatant.

[0090] (2) Resuspend the magnetic beads in washing solution and wash them at 50°C for 10 min. Repeat the washing process 3 times. Place the magnetic beads on a magnetic rack for 3 min and then completely discard the supernatant.

[0091] Step 4: Amplification Reaction

[0092] (1) Prepare the amplification reaction system (including amplification primers and amplification enzymes, etc.), add the above magnetic beads for magnetic bead resuspending, and the total system is 50 μL;

[0093] (2) The above amplification system was placed on a PCR instrument and the PCR amplification program was run: 95℃ for 1 min; then denatured at 95℃ for 30 s, annealed at 60℃ for 20 s, and extended at 72℃ for 30 s, for a total of 14 cycles. After the last cycle, it was incubated at 72℃ for 2 min to ensure sufficient extension.

[0094] Step 5: Magnetic Bead Recycling

[0095] (1) Take out the purified magnetic beads, mix them evenly, and equilibrate at room temperature for 30 min;

[0096] (2) After the amplification reaction is complete, add 55 μL of purified magnetic beads to the amplification product and mix thoroughly. Let stand at room temperature for 5 min.

[0097] (3) Centrifuge briefly, place it on a magnetic rack for 3 min, wait for the solution to become clear, and discard the supernatant;

[0098] (4) Add 200 μL of 80% ethanol solution to the PCR tube and let it stand for 30 s;

[0099] (5) Keep the PCR tube on the magnetic rack, discard the supernatant, wash once with 80% ethanol, and keep it on the magnetic rack to completely discard the supernatant;

[0100] (6) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 5 minutes to dry the magnetic beads;

[0101] (7) Add 25 μL Nuclease Free Water, remove the PCR tube from the magnetic rack, mix well by pipetting, and let stand at room temperature for 2 min;

[0102] (8) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, wait for the solution to become clear, aspirate the supernatant, transfer it to a new PCR tube, and obtain the capture library;

[0103] (9) Take 1 μL of the library for quantification and record the library concentration;

[0104] (10) Take 1 μL of the library for fragment quality control. The library length is approximately 220-320 bp.

[0105] Example 5: A probe hybridization capture method with adjustable rigor

[0106] Step 1. Hybridization reaction

[0107] The human whole genome library constructed in Example 3, the probes for the target region prepared, and the hybridization resolution-enhancing probes designed according to the method in Example 1 and prepared according to the method in Example 2 were mixed with the optimal hybridization reaction solution to form a hybridization reaction system. Hybridization reaction conditions: 5 min at 80°C, followed by 4 h at 50°C.

[0108] Step 2: Combining the capture magnetic bead with the probe

[0109] (1) The capture magnetic beads modified with streptavidin were taken out in advance and placed at room temperature for 30 min to equilibrate. The supernatant was then removed by placing them on a magnetic rack.

[0110] (2) Wash the magnetic beads with a binding reagent three times. Resuspend the magnetic beads with the binding reagent to complete the preparation for capturing the magnetic beads.

[0111] (3) Add the prepared capture magnetic beads to the hybridization product obtained from the above steps and mix thoroughly. Rotate slowly at room temperature for 30 minutes to fully combine.

[0112] Step 3: Wash the captured magnetic beads.

[0113] (1) Place the magnetic bead binding product obtained above on a magnetic rack for 3 minutes to bind, and then discard the supernatant.

[0114] (2) Resuspend the magnetic beads in washing solution and wash them at 50°C for 10 min. Repeat the washing process 3 times. Place the magnetic beads on a magnetic rack for 3 min and then completely remove the supernatant.

[0115] Step 4: Amplification Reaction

[0116] (1) Prepare the amplification reaction system (including amplification primers and amplification enzymes, etc.), add the above magnetic beads for magnetic bead resuspending, and the total system is 50 μL;

[0117] (2) The above amplification system was placed on a PCR instrument and the PCR amplification program was run: 95℃ for 1 min; then denatured at 95℃ for 30 s, annealed at 60℃ for 20 s, and extended at 72℃ for 30 s, for a total of 14 cycles. After the last cycle, it was incubated at 72℃ for 2 min to ensure sufficient extension.

[0118] Step 5: Magnetic Bead Recycling

[0119] (1) Take out the purified magnetic beads, mix them evenly, and equilibrate at room temperature for 30 min;

[0120] (2) After the amplification reaction is complete, add 55 μL of purified magnetic beads to the amplification product and mix thoroughly. Let stand at room temperature for 5 min.

[0121] (3) Centrifuge briefly, place it on a magnetic rack for 3 min, wait for the solution to become clear, and discard the supernatant;

[0122] (4) Add 200 μL of 80% ethanol solution to the PCR tube and let it stand for 30 s;

[0123] (5) Keep the PCR tube on the magnetic rack, discard the supernatant, repeat the washing with 80% ethanol once, and keep it on the magnetic rack to completely discard the supernatant;

[0124] (6) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 5 minutes to dry the magnetic beads;

[0125] (7) Add 25 μL Nuclease Free Water, remove the PCR tube from the magnetic rack, mix well by pipetting, and let stand at room temperature for 2 min;

[0126] (8) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, wait for the solution to become clear, aspirate the supernatant, transfer it to a new PCR tube, and obtain the capture library;

[0127] (9) Take 1 μL of the library for quantification and record the library concentration;

[0128] (10) Take 1 μL of the library for fragment quality control. The library length is approximately 220-320 bp.

[0129] Results Analysis

[0130] Both capture schemes in Examples 4 and 5 used human whole-genome libraries prepared in Example 3, and the probes were also from the same batch. The steps, reagents, consumables, and instruments used in both capture methods were identical, and the experiments were conducted using the same batch. The probes were biotin-labeled for subsequent binding to the capture beads, while the hybridization resolution enhancement probes were not biotin-labeled or otherwise labeled for binding to the capture beads. The only difference between the two capture methods was the composition of the hybridization capture system. Example 5 presented a controlled and rigorous probe hybridization capture method system, which, compared to the conventional capture method used in Example 4, added a hybridization resolution enhancement probe designed in Example 1 and synthesized in Example 2, while keeping other components of the system unchanged.

[0131] The capture libraries prepared by the two experimental methods were sequenced, and the data were analyzed using the same data analysis method to compare the sequencing depth of the target region and the non-target region. The results are shown in Table 2. In Example 4, the conventional capture method showed a certain capture depth in the non-target region for different probes, ranging from 250 to 490, while the capture depth in the target region ranged from 500 to 950. The difference between the capture depth of the non-target region and the target region was not obvious. However, in Example 5, the controllable and rigorous probe hybridization capture method added a hybridization resolution enhancement probe to the system. The capture depth in the target region was not less than 750, and the deepest was 1176. The overall capture depth in the target region was higher than that in Example 4. The capture depth in the non-target region was effectively reduced to between 4 and 40, which was much lower than that in Example 4. The capture depth in the non-target region and the target region of the method in Example 5 differed by almost two orders of magnitude, and the resolution was significantly higher than that in Example 4. It achieved flexible and uncoupled adjustment of the probe hybridization capture effect.

[0132] Table 3. Comparison of capture library data constructed using conventional capture methods and rigorous probe hybridization capture methods.

[0133]

[0134] The capture methods of Examples 4 and 5 were repeated four times each. The steps, reagents, consumables, and instruments used were kept consistent, and the experiments were conducted on the same batch to verify the capture efficiency of the two methods. Referring to Equations 1 to 4, the capture efficiency of the two methods was calculated, and the results are shown in Table 3. The capture rate of the method used in Example 5 was approximately 60%, with a mean of 59.62% and a standard deviation of approximately 0.4%; the capture rate of the method used in Example 4 was approximately 29%, with a mean of 29.51% and a standard deviation of approximately 0.4%. The stability of the capture rates of the two methods was basically the same, but the capture efficiency of the more rigorous and controllable method was higher than that of the conventional capture method.

[0135] Table 4. Comparison of capture efficiency between conventional capture methods and rigorously adjustable probe hybridization capture methods.

[0136] Serial Number Conventional capture method Rigor-adjustable probe hybridization capture method 1 29.42% 59.77% 2 29.99% 60.01% 3 28.9% 58.87% 4 29.74% 59.85%

[0137] In summary, the rigorous and adjustable capture method, by adding hybridization resolution-enhancing probes to the capture system, independently and decoupledly improves the capture depth of the target capture region, increases the capture efficiency of the target region, reduces the amount of sequencing data, and saves sequencing costs.

[0138] The above embodiments are only used to help understand the methods, principles and core ideas of the present invention; for those skilled in the art, there will be changes in specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A probe hybridization capture method with adjustable rigor, characterized in that, By adding hybridization resolution-enhancing probes to the probe hybridization capture system, the capture rigor of different regions can be adjusted independently and uncoupled.

2. The method according to claim 1, characterized in that, The hybridization resolution-enhancing probe binds to the potential nonspecific capture region with greater strength than the probe binds to the potential nonspecific capture region in the system.

3. The method according to claim 1, wherein the binding strength between the probe and the target capture region is higher than the binding strength between the hybridization resolution enhancement probe and the target capture region.

4. The method according to claim 1, characterized in that, The probes and hybridization resolution-enhancing probes include nucleic acids or nucleic acid analogs.

5. The method according to claim 1, specifically comprising the following steps: 1) Extract genomic DNA from the sample and construct a whole genome library; 2) Hybridize the whole genome library with probes of the target region sequence and hybridization resolution enhancement probes; 3) Incubate the hybridization reaction system of step 2) with a solid-phase vector to obtain a solid-phase vector with hybridization products; 4) Wash the solid-phase vector with hybridization products obtained in step 3) in washing solution to remove non-target libraries; 5) Perform an amplification reaction on the product obtained in step 4); 6) Purify.

6. The method according to claim 5, wherein the solid support comprises a solid support having reaction sites and providing sufficient connection points.

7. The method of claim 5, wherein the probe comprises a marker that can bind to a solid support, and the hybridization resolution enhancement probe does not comprise a marker that binds to a solid support.

8. The method according to claim 7, wherein the marker comprises one of biotin, digoxigenin, isotopes, or antibodies.

9. The method of claim 7, wherein the location of the marker includes the 3' end, 5' end, or interior of the probe.

10. A hybridization resolution-enhancing probe, characterized in that, The hybridization resolution-enhancing probe competitively binds to potential non-specific capture regions during the target region capture process.

11. The hybridization resolution-enhancing probe according to claim 10 has a binding capacity to potential non-target regions that is 2-50° higher than the binding capacity of the probe to non-target regions.

12. The hybridization resolution-enhancing probe according to claim 10, wherein the preparation method comprises: 1) Calculation: Calculate the binding strength of the probe to the specific capture region and the probe to the potential non-specific capture region, and denote them as Tm1 and Tm4, respectively. Calculate the binding strength of the hybridization resolution enhancement probe to the specific capture region of the corresponding probe and the binding strength of the hybridization resolution enhancement probe to the potential non-specific capture region of the corresponding probe, and denote them as Tm2 and Tm3, respectively. (2) Design: Design a hybridization resolution enhancement probe based on the calculation results, such that the binding strength of the hybridization resolution enhancement probe to the specific capture region of the probe is lower than the binding strength of the probe to the specific capture region of the probe; and the binding strength of the hybridization resolution enhancement probe to the potential non-specific capture region of the probe is higher than the binding strength of the probe to the potential non-specific capture region of the probe. That is, design a hybridization resolution enhancement probe that meets the conditions Tm1>Tm2 and Tm3>Tm4. (3) Synthesis.

13. The hybridization resolution enhancement probe according to claim 10, wherein the calculation process includes calculating the effects of base mismatch, base mutation type, base mutation position, and incorporation of base analogs on binding force in the optimal hybridization system.

14. The hybridization resolution enhancement probe according to claim 10, comprising a nucleic acid or nucleic acid analog fragment with a length greater than 5 nt, preferably, the length of the hybridization resolution enhancement probe is 50 nt-200 nt.

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