A targeted sequencing method and kit

Abstract

The present invention discloses a targeted sequencing method and a kit. The present invention greatly shortens the hybridization time by adding compounds and hybridization promoters. On the basis of maintaining high specificity and uniformity, the denaturation time is reduced from the traditional 10 minutes to 30 seconds, the hybridization time is reduced from more than 16 hours to 0.5 hours, and the probe enrichment time is shortened from 45 minutes to 20 minutes.

Description

[0001] This application is a divisional application with an application date of August 31, 2020, application number CN202010895989.2, and invention name as "Method and kit for rapid hybridization of probes to libraries." Technical Field

[0002] The present invention relates to the field of gene sequencing, and in particular to a targeted sequencing method and a kit. Background Art

[0003] High-throughput sequencing technology has the advantages of high throughput and high accuracy, and is gradually changing the traditional clinical diagnosis field, including auxiliary diagnosis of genetic diseases, companion diagnosis of tumor drugs, and pathogen detection. Among high-throughput sequencing technologies, targeted sequencing technology is one of the most widely used technologies, with multiple characteristics such as accuracy and economy.

[0004] Targeted sequencing technologies are divided into three categories, including amplicon technology based on the PCR principle, capture technology based on the hybridization principle, and molecular inversion probe technology (MIP) based on the hybridization, extension and connection principles. The above methods have their own application scenarios. Amplicon is limited by coverage uniformity and is suitable for targeted sequencing of smaller target areas, while Capture is not limited by the size of the target area and has been widely used in tumor companion diagnosis, non-invasive single gene disease diagnosis, pathogen detection and other fields.

[0005] Capture technology also has certain limitations, such as cumbersome operation and long experimental cycle. In the traditional experimental process of capture, the hybridization time of probe / library is more than 16 hours, accounting for more than 50% of the capture process; therefore, further shortening the denaturation, hybridization and probe enrichment time will fundamentally improve the timeliness of capture technology and better meet the needs of clinical diagnosis.

[0006] The occurrence of tumors is due to the accumulation of a series of gene mutations, which in turn lead to signal pathway disorders in cells, errors in the cell division cycle, and ultimately lead to malignant cell proliferation. Clinically, it is necessary to select drugs or therapies according to the individual patient's gene mutation status throughout the diagnosis and treatment process, which is companion diagnosis. Capture technology based on second-generation high-throughput sequencing has been widely used in clinical-level tumor gene companion diagnosis and has been approved by relevant departments, but the entire detection process is still relatively long. Therefore, optimizing the hybridization process of capture technology so that it can quickly complete the detection while maintaining detection performance has practical significance for further optimizing tumor gene companion diagnosis in the future. Summary of the invention

[0007] In view of the technical problems existing in the prior art, the present invention greatly shortens the hybridization time by adding compounds and hybridization promoters while maintaining high specificity and uniformity. Specifically, the present invention includes the following contents.

[0008] In a first aspect of the present invention, a method for rapid hybridization of a probe to a library is provided, comprising the following steps:

[0009] (1) contacting the probe with the library in a hybridization buffer;

[0010] (2) After the probe binds to the library to form a complex, the complex is captured by streptavidin using magnetic beads that have been washed and activated with a magnetic bead washing buffer;

[0011] (3) washing the complex bound to streptavidin using a washing solution;

[0012] (4) The target library bound to the probe is enriched by PCR reaction, then purified and subsequently subjected to high-throughput sequencing.

[0013] According to the method for rapid hybridization of a probe to a library of the present invention, preferably, the probe is added to the hybridization buffer in advance, rather than adding the hybridization buffer after the library is denatured.

[0014] According to the method for rapid hybridization of a probe with a library of the present invention, preferably, the denaturation temperature of the library and the probe is 92-98°C, such as 93, 95 or 97°C, and the denaturation time is 30 seconds to 2 minutes, such as 1 minute, preferably 2 minutes.

[0015] According to the method for rapid hybridization of a probe to a library of the present invention, preferably, the probe is a 5' biotin-modified single-stranded DNA with a length of 110-130 nt, preferably 120 nt.

[0016] According to the method for rapid hybridization of a probe with a library of the present invention, preferably, the hybridization buffer comprises sodium citrate buffer, 0.8-1.2M betaine, 0.8-1.2M tetramethylammonium chloride, 4-6% weight / volume ratio of dextran sulfate, and 18-44% by volume of formamide. Preferably, the sodium citrate buffer is 1X, the betaine is 1M, the tetramethylammonium chloride is 1M, the concentration of dextran sulfate is 5% weight / volume ratio, and the formamide is 20% by volume.

[0017] According to the method for rapid hybridization of a probe with a library of the present invention, preferably, the hybridization binding time of the probe with the library in the hybridization buffer is 0.5-4 hours, preferably 0.5 hours.

[0018] According to the method for rapid hybridization of a probe with a library of the present invention, preferably, in step (3), the components of the magnetic bead washing buffer include 3M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA and 0.1% Tween-20; and after washing, the streptavidin magnetic beads remain in the magnetic bead washing buffer without discarding the magnetic bead washing buffer. This formula facilitates the implementation of automated washing processes. For example, when the magnetic beads are kept in the magnetic bead washing buffer, they are not prone to drying and becoming ineffective; at the same time, liquid magnetic beads and a larger volume of reaction solution facilitate the process setting of the pipetting workstation, and the workflow is more stable.

[0019] According to the method for rapid hybridization of a probe with a library of the present invention, preferably, the cleaning solution comprises: cleaning solution S: 1X SSC, 0.1% Tween-20; cleaning solution I: 1X SSC, 0.1% sodium dodecyl sulfate (SDS); cleaning solution II: 0.5X SSC; cleaning solution III: 0.2X SSC.

[0020] The method for rapid hybridization of a probe with a library according to the present invention preferably comprises the following steps in sequence:

[0021] (a) mixing a pre-library, a linker blocker and a probe, and then evaporating to dryness, wherein the pre-library includes a pre-library suitable for MGI sequencers (MGISEQ-2000, MGISEQ-200, etc.), Illumina sequencers (Nextseq, Miseq and Miniseq, etc.), and Life sequencers (Ion Torrent PGM, etc.);

[0022] (b) adding a hybridization buffer to the product of step (a), mixing and incubating at room temperature for 5 minutes, followed by denaturation at 95° C. for 2 minutes and incubation at 65° C. for 30 minutes, wherein the hybridization buffer comprises 1X sodium citrate buffer (SSC), 2M betaine, 1M tetramethylammonium chloride, 5% (w / v) dextran sulfate, and 20% (v / v) formamide;

[0023] (c) After hybridization, the probe was bound to the streptavidin magnetic beads for 15 minutes, and the streptavidin magnetic beads were washed twice with magnetic bead washing buffer and present in 50 μL of magnetic bead washing buffer;

[0024] (d) washing the streptavidin beads in step (c) once at 65° C. using washing solution I at 65° C., with pipetting 10 times or vortex mixing for 3 seconds;

[0025] (e) Wash the beads twice at 65°C using washing solution S at 65°C. Washing conditions: vortex to mix, and incubate at 65°C for 5 minutes.

[0026] (f) Wash the beads by incubating with Wash Solution I at room temperature and vortexing for 2 minutes;

[0027] (g) Wash the beads by incubating with Wash Solution II at room temperature and vortexing for 1 minute;

[0028] (h) Wash the beads by incubating with washing solution III at room temperature and vortexing for 0.5 min;

[0029] (i) PCR enrichment and library purification;

[0030] (j) The purified library was subjected to high-throughput sequencing.

[0031] A second aspect of the present invention provides a kit for rapid hybridization of a probe with a library, the kit comprising the following components:

[0032] Component 1: Hybridization buffer;

[0033] Component 2: Magnetic beads washing buffer;

[0034] Component 3: cleaning solution S;

[0035] Component 4: cleaning solution I;

[0036] Component 5: Cleaning Solution II; and

[0037] Component 6: Cleaning solution III.

[0038] The present invention can be used for rapid hybridization and cleaning of probes / libraries for high-throughput targeted sequencing. By adding compounds and hybridization promoters, the present invention greatly shortens the hybridization time. On the basis of maintaining high specificity and uniformity, the denaturation time is reduced from the traditional 10 minutes to 30 seconds, the hybridization time is reduced from more than 16 hours to 0.5 hours, and the probe enrichment time is shortened from 45 minutes to 20 minutes, and EGFR, BRAF, KARS gene-related mutations can be accurately detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram comparing the hybridization process of the present invention with that of the traditional one. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

[0043] Example 1

[0044] This example is used to study the effect of the concentrations of betaine and tetramethylammonium chloride on short-time hybridization.

[0045] The prelibrary used in this example (insert size: ∼300 bp; PCR cycle number: 7) was constructed from NA12878 gDNA (Coriell) using a DNA library construction kit (Rapid DNA Lib Prep Kit, ABclonal). As shown in Table 1, hybridization was performed for 0.5 hours using hybridization buffers of different formulations according to the AI ​​steps described below (N=2).

[0046] Table 1

[0047]

[0048]

[0049] A. Library Pre-blocking

[0050] A-1. Add the reagents in Table 2 to a 0.2 mL low adsorption centrifuge tube (Eppendorf):

[0051] Table 2

[0052]

[0053] A-2. Evaporate the solution in the centrifuge tube to dryness using a vacuum concentrator (Eppendorf).

[0054] B. Hybridization of probes to libraries

[0055] Add 13 μL of hybridization buffer to the dry powder in step A-2, vortex to mix, and incubate at room temperature for 5 minutes. Denature at 95°C for 10 minutes, then add 4 μL of probe (280 Kb, 3 pmol, BOKE), vortex to mix, and incubate at 65°C for 0.5 hour.

[0056] C. Cleaning Solution Preparation

[0057] Various cleaning solutions were prepared as shown in Table 3, wherein cleaning solution S and part of cleaning solution I were preheated at 65° C. for 30 minutes before use.

[0058] Table 3

[0059]

[0060]

[0061] D. Streptavidin Magnetic Bead Preparation

[0062] D-1. Take the streptavidin magnetic beads out of the refrigerator (4°C) and return them to room temperature (about 30 minutes).

[0063] D-2. Vortex to mix for 15 seconds.

[0064] D-3. Take 100 μL of streptavidin magnetic beads and add them to a new 1.5 mL low-adsorption centrifuge tube.

[0065] D-4. Place the centrifuge tube on a magnetic stand until the solution becomes clear.

[0066] D-5. Aspirate and discard the supernatant without disturbing the magnetic beads.

[0067] D-6. Wash the streptavidin magnetic beads according to the following steps:

[0068] D-6-1. Remove the centrifuge tube from the magnetic stand, add 200 μL of magnetic bead washing buffer, and vortex for 10 seconds.

[0069] D-6-2. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear. Aspirate and discard the supernatant without disturbing the magnetic beads.

[0070] D-7. Repeat step D-6.

[0071] D-8. Remove the centrifuge tube from the magnetic stand and add 100 μL of magnetic bead washing buffer.

[0072] D-9. Transfer 100 μL of magnetic bead resuspension in the centrifuge tube to a new 0.2 mL low-adsorption centrifuge tube (Eppendorf) for later use.

[0073] D-10. Place the centrifuge tube on a magnetic stand until the solution becomes clear.

[0074] D-11. Aspirate and discard the supernatant without disturbing the magnetic beads and proceed to the next experimental steps immediately.

[0075] E. Probe enrichment

[0076] E-1. Add the hybridization mixture (step B-2) to the 0.2 mL low-binding centrifuge tube containing streptavidin magnetic beads (step D-11).

[0077] E-2. Mix by gently pipetting up and down 10 times.

[0078] E-3. Incubate at 65°C for 45 minutes using a PCR instrument (heat lid set to 75°C).

[0079] E-4. Vortex mix for 3 seconds every 12 minutes to ensure that the magnetic beads are in suspension.

[0080] F. Post-capture cleaning

[0081] F-1.65℃ cleaning

[0082] F-1-1. Add 100 μL of preheated washing solution I to the 0.2 mL low-adsorption centrifuge tube containing the hybridization mixture (step E-4).

[0083] F-1-2. After mixing by pipetting, transfer the reaction solution containing streptavidin magnetic beads to a new 1.5 mL low-adsorption centrifuge tube.

[0084] F-1-3. Place the centrifuge tube on a magnetic rack until the solution becomes clear and discard the supernatant.

[0085] F-1-4. Continue cleaning according to the following steps:

[0086] F-1-4-1. Add 200 μL of preheated cleaning solution S, pipette or vortex to mix, and incubate at 65°C for 5 minutes.

[0087] F-1-4-2. Centrifuge briefly and place the centrifuge tube on a magnetic rack until the solution becomes clear and discard the supernatant.

[0088] F-1-5. Repeat step F-1-4.

[0089] F-2. Room temperature cleaning

[0090] F-2-1. Add 200 μL of cleaning solution I and vortex to mix for 2 minutes.

[0091] F-2-2. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0092] F-2-3. Add 200 μL of cleaning solution II and vortex to mix for 1 minute.

[0093] F-2-4. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0094] F-2-5. Add 200 μL of cleaning solution III and vortex to mix for 30 seconds.

[0095] F-2-6. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0096] F-3. Resuspend the magnetic beads.

[0097] F-3-1. Immediately add 20 μL of enzyme-free sterile water.

[0098] F-3-2. Use a pipette to pipette up and down 10 times to resuspend the magnetic beads and proceed to the next experimental steps.

[0099] G. PCR amplification

[0100] G-1. Configure the PCR reaction system according to Table 4.

[0101] Table 4

[0102]

[0103] G-2. Mix by pipetting or vortexing at low speed to keep the magnetic beads suspended and immediately proceed to step G-3.

[0104] G-3. Use the PCR instrument to run according to the program in Table 5, with the heating cover temperature at 105°C.

[0105] Table 5

[0106]

[0107] H. PCR product purification

[0108] H-1. Add 75 μL AMPure XP purification magnetic beads (Beckman) to each PCR tube.

[0109] H-2. Purify the PCR product according to the AMPure XP operating manual.

[0110] H-3. Elution was performed using 22 μL of Tris-HCl (10 mM, pH 8.5).

[0111] H-4. Transfer 20 μL of the eluate containing the captured library to a new 1.5 mL low-binding centrifuge tube (Eppendorf).

[0112] I. Library Quality Control

[0113] I-1. The library concentration was measured using Qubit Fluorometer 3.0 (ThermoFisher).

[0114] I-2. The length of the library fragments was measured using Agilent 2100. The products were concentrated between 200-600 bp and there was no adapter dimerization.

[0115] H. High-throughput sequencing

[0116] Illumina Miniseq sequencer was used for PE150 sequencing.

[0117] Data analysis

[0118] Trimmomatic was used to remove adapters and low-quality sequences to obtain clean data, and then Samtools was used to extract reads of the target region. The on-target rate and average sequencing depth of the target region were counted according to the alignment position. The ratio of the depth of each target region to the average depth was used to calculate the proportion of the corresponding target region to obtain the proportion of target regions at 0.2 and 0.5x the average depth (a measure of capture uniformity).

[0119] As shown in Table 6, compared with the traditional hybridization buffer, the on-target rate was significantly improved by adding dextran sulfate, formamide, and increasing the concentration of betaine, but the target area proportion of 0.2 and 0.5x average depth gradually decreased; after adding tetramethylammonium chloride, as its concentration increased, the on-target rate gradually decreased, and the target area proportion of 0.2 and 0.5x average depth gradually increased. Among them, 2M betaine and 1M tetramethylammonium chloride can obtain the best on-target rate and uniformity.

[0120] Table 6

[0121]

[0122] Example 2

[0123] This example is used to study the effect of the co-denaturation time of the probe and the library on the capture uniformity.

[0124] The prelibrary was constructed using the method of Example 1, and hybridization capture (N=2) was performed using a hybridization reaction solution with a final concentration of 1X SSC, 2M betaine, 1.0M tetramethylammonium chloride, 5% (w / v) dextran sulfate, and 20% (v / v) formamide. The differences were the probe addition method (library denaturation alone, probe / library co-denaturation) and the denaturation time. The experimental design is shown in Table 7.

[0125] As shown in Table 7, as the probe / library co-denaturation time increases, the target rate remains unchanged, while the uniformity decreases significantly. When the co-denaturation time is 0.5 minutes and 2 minutes, the capture uniformity using the co-denaturation method is similar to the data of independent library denaturation, with no significant difference, while when the co-denaturation time is 5 and 10 minutes, the uniformity decreases significantly. The above results show that when the probe / library co-denaturation time is controlled at 0.5-2 minutes, data performance similar to independent library denaturation can be obtained.

[0126] Table 7

[0127]

[0128] Example 3

[0129] This example is used to study the effect of retention of magnetic bead washing buffer on probe enrichment

[0130] The library was denatured for 2 minutes by adding the probe (co-denaturation) before denaturation of the library in Example 2, followed by 0.5 hour hybridization capture (N=2), except that different formulations of magnetic bead washing buffer were used to wash the magnetic beads, and the effects of retaining (traditional solution) and not retaining the magnetic bead washing buffer on the capture data were compared. In the solution of retaining the magnetic bead washing buffer, after the second washing of the magnetic beads, 50 μL of magnetic bead washing buffer was added to resuspend the magnetic beads, and then 17 μL of hybridization reaction solution was added to the above 50 μL buffer containing magnetic beads. Among them, the traditional formula is as follows: 1M NaCl (ThermoFisher), 10mM Tris-HCl pH 7.5 (ThermoFisher), 1mM EDTA (ThermoFisher), 0.1% Tween-20 (v / v, Sigma).

[0131] As shown in Table 8, when the traditional formula of magnetic bead washing solution was retained for probe enrichment, the on-target rate in the capture data increased by about 3 percentage points, but the proportion of target areas at 0.2 and 0.5x average depths decreased by 3 and 4 percentage points, respectively, affecting the capture uniformity. When the NaCl concentration in the magnetic bead washing buffer gradually increased, its uniformity was significantly improved. At 3M NaCl, the target rate and uniformity reached the same level as the traditional solution without retaining the magnetic bead washing solution.

[0132] Table 8

[0133]

[0134] Example 4

[0135] This example is used to study the effect of probe enrichment time on capture data.

[0136] The library was denatured for 2 min by adding probes before denaturation of the library in Example 3 (co-denaturation), followed by 0.5 h of hybridization capture (N=2), and the magnetic beads were washed with magnetic bead washing buffer (3 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% Tween-20) and the magnetic bead washing buffer was retained for probe enrichment, with enrichment times of 5 min, 15 min, 30 min and 45 min, respectively.

[0137] As shown in Table 9, as the enrichment time decreases, the redundancy and on-target rate in the captured data gradually increase, while the uniformity is almost unaffected. When the enrichment time is 15-45 minutes, there is no significant difference in the parameters of the captured data. When the enrichment time is 5 minutes, the redundancy increases to about 7 times that of the 45-minute enrichment, and the sequencing depth decreases by 30%.

[0138] Table 9

[0139]

[0140] Example 4

[0141] This example is used to study the effect of probe enrichment time on capture data.

[0142] The prelibrary used in this example (insert size: ~300 bp; PCR cycle number: 7) was constructed using a DNA standard (Horizon) with known gene mutation frequency and a DNA library construction kit (Rapid DNALibPrep Kit, ABclonal). The gene mutation information is shown in Table 10. Using the traditional hybridization buffer and capture scheme in Example 1 as a control, the hybridization buffer provided by the present invention, the co-denaturation of the probe and the library, and the method of retaining the magnetic bead washing buffer for probe enrichment were used, and hybridization was performed for 0.5 hours according to the AI ​​steps described below (N=3)

[0143] Table 10

[0144] Gene mutation frequency AKT1 p.E17K 1.33％ BRAF p.V600E 2.42％ EGFR p.745_750del 0.91％ EGFR p.V769delinsVASV 1.05％ PIK3CA p.E545K 1.30％ PIK3CA p.H1047R 1.30％

[0145] As shown in Table 11, the traditional hybridization buffer and capture scheme in Example 1 were used as controls, and the hybridization buffer provided by the present invention, the probe and library co-denaturation, and the magnetic bead washing buffer were retained for probe enrichment. Hybridization was performed for 0.5 hours according to the AI ​​steps described below (N=3).

[0146] Table 11

[0147]

[0148] A. Library Pre-blocking

[0149] A-1. Add the reagents in Table 12 to a 0.2 mL low adsorption centrifuge tube (Eppendorf):

[0150] Table 12

[0151]

[0152] A-2. Evaporate the solution in the centrifuge tube to dryness using a vacuum concentrator (Eppendorf).

[0153] B. Hybridization of probes to libraries

[0154] Add 17 μL of hybridization buffer (1X SSC, 2M betaine, 1.0M tetramethylammonium chloride, 5% (w / v) dextran sulfate, 20% (v / v) formamide) to the dry powder in step A-2, vortex to mix, and incubate at room temperature for 5 minutes. Then denature at 95°C for 2 minutes and incubate at 65°C for 0.5 hours.

[0155] C. Preparation of cleaning solution

[0156] The cleaning solutions were prepared as shown in Table 13, wherein cleaning solution S and part of cleaning solution I were preheated at 65° C. for 30 minutes before use.

[0157] Table 13

[0158]

[0159] D. Streptavidin Magnetic Bead Preparation

[0160] D-1. Take the streptavidin magnetic beads out of the refrigerator (4°C) and return them to room temperature (about 30 minutes).

[0161] D-2. Vortex to mix for 15 seconds.

[0162] D-3. Take 100 μL of streptavidin magnetic beads and add them to a new 1.5 mL low-adsorption centrifuge tube.

[0163] D-4. Place the centrifuge tube on a magnetic stand until the solution becomes clear.

[0164] D-5. Aspirate and discard the supernatant without disturbing the magnetic beads.

[0165] D-6. Wash the streptavidin magnetic beads according to the following steps:

[0166] D-6-1. Remove the centrifuge tube from the magnetic stand, add 200 μL of magnetic bead washing buffer, and vortex for 10 seconds.

[0167] D-6-2. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear. Aspirate and discard the supernatant without disturbing the magnetic beads.

[0168] D-7. Repeat step D-6.

[0169] D-8. Remove the centrifuge tube from the magnetic stand, add 50 μL of magnetic bead washing buffer and set aside.

[0170] E. Probe enrichment

[0171] E-1. Add 50 μL of buffer containing streptavidin magnetic beads (step D-8) to the hybridization reaction solution (step B-2).

[0172] E-2. Mix by gently pipetting up and down 10 times.

[0173] E-3. Incubate at 65°C for 20 minutes using a PCR instrument (heat lid set to 75°C).

[0174] E-4. Vortex mix for 3 seconds every 6 minutes to ensure that the magnetic beads are in suspension.

[0175] F. Post-capture cleaning

[0176] F-1.65℃ cleaning

[0177] F-1-1. Add 100 μL of preheated cleaning solution I to the 0.2 mL low-adsorption centrifuge tube in the probe enrichment reaction solution (step E-4).

[0178] F-1-2. After mixing by pipetting, transfer the reaction solution containing streptavidin magnetic beads to a new 1.5 mL low-adsorption centrifuge tube.

[0179] F-1-3. Place the centrifuge tube on a magnetic rack until the solution becomes clear and discard the supernatant.

[0180] F-1-4. Continue cleaning according to the following steps:

[0181] F-1-4-1. Add 200 μL of preheated cleaning solution S, pipette or vortex to mix, and incubate at 65°C for 5 minutes.

[0182] F-1-4-2. Centrifuge briefly and place the centrifuge tube on a magnetic rack until the solution becomes clear and discard the supernatant.

[0183] F-1-5. Repeat step F-1-4.

[0184] F-2. Room temperature cleaning

[0185] F-2-1. Add 200 μL of cleaning solution I and vortex to mix for 2 minutes.

[0186] F-2-2. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0187] F-2-3. Add 200 μL of cleaning solution II and vortex to mix for 1 minute.

[0188] F-2-4. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0189] F-2-5. Add 200 μL of cleaning solution III and vortex to mix for 30 seconds.

[0190] F-2-6. Centrifuge the centrifuge tube briefly and place it on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0191] F-3. Resuspending magnetic beads

[0192] F-3-1. Immediately add 20 μL of enzyme-free sterile water.

[0193] F-3-2. Use a pipette to pipette up and down 10 times to resuspend the magnetic beads and proceed to the next experimental steps.

[0194] G. PCR amplification

[0195] G-1. Configure the PCR reaction system according to Table 14.

[0196] Table 14

[0197]

[0198] G-2. Mix by pipetting or vortexing at low speed to keep the magnetic beads suspended and immediately proceed to step G-3.

[0199] G-3. Use the PCR instrument to run according to the program in Table 15, with the hot cover temperature at 105°C.

[0200] Table 15

[0201]

[0202] H. PCR product purification

[0203] H-1. Add 75 μL AMPure XP purification magnetic beads (Beckman) to each PCR tube.

[0204] H-2. Purify the PCR product according to the AMPure XP operating manual.

[0205] H-3. Elution was performed using 22 μL of Tris-HCl (10 mM, pH 8.5).

[0206] H-4. Transfer 20 μL of the eluate containing the captured library to a new 1.5 mL low-binding centrifuge tube (Eppendorf).

[0207] I. Library Quality Control

[0208] I-1. The library concentration was measured using Qubit Fluorometer 3.0 (ThermoFisher).

[0209] I-2. The length of the library fragments was measured using Agilent 2100. The products were concentrated between 200-600 bp and there was no adapter dimerization.

[0210] H. High-throughput sequencing

[0211] Illumina Miniseq sequencer was used for PE150 sequencing.

[0212] Data analysis

[0213] Trimmomatic was used to remove adapters and low-quality sequences to obtain clean data, and then Samtools was used to extract reads of the target region. The on-target rate and average sequencing depth of the target region were counted according to the alignment position. The ratio of the depth of each target region to the average depth was used to calculate the proportion of the corresponding target region to obtain the proportion of target regions at 0.2 and 0.5x the average depth.

[0214] As shown in Table 16, under the same number of sequencing, the on-target rate of the present invention significantly exceeds that of the traditional scheme by nearly 20 percentage points, and a sequencing depth of about 1000x is obtained, while the traditional method is only about 600x. In addition, in terms of uniformity, the average depth ratio of 0.2 and 0.5x of the present invention is also significantly higher than that of the traditional method. Therefore, whether it is the on-target rate or uniformity, the present invention is significantly better than the traditional method.

[0215] Table 16

[0216]

[0217] GATK was used for quality value correction and local re-alignment, Freebayes was used to detect mutations, and after annovar was used for mutation annotation, the mutation frequencies of related genes were counted (less than 2 Unique positive Reads were judged as undetected-" / "). As shown in Table 17, using the scheme of the present invention, all mutation sites were detected, and the mutation frequency had a small deviation from the expected mutation frequency, while in the traditional scheme, only three repeated experiments of PIK3CA H1047R were all detected, and their mutation frequency had a large deviation from the expected frequency. The above results show that the scheme of the present invention can not only obtain good basic capture data, but also has a high mutation detection accuracy.

[0218] Table 17

[0219]

[0220] In summary, the present invention has the following advantages: 1) The capture process is short in time, and the entire hybridization, washing, probe enrichment and washing only take 1 hour, which is much shorter than the traditional method. 2) The operation is simple, the probe and library are co-denatured, and the magnetic bead washing buffer is retained, which greatly simplifies the operation and is more conducive to automated workstation operation; 3) The buffer component cost is low. 4) The data quality is high, and the specificity and coverage uniformity are excellent; 5) Gene mutation detection is accurate. With the development of high-throughput sequencing, clinical practice has also put forward higher requirements for it. The rapid hybridization method provided by the present invention will greatly shorten the time of the hybridization capture sequencing method, provide diagnostic results in a timely manner, and buy more time for patient treatment.

[0221] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. Use of a kit for rapid hybridization of a probe with a library, characterized in that: The application is a method for rapid hybridization of a probe with a library for non-disease diagnosis purposes, and the kit comprises the following components: Component 1: hybridization buffer, comprising sodium citrate buffer, 0.8-1.2 M betaine, 0.8-1.2 M tetramethylammonium chloride, 4-6% weight / volume dextran sulfate, and 18-44% volume % formamide; Component 2: magnetic bead washing buffer, the magnetic bead washing buffer comprising 3 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 0.1% Tween-20; Component 3: Washing solution S: 1X SSC, 0.1% Tween-20; Component 4: cleaning solution I: 1X SSC, 0.1% sodium dodecyl sulfate; Component 5: Washing solution II: 0.5X SSC; Component 6: Washing solution III: 0.2X SSC; The method comprises the following steps: (1) contacting the probe with the library in a hybridization buffer, wherein the probe is added to the hybridization buffer in advance rather than after the library is denatured, the denaturation temperature of the library and the probe is 92-98° C., the denaturation time is 30 seconds to 2 minutes, and the hybridization binding time of the probe and the library in the hybridization buffer is 0.5-4 hours; (2) After the probe binds to the library to form a complex, the complex is captured by streptavidin using magnetic beads that have been washed and activated with a magnetic bead washing buffer, and after washing, the streptavidin magnetic beads remain in the magnetic bead washing buffer without discarding the magnetic bead washing buffer; (3) washing the complex bound to streptavidin using a washing solution; (4) The target library bound to the probe is enriched by PCR reaction, then purified and subsequently subjected to high-throughput sequencing.

2. The use according to claim 1, characterized in that: The probe is a 5' biotin-modified single-stranded DNA with a length of 110-130 nt.

3. The use according to claim 1, characterized in that: The steps include: (a) mixing a pre-library, an adapter blocker and a probe, and then evaporating to dryness, wherein the pre-library includes a pre-library suitable for an MGI sequencer, an Illumina sequencer, and a Life sequencer; (b) adding hybridization buffer to the product of step (a), mixing and incubating at room temperature for 5 minutes, followed by denaturation at 95°C for 2 minutes and incubation at 65°C for 30 minutes; (c) After hybridization, the probe was bound to the streptavidin magnetic beads for 15 minutes, and the streptavidin magnetic beads were washed twice with magnetic bead washing buffer and present in 50 μL magnetic bead washing buffer; (d) washing the streptavidin magnetic beads in step (c) once at 65°C using washing solution I at 65°C; (e) Wash the beads twice at 65 °C using 65 °C washing solution S; (f) Wash the beads by incubating with Wash Solution I at room temperature and vortexing for 2 minutes; (g) Wash the beads by vortexing and incubating with Wash Buffer II at room temperature for 1 min. (h) Wash the beads by incubating with Wash Solution III at room temperature and vortexing for 0.5 min. (i) PCR enrichment and library purification; (j) The purified library was subjected to high-throughput sequencing.

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