WO2020113460A1 - 滚环扩增方法、测序文库制备方法及制得的dna纳米球 - Google Patents

滚环扩增方法、测序文库制备方法及制得的dna纳米球 Download PDF

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WO2020113460A1
WO2020113460A1 PCT/CN2018/119335 CN2018119335W WO2020113460A1 WO 2020113460 A1 WO2020113460 A1 WO 2020113460A1 CN 2018119335 W CN2018119335 W CN 2018119335W WO 2020113460 A1 WO2020113460 A1 WO 2020113460A1
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dna
stranded dna
ligase
rolling circle
circle amplification
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PCT/CN2018/119335
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English (en)
French (fr)
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廖莎
陈奥
章文蔚
徐崇钧
沈寒婕
何琳
许军强
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深圳华大智造极创科技有限公司
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Priority to EP18942297.5A priority Critical patent/EP3892735A4/en
Priority to US17/299,323 priority patent/US20220025429A1/en
Priority to KR1020217017811A priority patent/KR102618147B1/ko
Priority to AU2018452059A priority patent/AU2018452059B2/en
Priority to CN201880098239.3A priority patent/CN112805390B/zh
Priority to SG11202105957YA priority patent/SG11202105957YA/en
Priority to CA3122127A priority patent/CA3122127C/en
Priority to JP2021532178A priority patent/JP7393429B2/ja
Priority to PCT/CN2018/119335 priority patent/WO2020113460A1/zh
Priority to CN202410288036.8A priority patent/CN118222672A/zh
Publication of WO2020113460A1 publication Critical patent/WO2020113460A1/zh

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    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
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    • C12Q1/6869—Methods for sequencing
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    • C40—COMBINATORIAL TECHNOLOGY
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/125—Rolling circle

Definitions

  • the invention relates to the field of sequencing technology, in particular to a rolling circle amplification method for preparing DNA nanospheres, a method for preparing a sequencing library, and the prepared DNA nanospheres.
  • Rolling circle amplification uses single-stranded circular DNA/RNA as a template to replicate DNA/RNA in large quantities. Because of its fast, accurate, and small amount of required templates, it has become an important research method in the field of biomedical technology and bio-nanotechnology.
  • the sequencing method of DNA nanospheres (DNB) formed by BGI based on RCA method does not accumulate the error rate of amplification, so it is superior to other sequencing platforms at the source of sequencing accuracy, and has been getting more and more Widely used.
  • DNB needs to be prepared using single-stranded circular DNA as a template, the preparation process of single-stranded circularization needs to be additionally added in library preparation, which increases the complexity of library preparation.
  • single-stranded circular DNA in sample preparation, single-stranded circular DNA must be obtained first, and its library preparation process includes steps such as DNA extraction, interruption, addition of adaptors, PCR amplification (optional), purification, single-stranded circularization, and purification.
  • the double-stranded DNA is denatured at both ends of the single-stranded DNA and complements with a mediator sequence (Splint), circularizes under the action of T4 ligase, and then digests the linear DNA Magnetic beads were purified, and then DNB was obtained by rolling circle amplification (RCA) under the action of Phi29 polymerase.
  • Splint mediator sequence
  • the sequencing method based on DNB has two more steps of late circularization and purification, it increases the difficulty and time of sample preparation, and at the same time increases the cost of sample preparation.
  • the efficiency of these two steps is only 10% to 30%, which greatly increases the amount of sample input. Therefore, the current DNB-based sequencing technology requires a large number of samples, which limits its use in the sequencing of rare samples.
  • PCR amplification can be used to make up for the lack of sample volume, PCR will introduce errors and is increasingly recognized by industry players.
  • the invention provides a rolling circle amplification method, a sequencing library preparation method and the prepared DNA nanosphere.
  • the method no longer uses single-stranded circularized DNA as a template, but directly uses double-stranded DNA as a template for DNB preparation, saving
  • the single-chain cyclization process in the de-DNB preparation process simplifies the DNB preparation process, shortens the preparation time, saves the preparation cost, and reduces the amount of samples used.
  • an embodiment provides a rolling circle amplification method, which includes: sequentially denaturing and annealing double-stranded DNA and a mediating sequence (Splint oligo) in the same system to make the mediating sequence and the denatured
  • the two ends of the single-stranded DNA are complementary paired; in the above system, the ligase and the polymerase are introduced at the same time, the two ends of the single-stranded DNA are connected under the action of the ligase, and the mediating sequence is used as a primer under the action of the polymerase.
  • a rolling circle amplification reaction is performed to obtain an amplified product, which in a preferred embodiment is a DNA nanosphere.
  • the double-stranded DNA is selected from double-stranded DNA amplified by PCR and a ligation product, wherein the ligation product is DNA with a linker after the DNA is interrupted.
  • the above-mentioned mediating sequence is complementary paired with only two ends of one of the two single-stranded DNAs of the double-stranded DNA after denaturation.
  • the aforementioned ligase is T4 DNA ligase.
  • the above polymerase is Phi 29 polymerase.
  • the above denaturation is performed at 95°C.
  • the above annealing is performed at 40°C.
  • the reaction under the action of the above-mentioned ligase and polymerase is carried out at 30°C.
  • the reaction under the action of the above ligase and polymerase is carried out for more than 20 minutes.
  • ATP is also introduced into the above system to provide energy for the above ligase.
  • the amount of the above double-stranded DNA added to the system is femtomol (fmol) level.
  • the amount of the double-stranded DNA added to the system is 1 femtomol or more, preferably 10 femtomol or more, and more preferably 40 femtomol or more.
  • the above method is a rolling circle amplification method used on a BGI sequencing platform.
  • an embodiment provides a method for preparing a sequencing library, which includes the steps of preparing DNA nanospheres.
  • the steps include: sequentially denaturing double-stranded DNA and a mediating sequence (Splint oligo) in the same system and Annealing, so that the mediating sequence complements the denatured single-stranded DNA at both ends; in the above system, ligase and polymerase are simultaneously introduced, under the action of the ligase, the two ends of the single-stranded DNA are connected, and at the same time, the polymerase Next, using the above-mentioned mediating sequence as a primer and the above-mentioned single-stranded DNA as a template to perform a rolling circle amplification reaction to obtain DNA nanospheres.
  • a mediating sequence Splint oligo
  • the double-stranded DNA is selected from double-stranded DNA amplified by PCR and a ligation product, wherein the ligation product is DNA with a linker after the DNA is interrupted.
  • the above method before the step of preparing DNA nanospheres, further includes a step of DNA interruption and adding a linker to obtain the above double-stranded DNA, and optionally, a PCR step after adding the linker.
  • the above-mentioned mediating sequence is complementary paired with only two ends of one of the two single-stranded DNAs of the double-stranded DNA after denaturation.
  • the ligase is T4 DNA ligase; the polymerase is Phi 29 polymerase.
  • the aforementioned denaturation is performed at 95°C; the aforementioned annealing is performed at 40°C.
  • the reaction under the action of the above-mentioned ligase and polymerase is carried out at 30°C.
  • the reaction under the action of the above ligase and polymerase is carried out for more than 20 minutes.
  • ATP is also introduced into the above system to provide energy for the above ligase.
  • the amount of the above double-stranded DNA added to the system is femtomol.
  • the amount of the above double-stranded DNA added to the system is 1 femtomol or more, preferably 10 femtomol or more, and more preferably 40 femtomol or more.
  • the above method is a method for preparing a sequencing library on a BGI sequencing platform.
  • an embodiment provides a DNA nanosphere obtained by the rolling circle amplification method of the first aspect or the sequencing library preparation method of the second aspect.
  • an embodiment provides a use of the DNA nanosphere of the third aspect in sequencing.
  • the rolling circle amplification method of the first aspect or the sequencing library preparation method of the second aspect is used to reduce the initial amount of DNA in library preparation.
  • the rolling circle amplification method of the present invention uses double-stranded DNA as a template, and the double-stranded DNA after PCR purification or adding a linker can be used for DNB preparation without the need for single-strand circularization and purification steps, simplifying the sample preparation process and saving time And cost.
  • the advantage is that, because there is no need for subsequent single-stranded cyclization and purification steps, the amount of sample required is greatly reduced. Even without PCR amplification, it can meet the needs of subsequent sequencing and avoid errors introduced by PCR amplification. Therefore, this method is conducive to the development of PCR-free sample preparation methods. For the preparation of DNB-free samples and rare samples, the advantages of this method are more obvious.
  • FIG. 1 is a schematic diagram of the principle of the rolling circle amplification method for preparing DNA nanospheres in the prior art
  • FIG. 2 is a schematic diagram of the principle of a rolling circle amplification method for preparing DNA nanospheres in an embodiment of the present invention
  • Fig. 3 shows the loading efficiency (BIC), loading quality (Fit) and ESR value reflecting the quality value of DNB in the first cycle (cycle 1) sequencing of DNB prepared by the prior art and the method of the present invention;
  • FIG. 4 is a graph showing four types of base distribution curves prepared by DNB sequencing prepared in the prior art, where curves 1-4 represent the percentages of bases A, T, C, and G, respectively;
  • Fig. 5 is a graph showing four types of base distribution curves prepared by DNB sequencing prepared by the method of the present invention, wherein curves 1-4 represent the percentages of bases A, T, C, and G, respectively.
  • the rolling circle amplification method for preparing DNA nanospheres in the embodiment of the present invention includes: sequentially denaturing and annealing double-stranded DNA and a splicing sequence (Splint) in the same system to make the splicing sequence Complementary pairing with denatured single-stranded DNA at both ends; introduce ligase and polymerase into the system at the same time, connect the two ends of single-stranded DNA under the action of ligase, and at the same time use the mediator sequence as primer and single Strand DNA is used as a template to perform rolling circle amplification reaction to obtain DNA nanospheres.
  • Splint splicing sequence
  • the two ends of the mediating sequence are complementary paired with the denatured single-stranded DNA respectively, so the two ends of the denatured single-stranded DNA can be pulled together, and then the two ends of the single-stranded DNA are connected under the action of the ligase Circular DNA is formed as a template for rolling circle amplification reactions.
  • the mediating sequence may be a sequence complementary to only the two ends of one single-stranded DNA in the two single-stranded DNAs after denaturation of the double-stranded DNA, and one of the two single-stranded strands is selected for circularization , And use this circularized single strand as a template for rolling circle amplification reaction to generate DNA nanospheres.
  • the mediator sequence may be two or more sequences, which can be complementary paired with the two single-stranded DNAs of the double-stranded DNA after denaturation, so that both single-stranded strands can be circularized and used as rolling circles Amplification reaction template.
  • the double-stranded DNA may be double-stranded DNA from any source.
  • the double-stranded DNA is the double-stranded DNA obtained by PCR amplification, especially the double-stranded DNA obtained from the PCR amplification step in the library building process (such as the sequencing library building process).
  • the method uses double-stranded DNA as a template, and the double-stranded DNA purified by PCR can be used for DNB preparation without the need for single-stranded cyclization and purification steps, simplifying the sample preparation process, saving time and cost.
  • the ligase used to connect the two ends of the single-stranded DNA may be any ligase suitable for the circular connection of single-stranded DNA, for example, T4 DNA ligase (ligase), T3 DNA ligase (ligase), T7 DNA ligase, Taq DNA ligase, etc.
  • the ligase is T4 DNA ligase.
  • the polymerase used to catalyze the rolling circle amplification reaction may be any polymerase suitable for the rolling circle amplification reaction, for example, phi29 polymerase, bst polymerase, etc.
  • the polymerase is Phi 29 polymerase.
  • the double-stranded DNA may be denatured in any suitable manner, including but not limited to thermal denaturation and alkali denaturation.
  • thermal denaturation is used to denature the double-stranded DNA.
  • the denaturation is performed at 95°C.
  • the denaturation time may be 1 minute or more, preferably 3 minutes or more, and particularly preferably 3 minutes.
  • the annealing of the mediating sequence and the denatured single-stranded DNA can be performed at an appropriate temperature, which can be specifically based on the length and base of the base sequence of the complementary pairing of both ends of the mediating sequence and the denatured single-stranded DNA.
  • the composition and other factors determine that, in general, the longer the length of the complementary paired base sequence, the corresponding annealing temperature can be relatively higher; the higher the GC content of the complementary paired bases, the corresponding annealing temperature can be high.
  • the annealing is performed at 40°C.
  • the conditions of the reaction under the action of ligase and polymerase can be determined according to the type of ligase and polymerase selected.
  • the optimal reaction conditions (especially the reaction temperature) of the selected ligase and polymerase are basically the same or as close as possible.
  • T4 DNA ligase is used as the ligase
  • Phi 29 polymerase is used as the polymerase.
  • the reaction is better at 30°C.
  • the reaction time can also be determined according to the types of ligase and polymerase.
  • T4 DNA ligase is used as the ligase, and ATP is introduced into the system to provide energy for T4 DNA ligase.
  • the method of the present invention does not need to perform single-stranded cyclization and purification steps, it simplifies the sample preparation process, and has the advantage that the amount of sample required is greatly reduced, and even without PCR amplification, it can meet the needs of subsequent sequencing. To avoid errors introduced by PCR amplification.
  • the amount of double-stranded DNA added to the system can be as low as femtomolar (fmol) levels.
  • the amount of double-stranded DNA added to the system is more than 1 femtomolar, preferably 10 femtomole or more, more preferably 40 femtomole or more.
  • the method of the present invention is conducive to the development of a PCR-free sample preparation method. For the preparation of DNB-free samples and rare samples, the advantages of this method are more obvious.
  • the method of the present invention is widely applicable to various DNB-based sequencing platforms, especially to the BGI sequencing platform.
  • the sequencing strategy may be BGISEQ-500 SE50 sequencing and the like.
  • An embodiment of the present invention provides a method for preparing a sequencing library, which includes the steps of preparing DNA nanospheres.
  • the steps include: sequentially denaturing and annealing double-stranded DNA and a mediating sequence (Splint oligo) in the same system, The complementary sequence of the mediating sequence and the denatured single-stranded DNA is paired; in the above system, ligase and polymerase are simultaneously introduced, and the two ends of the single-stranded DNA are connected under the action of the ligase, and under the action of the polymerase
  • the mediating sequence is a primer, and the single-stranded DNA is used as a template to perform a rolling circle amplification reaction to obtain DNA nanospheres.
  • the double-stranded DNA is double-stranded DNA amplified by PCR.
  • the above method before the step of preparing DNA nanospheres, further includes steps of DNA interruption, adding a linker and PCR to obtain the above double-stranded DNA.
  • the above-mentioned mediating sequence is complementary paired with only two ends of one of the two single-stranded DNAs of the double-stranded DNA after denaturation.
  • the ligase is T4 DNA ligase; the polymerase is Phi 29 polymerase.
  • the aforementioned denaturation is performed at 95°C; the aforementioned annealing is performed at 40°C.
  • the reaction under the action of the above ligase and polymerase is carried out at 30°C for more than 20 min.
  • ATP is also introduced into the above system to provide energy for the above ligase.
  • the amount of the above double-stranded DNA added to the system is femtomol. In a preferred embodiment, the amount of the double-stranded DNA added to the system is 1 femtomol or more, preferably 10 femtomol or more, and more preferably 40 femtomol or more.
  • the above method is a method for preparing a sequencing library on a BGI sequencing platform.
  • the DNA nanospheres prepared in the embodiments of the present invention can be directly sequenced on the computer. Therefore, in one embodiment of the present invention, the use of the DNA nanospheres prepared in the embodiments of the present invention in sequencing is provided.
  • E. coli samples according to the BGISEQ-500 SE50 sequencing library preparation kit (Shenzhen Huada Zhizao Technology Co., Ltd.) (average inserts 170bp) instructions, DNA extraction, interruption, add adapters, PCR amplification , Purification, quantitative and other processes.
  • PCR products of 120 fmol, 80 fmol, and 40 fmol are respectively taken, and DNB preparation is performed according to the following steps.
  • the existing method requires 1.5h to 2h, and the method of the present invention can obtain the DNB in only 30 minutes, and shortens a lot of manual operations and reduces manual errors.
  • the amount of DNB obtained by the method of the present invention using 40 fmol PCR products is equivalent to the amount of DNB obtained by the existing method using 160 fmol PCR products, that is, the amount of samples required by the method of the present invention is only 25% of the prior art.
  • the DNB obtained by the existing DNB preparation method and the DNB obtained by the DNB preparation method of the present invention are loaded according to the instructions of the BGISEQ500SE50 sequencing kit, and the first cycle (cycle1) sequencing is performed. Compare the loading conditions of DNB obtained by the two methods, and analyze the loading efficiency and quality of the DNB by the two methods. As shown in Figure 3, there is no significant difference between the two methods in loading efficiency (BIC) and loading quality (Fit), while comparing ESR (reflecting the DNB quality value), there is no significant difference. It shows that the preparation method of DNB of the present invention will not affect the quality and loading efficiency of DNB.
  • Example 1 uses an E. coli library with an average insert of 170 bp.
  • the human library NA12878 was used to prepare an average insert of 320 bp and 400 bp for testing.
  • BGISEQ500PE100 and PE150 library preparation kits DNA extraction, interruption, addition of adaptors, PCR amplification, purification, quantification and other processes are performed.
  • the two libraries were taken at 40 fmol each, and DNB preparation and quantification were performed according to the DNB preparation method of the present invention in Example 1, labeled as H400 library and H320 library, respectively, and then SE50 sequencing was performed according to the sequencing instructions.
  • the BGI500 off-machine report shows that the DNB prepared by this method, whether it is H400 or H320, has good sequencing quality, Q30 is greater than 90% and ESR is greater than 85%, and the chip output rate is greater than 85%, which exceeds the library sequencing.
  • the standard parameters ⁇ 90% Q30, ⁇ 80% ESR, ⁇ 80% chip output rate), and other sequencing indicators (such as lag, runon) also meet the basic parameters ( ⁇ 0.1).
  • the H400 library was used, and PE100 sequencing was performed using the existing method and the method of the present invention, respectively.
  • the DNB preparation method is as in Example 1.
  • the DNB prepared by the two methods are respectively loaded into two sequencing lanes of a chip, and PE100 sequencing is loaded and sequenced according to the BGISEQ500 PE100 kit.
  • the statistical results of sequencing data of existing methods are shown in Table 3 and FIG. 4; the statistical results of sequencing data of the method of the present invention are shown in Table 4 and FIG. 5.

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Abstract

提供了一种滚环扩增方法、测序文库制备方法及制得的DNA纳米球，该滚环扩增方法，包括：将双链DNA和介导序列在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在体系中同时引入连接酶和聚合酶，在连接酶作用下使所述单链DNA两端连接，同时在聚合酶作用下以介导序列为引物、以单链DNA为模板进行滚环扩增反应，得到DNA纳米球。

Description

滚环扩增方法、测序文库制备方法及制得的DNA纳米球 技术领域
本发明涉及测序技术领域，具体涉及一种制备DNA纳米球的滚环扩增方法、测序文库制备方法及制得的DNA纳米球。
背景技术
滚环扩增(RCA)是以单链环状DNA/RNA为模版，将DNA/RNA进行大量复制的过程。因其具有快速、准确、所需模板量少等特点，目前已成为生物医学技术和生物纳米技术领域重要的研究手段。华大基因(BGI)基于RCA方法形成的DNA纳米球(DNB)的测序方法，扩增发生的错误率不累积，从而在测序准确性的源头上就优于其他测序平台，得到了越来越广泛的应用。但是，由于DNB需要以单链环状DNA为模板制备而成，在文库制备中需要额外增加单链环化的制备过程，增加了文库制备的复杂度。具体而言，在样品制备中，必须先得到单链环状DNA，其文库制备流程包括DNA提取，打断，加接头，PCR扩增(可选)，纯化，单链环化，纯化等步骤。如图1所示，现有制备DNB的技术中，双链DNA经变性后的单链两端与介导序列(Splint oligo)互补配对，在T4连接酶的作用下环化，然后消化线性DNA并进行磁珠纯化，之后再在Phi29聚合酶的作用下滚环扩增(RCA)得到DNB。
由于基于DNB的测序方法多了后期环化和纯化两个步骤，增加了样品制备难度及时间，同时增加了样品制备的成本。另外，由于环化效率和纯化效率等综合因素影响，此两步的效率仅10％～30％，大大增加了样品投入量。因而目前基于DNB的测序技术所需要的样品量偏多，限制了其在珍稀样品测序的使用范围。虽然可以通过PCR扩增来弥补样品量不足的缺陷，但是PCR会引入错误，越来越不被业界人士所认可。
发明内容
本发明提供一种滚环扩增方法、测序文库制备方法及制得的DNA纳米球，该方法不再以单链环化DNA为模版，而是直接以双链DNA为模板进行DNB制备，省去DNB制备过程中单链环化过程，简化了DNB制备流程，缩短了制备时间，节约了制备成本，且降低了样品的使用量。
根据第一方面，一种实施例中提供一种滚环扩增方法，包括：将双链DNA和介导序列(Splint oligo)在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在上述体系中同时引入连接酶和聚合酶，在上述连接酶作用下使上述单链DNA两端连接，同时在上述聚合酶作用下以上述介导序列为引物、以上述单链DNA为模板进行滚环扩增反应，得到扩增产物，在优选实施例中为DNA纳米球。
在优选实施例中，上述双链DNA选自PCR扩增得到的双链DNA、连接产物，其中连接产物为DNA打断后加上接头的DNA。
在优选实施例中，上述介导序列仅与上述双链DNA变性后的两条单链中的一条单链DNA两端互补配对。
在优选实施例中，上述连接酶是T4DNA连接酶。
在优选实施例中，上述聚合酶是Phi 29聚合酶。
在优选实施例中，上述变性在95℃下进行。
在优选实施例中，上述退火在40℃下进行。
在优选实施例中，上述连接酶和聚合酶作用下的反应在30℃下进行。
在优选实施例中，上述连接酶和聚合酶作用下的反应进行20min以上。
在优选实施例中，上述体系中还引入ATP为上述连接酶提供能量。
在优选实施例中，上述双链DNA在体系中的加入量是飞摩尔(fmol)水平。
在优选实施例中，上述双链DNA在体系中的加入量是1飞摩尔以上，优选10飞摩尔以上，更优选40飞摩尔以上。
在优选实施例中，上述方法是用于BGI测序平台上的滚环扩增方法。
根据第二方面，一种实施例中提供一种测序文库制备方法，包括制备DNA纳米球的步骤，该步骤包括：将双链DNA和介导序列(Splint oligo)在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在上述体系中同时引入连接酶和聚合酶，在上述连接酶作用下使上述单链DNA两端连接，同时在上述聚合酶作用下以上述介导序列为引物、以上述单链DNA为模板进行滚环扩增反应，得到DNA纳米球。
在优选实施例中，上述双链DNA选自PCR扩增得到的双链DNA、连接产物，其中连接产物为DNA打断后加上接头的DNA。
在优选实施例中，上述方法在制备DNA纳米球的步骤之前，还包括DNA打断、加接头步骤以得到上述双链DNA，任选地，在加接头之后还包括PCR步骤。
在优选实施例中，上述介导序列仅与上述双链DNA变性后的两条单链中的一条单链DNA两端互补配对。
在优选实施例中，上述连接酶是T4DNA连接酶；上述聚合酶是Phi 29聚合酶。
在优选实施例中，上述变性在95℃下进行；上述退火在40℃下进行。
在优选实施例中，上述连接酶和聚合酶作用下的反应在30℃下进行。
在优选实施例中，上述连接酶和聚合酶作用下的反应进行20min以上。
在优选实施例中，上述体系中还引入ATP为上述连接酶提供能量。
在优选实施例中，上述双链DNA在体系中的加入量是飞摩尔水平。
在优选实施例中，上述双链DNA在体系中的加入量是1飞摩尔以上，优选 10飞摩尔以上，更优选40飞摩尔以上。
在优选实施例中，上述方法是用于BGI测序平台上的测序文库制备方法。
根据第三方面，一种实施例中提供一种第一方面的滚环扩增方法或第二方面的测序文库制备方法得到的DNA纳米球。
根据第四方面，一种实施例中提供一种第三方面的DNA纳米球在测序中的用途。
在优选实施例中，第一方面的滚环扩增方法或第二方面的测序文库制备方法在文库制备中用于降低DNA的起始量。
本发明的滚环扩增方法，以双链DNA为模板，PCR纯化后或加接头的双链DNA即可用于DNB制备，无需进行单链环化和纯化步骤，简化了样品制备流程，节省时间和成本。更具优势的地方在于，由于无需后续单链环化和纯化的步骤，样品所需量大大减少，即使不做PCR扩增，也能满足后续测序所需，避免了PCR扩增引入的错误。因此，此方法利于发展无需PCR(PCR free)的样品制备方法，针对无需PCR的样品以及珍稀样品的DNB制备，此方法的优势更为明显。
附图说明
图1为现有技术中制备DNA纳米球的滚环扩增方法原理示意图；
图2为本发明实施例中制备DNA纳米球的滚环扩增方法原理示意图；
图3为现有技术和本发明方法制备的DNB在第一循环(cycle1)测序中装载效率(BIC)、装载质量(Fit)和反映DNB质量值的ESR值的情况；
图4为现有技术制备的DNB测序显示的四种碱基分布曲线图，其中曲线1-4分别表示碱基A、T、C、G百分含量；
图5为本发明方法制备的DNB测序显示的四种碱基分布曲线图，其中曲线 1-4分别表示碱基A、T、C、G百分含量。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
如图2所示，本发明实施例中制备DNA纳米球的滚环扩增方法，包括：将双链DNA和介导序列(Splint oligo)在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在体系中同时引入连接酶和聚合酶，在连接酶作用下使单链DNA两端连接，同时在聚合酶作用下以介导序列为引物、以单链DNA为模板进行滚环扩增反应，得到DNA纳米球。
本发明实施例中，介导序列两端分别与变性的单链DNA两端互补配对，因此能够将变性的单链DNA两端拉在一起，然后在连接酶的作用下单链DNA两端连接形成环状DNA，作为滚环扩增反应的模板。本发明实施例中，介导序列可以是一条仅与双链DNA变性后的两条单链中的一条单链DNA两端互补配对的序列，选择两条单链中的一条单链进行环化，并以此环化单链作为滚环扩增反应的模板生成DNA纳米球。当然，作为优选方式，介导序列可能是两种或以上的序列，能够与双链DNA变性后的两条单链DNA两端互补配对，这样两条单链均能发生环化并作为滚环扩增反应的模板。
本发明实施例中，双链DNA可以是任何来源的双链DNA。作为优选实施例，双链DNA是PCR扩增得到的双链DNA，尤其是来源于建库过程(如测序文库建库过程)中PCR扩增步骤得到的双链DNA。该方法以双链DNA为模板，PCR纯化后的双链DNA即可用于DNB制备，无需进行单链环化和纯化步骤，简化了样品制备流程，节省时间和成本。
本发明实施例中，用于使单链DNA两端连接的连接酶可以是任何适合单链DNA环化连接的连接酶，例如，T4 DNA ligase(连接酶)，T3 DNA ligase(连接酶)，T7 DNA ligase(连接酶)，Taq DNA ligase(连接酶)等，在优选实施例中，连接酶是T4 DNA连接酶。
本发明实施例中，用于催化滚环扩增反应的聚合酶可以是任何适合滚环扩增反应的聚合酶，例如，phi29聚合酶，bst聚合酶等，在优选实施例中，聚合酶是Phi 29聚合酶。
本发明实施例中，双链DNA可以通过任何合适的方式变性，包括但不限于热变性和碱变性等。但是从操作的便捷性和不对后续反应产生负面影响的角度，在优选实施例中，选用热变性对双链DNA进行变性处理。例如，在一个优选实施例中，变性在95℃下进行。变性时间可以是1分钟以上，优选3分钟以上，特别优选3分钟。
本发明实施例中，介导序列与变性的单链DNA的退火可以在合适的温度进行，具体可以根据介导序列两端和变性的单链DNA两端互补配对的碱基序列长度、碱基组成等因素确定，一般而言，两端互补配对的碱基序列长度越长，相应的退火温度可以相对越高；两端互补配对的碱基中GC含量越高，相应的退火温度可以相对越高。在一个优选实施例中，退火在40℃下进行。
本发明实施例中，连接酶和聚合酶作用下的反应的条件，如反应温度和时间等，可以根据选用的连接酶和聚合酶的种类来确定。为保证反应效率，尽量保证所选用的连接酶和聚合酶的最适反应条件(特别是反应温度)基本一致或 尽量接近。例如，在一个优选实施例中，连接酶选用T4 DNA连接酶，聚合酶选用Phi 29聚合酶，反应在30℃下进行效果较好。相应地，反应时间也可以根据连接酶和聚合酶的种类来确定，一般而言，尽量保证连接酶和聚合酶催化的反应都能获得比较充分的反应程度。在一个优选实施例中，在连接酶选用T4DNA连接酶、聚合酶选用Phi 29聚合酶的情况下，反应时间20min以上效果较好，特别是在30℃下进行20min的反应效果较佳。
本发明实施例中，根据选用的连接酶的具体种类，还可能需要加入ATP为连接酶提供能量。例如，在一个优选实施例中，连接酶选用T4 DNA连接酶，体系中引入ATP为T4 DNA连接酶提供能量。
由于本发明方法无需进行单链环化和纯化步骤，简化了样品制备流程的同时，更具优势的地方在于，样品所需量大大减少，即使不做PCR扩增，也能满足后续测序所需，避免了PCR扩增引入的错误。在优选实施例中，双链DNA在体系中的加入量可以低至飞摩尔(fmol)水平，例如，在一些优选实施例中，双链DNA在体系中的加入量是1飞摩尔以上，优选10飞摩尔以上，更优选40飞摩尔以上。本发明方法利于发展无需PCR(PCR free)的样品制备方法，针对无需PCR的样品以及珍稀样品的DNB制备，此方法的优势更为明显。
本发明方法广泛适用于各种基于DNB的测序平台，特别是适用于华大基因(BGI)测序平台，测序策略可以是BGISEQ-500 SE50测序等。
本发明的一种实施例中提供一种测序文库制备方法，包括制备DNA纳米球的步骤，该步骤包括：将双链DNA和介导序列(Splint oligo)在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在上述体系中同时引入连接酶和聚合酶，在上述连接酶作用下使上述单链DNA两端连接，同时在上述聚合酶作用下以上述介导序列为引物、以上述单链DNA为模板进行滚环扩增反应，得到DNA纳米球。
在优选实施例中，上述双链DNA是PCR扩增得到的双链DNA。在优选实 施例中，上述方法在制备DNA纳米球的步骤之前，还包括DNA打断、加接头和PCR步骤以得到上述双链DNA。在优选实施例中，上述介导序列仅与上述双链DNA变性后的两条单链中的一条单链DNA两端互补配对。在优选实施例中，上述连接酶是T4DNA连接酶；上述聚合酶是Phi 29聚合酶。在优选实施例中，上述变性在95℃下进行；上述退火在40℃下进行。在优选实施例中，上述连接酶和聚合酶作用下的反应在30℃下进行20min以上。在优选实施例中，上述体系中还引入ATP为上述连接酶提供能量。在优选实施例中，上述双链DNA在体系中的加入量是飞摩尔水平。在优选实施例中，上述双链DNA在体系中的加入量是1飞摩尔以上，优选10飞摩尔以上，更优选40飞摩尔以上。在优选实施例中，上述方法是用于BGI测序平台上的测序文库制备方法。
本发明实施例制备得到的DNA纳米球，可以直接上机测序，因此在本发明的一个实施例中，提供本发明实施例制备得到的DNA纳米球在测序中的用途。
以下通过实施例对本发明进行详细描述，需要说明的是，该实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例1：两种方法比较
(1)DNB制备
大肠杆菌(E.coli)样品，按照BGISEQ-500 SE50测序的文库制备试剂盒(深圳华大智造科技有限公司)(插入片段平均170bp)说明书，进行DNA提取，打断，加接头，PCR扩增，纯化，定量等过程。
取1pmol PCR产物，按照文库制备试剂盒说明书，采用介导序列(Splint oligo)进行环化及以及磁珠(beads)纯化，qubit定量，此过程用时约1h至1.5h，获得250fmol单链环化DNA。按照BGISEQ-500 SE50测序试剂盒的说明书，取其中40fmol单链环化DNA制备DNB。使用qubit进行定量，DNB浓度为12ng/ul，DNB命名为A待用。
按照本发明方法，分别取120fmol、80fmol、40fmol PCR产物，按照以下步 骤进行DNB制备。
(a)配置以下表1的反应体系：
表1
Figure PCTCN2018119335-appb-000001
(b)将表1的反应体系置于PCR仪中，按照如下表2的程序进行变性及退火。当程序进入第一个4℃时，按照BGISEQ-500 SE50测序试剂盒的说明书加入酶混合物(Enzyme mix)I和II，其中含有Phi 29聚合酶，并加入1ul BGI的T4 DNA连接酶(60U/ul)和1ul ATP(Thermo)，混匀后按“skip(跳过)”键，进入30℃进行20min，进入4℃后加入DNB制备试剂盒中的终止缓冲液，进行qubit定量。120fmol、80fmol和40fmol PCR产物得到的DNB浓度分别是37ng/ul、23ng/ul、11ng/ul，分别命名为B、C、D待用。
表2
温度 时间
95℃ 3min
40℃ 2min
4℃ ∞
30℃ 20min
4℃ ∞
比较上述不同的DNB制备方法，从PCR产物到DNB，现有方法需要1.5h至2h，而本发明方法仅需30min内即可得到DNB，且缩短了很多的手动操作，减少了人工误差。从样品投入量来计算，本发明方法使用40fmol PCR产物得到的DNB量与现有方法使用160fmol PCR产物得到的DNB量相当，即本发明方法样品所需量仅为现有技术的25％。
(2)DNB装载
将现有DNB制备方法所得DNB A和本发明方法DNB制备方法所得DNB D按照BGISEQ500SE50测序试剂盒说明书进行DNB装载，并进行第一循环(cycle1)测序。比较根据两种方法所得DNB的装载情况，分析两种方法的DNB装载效率和质量。如图3所示，两种方法在装载效率(BIC)和装载质量(Fit)上无明显差异，同时比较ESR(反映DNB质量值)，也无明显差异。说明本发明的DNB制备方法不会影响DNB质量和装载效率。
实施例2：本发明方法的适用性
实施例1使用大肠杆菌(E.coli)文库，平均插入片段为170bp，为了证明本发明方法的广泛适用性，使用人的文库NA12878制备平均插入片段320bp和400bp进行测试。按照BGISEQ500PE100和PE150文库制备试剂盒，进行DNA提取，打断，加接头，PCR扩增，纯化，定量等过程。
两种文库各取40fmol，按照实施例1中本发明的DNB制备方法进行DNB制备与定量，分别标记为H400文库和H320文库，然后按照测序说明书进行SE50测序。BGI500下机报告显示，此方法制备的DNB，无论是H400还是H320，其测序质量都表现良好，Q30大于90％且ESR大于85％，芯片的产出率大于85％，都超出了该文库测序的标准参数(≥90％Q30，≥80％ESR，≥80％芯片产出率)，其他测序指标(如lag，runon)，也都符合基础参数(≤0.1)。这些结果表明，本发明的DNB制备方法能够适用于不用插入片段及不同物种的文库。以上结果显示本发明方法不影响测序质量。
为测试本发明方法是否会引入CG和AT偏好(bias)，使用H400文库，用现有方法和本发明方法分别进行PE100测序。其中DNB制备方法如实施例1，两种方法所制备的DNB分别装载到一张芯片的两条测序泳道(lane)，PE100测序按照BGISEQ500 PE100试剂盒进行装载和测序。现有方法测序数据统计结果如表3和图4所示；本发明方法测序数据统计结果如表4和图5所示。二者在Q30，错误率(error rate)和GC含量上都无明显区别，但是比较碱基分布的情况，现有方法存在AT偏好(bias)，尤其是读长2(read2)表现更为明显，而本发明方法几乎不存在AT偏好。
表3
Figure PCTCN2018119335-appb-000002
表4
Figure PCTCN2018119335-appb-000003
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (26)

  1. 一种滚环扩增方法，其特征在于，所述方法包括：将双链DNA和介导序列在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在所述体系中同时引入连接酶和聚合酶，在所述连接酶作用下使所述单链DNA两端连接，同时在所述聚合酶作用下以所述介导序列为引物、以所述单链DNA为模板进行滚环扩增反应，得到扩增产物。
  2. 根据权利要求1所述的滚环扩增方法，其特征在于，所述双链DNA选自PCR扩增得到的双链DNA、连接产物，其中所述连接产物为DNA打断后加上接头的DNA。
  3. 根据权利要求1所述的滚环扩增方法，其特征在于，所述介导序列仅与所述双链DNA变性后的两条单链中的一条单链DNA两端互补配对。
  4. 根据权利要求1所述的滚环扩增方法，其特征在于，所述连接酶是T4DNA连接酶。
  5. 根据权利要求1所述的滚环扩增方法，其特征在于，所述聚合酶是Phi 29聚合酶。
  6. 根据权利要求1所述的滚环扩增方法，其特征在于，所述变性在95℃下进行。
  7. 根据权利要求1所述的滚环扩增方法，其特征在于，所述退火在40℃下进行。
  8. 根据权利要求1所述的滚环扩增方法，其特征在于，所述连接酶和聚合酶作用下的反应在30℃下进行；
    任选地，所述连接酶和聚合酶作用下的反应进行20min以上。
  9. 根据权利要求1所述的滚环扩增方法，其特征在于，所述体系中还引入ATP为所述连接酶提供能量。
  10. 根据权利要求1所述的滚环扩增方法，其特征在于，所述双链DNA在 体系中的加入量是飞摩尔水平。
  11. 根据权利要求10所述的滚环扩增方法，其特征在于，所述双链DNA在体系中的加入量是1飞摩尔以上，优选10飞摩尔以上，更优选40飞摩尔以上。
  12. 根据权利要求1所述的滚环扩增方法，其特征在于，所述方法是用于BGI测序平台上的滚环扩增方法。
  13. 一种测序文库制备方法，其特征在于，所述方法包括制备DNA纳米球的步骤，该步骤包括：将双链DNA和介导序列在同一体系中依次进行变性和退火，使介导序列与变性的单链DNA两端互补配对；在所述体系中同时引入连接酶和聚合酶，在所述连接酶作用下使所述单链DNA两端连接，同时在所述聚合酶作用下以所述介导序列为引物、以所述单链DNA为模板进行滚环扩增反应，得到DNA纳米球。
  14. 根据权利要求13所述的测序文库制备方法，其特征在于，所述双链DNA选自PCR扩增得到的双链DNA、连接产物，其中所述连接产物为DNA打断后加上接头的DNA。
  15. 根据权利要求13所述的测序文库制备方法，其特征在于，所述方法在制备DNA纳米球的步骤之前，还包括DNA打断、加接头步骤以得到所述双链DNA，任选地，加接头后还包括PCR扩增步骤。
  16. 根据权利要求13所述的测序文库制备方法，其特征在于，所述介导序列仅与所述双链DNA变性后的两条单链中的一条单链DNA两端互补配对。
  17. 根据权利要求13所述的测序文库制备方法，其特征在于，所述连接酶是T4DNA连接酶；所述聚合酶是Phi 29聚合酶。
  18. 根据权利要求13所述的测序文库制备方法，其特征在于，所述变性在95℃下进行；所述退火在40℃下进行。
  19. 根据权利要求13所述的测序文库制备方法，其特征在于，所述连接酶和聚合酶作用下的反应在30℃下进行；
    任选地，所述连接酶和聚合酶作用下的反应进行20min以上。
  20. 根据权利要求13所述的测序文库制备方法，其特征在于，所述体系中还引入ATP为所述连接酶提供能量。
  21. 根据权利要求13所述的测序文库制备方法，其特征在于，所述双链DNA在体系中的加入量是飞摩尔水平。
  22. 根据权利要求21所述的测序文库制备方法，其特征在于，所述双链DNA在体系中的加入量是1飞摩尔以上，优选10飞摩尔以上，更优选40飞摩尔以上。
  23. 根据权利要求13所述的测序文库制备方法，其特征在于，所述方法是用于BGI测序平台上的测序文库制备方法。
  24. 根据权利要求1至12所述的滚环扩增方法或权利要求13至23所述的测序文库制备方法得到的DNA纳米球。
  25. 权利要求24所述的DNA纳米球在测序中的用途。
  26. 根据权利要求1至12所述的滚环扩增方法或权利要求13至23所述的测序文库制备方法在文库制备中用于降低DNA的起始量。
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