WO2023077576A1 - 低频引入突变的酶切打断建库方法和试剂盒 - Google Patents

低频引入突变的酶切打断建库方法和试剂盒 Download PDF

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WO2023077576A1
WO2023077576A1 PCT/CN2021/133239 CN2021133239W WO2023077576A1 WO 2023077576 A1 WO2023077576 A1 WO 2023077576A1 CN 2021133239 W CN2021133239 W CN 2021133239W WO 2023077576 A1 WO2023077576 A1 WO 2023077576A1
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fragmentation
dna polymerase
enzyme
dna
mutations
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PCT/CN2021/133239
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French (fr)
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曲燕
胡玉刚
吴强
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纳昂达(南京)生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

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  • the present application relates to the technical field of high-throughput sequencing library construction, in particular, to a method and kit for constructing a library by enzyme cutting and fragmentation with low-frequency introduction of mutations.
  • high-throughput sequencing has played an irreplaceable role in auxiliary diagnosis.
  • the library construction link of high-throughput sequencing is an important link in the high-throughput sequencing process.
  • two important aspects of library construction automation and cost control are usually controlled and optimized.
  • the fragmentation of library construction was generally carried out by ultrasonic fragmentation, but this method has two obvious disadvantages, one is that the fragmentation cost is too high, and the other is not conducive to automatic operation.
  • Enzyme digestion library construction can be better integrated into automated operations, and since ultrasonic disruption is not required, this part of the cost can also be saved.
  • library construction by enzyme digestion can solve some of the disadvantages of ultrasonic fragmentation, it will also introduce many point mutations (SNP) and double-directional false fusion site sequences (Dual Stranded Artifact sequence, https://doi.org/10.1101/2020.01. 30.927491). Since the core idea of enzyme digestion library construction is to use a gap-generating enzyme and a polymerase to work together to achieve interruption, only the purpose of interruption is achieved, and the problem of introducing mutations is not fully considered.
  • Nt.CviPII or NciI is a mutant of a restriction endonuclease, which can only cut one strand of the double strand, and completes it with E. coli polymerase (20U) broken.
  • CN111763664B is also based on the principle of acquaintance, which can realize the interruption of DNA fragments, and the dosage of E. coli polymerase is 0.05-1.0U/ ⁇ L.
  • DNA fragmentation methods as shown in Figure 1, are the first step to generate a gap, the second step is to break through the continuous displacement of the polymerase at room temperature from the gap, and the third step is to inactivate the high-temperature inactivation of the interrupted enzyme. At the same time add A at the end.
  • the purpose of this application is to provide a method and kit for library construction by enzyme digestion and fragmentation with low-frequency introduction of mutations, so as to solve the technical problem of introducing more mutations in library construction by enzyme digestion in the prior art.
  • a method for constructing a library by enzyme cutting and fragmentation for low-frequency introduction of mutations includes the steps of DNA enzymatic fragmentation, filling and adding A, linker ligation, PCR amplification and library purification, wherein, in the step of DNA enzymatic cutting and fragmentation, filling and adding A, by controlling normal temperature The amount of DNA polymerase used controls repeated cutting and strand displacement.
  • the amount of normal temperature DNA polymerase is 0.001 ⁇ 0.045U/ ⁇ L; preferably, the normal temperature DNA polymerase is selected from E. coli polymerase I, Klenow large fragment or One or more of Bst DNA polymerases.
  • the amount of heat-resistant DNA polymerase is 0.05-0.2 U/ ⁇ L; preferably, the heat-resistant DNA polymerase is Taq DNA polymerase.
  • the reaction temperature of DNA polymerase at room temperature is 25-32°C; preferably 25°C; preferably, the inactivation temperature of DNA polymerase at room temperature is 65°C; preferably Preferably, the reaction time of the DNA polymerase at room temperature is 25-30 minutes; preferably, the inactivation reaction time of the DNA polymerase at room temperature is 25-30 minutes.
  • the interrupting enzyme solution includes T4GP32 0.01-0.03 ⁇ g/ ⁇ L, DNaseI 0.004-0.01U/ ⁇ L, Klenow DNA polymerase 0.001-0.045U/ ⁇ L, Taq DNA Polymerase 0.05 ⁇ 0.2U/ ⁇ L and 50% glycerol; preferably, the interrupting enzyme solution includes T4GP32 0.01 ⁇ g/ ⁇ L, DNase I 0.004U/ ⁇ L, Klenow DNA polymerase 0.045U/ ⁇ L, Taq DNA polymerase 0.2U/ ⁇ L ⁇ L and 50% glycerol.
  • the interrupting enzyme buffer used includes Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM
  • the interrupting enzyme buffer includes Tris-HCl 5-15mM, NaCl 20-40mM, MgCl 2 3-8mM, CaCl 2 1-3mM, MnCl 2 1-3mM, dNTPs 0.6-1mM, ATP 5-15mM.
  • the method of enzymatic digestion and fragmentation library construction includes: mixing the fragmentation enzyme buffer and fragmentation enzyme solution to obtain a fragmentation system, adding the DNA sample to be fragmented into the fragmentation system, and reacting at 25-32°C for 25-30 minutes , and then react at 65°C for 25-30min.
  • a library construction kit for enzyme cutting and fragmentation for low-frequency introduction of mutations includes an interrupting enzyme solution and an interrupting enzyme buffer: the interrupting enzyme solution includes a normal temperature DNA polymerase, and the final reaction concentration of the normal temperature DNA polymerase is 0.001-0.045U/ ⁇ L; more preferably, the normal temperature DNA polymerase is One or more selected from Escherichia coli polymerase I, Klenow large fragment or Bst DNA polymerase; preferably, the interrupting enzyme solution also includes heat-resistant DNA polymerase, and the reaction final concentration of heat-resistant DNA polymerase is 0.05 ⁇ 0.2U/ ⁇ L; more preferably, the thermostable DNA polymerase is Taq DNA polymerase.
  • the breaking enzyme solution includes T4GP32 0.01 ⁇ g/ ⁇ L, DNaseI 0.004U/ ⁇ L, Klenow DNA polymerase 0.001 ⁇ 0.045U/ ⁇ L, Taq DNA polymerase 0.05 ⁇ 0.2U/ ⁇ L and 50% glycerol; Enzyme breaking solution includes T4GP32 0.01 ⁇ g/ ⁇ L, DNase I 0.004U/ ⁇ L, Klenow DNA polymerase 0.045U/ ⁇ L, Taq DNA polymerase 0.2U/ ⁇ L and 50% glycerol.
  • the interrupting enzyme buffer used includes Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM ;
  • the interrupting enzyme buffer includes Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM;
  • the enzyme cutting library construction kit also includes Reagents for adapter ligation, PCR amplification, and library purification.
  • Figure 1 shows a schematic flow diagram of enzyme cutting and breaking, filling in and adding A
  • Figure 2 shows a schematic diagram of homozygous mutations produced after multiple rounds of restriction digestion
  • Figure 3 shows a schematic diagram of pseudofusion mutations with reverse order generated after multiple rounds of restriction digestion
  • Figure 4 shows how to precisely control each link to reduce the introduction of mutations for enzyme digestion library construction
  • Figure 5 shows the impact of different stages of enzyme amount on the introduction of false fusion mutations
  • Figure 6 shows the impact of different temperatures on the introduction of false fusion mutations
  • Figure 7 shows the library output of 6 cycles of 100ng input
  • FIG. 8 shows the comparison between different enzyme digestion schemes and ultrasonic disruption in the introduction of SNP mutations
  • Figure 9 shows the ratio of palindromic misjoins introduced by different database construction schemes.
  • Single base polymorphism Single nucleotide polymorphism mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level.
  • Fusion gene refers to connecting the coding regions of two or more genes end to end and placing them under the control of the same set of regulatory sequences (including promoters, enhancers, ribosome binding sequences, terminators, etc.) to form chimeric genes.
  • Dual Stranded Artifact sequence a quadrant in which the direction of the sequence near the reverse complementary sequence is reversed due to enzyme digestion, similar to a false fusion site, which is not a true fusion in essence, but The wrong sequence inversion product caused by enzyme digestion.
  • next-generation sequencing process in order to be able to carry out sequencing on the machine, in addition to cfDNA or fragmented samples that meet the second-generation library construction, they must be fragmented into 200-400bp fragments before library construction.
  • DNA fragmentation is generally interrupted by ultrasound.
  • the advantage of ultrasonic fragmentation is that the length of fragments is moderate and mutations are rarely introduced. Therefore, ultrasonic fragmentation is widely used, but ultrasonic fragmentation is also widely used.
  • the problem is that the cost of ultrasonic instruments and consumables is relatively high, and the ultrasonic method is difficult to integrate with automation. Therefore, many people think of using enzyme cutting and interruption to solve the high cost and adapt to automation solutions.
  • the inventors of the present application have found that in addition to the effective interruption of DNA by enzymatic digestion, the amount of enzyme and the reaction temperature are two key factors. Between 25 and 32°C, the speed of interruption can be significantly slowed down, thereby reducing the risk of mutation. The amount introduced is obviously less at 25°C than at 37°C. During the first step of interrupting, the amount of polymerase should be controlled so that the interrupting does not introduce too many SNPs and false fusion mutations repeatedly.
  • the inventors have found through testing that controlling the amount of breaking enzyme in the first step and reacting under appropriate reaction conditions (for example, 25 ° C conditions), even this step only needs breaking and partial strand displacement reaction, and the second step high-temperature polymerase
  • the reaction makes up for the unfinished chain displacement reaction in the first step. Since the interrupting enzyme has been inactivated in the second step, this step can make up for the unfinished chain displacement reaction.
  • the increase in the amount of enzyme in the second step will not introduce too many mutations , the detailed logic is shown in Figure 4.
  • the path of this application controls the introduction of few mutations through two conditions.
  • the first condition is to control the amount of enzyme for the first interruption reaction, and to control the amount of polymerase in this step.
  • the enzyme in this step can be E.
  • the concentration is in the range of 0.001-0.045U/ ⁇ L, and the concentration of DNase I can be in the range of 0.01-0.05U/ ⁇ L.
  • the control of this step is to avoid excessive repeated cutting and strand displacement leading to the introduction of too many mutations.
  • the polymerase concentration is increased (traditional scheme)
  • the corresponding mutations introduced will increase significantly.
  • schemes 1 to 3 will significantly reduce the introduction of false fusion mutations compared with the traditional scheme.
  • scheme 4 In the traditional scheme, a large number of false fusion mutations will be introduced (see Example 1 for details).
  • this step will add more ordinary Taq enzyme to complete the unfinished strand replacement process and A process in the first step.
  • the enzyme concentration in this step is 0.05 ⁇ 0.2U/ ⁇ L scope.
  • reaction temperature the inventors found that 25°C was significantly better than a higher reaction temperature during testing.
  • the first step reaction temperature of 25°C to 32°C was obviously better than the temperature of 37°C, 25°C °C reaction temperature can introduce fewer spurious fusion mutations (see Example 2 for details). Therefore, the common purpose of controlling the amount of enzyme in the first step and the reaction temperature in the first step is to control the reaction speed and avoid a large number of mutations introduced due to repeated enzyme digestion caused by excessive disruption.
  • Enzyme digestion library construction can solve the cost of ultrasonic fragmentation library construction and is more suitable for automation.
  • the efficiency of enzyme digestion library construction is also significantly higher than ultrasonic fragmentation library construction.
  • the efficiency of enzyme digestion library construction is higher than that of ultrasonic disruption library construction, the higher the efficiency, the better, and some enzyme digestion library construction efficiency is not high.
  • V Company Vazmye Company
  • K Company Kapa Company
  • a method for constructing a library by restriction enzyme cutting and fragmentation for introducing mutations at a low frequency includes the steps of DNA enzymatic fragmentation, filling and adding A, linker ligation, PCR amplification and library purification, wherein, in the step of DNA enzymatic cutting and fragmentation, filling and adding A, by controlling normal temperature The amount of DNA polymerase used controls repeated cutting and strand displacement.
  • the amount of DNA polymerase at room temperature is 0.001-0.045U/ ⁇ L, for example, 0.001, 0.005U/ ⁇ L, 0.01U/ ⁇ L , 0.015U/ ⁇ L, 0.02U/ ⁇ L, 0.025U/ ⁇ L, 0.03U/ ⁇ L, 0.035U/ ⁇ L, 0.04U/ ⁇ L, 0.045U/ ⁇ L; preferably, the room temperature DNA polymerase is selected from Escherichia coli polymerase One or more in 1, Klenow large fragment or Bst DNA polymerase.
  • the amount of heat-resistant DNA polymerase is 0.05-0.2U/ ⁇ L, for example, 0.05U/ ⁇ L, 0.06U/ ⁇ L, 0.07U/ ⁇ L, 0.08U/ ⁇ L, 0.09U/ ⁇ L, 0.1U/ ⁇ L, 0.12U/ ⁇ L, 0.14U/ ⁇ L, 0.15U/ ⁇ L, 0.17U/ ⁇ L, 0.18U/ ⁇ L; preferably, the heat-resistant DNA polymerase is Taq DNA polymerase.
  • the reaction temperature of normal temperature DNA polymerase is 25-32°C, for example, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C; preferably 25°C; preferably, the inactivation temperature of DNA polymerase at room temperature is 65°C; preferably, the reaction time of DNA polymerase at room temperature is 25-30min; preferably, the inactivation temperature of DNA polymerase at room temperature The active reaction time is 25-30 minutes.
  • the interrupting enzyme solution in the steps of DNA enzymatic cleavage and fragmentation, filling and adding A, includes T4GP32 0.01-0.03 ⁇ g/ ⁇ L, DNaseI 0.004-0.01U/ ⁇ L, Klenow DNA polymerase 0.001- 0.045U/ ⁇ L, Taq DNA polymerase 0.05 ⁇ 0.2U/ ⁇ LU/ ⁇ L and 50% glycerol; preferably, the disrupting enzyme solution includes T4GP32 0.01 ⁇ g/ ⁇ L, DNase I 0.004U/ ⁇ L, Klenow DNA polymerase 0.045U/ ⁇ L ⁇ L, Taq DNA polymerase 0.2U/ ⁇ L and 50% glycerol.
  • the interrupting enzyme buffer used includes Tris-HCl 5-15mM, NaCl 20-40mM, MgCl 2 3-8mM, CaCl 2 1 ⁇ 3mM, MnCl 2 1 ⁇ 3mM, dNTPs 0.6 ⁇ 1mM, ATP 5 ⁇ 15mM; preferably, the interrupting enzyme buffer includes Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM.
  • the enzyme digestion and fragmentation method for building a library includes: mixing the fragmentation enzyme buffer and fragmentation enzyme solution to obtain a fragmentation system, adding the DNA sample to be fragmented into the fragmentation system, 25-32 °C for 25-30 minutes, then 65 °C for 25-30 minutes.
  • a library construction kit for enzyme cutting and fragmentation for low-frequency introduction of mutations includes interrupting enzyme solution and interrupting enzyme buffer: the interrupting enzyme solution includes normal temperature DNA polymerase, and the final reaction concentration of normal temperature DNA polymerase is 0.001 ⁇ 0.045U/ ⁇ L, for example, 0.001, 0.005U/ ⁇ L, 0.01U/ ⁇ L, 0.015U/ ⁇ L, 0.02U/ ⁇ L, 0.025U/ ⁇ L, 0.03U/ ⁇ L, 0.035U/ ⁇ L, 0.04U/ ⁇ L, 0.045U/ ⁇ L; more preferably, room temperature DNA polymerase is selected One or more of Escherichia coli polymerase I, Klenow large fragment or Bst DNA polymerase; preferably, the interrupting enzyme solution also includes heat-resistant DNA polymerase, and the reaction final concentration of heat-resistant DNA polymerase is 0.05 ⁇ 0.2U/ ⁇ L, for example, 0.05U/ ⁇ L, 0.06U/ ⁇
  • the breaking enzyme solution includes T4GP32 0.01 ⁇ g/ ⁇ L, DNaseI 0.004U/ ⁇ L, Klenow DNA polymerase 0.001 ⁇ 0.045U/ ⁇ L, Taq DNA polymerase 0.05 ⁇ 0.2U/ ⁇ L and 50 % glycerol; preferably, the breaking enzyme solution includes T4GP32 0.01 ⁇ g/ ⁇ L, DNase I 0.004U/ ⁇ L, Klenow DNA polymerase 0.045U/ ⁇ L, Taq DNA polymerase 0.2U/ ⁇ L and 50% glycerol.
  • the interrupting enzyme buffer used includes Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM; preferably, the interrupting enzyme buffer includes Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM; Library kits also include reagents for adapter ligation, PCR amplification, and library purification.
  • the main innovation of this application is to achieve efficient library construction through two-step reaction enzyme amount control and temperature control, and at the same time control the introduction level of mutations to be the same as ultrasonic disruption.
  • This application can make full use of enzyme cutting and cutting to save the cost of breaking DNA, is more suitable for automatic and efficient library construction, and at the same time avoids the introduction of too many mutations, and realizes the detection of low-frequency SNPs and fusion mutations in clinical samples.
  • this application solves the technical problem that the introduction of too many mutations due to enzyme cutting interferes with the detection of real mutations in clinical samples.
  • FFPE DNA or gDNA is digested and fragmented by enzymes and filled with A---adapter ligation---PCR amplification---library purification.
  • Interrupt kit composition 1 times buffer composition and concentration: Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM; reaction enzyme composition: T4GP32 0.01 ⁇ g/ ⁇ L, DNaseI 0.004U/ ⁇ L, Klenow DNA polymerase 0.001U/ ⁇ L, Taq DNA polymerase 0.2U/ ⁇ L and glycerol.
  • Interrupt kit composition 1 times buffer composition and concentration: Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM; reaction enzyme composition: T4GP32 0.01 ⁇ g/ ⁇ L, DNaseI 0.004U/ ⁇ L, Klenow DNA polymerase 0.005U/ ⁇ L, Taq DNA polymerase 0.1U/ ⁇ L and glycerol.
  • Interrupt kit composition 1 times buffer composition and concentration: Tris-HCl 10mM, NaCl 30mM, MgCl 2 5mM, CaCl 2 2mM, MnCl 2 2mM, dNTPs 0.6mM, ATP 10mM; reaction enzyme composition: T4GP32 0.01 ⁇ g/ ⁇ L, DNaseI 0.004U/ ⁇ L, Klenow DNA polymerase 0.045U/ ⁇ L, Taq DNA polymerase 0.05U/ ⁇ L and glycerol.
  • the Option One Option II third solution Option four Klenow DNA polymerase 0.001U/ ⁇ L 0.005U/ ⁇ L 0.045U/ ⁇ L 0.1U/ ⁇ L Taq DNA polymerase 0.2U/ ⁇ L 0.1U/ ⁇ L 0.05U/ ⁇ L 0.01U/ ⁇ L
  • Step 1 DNA fragmentation and repair plus A
  • this sample addition step must be performed on ice.
  • the heated lid temperature is set to 70 °C.
  • Step 2 Connect the connector
  • reaction program Cycling Program III reaction program shown in Table 6
  • reaction program shown in Table 6 reaction program shown in Table 6
  • Step 4 PCR amplification
  • Step 3 Purify and recover the product (with magnetic beads) 20 ⁇ L 2X HiFi PCR Master Mix 25 ⁇ L Cat. No. 1004102 Universal Amplification Primer 5 ⁇ L total 50 ⁇ L
  • scheme 4 which is a traditional scheme, a large number of false fusion mutations will be introduced.
  • the second step will add more ordinary Taq enzyme to complete the unfinished strand replacement process and A process in the first step.
  • the enzyme concentration in this step is 0.05 ⁇ 0.1U/ ⁇ L range.
  • schemes 1 to 3 are significantly better than scheme 4
  • schemes 1 to 3 are the optimization schemes of this application, and scheme 4 is to simulate other major patents program results.
  • Example 1 The process steps of this program are the same as in Example 1, the only difference is that the interruption temperature is different, and the interruption temperature was tested at 25°C, 28°C, 32°C and 37°C respectively, and the main reagent composition uses the optimization scheme of this application (scheme Two), the difference with Example 1 is mainly in the table below, that is, the reaction temperature of the first step enzyme digestion, except that, other steps are exactly the same.
  • the three representative products selected in this example were compared with our company’s ultrasonic disruption program for library construction. They were operated according to their respective instructions, and a 100ng standard product was used to interrupt and build a library.
  • the interruption program of KAPA Company was in the same The interruption is completed under the same conditions, that is, the interruption and chain replacement process is completed in one step at 37°C; although Vazyme’s products are interrupted, chain replacement and A addition are completed in two steps, but because they only focus on production, it leads to excessive breaking. It is concluded that the library yield is the highest, and the SNPs and false fusion mutations introduced are also the most, as shown in Figure 7-9.
  • the solution of this application is an enzyme digestion library construction solution closest to the ultrasonic fragmentation solution, and the products of Company V and Company K are introducing a large number of SNP mutations. Also in terms of introducing false fusion mutations, the solution of this application is also close to ultrasonic disruption, and the products of V and K companies are also very high in introducing false fusion mutations, as shown in Figure 9.
  • this application optimizes an enzyme cutting and fragmentation kit, which solves the problem of sample fragmentation. Compared with ultrasonic fragmentation, it solves the high cost and unsuitable automation problems of ultrasonic fragmentation.
  • This application guarantees that While improving the efficiency of disruption, focus on optimizing and controlling the level of mutation introduction, mainly by controlling the amount of enzyme and disruption temperature during disruption, so that disruption can be completed without introducing a large number of mutations caused by excessive disruption.
  • the enzyme concentration in this step is in the range of 0.05-0.1U/ ⁇ L. Controlling the blocking temperature at 25-32°C will be more beneficial to avoid primers with a large number of mutations.
  • This application solves the core problem of large-scale application of enzymatic cutting and interruption. A large number of primers are not real mutations. This application optimizes the primers for controlled mutations, so that the solution of this application can solve the detection of low-frequency mutation samples, such as a large number of mutations. Detection of clinical samples FFPE samples.

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Abstract

一种低频引入突变的酶切打断建库方法和试剂盒。其中，酶切打断建库方法包括DNA酶切片段化和补平加A、接头连接、PCR扩增和文库纯化的步骤，其中，DNA酶切片段化和补平加A步骤中，通过控制常温DNA聚合酶的用量控制反复切割和链置换。低频引入突变的酶切打断建库方法除了保证打断的效率和稳定性，同时也避免了由于打断引入大量的突变导致影响酶切打断在NGS测序过程中的应用，做到打断水平和超声相当的水平，使酶切打断能够在临床检测过程中得到很好的应用。

Description

低频引入突变的酶切打断建库方法和试剂盒 技术领域
本申请涉及高通量测序文库构建技术领域，具体而言，涉及一种低频引入突变的酶切打断建库方法和试剂盒。
背景技术
目前，高通量测序已在辅助诊断方面有不可替代的作用。高通量测序的建库环节是高通测序过程中的重要环节，为了未来更好的在临床方向的应用，通常在建库自动化和成本控制两个重要方面进行控制和优化。以前文库构建的打断一般是用超声打断的方式进行，但这种方式有两个明显的缺点，一个是打断成本过高，另一个是不利于自动化操作。
当前，解决上述两个问题的方案通常是利用酶切建库的方法，酶切建库可以更好的整合到自动化的操作中，且由于不需要超声打断，这部分成本也可以节省。虽然酶切建库可以解决超声打断的部分弊端，但也会引入很多点突变(SNP)和双方向假融合位点序列(Dual Stranded Artifact sequence，https://doi.org/10.1101/2020.01.30.927491)。由于酶切建库的核心思想是利用一种产生缺口的酶和一种聚合酶共同作用实现打断，只是实现了打断的目的，对引入突变的问题没有充分考虑。
一般的酶切打断方式主要有两种，一种是由一种酶切出缺口，之后用T7内切酶把这个缺口对面的序列切断(US8703462B2)，这种打断方式是能打断DNA，并且也很少引入突变，但是由于此种打断方式DNA损失严重，在效率上甚至还不如超声打断的方式，并且打断的长度不集中，也没有很好的得到应用。另一种打断方式是利用一种酶切出缺口，再用另一种聚合酶进行连置换的方式对DNA片段化，这种方式已经存在了很多年(Methods in enzymology,152,330-335 1987-01-01)，并且最近也不断有相似的专利出现。例如，US 2019/0153453A1是利用Nt.CviPII或NciI，Nt.CviPII或NciI是一种限制性内切酶的突变体，只能切割双链的一条链，搭配大肠杆菌聚合酶(20U)完成打断。CN111763664B也是相识的原理，都能实现DNA片段的打断，其大肠杆菌聚合酶的用量为0.05-1.0U/μL。可以理解，这些DNA打断方式，如图1所示，都是第一步产生缺口，第二步是经过常温聚合酶从缺口进行连置换的打断，第三步高温失活打断酶的同时末端加A。
由于以前在酶切打断时没有人重视酶切打断的引入突变问题，酶切过程中存在着大量的反复打断过程，由于链置换过程中会不断的引入突变，所以当第一次链置换的时候引入杂合突变，在后续的反复打断和链置换过程中另一个条链也被置换成突变的序列，所以经过长时间的打断，就会引入纯合的突变，如图2所示，经过多轮打断不可避免的产生大量的SNP突变。同样的道理，由于基因组上存在比较近的反向重复序列，在打断的过程中，由于在链置换的过程中有单链状态存在，所以反向重复区域会形成自己互补结构，这样自己会形成局部互补驱动链置换过程，这种结构在后续的打断和链置换过程中会形成颠倒的假的融合形式 (https://doi.org/10.1101/2020.01.30.927491)，如图3所示，这种形式如果大量引入，会影响融合突变的准确检出。一般融合的发生也是有一段重复序列导致，重复序列被转座酶识别发生片段错接，准确的融合突变和SNP检测有重要的临床意义，但是酶切打断会大量的产生SNP和假的融合突变，所以目前虽然酶切建库比较节省成本和适应自动化，但是由于引入大量的突变也限制了酶切打断的大规模应用。
目前，应用者对酶切打断的效率都比较认可，因为和超声打断相比，其节省成本，且效率比较高。但影响酶切打断大规模应用的因素主要是酶切会引入大量突变，在检测体细胞突变时酶切建库引入的突变会影响检测这些突变的准确性。
发明内容
本申请旨在提供一种低频引入突变的酶切打断建库方法和试剂盒，以解决现有技术中酶切建库引入较多突变的技术问题。
为了实现上述目的，根据本申请的一个方面，提供了一种低频引入突变的酶切打断建库方法。该酶切打断建库方法包括DNA酶切片段化和补平加A、接头连接、PCR扩增和文库纯化的步骤，其中，DNA酶切片段化和补平加A步骤中，通过控制常温DNA聚合酶的用量控制反复切割和链置换。
进一步地，DNA酶切片段化和补平加A步骤中，常温DNA聚合酶的用量为0.001～0.045U/μL；优选的，常温DNA聚合酶为选自大肠杆菌聚合酶I、Klenow大片段或Bst DNA聚合酶中的一种或多种。
进一步地，DNA酶切片段化和补平加A步骤中，耐热DNA聚合酶的用量为0.05～0.2U/μL；优选的，耐热DNA聚合酶为Taq DNA聚合酶。
进一步地，DNA酶切片段化和补平加A步骤中，常温DNA聚合酶的反应温度为25～32℃；优选为25℃；优选的，常温DNA聚合酶的灭活温度为65℃；优选的，常温DNA聚合酶的反应时间为25～30min；优选的，常温DNA聚合酶的灭活反应时间为25～30min。
进一步地，DNA酶切片段化和补平加A步骤中，打断酶溶液包括T4GP32 0.01～0.03μg/μL、DNaseI 0.004～0.01U/μL、Klenow DNA聚合酶0.001～0.045U/μL、Taq DNA聚合酶0.05～0.2U/μL和50％甘油；优选的，打断酶溶液包括T4GP32 0.01μg/μL、DNase I 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.2U/μL和50％甘油。
进一步地，DNA酶切片段化和补平加A步骤中，所用的打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；优选的，打断酶缓冲液包括Tris-HCl 5～15mM、NaCl 20～40mM、MgCl 2 3～8mM、CaCl 2 1～3mM、MnCl 2 1～3mM、dNTPs 0.6～1mM、ATP 5～15mM。
进一步地，酶切打断建库方法包括：取打断酶缓冲液和打断酶溶液混合得到打断体系，向打断体系中加入待打断的DNA样本，25～32℃反应25～30min，然后65℃反应25～30min。
根据本发明的另一个方面，提供一种低频引入突变的酶切打断建库试剂盒。该试剂盒包括打断酶溶液和打断酶缓冲液：打断酶溶液包括常温DNA聚合酶，常温DNA聚合酶的反应终浓度为0.001～0.045U/μL；更优选的，常温DNA聚合酶为选自大肠杆菌聚合酶I、Klenow大片段或Bst DNA聚合酶中的一种或多种；优选的，打断酶溶液还包括耐热DNA聚合酶，耐热DNA聚合酶的反应终浓度为0.05～0.2U/μL；更优选的，耐热DNA聚合酶为Taq DNA聚合酶。
进一步地，打断酶溶液包括T4GP32 0.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.001～0.045U/μL、Taq DNA聚合酶0.05～0.2U/μL和50％甘油；优选的，打断酶溶液包括T4GP32 0.01μg/μL、DNase I 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.2U/μL和50％甘油。
进一步地，DNA酶切片段化和补平加A步骤中，所用的打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；优选的，打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 22mM、dNTPs 0.6mM、ATP 10mM；优选的，酶切打断建库试剂盒还包括接头连接、PCR扩增和文库纯化相关的试剂。
应用本申请的技术方案，除了保证打断的效率和稳定性，同时也避免了由于打断引入大量的突变导致影响酶切打断在NGS测序过程中的应用，做到打断水平和超声相当的水平，使本申请的酶切打断能够在临床检测过程中得到很好的应用。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1示出了酶切打断，补平和加A的流程示意图；
图2示出了多轮酶切后产生纯合突变的示意图；
图3示出了多轮酶切后产生顺序颠倒的假融合突变的示意图；
图4示出了如何精确控制各环节降低酶切建库的突变引入；
图5示出了不同阶段酶量对引入假融合突变的影响；
图6示出了不同的温度对引入假融合突变的影响；
图7示出了100ng投入量6个循环的文库产出；
图8示出了不同的酶切方案与超声打断在引入SNP突变的对比；
图9示出了不同建库方案引入回文错接的比率。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本申请。
术语解释：
单碱基多态性(SNP)：单核苷酸多态性主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。
融合基因：所谓融合基因，是指将两个或多个基因的编码区首尾相连.置于同一套调控序列(包括启动子、增强子、核糖体结合序列、终止子等)控制之下，构成的嵌合基因。
双方向假融合位点序列(Dual Stranded Artifact sequence)：由酶切导致的反向互补序列的附近序列方向反转的一种象限，类似一种假的融合位点，实质不是真的融合，只是酶切导致的错误序列反转产物。
在二代测序过程中，为了能够实现测序上机，除了cfDNA或符合二代建库的片段化样本，都要打断成200～400bp的片段再进行文库构建。传统的建库过程中，DNA的打断一般会用超声的方式打断，超声打断的好处是打断的片段长度适中，同时很少引入突变，所以超声打断被广泛应用，但是超声也问题，一个是超声的仪器和耗材的成本比较高，还有就是超声的方式很难和自动化整合到一起。所以很多人想到了用酶切打断的方式来解决成本过高和适应自动化的方案。
本申请的发明人研究发现，酶切打断DNA除了有效打断，酶量和反应温度是两个关键的因素，在25～32℃之间，可以明显减缓打断的速度，从而减少突变的引入量，且在25℃时明显要比在37℃时少。在第一步打断的过程中要控制聚合酶的量，使打断不要反复多次引入过多的SNP和假的融合突变。发明人通过测试发现，控制第一步的打断酶量和在合适的反应条件(例如，25℃条件反应)，甚至此步骤只需打断和部分链置换反应，通过第二步高温聚合酶反应弥补第一步中未完成的链置换反应，由于第二步已经把打断酶失活，此步骤可以弥补未完成的链置反应，第二步骤的酶量增加不会引入过多的突变，详细的逻辑如图4所示。本申请的路径通过两个条件来控制引入少的突变，第一个条件是控制第一个打断反应的酶量，控制此步骤的聚合酶量，此步骤的酶可以是大肠杆菌聚合酶I或Klenow大片段或Bst DNA聚合酶等常温聚合酶，浓度在0.001～0.045U/μL范围，DNase I的使用浓度可以在0.01～0.05U/μL范围。此步骤的控制就是避免过度反复切割和链置换导致引入过多的突变。相对应的，如果把聚合酶浓度调高(传统的方案)，对应引入的突变明显增多，如图5所示方案一至方案三在引入假融合突变时相对传统方案要明显减少，对比方案四是传统方案，会引入大量的假融合突变(详见实施例1)。在后续的高温失活第一步的打断酶的同时，此步骤会多加普通Taq酶完成第一步没有完成的链置换过程和加A过程，此步骤的酶浓度在0.05～0.2U/μL范围。同时反应温度方面，本发明人测试时发现，25℃要明显优于更高的反应温度，如图6所示，25℃～32℃的第一步反应温度明显要好于37℃的温度，25℃反应温度可以引入更少的假的融合突变(详见实施例2)。因此，控制第一步的酶量和第一步反应温度的共同目的是控制反应速度，避免 过度打断导致的由于反复酶切大量引入突变。
酶切建库除了可以解决超声打断建库成本和更适应自动化外，酶切的建库效率也明显高于超声打断建库。虽然酶切建库效率要高于超声打断建库，但是不是效率越高越好，并且有的酶切建库效率也不高。例如：如图7-9所示，采用Vazmye公司(简称V公司)的产品进行酶切建库，文库产量极高，但同时引入突变最多；Kapa公司(简称K公司)的建库产品进行DNA打断都是在第一步完成的，所以引入的突变也特别多。也有更依赖第二步打断的产品，如Swift公司(简称S公司)的产品，主要是的链置换是在高温条件下完成，如文章《Fragmentation Through Polymerization(FTP):A new method to fragment DNA for next-generation sequencing》(PLOS ONE|https://doi.org/10.1371/journal.pone.0210374April 1,2019)所述，虽然引入突变比K和V公司的产品要低很多，但是还是没有本申请方案的更低，同时S公司的建库效率很低，不能很好的发挥酶切高效建库的优势。
根据本发明一种典型的实施方式，提供一种低频引入突变的酶切打断建库方法。该酶切打断建库方法包括DNA酶切片段化和补平加A、接头连接、PCR扩增和文库纯化的步骤，其中，DNA酶切片段化和补平加A步骤中，通过控制常温DNA聚合酶的用量控制反复切割和链置换。应用本申请的技术方案，除了保证打断的效率和稳定性，同时也避免了由于打断引入大量的突变导致影响酶切打断在NGS测序过程中的应用，做到打断水平和超声相当的水平，使本申请的酶切打断能够在临床检测过程中得到很好的应用。
在本发明一典型的实施方式中，DNA酶切片段化和补平加A步骤中，常温DNA聚合酶的用量为0.001～0.045U/μL，例如，0.001、0.005U/μL、0.01U/μL、0.015U/μL、0.02U/μL、0.025U/μL、0.03U/μL、0.035U/μL、0.04U/μL、0.045U/μL；优选的，常温DNA聚合酶为选自大肠杆菌聚合酶I、Klenow大片段或Bst DNA聚合酶中的一种或多种。DNA酶切片段化和补平加A步骤中，耐热DNA聚合酶的用量为0.05～0.2U/μL，例如，0.05U/μL、0.06U/μL、0.07U/μL、0.08U/μL、0.09U/μL、0.1U/μL、0.12U/μL、0.14U/μL、0.15U/μL、0.17U/μL、0.18U/μL；优选的，耐热DNA聚合酶为Taq DNA聚合酶。
在本发明一典型的实施方式中，DNA酶切片段化和补平加A步骤中，常温DNA聚合酶的反应温度为25～32℃，例如，26℃、27℃、28℃、29℃、30℃、31℃；优选为25℃；优选的，常温DNA聚合酶的灭活温度为65℃；优选的，常温DNA聚合酶的反应时间为25～30min；优选的，常温DNA聚合酶的灭活反应时间为25～30min。
在本发明一典型的实施方式中，DNA酶切片段化和补平加A步骤中，打断酶溶液包括T4GP32 0.01～0.03μg/μL、DNaseI 0.004～0.01U/μL、Klenow DNA聚合酶0.001～0.045U/μL、Taq DNA聚合酶0.05～0.2U/μLU/μL和50％甘油；优选的，打断酶溶液包括T4GP32 0.01μg/μL、DNase I 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.2U/μL和50％甘油。
在本申请一实施例中，DNA酶切片段化和补平加A步骤中，所用的打断酶缓冲液包括Tris-HCl 5～15mM、NaCl 20～40mM、MgCl 2 3～8mM、CaCl 2 1～3mM、MnCl 2 1～3mM、dNTPs0.6～1mM、ATP 5～15mM；优选的，打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM。
在本发明一实施方式中，酶切打断建库方法包括：取打断酶缓冲液和打断酶溶液混合得到打断体系，向打断体系中加入待打断的DNA样本，25～32℃反应25～30min，然后65℃反应25～30min。
根据本发明的另一种典型的实施方式，提供一种低频引入突变的酶切打断建库试剂盒。该试剂盒包括打断酶溶液和打断酶缓冲液：打断酶溶液包括常温DNA聚合酶，常温DNA聚合酶的反应终浓度为0.001～0.045U/μL，例如，0.001、0.005U/μL、0.01U/μL、0.015U/μL、0.02U/μL、0.025U/μL、0.03U/μL、0.035U/μL、0.04U/μL、0.045U/μL；更优选的，常温DNA聚合酶为选自大肠杆菌聚合酶I、Klenow大片段或Bst DNA聚合酶中的一种或多种；优选的，打断酶溶液还包括耐热DNA聚合酶，耐热DNA聚合酶的反应终浓度为0.05～0.2U/μL，例如，0.05U/μL、0.06U/μL、0.07U/μL、0.08U/μL、0.09U/μL、0.1U/μL、0.12U/μL、0.14U/μL、0.15U/μL、0.17U/μL、0.18U/μL；更优选的，耐热DNA聚合酶为Taq DNA聚合酶。
在本发明一典型的实施方式中，打断酶溶液包括T4GP32 0.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.001～0.045U/μL、Taq DNA聚合酶0.05～0.2U/μL和50％甘油；优选的，打断酶溶液包括T4GP32 0.01μg/μL、DNase I 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.2U/μL和50％甘油。
在本申请一实施例中，DNA酶切片段化和补平加A步骤中，所用的打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；优选的，打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 22mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；优选的，酶切打断建库试剂盒还包括接头连接、PCR扩增和文库纯化相关的试剂。综上所述，本申请主要的创新点在于通过两步反应酶量控制和温度控制实现高效建库的同时，还能控制突变的引入水平和超声打断相同。本申请可以充分发挥酶切打断的节省打断DNA成本、更适合自动化和高效建库的同时，避免过多突变的引入，实现在临床样本低频SNP和融合突变的检测，相较其它发明酶切产品，本申请解决了由于酶切引入过多突变干扰临床样本的真实突变检测的技术问题。
下面将结合具体的实施例来进一步说明本申请的有益效果。
需要说明的是，以下实施例仅是示例性说明，并不限定本申请的方法仅能采用如下方法。具体流程简述如下：
本申请的建库过程可以简要概括：FFPE DNA或gDNA经过酶切片段化和补平加A---接头连接---PCR扩增---文库纯化。
实施例1
打断环节控制聚合酶量会降低酶切引入突变的影响
方案一：打断试剂盒组成：1倍缓冲液组成和浓度：Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；反应酶组成：T4GP32 0.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.001U/μL、Taq DNA聚合酶0.2U/μL和甘油。
方案二：打断试剂盒组成：1倍缓冲液组成和浓度：Tris-HCl 10mM、NaCl 30mM、MgCl 25mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；反应酶组成：T4GP32 0.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.005U/μL、Taq DNA聚合酶0.1U/μL和甘油。
方案三：打断试剂盒组成：1倍缓冲液组成和浓度：Tris-HCl 10mM、NaCl 30mM、MgCl 25mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；反应酶组成：T4GP32 0.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.05U/μL和甘油。
方案四：打断试剂盒组成：1倍缓冲液组成和浓度：Tris-HCl 10mM、NaCl 30mM、MgCl 2 5mM、CaCl 2 2mM、MnCl 2 2mM、dNTPs 0.6mM、ATP 10mM；反应酶组成：T4GP320.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.1U/μL、Taq DNA聚合酶0.01U/μL和甘油。
方案之间的区别见表1：
表1
  方案一 方案二 方案三 方案四
Klenow DNA聚合酶 0.001U/μL 0.005U/μL 0.045U/μL 0.1U/μL
Taq DNA聚合酶 0.2U/μL 0.1U/μL 0.05U/μL 0.01U/μL
具体操作步骤如下：
步骤一：DNA片段化及修复加A
1.1取出打断酶buffer(上述1倍缓冲液)常温融解，混合均匀，置于冰上备用。
1.2取出打断酶(上述反应酶)置于冰上，混合均匀，瞬时离心备用。
1.3按照下表2，在冰上配制片段化Master Mix(乘以反应数，并多配制10％富余量)：
表2
打断酶Buffer 5μL/反应
打断酶 5μL/反应
1.4混合均匀，瞬时离心放置冰上备用。
1.5在已标记的0.2mL PCR管中，分别加入总量为100ng gDNA，并置于冰上。如gDNA不足40μL，可用Nuclease Free Water补足至40μL。
1.6向置于冰上的每个PCR管中加入10μL片段化Master Mix，配制为50μL终体积的反应混合液，见表3。
表3
gDNA 40μL
片段化Master Mix 10μL
注意：为避免时间差异引起的片段化不均一，此加样步骤务必在冰上进行。
1.7混合均匀，瞬时离心使全部反应液置于PCR管底部。
1.8在PCR仪上启动反应程序Cycling Program I，待温度稳定至25℃时将反应管放进PCR仪，进行如表4的反应程序：
表4
25℃ 25-30min
65℃ 30min
4℃ Hold
注意：热盖温度设置为70℃。
步骤二：接头连接
2.1取出T4DNA Ligation Buffer(NEB)常温融解，混合均匀，置于冰上备用。
2.2取出T4DNA Ligase(NEB)置于冰上，混合均匀，瞬时离心备用。
2.3从PCR仪上取出步骤一PCR反应管，置于冰上，按照下表5进行反应体系配制：
表5
Figure PCTCN2021133239-appb-000001
2.4混合均匀，瞬时离心使全部反应液置于PCR管底部。
表6
20℃ 15min
4℃ Hold
2.5在PCR仪上启动反应程序Cycling Program III(如表6所示的反应程序)，等温度稳定至20℃时将反应管放进PCR仪。
步骤三：连接产物纯化
3.1提前将
Figure PCTCN2021133239-appb-000002
SP Beads取出涡旋混匀，室温平衡30min后使用。
3.2向步骤二连接反应产物中加入40μL
Figure PCTCN2021133239-appb-000003
SP Beads，混合均匀，25℃孵育5–10min。
3.3将PCR管瞬时离心后放置于磁力架上5min至液体完全澄清，使用移液器吸取移弃上清。
3.4沿PCR管侧壁缓慢加入150μL 80％乙醇，注意勿扰动磁珠，静置30s，使用移液器吸取移弃上清。
3.5重复步骤3.4一次。
3.6将PCR管瞬时离心后放置于磁力架上，使用10μL吸头移去少量残留乙醇，注意勿吸到磁珠。
3.7打开PCR管盖，并于室温静置约2～3min，至乙醇挥发完全。
3.8从磁力架上移出PCR管，向PCR管中加入20μL Nuclease Free Water，带磁珠进入下一步PCR扩增。
步骤四：PCR扩增
4.1取出2XHiFi PCR Master Mix(纳昂达)和104102货号(纳昂达)通用扩增引物置于冰上自然融解，混合均匀，瞬时离心备用。
4.2按照下表7在置于冰上的0.2mL PCR管中进行反应体系配制：
表7
步骤三纯化回收产物(带磁珠) 20μL
2X HiFi PCR Master Mix 25μL
1004102货号通用扩增引物 5μL
总计 50μL
4.3将PCR管放置在PCR仪中启动如下表8程序(Cycling Program IV)：
表8
Figure PCTCN2021133239-appb-000004
步骤五：扩增文库纯化
5.1根据引物类型进行扩增产物纯化。加入50μL
Figure PCTCN2021133239-appb-000005
SP Beads，混合均匀，25℃孵育5–10min。
5.2将PCR管瞬时离心后放置于磁力架上5min至液体完全澄清，使用移液器吸取移弃上清。
5.3沿PCR管侧壁缓慢加入150μL 80％乙醇，注意勿扰动磁珠，静置30s，使用移液器吸取移弃上清。
5.4重复步骤5.3一次。
5.5将PCR管瞬时离心后放置于磁力架上，使用10μL吸头移去少量残留乙醇，注意勿吸到磁珠。
5.6打开PCR管管盖，并于室温静置约5min，至乙醇挥发完全。
5.7移出PCR管，向PCR管中加入30μL TE Solution，涡旋振荡混匀，室温孵育5min。
5.8将PCR管瞬时离心后放置于磁力架上2min至液体完全澄清，使用移液器小心将上清转移至一个新的0.2mL PCR管中进行保存，注意勿吸到磁珠。
实施例1实验结果总结，通过方案一至方案四对比，本申请测试发现第一步的酶(例如：大肠杆菌聚合酶I或Klenow大片段或Bst DNA聚合酶等常温聚合酶)浓度在0.001-0.045U/μL范围较为合适。此步骤的控制就是避免过度反复切割和链置换导致的引入过多的突变，相对应的，如果把聚合酶浓度调高(传统的方案)，对应引入的突变明显增多，如图5所示方案一至方案三在引入假融合突变时相对传统方案要明显减少，对比方案四是传统方案，会引入大量的假融合突变。在后续的高温失活第一步的打断酶的同时，第二步会多加普通Taq酶完成第一步没有完成的链置换过程和加A过程，此步骤的酶浓度在0.05～0.1U/μL范围。如图5所示，不同功能的酶量对引入突变的影响，从结果上看方案一至方案三明显好于方案四，方案一至方案三是本申请的优化方案，方案四是模拟其它主要专利的方案结果。
实施例2
酶切打断时的温度控制对酶切引入突变的影响
本方案的流程步骤和实施例1相同，唯一的区别是打断温度不同，打断温度分别测试了25℃，28℃，32℃和37℃，主要的试剂组成用本申请的优化方案(方案二)，与实施例1的区别主要在下面表，即第一步酶切的反应温度，除此之外，其它步骤完全相同。
表9
25/28/32/37℃ 25-30min
65℃ 30min
4℃ Hold
实验结果发现，在测试25℃、28℃、32℃和37℃的反应条件下，在25～32℃在引入突变时明显少于37℃反应，见图6，这个优化在检测融合时消除酶切产生的背景分析到真实融合突变意义重大。
实施例3
对比市面上的主要竞品试剂盒的数据表现
本申请在优化出方案时，也分别购买了下列产品进行了对比测试，具体竞品名称和货号如下表10：
表10
Figure PCTCN2021133239-appb-000006
本实施例选择的三个代表性产品和本公司超声打断的建库方案进行对比，分别按照各自说明书操作，用100ng的标准品进行打断建库，KAPA公司的打断方案是在同一个条件下打断完成，即在37℃一步完成所以的打断和链置换过程；Vazyme公司的产品虽然是打断、链置换和加A是在两步完成，但是由于只注重产量，导致过度打断，文库产偏最高，同时引入的SNP和假的融合突变也最多，如图7-9所示数据。Swift公司打断虽然没有过度打断，但是文库产量也很低，说明第一步的聚合酶的量不能太多。同时Swift公司的突变引入也多于本申请的优化方案。本申请的技术方案的打断效率明显高于超声打断，同时引入突变水平也控制的比其它公司的打断方案要低很多。如图7-9所示，图7展示的是投入100ng DNA，按照各自 的说明书操作，扩增6个循环的文库产量，本申请方案在产量方面明显优于超声打断和S公司的文库产量。在引入SNP突变方面，如图8所示，本申请方案是最接近超声打断方案的一种酶切建库方案，V公司和K公司产品在引入大量的SNP突变。同样在引入假的融合突变方面，本申请方案也和超声打断接近，V和K公司的产品在引入假融合突变的方面也很高，如图9所示。
综上所述，本申请优化了一种酶切打断试剂盒，解决了样本打断的问题，与超声打断对比，解决了超声打断的高成本和不适应自动化问题，本申请保证打断效率的同时，重点优化控制突变的引入水平，主要通过控制打断时的酶量和打断温度，使打断在能完成的同时不过度打断导致的引入大量突变。本发明人测试发现在打断时大肠杆菌聚合酶I或Klenow大片段或Bst DNA聚合酶等常温聚合酶，浓度在0.001～0.045U/μL范围。在后续的高温失活第一步的打断酶的同时，多加普通Taq酶完成第一步没有完成的链置换过程和加A过程，此步骤的酶浓度在0.05～0.1U/μL范围，打断温度控制在25～32℃会比较有利于避免大量突变的引物。本申请解决了困扰酶切打断能够大规模应用的核心问题，大量引物非真实的突变，本申请通过优化控制的突变的引物，使得本申请的方案可以解决低频突变样本的检测，比如大量的临床样本FFPE样本的检测。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 一种低频引入突变的酶切打断建库方法，其特征在于，包括DNA酶切片段化和补平加A、接头连接、PCR扩增和文库纯化的步骤，其中，所述DNA酶切片段化和补平加A步骤中，通过控制常温DNA聚合酶的用量控制反复切割和链置换。
  2. 根据权利要求1所述的酶切打断建库方法，其特征在于，所述DNA酶切片段化和补平加A步骤中，所述常温DNA聚合酶的用量为0.001～0.045U/μL；
    优选的，所述常温DNA聚合酶为选自大肠杆菌聚合酶I、Klenow大片段或Bst DNA聚合酶中的一种或多种。
  3. 根据权利要求1所述的酶切打断建库方法，其特征在于，所述DNA酶切片段化和补平加A步骤中，耐热DNA聚合酶的用量为0.05～0.2U/μL；
    优选的，所述耐热DNA聚合酶为Taq DNA聚合酶。
  4. 根据权利要求1至3中任一项所述的酶切打断建库方法，其特征在于，所述DNA酶切片段化和补平加A步骤中，所述常温DNA聚合酶的反应温度为25～32℃；优选为25℃；
    优选的，所述常温DNA聚合酶的灭活温度为65℃；
    优选的，所述常温DNA聚合酶的反应时间为25～30min；
    优选的，所述常温DNA聚合酶的灭活反应时间为25～30min。
  5. 根据权利要求1所述的酶切打断建库方法，其特征在于，所述DNA酶切片段化和补平加A步骤中，打断酶溶液包括T4 GP32 0.01～0.03μg/μL、DNaseI 0.004～0.01U/μL、Klenow DNA聚合酶0.001～0.045U/μL、Taq DNA聚合酶0.05～0.2U/μLU/μL和50％甘油；
    优选的，所述打断酶溶液包括T4 GP32 0.01μg/μL、DNase I 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.2U/μL和50％甘油。
  6. 根据权利要求1所述的酶切打断建库方法，其特征在于，所述DNA酶切片段化和补平加A步骤中，所用的打断酶缓冲液包括Tris-HCl 5～15mM、NaCl 20～40mM、MgCl 23～8mM、CaCl 21～3mM、MnCl 21～3mM、dNTPs 0.6～1mM、ATP 5～15mM；
    优选的，所述打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 25mM、CaCl 22mM、MnCl 22mM、dNTPs 0.6mM、ATP 10mM。
  7. 根据权利要求5或6所述的酶切打断建库方法，其特征在于，所述酶切打断建库方法包括：取所述打断酶缓冲液和所述打断酶溶液混合得到打断体系，向所述打断体系中加入待打断的DNA样本，25～32℃反应25～30min，然后65℃反应25～30min。
  8. 一种低频引入突变的酶切打断建库试剂盒，其特征在于，包括打断酶溶液和打断酶缓冲液：所述打断酶溶液包括常温DNA聚合酶，所述常温DNA聚合酶的反应终浓度为0.001～0.045U/μL；
    更优选的，所述常温DNA聚合酶为选自大肠杆菌聚合酶I、Klenow大片段或Bst DNA聚合酶中的一种或多种；
    优选的，所述打断酶溶液还包括耐热DNA聚合酶，所述耐热DNA聚合酶的反应终浓度为0.05～0.2U/μL；
    更优选的，所述耐热DNA聚合酶为Taq DNA聚合酶。
  9. 根据权利要求8所述的酶切打断建库试剂盒，其特征在于，打断酶溶液包括T4 GP32 0.01μg/μL、DNaseI 0.004U/μL、Klenow DNA聚合酶0.001～0.045U/μL、Taq DNA聚合酶0.05～0.2U/μL和50％甘油；
    优选的，所述打断酶溶液包括T4 GP32 0.01μg/μL、DNase I 0.004U/μL、Klenow DNA聚合酶0.045U/μL、Taq DNA聚合酶0.2U/μL和50％甘油。
  10. 根据权利要求8所述的酶切打断建库试剂盒，其特征在于，所述DNA酶切片段化和补平加A步骤中，所用的打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 25mM、CaCl 22mM、MnCl 22mM、dNTPs 0.6mM、ATP 10mM；
    优选的，所述打断酶缓冲液包括Tris-HCl 10mM、NaCl 30mM、MgCl 25mM、CaCl 22mM、MnCl 22mM、dNTPs 0.6mM、ATP 10mM；
    优选的，所述酶切打断建库试剂盒还包括接头连接、PCR扩增和文库纯化相关的试剂。
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