WO2021007724A1 - 一种全基因组全流程微流控自动化建库方法和装置 - Google Patents

一种全基因组全流程微流控自动化建库方法和装置 Download PDF

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WO2021007724A1
WO2021007724A1 PCT/CN2019/095868 CN2019095868W WO2021007724A1 WO 2021007724 A1 WO2021007724 A1 WO 2021007724A1 CN 2019095868 W CN2019095868 W CN 2019095868W WO 2021007724 A1 WO2021007724 A1 WO 2021007724A1
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microliters
tween
dna
stranded
reaction
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PCT/CN2019/095868
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French (fr)
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崔淼
任悍
汪元涛
陈杨帆
江媛
陈奥
章文蔚
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深圳华大生命科学研究院
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Priority to CN201980096864.9A priority Critical patent/CN113891961A/zh
Priority to PCT/CN2019/095868 priority patent/WO2021007724A1/zh
Publication of WO2021007724A1 publication Critical patent/WO2021007724A1/zh

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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
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

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  • the invention relates to the technical field of library construction, in particular to a method and device for automatic library construction of a whole genome and whole process microfluidic control.
  • the platform is mainly for NGS products of small and medium-sized clinical testing institutions such as hospitals and regional testing centers.
  • the MiniSeq platform makes up for the shortcomings of Illumina's series of instruments that are not flexible enough to face the sequencing needs of a small number of samples.
  • the common miniaturized NGS database construction and sequencing platforms on the market can cover most of the existing sequencing applications.
  • the miniaturized system itself can provide flexibility that HiSeq does not have in sequencing factory-level products.
  • QIAGEN has launched the GeneReader series of fully automated sequencing systems.
  • Thermo and Illumina are also improving the automation of their sequencing platforms, gradually realizing the entire process automation from sample processing, library construction, sequencing to data interpretation. This automated upgrade has created conditions for the standardization and popularization of clinical applications, and has also clarified the different solutions corresponding to the different needs of the clinical examination field and scientific research users.
  • the first three are all based on the microfluidic library construction platform supporting the Illumina company's sequencer and are suitable for second-generation sequencing; the Voltrax platform is based on the microfluidic automated library construction platform supporting the third-generation sequencer of Oxford Nanopore.
  • 10X genomics Chromium genome sequencing solution uses high-throughput droplet microfluidics to wrap high-molecular-weight genomic DNA and coded magnetic beads in microdroplets for hybridization and PCR amplification. Amplicons with 10X tags are obtained, which is the library of the Illumina platform.
  • the automated Juno TM system introduced by Fluidigm is to manually process the sample, use high-throughput microdroplets on the chip to obtain tagged amplicon products, and then use manual purification and the second round of PCR amplification.
  • the Voltrax launched by Oxford Nanopore also realizes the automatic construction of the third-generation sequencing library by means of digital microfluidics, that is, starting with genomic DNA, breaking the genomic DNA, adding a linker, and then adding a fixed strand to complete the third-generation sequencing library. Construct.
  • the magnetic bead purification steps of 10X genomics' Chromium genome sequencing solution need to be performed manually, and the magnetic bead purification and adapter addition of Fluidigm's automated Juno TM system also requires manual operations, which cannot realize the entire process of automated library building.
  • the third-generation sequencing realized by the library constructed on the Voltrax platform has obvious advantages in long read length, the error rate is still relatively high, and it cannot truly replace the second-generation sequencing solution at present.
  • the automated liquid handling system is currently also used in the automated library building process, but this solution has a larger reaction system than the microfluidic system, higher reagent costs, and the entire system cannot be upgraded to a portable device.
  • the present invention uses digital microfluidics to realize rapid and automated database construction to serve personalized sequencing and precision medical systems.
  • the invention controls the movement of droplets by the principle of electrowetting, realizes the automatic construction of microfluidic database of the whole genome and the whole process, with low cost and no pollution.
  • the present invention provides a whole-genome whole-process microfluidic automated library construction method, including: using genomic DNA as the starting material for library construction on a digital microfluidic chip, and controlling droplets through electrowetting principles Moving on the above-mentioned digital microfluidic chip, the reactions of genomic DNA interruption, end repair, adaptor connection, single-strand circularization and DNA nanosphere preparation are carried out in sequence.
  • the reaction system of each step does not exceed 10 microliters.
  • the reaction system contains reaction components and surfactant components for reducing surface tension.
  • the above-mentioned method further comprises performing PCR amplification between the above-mentioned adaptor connection and single-strand circularization.
  • 10-500 ng genomic DNA is used as the starting material for library construction.
  • the above-mentioned surfactant component is Tween 20.
  • the concentration of the Tween 20 in the reaction system is 0.01% to 0.1%.
  • the above-mentioned digital microfluidic chip is provided with a Peltier plate to realize temperature control of the reagent reaction zone.
  • the above-mentioned genomic DNA interruption reaction system includes 1 to 5 microliters of genome interruption buffer and 1 to 3 microliters of genome interruption enzyme.
  • the above method further comprises: after the genomic DNA is interrupted, 1 microliter of 0.1-1M ethylenediaminetetraacetic acid is moved to the previous step of the mixed solution by electrowetting to stop the interruption reaction.
  • 10-20 microliters of magnetic beads are mixed with the disrupted product and incubated for product purification.
  • the above-mentioned end repair reaction system includes 1 to 3 microliters of end repair buffer and 1 to 3 microliters of end repair enzyme mixture, wherein the above end repair buffer includes 0.1 to 1M dATP, 0.03 to 0.2M dNTPs, 0.01 to 0.1% Tween 20; the above-mentioned end repair enzyme mixture includes 0.05 to 0.5 U/ ⁇ L T4 PNK, 0.05 to 0.5 U/ ⁇ L T4 DNA polymerase, 0.005 to 0.05 U/ ⁇ L Klenow polymerase, 0.005 to 0.05 U/ ⁇ L rTaq polymerase, 0.01 ⁇ 0.1% Tween 20.
  • the reaction system for linker connection includes 1 to 3 microliters of linker connection buffer and 1 to 3 microliters of linker ligase mixture, wherein the linker connection buffer includes 0.1 to 1 ⁇ M linker, 0.1 to 10 mM adenosine triphosphate , 3% ⁇ 20% polyethylene glycol 8000, 0.1 ⁇ 10mM hexaammine cobalt trichloride, 0.01 ⁇ 0.1% Tween 20; the above-mentioned joint ligase mixture includes 1 ⁇ 20U/ ⁇ L T4 ligase, 0.01 ⁇ 0.1 % Tween 20.
  • the above-mentioned single-stranded cyclization reaction system includes the purified product of the previous step, and 1 to 3 microliters of single-stranded cyclization buffer and 1 to 3 microliters of single-stranded cyclase mixture, wherein
  • the chain cyclization buffer includes 0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween 20;
  • the above single-chain cyclase mixture includes 0.1 ⁇ 5U/ ⁇ L T4 ligase, 0.01 ⁇ 0.1% Tween 20.
  • the above method further includes: after the single-stranded cyclization, adding 1 to 3 microliters of digestive enzyme mixture for digestion, and then adding 1 to 3 microliters of digestion termination solution to terminate the reaction, wherein the above digestive enzymes are mixed
  • the solution includes 0.1 ⁇ 1U/ ⁇ L exonuclease I, 0.1 ⁇ 10U/ ⁇ L exonuclease III, 0.01 ⁇ 0.1% Tween 20; the above digestion termination solution includes 0.1 ⁇ 1mM ethylenediaminetetraacetic acid, 0.01 ⁇ 0.1% Tween 20.
  • the reaction system for the preparation of DNA nanospheres includes 1 to 5 microliters of DNA nanosphere preparation buffer and 1 to 3 microliters of DNA nanosphere preparation enzyme mixture, wherein the DNA nanosphere preparation enzyme mixture includes 0.1 ⁇ 1U/ ⁇ L phi29 polymerase, 0.01 ⁇ 0.1% Tween 20.
  • DNA nanosphere preparation stop solution is added to terminate the rolling circle linear amplification.
  • the PCR amplification reaction system includes 3 to 5 microliters of PCR amplification enzyme mixture, and the PCR amplification enzyme mixture includes PCR enzyme, 0.1-2 ⁇ M primers, and 0.01-0.1% Tween 20.
  • the present invention provides a full-genome full-process microfluidic automated library building device, including: a microfluidic chip, the microfluidic chip includes a chip substrate and electrode distribution and electrode switches arranged on the chip substrate Combined control, the above-mentioned microfluidic chip is used to implement the method of the first aspect.
  • the solution of the present invention can realize a fully automated database construction process, the manual operation time is less than 5 minutes, and at the same time, it can reduce the pollution of exogenous nucleic acid to the database construction process.
  • the solution of the present invention can realize the library building reaction of each step in a small volume, and reduce the reagent cost by about 10 times.
  • the instruments and chips used in the solution of the present invention are all portable devices, which can realize decentralized applications in remote and low-equipment storage areas.
  • the speed of library construction is twice that of traditional library construction methods, and it does not affect the sensitivity of low-abundance nucleic acid detection.
  • FIG. 1 is a schematic diagram of a low-initiated volume, low-cost, fast and portable whole-genome PCR full-process automated library construction process in an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of a low-initiated quantity, low-cost, fast and portable PCR free full-process automated database construction process in an embodiment of the present invention.
  • FIG. 3 is a schematic diagram of the structure and functional modules of a digital microfluidic automated library building chip in an embodiment of the present invention.
  • FIG. 4 is a comparison diagram of the detection effect of using the metagenomic sequencing scheme to realize the construction and sequencing of the original text library of different abundance diseases, and the detection effect of manual library construction and digital microfluidic library construction in the embodiment of the present invention.
  • a whole-genome whole-process microfluidic automated library building method includes: using genomic DNA as the starting material for library building on a digital microfluidic chip, and controlling droplets through electrowetting principles Moving on the above-mentioned digital microfluidic chip, the reactions of genomic DNA interruption, end repair, adaptor connection, single-strand circularization and DNA nanosphere preparation are carried out in sequence.
  • the reaction system of each step does not exceed 10 microliters.
  • the reaction system contains reaction components and surfactant components for reducing surface tension.
  • the method of the present invention realizes the whole-genome whole-process library construction on the digital microfluidic chip, from genomic DNA interruption, end repair and joint connection, to single-stranded circularization and DNA nanosphere preparation, the whole process is in digitalization It is carried out on a microfluidic chip, basically without manual manual operation, which can reduce the contamination of foreign nucleic acids in the process of library construction.
  • the reaction system for each step of the reaction does not exceed 10 microliters.
  • the solution of the present invention can realize the library building reaction of each step in a small volume and reduce the reagent cost by about 10 times.
  • the present invention controls the movement of liquid droplets (reaction reagents) on the digital microfluidic chip by the principle of Electrowetting (EW).
  • EW Electrowetting
  • the reaction system includes a reaction component and a surfactant component, where the reaction component refers to an effective component participating in a chemical reaction, including buffers and enzymes, etc.; the surfactant component is used to reduce droplets The surface tension.
  • the surfactant component is Tween 20, especially Tween 20 at a concentration of 0.01 to 0.1%.
  • the full-genome full-process microfluidic automated library construction method of the present invention can be implemented in two ways: PCR amplification or PCR-free amplification (PCR free).
  • PCR amplification is performed between the above-mentioned linker connection and single-strand circularization.
  • all the reaction reagents are loaded into the reagent loading area of the digital microfluidic chip, and the driving, separation and mixing of the reaction reagents on the chip are realized by the electrowetting method; the Peltier plate is used The temperature control of the reaction zone is realized, and the magnetic bead purification process is realized by using a mechanical lifting magnet.
  • micro-volume genomic DNA interruption, end repair, adaptor connection, PCR amplification, single-strand circularization, and DNA nanosphere preparation are realized on the chip.
  • it also includes magnetic bead purification and concentration detection.
  • the principles and features of the low-initiated, low-cost, fast and portable whole-genome PCR full-process automated library construction method of the present invention are as follows:
  • the automated library construction process can also save labor costs, while reducing the contamination of the sample library by foreign nucleic acids during the library construction process.
  • the principle of electrowetting is used to realize the driving, separation, mixing and mixing of all reaction reagents and reaction systems on the chip; the Peltier plate is used to realize the temperature control and rise and fall temperature changes of the reaction zone, and the mechanical lifting magnet is used to achieve Magnetic bead purification process to realize digital microfluidic control, low-cost, rapid and automated whole-genome PCR library construction.
  • the library preparation method is as follows:
  • the reagent is the volume used in the micro-reaction system and added with a proper concentration of surfactant to reduce the surface tension of the reaction reagent.
  • the broken genomic DNA is first repaired and adenine nucleotides are added, and then the linker is connected.
  • the ligated mixture is purified by magnetic beads to obtain the adapter ligation product and perform PCR amplification.
  • the PCR-amplified mixture is purified by magnetic beads to obtain the amplified adapter ligation product for double-strand fluorescence quantification and homogenization.
  • Two schemes can be adopted for the preparation of single-stranded loops and DNA nanospheres. The first scheme is to continue single-stranded circularization and digestion using the homogenized amplified product. After the magnetic beads are purified, the single-stranded ring can be used for rolling circle amplification to obtain the final product DNA nanospheres; the second solution is to use the homogenized amplified product to continue the single-stranded circularization and then directly prepare the DNA nanospheres. Finally, single-stranded fluorescent quantitative reagents are used to quantify and inspect the concentration of DNA nanospheres. Except for reagent loading, the entire process can be automated on the chip without any manual operation, and all reaction systems are less than 10 microliters.
  • micro-volume genomic DNA interruption, end repair, linker connection, single-strand circularization, and DNA nanosphere preparation are realized on the chip.
  • it also includes magnetic bead purification and concentration detection.
  • the principles and features of the low-initiated, low-cost, fast, portable, whole-genome PCR-free full-process automated library construction method of the present invention are as follows:
  • genomic DNA can be used as the starting material for library construction, which is suitable for precious and limited-source samples
  • the library preparation requirements can also save labor costs and reduce the contamination of the sample library by foreign nucleic acids during the library construction process.
  • the principle of electrowetting is used to realize the driving, separation, mixing and mixing of all reaction reagents and reaction systems on the chip; the Peltier plate is used to realize the temperature control and rise and fall of the reaction zone, and the mechanical lifting magnet is used Realize the magnetic bead purification process to realize digital microfluidic control, low-cost, rapid and automated whole-genome PCR library construction.
  • the preparation method is as follows:
  • the first scheme is to use the purified linker ligation product to continue single-stranded circularization and digestion. After the magnetic beads are purified, the single-stranded ring can be used for rolling circle amplification to obtain the final product DNA nanospheres; the second solution is to use the purified linker ligation product to continue the single-stranded circularization and then directly prepare the DNA nanospheres. Finally, single-stranded fluorescent quantitative reagents are used to quantify and inspect the concentration of DNA nanospheres. Except for reagent loading, the entire process can be automated on the chip without any manual operation, and all reaction systems are less than 10 microliters.
  • Example 1 Low starting amount, low cost, fast and portable whole-genome PCR full-process automated library construction
  • FIG. 3 The structure and functional modules of the microfluidic chip used in this embodiment are shown in FIG. 3.
  • Reagent storage is divided into three types of reagent tanks: large (L), medium (M), and small (S), which store reagents of less than 20 microliters, less than 5 microliters, and less than 3 microliters.
  • L1 and L2 are loaded with single-stranded fluorescent quantitative reagent and double-stranded fluorescent quantitative reagent;
  • L4 to L7 are respectively loaded with magnetic beads 1, eluent, magnetic bead purification washing solution and magnetic beads 2.
  • S1 to S10 are loaded with genomic DNA, end repair buffer, end repair enzyme mixture, adaptor ligase mixture, single-stranded cyclase mixture, digestion stop solution, fluorescence quantitative standard solution 1, double-stranded fluorescence quantitative standard solution 2 , Single-stranded fluorescence quantitative standard solution 2 and genome interrupting enzyme.
  • M1 to M10 are loaded with eluent, DNA nanosphere preparation enzyme mixture, adapter connection buffer, digestive enzyme mixture, single-stranded circularization buffer, DNA nanosphere preparation stop solution, DNA nanosphere preparation buffer, PCR amplification Enzyme Enzyme Mix, Elution and Genome Interruption Buffer.
  • end repair buffer 0.1 to 1M dATP, 0.03 to 0.2M dNTPs, 0.01 to 0.1% Tween 20
  • end repair enzyme mixture 0.05 to 0.5U/ ⁇ L T4 PNK, 0.05 ⁇ 0.5U/ ⁇ L T4 DNA polymerase, 0.005 ⁇ 0.05U/ ⁇ L Klenow polymerase, 0.005 ⁇ 0.05U/ ⁇ L rTaq polymerase, 0.01 ⁇ 0.1% Tween 20
  • electrowetting Principle Move to the position of heating plate 1 (H1) to mix and mix. Use a gradient reaction at 37°C and 65°C for 45 minutes to complete the end repair.
  • adapter connection buffer 0.1 ⁇ 1 ⁇ M adapter, 0.1 ⁇ 10mM adenosine triphosphate, 3% ⁇ 20% polyethylene glycol 8000, 0.1 ⁇ 10mM hexaammine cobalt trichloride, 0.01 ⁇ 0.1% Tween 20
  • adaptor ligase mixture (1 ⁇ 20U/ ⁇ L T4 ligase, 0.01 ⁇ 0.1% Tween 20)
  • Solution 1 Add 1-3 microliters of single-stranded circularization buffer (0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween) to the amplified fragment after purification and homogenization in step (7) 20), move to the position of heating plate 3 (H3), mix and mix, heat to 95°C for high temperature denaturation for 1-10 minutes, quickly cool and refold, add 1 to 3 microliters of single-stranded cyclase mixture (0.1 ⁇ 5U) / ⁇ L T4 ligase, 0.01 ⁇ 0.1% Tween 20), react at 37°C for 5 ⁇ 60 minutes.
  • single-stranded circularization buffer 0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween
  • Scheme 2 Add 1-3 microliters of single-stranded circularization buffer (0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween) to the amplified fragment after purification and homogenization in step (7) 20), move to the position of heating plate 3 (H3), mix and mix, heat to 95°C for high temperature denaturation for 1-10 minutes, quickly cool and refold, add 1 to 3 microliters of single-stranded cyclase mixture (0.1 ⁇ 5U) / ⁇ L T4 ligase, 0.01 to 0.1% Tween 20), react at 37°C for 5 to 60 minutes.
  • single-stranded circularization buffer 0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween
  • Example 2 Low starting amount, low cost, fast, portable, whole genome PCR-free whole process automated library construction
  • FIG. 3 The structure and functional modules of the microfluidic chip used in this embodiment are shown in FIG. 3.
  • Reagent storage is divided into three types of reagent tanks: large (L), medium (M), and small (S), which store reagents of less than 20 microliters, less than 5 microliters, and less than 3 microliters.
  • L1 and L2 are loaded with single-stranded fluorescent quantitative reagent and double-stranded fluorescent quantitative reagent;
  • L4 to L7 are respectively loaded with magnetic beads 1, eluent, magnetic bead purification washing solution and magnetic beads 2.
  • S1 to S10 are loaded with genomic DNA, end repair buffer, end repair enzyme mixture, adaptor ligase mixture, single-stranded cyclase mixture, digestion stop solution, fluorescence quantitative standard solution 1, double-stranded fluorescence quantitative standard solution 2 , Single-stranded fluorescence quantitative standard solution 2 and genome interrupting enzyme.
  • M1 to M10 are loaded with eluent, DNA nanosphere preparation enzyme mixture, adapter connection buffer, digestive enzyme mixture, single-stranded circularization buffer, DNA nanosphere preparation stop solution, DNA nanosphere preparation buffer, PCR amplification Enzyme Enzyme Mix, Elution and Genome Interruption Buffer.
  • end repair buffer 0.1 to 1M dATP, 0.03 to 0.2M dNTPs, 0.01 to 0.1% Tween 20
  • end repair enzyme mixture 0.05 to 0.5U/ ⁇ L T4 PNK, 0.05 ⁇ 0.5U/ ⁇ L T4 DNA polymerase, 0.005 ⁇ 0.05U/ ⁇ L Klenow polymerase, 0.005 ⁇ 0.05U/ ⁇ L rTaq polymerase, 0.01 ⁇ 0.1% Tween 20
  • electrowetting Principle Move to the position of heating plate 1 (H1) to mix and mix. Use a gradient reaction at 37°C and 65°C for 45 minutes to complete the end repair.
  • adapter connection buffer 0.1 ⁇ 1 ⁇ M adapter, 0.1 ⁇ 10mM adenosine triphosphate, 3% ⁇ 20% polyethylene glycol 8000, 0.1 ⁇ 10mM hexaammine cobalt trichloride, 0.01 ⁇ 0.1% Tween 20
  • adaptor ligase mixture (1 ⁇ 20U/ ⁇ L T4 ligase, 0.01 ⁇ 0.1% Tween 20)
  • Solution 1 Add 1-3 microliters of single-stranded cyclization buffer (0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween 20) to the linker ligation product purified in step (5) , Move to the position of heating plate 3 (H3), mix and mix, heat to 95°C for high temperature denaturation for 1-10 minutes, quickly cool and refold, add 1 to 3 microliters of single-stranded cyclase mixture (0.1 ⁇ 5U/ ⁇ L) T4 ligase, 0.01 ⁇ 0.1% Tween 20), react at 37°C for 5 ⁇ 60 minutes.
  • single-stranded cyclization buffer 0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween 20
  • Scheme 2 Add 1-3 microliters of single-stranded cyclization buffer (0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween 20) to the linker ligation product purified in step (5) , Move to the position of heating plate 3 (H3), mix and mix, heat to 95°C for high temperature denaturation for 1-10 minutes, quickly cool and refold, add 1 to 3 microliters of single-stranded cyclase mixture (0.1 ⁇ 5U/ ⁇ L) T4 ligase, 0.01 to 0.1% Tween 20), react at 37°C for 5 to 60 minutes.
  • single-stranded cyclization buffer 0.1 ⁇ 5 ⁇ M anchor nucleotide, 0.1 ⁇ 10mM adenosine triphosphate, 0.01 ⁇ 0.1% Tween 20
  • DNA nanosphere preparation buffer in the M7 reagent tank to the single-stranded ring prepared in step (6), incubate at 95°C for 1 minute, 65°C for 1 minute, and 40°C for 1 minute, and then add 1 ⁇ 3 microliters of DNA nanospheres in the M2 reagent tank to prepare enzyme mixture (0.1 ⁇ 1U/ ⁇ L phi29 polymerase, 0.01 ⁇ 0.1% Tween 20), mix well at room temperature on heating plate 3 (H3) at 30°C After incubating for 10-60 minutes, add 1 to 3 microliters of DNA nanospheres in the M6 reagent tank to prepare a stop solution to terminate the rolling circle linear amplification.
  • enzyme mixture 0.1 ⁇ 1U/ ⁇ L phi29 polymerase, 0.01 ⁇ 0.1% Tween 20
  • example 1 is the automatic library construction of the whole genome PCR with low starting quantity and low cost, fast and portable
  • example 2 is the automatic library construction of the whole process of PCR free (PCR free), and the resulting DNA nano
  • Table 1 The results of the ball quality inspection concentration are shown in Table 1 below.
  • the metagenomic sequencing scheme is used to realize the construction and sequencing of the low-abundance disease original text library.
  • Figure 4 shows the comparison results of the detection effect of manual library construction and digital microfluidic library construction. The results show: (1) For samples With the eight microbial species mixed in, both digital microfluidic library construction and manual library construction can detect equivalent abundance values of each species, indicating that the digital microfluidic fully automated library construction can obtain library quality consistent with manual library construction (2) The library obtained by using the microfluidic small-volume solution has less sample contamination than the conventional large-volume library.

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Abstract

一种全基因组全流程微流控自动化建库方法和装置，其中建库方法包括：在数字化微流控芯片上，以基因组DNA作为建库起始原料，通过电润湿原理控制液滴在数字化微流控芯片上的移动，依次进行基因组DNA打断、末端修复、接头连接、单链环化和DNA纳米球制备的反应，其中每一步反应的反应体系不超过10微升，反应体系包含反应组分和用于降低表面张力的表面活性剂成分。利用数字化微流控实现快速自动化建库以服务个性化测序和精准医疗体系。本发明通过电润湿原理控制液滴移动，实现全基因组全流程微流控自动化建库，成本低，无污染。

Description

一种全基因组全流程微流控自动化建库方法和装置 技术领域
本发明涉及文库构建技术领域，具体涉及一种全基因组全流程微流控自动化建库方法和装置。
背景技术
自2003年人类基因组计划完成以来，测序技术发展迅猛，多种测序原理产品在市场上出现，接受市场的检验。测序读长不断加长、通量不断提升、时间不断缩短，促进测序成本以“超摩尔定律”的模式急剧下降。国内外企业争相推出应用于不同需求的NGS平台及配套产品，例如BGI公司的BGISEQ-500和Illumina公司的NextSeq-500等。自2015年起，主要的二代测序(NGS)企业均陆续推出小型化台式设备。2016年初，Illumina公司在正在进行的34届J.P.摩根健康产业大会(J.P.Morgan Healthcare Conference)上公布了最新型的MiniSeq平台。相较于高数据产量，该平台主要面向医院、地区性检验中心等中小型临床检测机构的NGS产品，MiniSeq平台弥补了Illumina公司的系列仪器面对少量样本的测序需求时不够灵活的缺点。目前，市场上常见的小型化NGS建库及测序平台已经可以覆盖现有的大部分测序应用。同时，小型化系统本身又可以提供HiSeq这类面向测序工厂级别的产品所不具有的灵活性。此外，QIAGEN公司推出了GeneReader系列全自动化测序系统，Thermo和Illumina公司也在提高各自测序平台的自动化程度，逐步实现从样本处理、建库、测序到数据解读的全流程自动化。这种自动化升级为临床应用的标准化和普及化创造了条件，也更加明确了临检领域与科研用户不同需求所对应的不同方案。
综上，通过技术革新和升级，实现快速、低成本、自动化样本处理和建库，使基因科技以及个性化诊断及精准医疗惠及普通大众，已成为各大测序公司抢占测序市场的新热点。随着基因行业的发展和精准医疗方案的逐渐落地，个性化测序需求会成为全新的市场增长点。针对各大测序公司关注的新热点，蓄力发展小型化、自动化建库仪则至关重要。目前传统的建库方法存在的限制有样本起始需求量及建库试剂用量比较大，导致一些珍贵样本及建库试剂的浪费；建库过程人力消耗较多且 需要专业人士操作完成，而人工操作的同时也会引入一些随机误差和外源污染等。
此外，很多研究发现微流控体系下的非特异性扩增会显著降低。目前，市面上有多种不同的适应于二代测序及三代测序的微流控建库平台，包括数字生物系统(Digital Biosystems)公司正在研发的DBS-LibPrep DMF Device，10X genomics公司推出的Chromium基因组测序解决方案(Chromium Genome Sequencing Solution)，Fluidigm公司推出的自动化Juno TM系统(automated Juno TM system)以及Oxford Nanopore公司推出的Voltrax平台。前三者都是基于Illumina公司测序仪配套的微流控建库平台，适应于二代测序；Voltrax平台是基于Oxford Nanopore公司的三代测序仪配套的微流控自动化建库平台。其中，10X genomics公司的Chromium基因组测序解决方案，是利用高通量液滴微流控的方式将高分子量的基因组DNA与带有编码的磁珠包裹在微液滴中进行杂交和PCR扩增，得到带有10X标签的扩增子，即Illumina平台的文库。Fluidigm公司推出的自动化Juno TM系统是手动处理后的样本后，在芯片上利用高通量微液滴的方式得到带有标签的扩增子产物，再利用手动纯化及第二轮PCR扩增的方式加入接头序列，从而完成Illumina文库的构建。Oxford Nanopore公司推出的Voltrax同样是以数字化微流控的方式实现自动化三代测序文库的构建，即以基因组DNA为起始，打断基因组DNA后加接头，之后加入固定链，即完成三代测序文库的构建。
现有技术方案存在如下缺陷：目前大部分二代测序自动化建库方案都是针对Illumina平台设计的，无法广泛适应其他更多的测序平台。针对Illumina平台的文库，测序前需完成桥式扩增，相较线性扩增容易产生错误积累。目前大部分二代测序自动化建库方案都需要至少一步PCR扩增步骤，成本较高，重复序列增加，错误率升高，同时存在扩增偏好性等问题。10X genomics公司的Chromium基因组测序解决方案的磁珠纯化步骤需要手动进行，Fluidigm公司的自动化Juno TM系统的磁珠纯化和加接头同样需要手动操作，无法实现全流程自动化建库。Voltrax平台构建出的文库所实现的三代测序虽然在长读长方面具有明显优势，但是错误率还是比较高，目前无法真正替代二代测序方案。自动化液体处理系统目前同样被应用于自动化建库流程中，但是这种方案与微流控体系相比反应体系较大，试剂成本较高，同时无法将整个体系升级成便携式设备。
发明内容
为解决上述问题并利用微流控技术的优势，本发明利用数字化微流控实现快速自动化建库以服务个性化测序和精准医疗体系。本发明通过电润湿原理控制液滴移动，实现全基因组全流程微流控自动化建库，成本低，无污染。
本发明的技术方案如下：
根据第一方面，本发明提供一种全基因组全流程微流控自动化建库方法，包括：在数字化微流控芯片上，以基因组DNA作为建库起始原料，通过电润湿原理控制液滴在上述数字化微流控芯片上的移动，依次进行基因组DNA打断、末端修复、接头连接、单链环化和DNA纳米球制备的反应，其中每一步反应的反应体系不超过10微升，上述反应体系包含反应组分和用于降低表面张力的表面活性剂成分。
在优选实施例中，上述方法还包括在上述接头连接和单链环化之间进行PCR扩增。
在优选实施例中，以10～500纳克基因组DNA作为建库起始原料。
在优选实施例中，上述表面活性剂成分是吐温20。
在优选实施例中，上述吐温20在上述反应体系中的浓度是0.01～0.1％。
在优选实施例中，上述数字化微流控芯片上具有帕尔帖板，以实现试剂反应区的温度控制。
在优选实施例中，上述基因组DNA打断的反应体系包括1～5微升基因组打断缓冲液和1～3微升基因组打断酶。
在优选实施例中，上述方法还包括：在上述基因组DNA打断之后，通过电润湿原理移动1微升0.1～1M乙二胺四乙酸到上一步混合液中混匀以终止打断反应。
在优选实施例中，上述基因组DNA打断之后，取10～20微升磁珠与打断产物混合并孵育进行产物纯化。
在优选实施例中，上述末端修复的反应体系包括1～3微升末端修复缓冲液和1～3微升末端修复酶混合液，其中上述末端修复缓冲液包括0.1～1M dATP，0.03～0.2M dNTPs，0.01～0.1％吐温20；上述末端修复酶混合液包括0.05～0.5U/μL T4 PNK，0.05～0.5U/μL T4 DNA聚合酶，0.005～0.05U/μL Klenow聚合酶，0.005～0.05U/μL rTaq聚合酶，0.01～0.1％吐温20。
在优选实施例中，上述接头连接的反应体系包括1～3微升接头连接缓冲液和1～3微升接头连接酶混合液，其中上述接头连接缓冲液包括0.1～1μM接头，0.1～10mM三磷酸腺苷，3％～20％聚乙二醇8000，0.1～10mM三氯化六氨合钴，0.01～0.1％吐温20；上述接头连接酶混合液包括1～20U/μL T4连接酶，0.01～0.1％吐温20。
在优选实施例中，上述接头连接之后，取3～10微升磁珠与接头连接产物混合并孵育进行产物纯化。
在优选实施例中，上述单链环化的反应体系包括上一步的纯化产物，以及1～3微升单链环化缓冲液和1～3微升单链环化酶混合液，其中上述单链环化缓冲液包括0.1～5μM锚定核苷酸，0.1～10mM三磷酸腺苷，0.01～0.1％吐温20；上述单链环化酶混合液包括0.1～5U/μL T4连接酶，0.01～0.1％吐温20。
在优选实施例中，上述方法还包括：在上述单链环化之后，加入1～3微升消化酶混合液进行消化，然后加入1～3微升消化终止液终止反应，其中上述消化酶混合液包括0.1～1U/μL外切酶I，0.1～10U/μL外切酶III，0.01～0.1％吐温20；上述消化终止液包括0.1～1mM乙二胺四乙酸，0.01～0.1％吐温20。
在优选实施例中，上述单链环化之后，取10～20微升磁珠与单链环化产物混合并孵育进行产物纯化。
在优选实施例中，上述DNA纳米球制备的反应体系包括1～5微升DNA纳米球制备缓冲液以及1～3微升DNA纳米球制备酶混合液，其中上述DNA纳米球制备酶混合液包括0.1～1U/μL phi29聚合酶，0.01～0.1％吐温20。
在优选实施例中，上述DNA纳米球制备反应之后，加入1～3微升DNA纳米球制备终止液来终止滚环线性扩增。
在优选实施例中，上述PCR扩增的反应体系包括3～5微升PCR扩增酶混合液，上述PCR扩增酶混合液包括PCR酶，0.1～2μM引物，0.01～0.1％吐温20。
根据第二方面，本发明提供一种全基因组全流程微流控自动化建库装置，包括：微流控芯片，该微流控芯片包括芯片基底和设置在芯片基底上的电极分布及电极开关的组合控制，上述微流控芯片用于实现第一方面的方法。
相较于市面上诸多微流控建库方案，本发明的方案可实现全自动化建库流程， 手动操作时间小于5min，同时可降低外源核酸对建库过程中的污染。相较于自动化液剂处理系统，本发明的方案可实现微小体积的各步骤建库反应，降低10倍左右的试剂成本。本发明的方案所用仪器及芯片皆为便携式设备，可实现去中心化的偏远、低设备储备量区域的应用。建库速度比传统建库方法提高一倍，且并不影响低丰度核酸检测的灵敏度。通过替换使用接头的种类即可适用于大部分二代测序平台，适用范围更广。利用DNA纳米球测序技术的滚环扩增方案替代桥式扩增方案会减少错误积累，可实现更低丰度的突变检测。可实现低起始量全基因组免PCR(PCR free)的文库构建，相较于PCR文库构建可以降低成本，减少重复序列，降低错误率，同时解决扩增偏好性等问题。
附图说明
图1为本发明实施例中低起始量低成本快速便携式全基因组PCR全流程自动化建库流程示意图。
图2为本发明实施例中低起始量低成本快速便携式全基因组免PCR(PCR free)全流程自动化建库流程示意图。
图3为本发明实施例中数字化微流控自动化建库芯片的结构和功能模块示意图。
图4为本发明实施例中利用宏基因组测序方案实现不同丰度病原文库的构建和测序及手动建库与数字化微流控建库检测效果对比图。
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。
在本发明一个实施例中，一种全基因组全流程微流控自动化建库方法，包括：在数字化微流控芯片上，以基因组DNA作为建库起始原料，通过电润湿原理控制液滴在上述数字化微流控芯片上的移动，依次进行基因组DNA打断、末端修复、接头连接、单链环化和DNA纳米球制备的反应，其中每一步反应的反应体系不超过10微升，上述反应体系包含反应组分和用于降低表面张力的表面活性剂成分。
本发明的方法实现了在数字化微流控芯片上进行全基因组全流程的文库构建，从基因组DNA打断，到末端修复和接头连接，直至单链环化和DNA纳米球制备全过程均在数字化微流控芯片上进行，基本上无需人工手动操作，可降低外源核酸对建库过程中的污染。
本发明的方法中，每一步反应的反应体系不超过10微升，相较于自动化液剂处理系统，本发明的方案可实现微小体积的各步骤建库反应，降低10倍左右的试剂成本。
为了实现微小体积的各步骤建库反应，本发明通过电润湿(Electrowetting，EW)原理控制液滴(反应试剂)在数字化微流控芯片上的移动。通过改变液滴与芯片基板之间的电压，来改变液滴在芯片基板上的润湿性，即改变接触角，使液滴发生形变和位移。
为了通过电润湿原理控制液滴在数字化微流控芯片上的移动，需要降低液滴的表面张力。在本发明一个实施例中，反应体系包含反应组分和表面活性剂成分，其中反应组分是指参与化学反应的有效组分，包括缓冲液和酶等；表面活性剂成分用于降低液滴的表面张力。本发明实施例中，表面活性剂有多种选择。在优选实施例中，表面活性剂成分是吐温20，特别是浓度为0.01～0.1％的吐温20。
本发明的全基因组全流程微流控自动化建库方法，可以通过PCR扩增或免PCR扩增(PCR free)两种方式实现。在PCR扩增的方式中，在上述接头连接和单链环化之间进行PCR扩增。
在优选方案中，在文库制备前，将所有反应试剂加载到数字化微流控芯片的试剂装载区，利用电润湿的方法实现反应试剂在芯片上的驱动、分离和混合；利用帕尔帖板实现反应区的温控，利用机械升降磁铁实现磁珠纯化过程。
在PCR扩增的方案中，在芯片上实现微量体积的基因组DNA打断，末端修复，接头连接，PCR扩增，单链环化，以及DNA纳米球制备。在优选方案中，还包括磁珠纯化，以及浓度检测等过程。
具体而言，作为第一种方案，本发明的低起始量低成本快速便携式的全基因组PCR全流程自动化建库方法的原理及特点如下：
利用数字化液滴微流控的方式完成基于DNA纳米球测序的自动化建库流程。同时，建库流程稍加改动及组合即可完成其他基于不同的二代或三代测序技术的建库流程以及其他体外诊断或床旁诊断的应用。所有的反应流程皆可在小于10微升及存在表面活性剂的体系下实现，反应效率与手工建库相当，建库时间为手工建库的一半。此微小体系的反应流程不但能够降低试剂成本，同时能够降低起始样本的需求量，一般而言，10～500纳克基因组DNA作为建库起始原料即可，适合珍贵及来源有限的样本的文库制备需求。此外，该自动化建库流程也可节省人力成本，同时减少建库过程中外源核酸对样本文库的污染。该种方案中利用电润湿原理实现所有反应试剂及反应体系在芯片上的驱动、分离、混合和混匀；利用帕尔帖板实现反应区的温控及升降温变化，利用机械升降磁铁实现磁珠纯化过程，以实现数字化微流控低成本快速自动化全基因组PCR文库构建。其中，文库制备方法如下：
如图1所示，首先在装载好的芯片上注满低粘度硅油，并将所有反应试剂加载到试剂存储区，该存储区域在整个建库过程中维持4℃，装载的全基因组建库反应试剂为微反应体系所用体积且加有适量浓度的表面活性剂以降低反应试剂的表面张力。试剂装载完成后首先将打断好的基因组DNA进行末端修复及加入腺嘌呤核苷酸，然后进行接头连接。连接后的混合液进行磁珠纯化得到接头连接产物并进行PCR扩增。PCR扩增后的混合液进行磁珠纯化得到扩增后的接头连接产物进行双链荧光定量及均一化。制备单链环和DNA纳米球时可以采取两种方案，第一种方案为利用均一化的扩增产物继续进行单链环化及消化。磁珠纯化后利用单链环可进行滚环扩增得到最终产物DNA纳米球；第二种方案为利用均一化的扩增产物继续进行单链环化后直接进行DNA纳米球的制备。最后利用单链荧光定量的试剂对DNA纳米球浓度进行定量及质检。除试剂装载外，整个过程可以在芯片上自动化进行而不需要任何手动操作，所有反应体系都小于10微升。
在免PCR扩增的方案中，在芯片上实现微量体积的基因组DNA打断，末端修 复，接头连接，单链环化，以及DNA纳米球制备。在优选方案中，还包括磁珠纯化，以及浓度检测等过程。
具体而言，作为第二种方案，本发明的低起始量低成本快速便携式全基因组免PCR扩增全流程自动化建库方法的原理及特点如下：
利用数字化液滴微流控的方式完成基于DNA纳米球测序的自动化建库流程。同时，建库流程稍加改动及组合即可完成其他基于不同的二代或三代测序技术的建库流程以及其他体外诊断或床旁诊断的应用。所有的反应流程皆可在小于10微升及存在表面活性剂的体系下实现，反应效率与手工建库相当，建库时间为手工建库的一半。此微小体系的反应流程不但可以降低试剂成本，同时能够降低起始样本的需求量，一般而言，50～500纳克的基因组DNA作为建库起始原料即可，适合珍贵及来源有限的样本的文库制备需求。此外，此自动化建库流程也可节省人力成本，同时减少建库过程中外源核酸对样本文库的污染。该种方案中利用电润湿的原理实现所有反应试剂及反应体系在芯片上的驱动、分离、混合和混匀；利用帕尔帖板实现反应区的温控及升降温变化，利用机械升降磁铁实现磁珠纯化过程，以实现数字化微流控低成本快速自动化全基因组PCR文库构建。其中，制备方法如下：
如图2所示，首先在装载好的芯片上注满低粘度硅油，并将所有反应试剂加载到试剂存储区，该存储区域在整个建库过程中维持4℃，装载的全基因组建库反应试剂为微反应体系所用体积且加有适量浓度的表面活性剂以降低反应试剂的表面张力。试剂装载完成后首先将打断好的基因组DNA进行末端修复及加入腺嘌呤核苷酸，然后进行接头连接。连接后的混合液进行磁珠纯化得到接头连接产物。制备单链环和DNA纳米球时可以采取两种方案，第一种方案为利用纯化后的接头连接产物继续进行单链环化及消化。磁珠纯化后利用单链环可进行滚环扩增得到最终产物DNA纳米球；第二种方案为利用纯化后的接头连接产物继续进行单链环化后直接进行DNA纳米球的制备。最后利用单链荧光定量的试剂对DNA纳米球浓度进行定量及质检。除试剂装载外，整个过程可以在芯片上自动化进行而不需要任何手动操作，所有反应体系都小于10微升。
以下通过具体实施例详细说明本发明的技术方案，应当理解，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：低起始量低成本快速便携式全基因组PCR全流程自动化建库
本实施例中使用的微流控芯片的结构和功能模块如图3所示。
(1)自动化建库前试剂准备与装载：
在装载好的芯片上注满低粘度硅油，并将所有反应试剂加载到试剂存储区，该存储区域在整个建库过程中维持4℃，装载的全基因组建库反应试剂为微反应体系所用体积。试剂存储区分为大(L)、中(M)及小(S)三类试剂槽，分别存储20微升以下、5微升以下及3微升以下的反应试剂。L1和L2分别装载单链荧光定量试剂及双链荧光定量试剂；L4至L7分别装载磁珠1、洗脱液、磁珠纯化洗涤液和磁珠2。S1至S10分别装载基因组DNA、末端修复缓冲液、末端修复酶混合液、接头连接酶混合液、单链环化酶混合液、消化终止液、荧光定量标准液1、双链荧光定量标准液2、单链荧光定量标准液2和基因组打断酶。M1至M10分别装载洗脱液、DNA纳米球制备酶混合液、接头连接缓冲液、消化酶混合液、单链环化缓冲液、DNA纳米球制备终止液、DNA纳米球制备缓冲液、PCR扩增酶混合液、洗脱液和基因组打断缓冲液。
(2)基因组DNA片段化的制备方法：
将10～500纳克的基因组DNA，加入1～5微升基因组打断缓冲液及1～3微升基因组打断酶通过电润湿原理移动到加热板1(H1)的位置混合并混匀，利用32℃及65℃的梯度反应45分钟完成基因组打断。再移动1微升0.1～1M的乙二胺四乙酸到上一步混合液中混合并混匀，终止打断反应。
(3)打断基因组DNA片段纯化方法：
DNA片段化后，从磁珠存储区L7分出10～20微升的磁珠并将其与打断的产物混合并常温孵育，待目标片段结合到磁珠上后将混合物移动到磁珠吸附区1，利用外加磁铁捕获磁珠并去除废液到W1。在芯片上将L6内的洗涤液运送到磁珠吸附区洗涤后，运输L5内的洗脱液到磁珠吸附区，放开磁珠进行洗脱。
(4)带有接头序列的基因组DNA片段的制备方法：
在上步反应液中加入1～3微升末端修复缓冲液(0.1～1M dATP，0.03～0.2M dNTPs，0.01～0.1％吐温20)及1～3微升末端修复酶混合液(0.05～0.5U/μL T4 PNK，0.05～0.5U/μL T4 DNA聚合酶，0.005～0.05U/μL Klenow聚合酶，0.005～0.05U/μL rTaq聚合酶，0.01～0.1％吐温20)通过电润湿原理移动到加热板1(H1)的位置混 合并混匀，利用37℃及65℃的梯度反应45分钟完成末端修复。再移动1～3微升接头连接缓冲液(0.1～1μM接头，0.1～10mM三磷酸腺苷，3％～20％聚乙二醇8000，0.1～10mM三氯化六氨合钴，0.01～0.1％吐温20)及1～3微升接头连接酶混合液(1～20U/μL T4连接酶，0.01～0.1％吐温20)到上一步混合液中混合并混匀，23℃反应5～60分钟完成接头连接。
(5)连接产物纯化方法：
接头连接完成后，从磁珠存储区L7分出3～10微升的磁珠并将其与接头连接产物混合并常温孵育，待目标片段结合到磁珠上后将混合物移动到磁珠吸附区1，利用外加磁铁捕获磁珠并去除废液到W1。在芯片上将L6内的洗涤液运送到磁珠吸附区洗涤后，运输L5内的洗脱液到磁珠吸附区，放开磁珠进行洗脱。
(6)连接产物扩增方法：
洗脱完成后将3～5微升M8试剂槽中PCR扩增酶混合液(1X PCR酶，0.1～2μM引物，0.01～0.1％吐温20)移动到上一步的洗脱液中混合并混匀，将混合液移动到加热板2(H2)，首先进行98℃3分钟的预变性，然后进行7～15个循环的98℃20秒，60℃15秒，72℃30秒，最后进行72℃10分钟终延伸。快速升降温控制都由加热板2(H2)实现。
(7)扩增产物的定量及均一化方法：
分别将S7试剂槽中的荧光定量标准液1，S8试剂槽中的双链荧光定量标准液2与L2试剂槽中的双链荧光定量试剂移动到加热板4(H4)的位置常温混合混匀，利用外加荧光镜头获取荧光强度并做标准曲线。取1微升扩增后产物到加热板4(H4)的位置与双链荧光定量试剂常温混合混匀并利用荧光镜头读取相应的荧光强度，计算所得扩增后所得产物的量并利用L5中的洗脱液将10～50纳克扩增产物均一化到1微升洗脱液中。
(8)两种单链环制备方法：
方案一：将步骤(7)纯化均一化后的扩增片段中加入1～3微升单链环化缓冲液(0.1～5μM锚定核苷酸，0.1～10mM三磷酸腺苷，0.01～0.1％吐温20)，移动到加热板3(H3)的位置混合混匀，加热到95℃高温变性1～10分钟，快速冷却复性后加入1～3微升单链环化酶混合液(0.1～5U/μL T4连接酶，0.01～0.1％吐温20)， 37℃反应5～60分钟，反应结束后加入1～3微升消化酶混合液(0.1～1U/μL外切酶I，0.1～10U/μL外切酶III，0.01～0.1％吐温20)混合均匀，37℃反应5～60分钟，最后加入1～3微升消化终止液(0.1～1mM乙二胺四乙酸，0.01～0.1％吐温20)混合均匀。单链环制备完成后，从磁珠存储区L7分出10～20微升的磁珠并将其与单链环化的产物混合并常温孵育，待目标片段结合到磁珠上后将混合物移动到磁珠吸附区2，利用外加磁铁捕获磁珠并去除废液到W2。在芯片上将L6内的洗涤液运送到磁珠吸附区洗涤后，运输L5内的洗脱液到磁珠吸附区，放开磁珠进行洗脱。
方案二：将步骤(7)纯化均一化后的扩增片段中加入1～3微升单链环化缓冲液(0.1～5μM锚定核苷酸，0.1～10mM三磷酸腺苷，0.01～0.1％吐温20)，移动到加热板3(H3)的位置混合混匀，加热到95℃高温变性1～10分钟，快速冷却复性后加入1～3微升单链环化酶混合液(0.1～5U/μL T4连接酶，0.01～0.1％吐温20)，37℃反应5～60分钟。
(9)DNA纳米球的制备方法：
向步骤(8)制备的单链环中加入1～5微升M7试剂槽中的DNA纳米球制备缓冲液，进行95℃1分钟，65℃1分钟，40℃1分钟的孵育，然后加入1～3微升M2试剂槽中的DNA纳米球制备酶混合液(0.1～1U/μL phi29聚合酶，0.01～0.1％吐温20)，在加热板3(H3)的位置常温混合均匀后30℃孵育10～60分钟后，加入1～3微升M6试剂槽中的DNA纳米球制备终止液来终止滚环线性扩增。
(10)DNA纳米球定量质检方法：
分别将S7试剂槽中的荧光定量标准液1，S9试剂槽中的单链荧光定量标准液2与L1试剂槽中的单链荧光定量试剂移动到加热板4(H4)的位置常温混合混匀，利用外加荧光镜头获取荧光强度并做标准曲线。取1微升DNA纳米球溶液到加热板4(H4)的位置与双链荧光定量试剂常温混合混匀并利用荧光镜头读取相应的荧光强度，计算所得DNA纳米球的浓度。
实施例2：低起始量低成本快速便携式全基因组免PCR全流程自动化建库
本实施例中使用的微流控芯片的结构和功能模块如图3所示。
(1)自动化建库前试剂准备与装载：
在装载好的芯片上注满低粘度硅油，并将所有反应试剂加载到试剂存储区，该存储区域在整个建库过程中维持4℃，装载的全基因组建库反应试剂为微反应体系所用体积。试剂存储区分为大(L)、中(M)及小(S)三类试剂槽，分别存储20微升以下、5微升以下及3微升以下的反应试剂。L1和L2分别装载单链荧光定量试剂及双链荧光定量试剂；L4至L7分别装载磁珠1、洗脱液、磁珠纯化洗涤液和磁珠2。S1至S10分别装载基因组DNA、末端修复缓冲液、末端修复酶混合液、接头连接酶混合液、单链环化酶混合液、消化终止液、荧光定量标准液1、双链荧光定量标准液2、单链荧光定量标准液2和基因组打断酶。M1至M10分别装载洗脱液、DNA纳米球制备酶混合液、接头连接缓冲液、消化酶混合液、单链环化缓冲液、DNA纳米球制备终止液、DNA纳米球制备缓冲液、PCR扩增酶混合液、洗脱液和基因组打断缓冲液。
(2)基因组DNA片段化的制备方法：
将50～500纳克的基因组DNA，加入1～5微升基因组打断缓冲液及1～3微升基因组打断酶通过电润湿原理移动到加热板1(H1)的位置混合并混匀，利用32℃及65℃的梯度反应45分钟完成基因组打断。再移动1微升0.1～1M的乙二胺四乙酸到上一步混合液中混合并混匀，终止打断反应。
(3)打断基因组DNA片段纯化方法：
DNA片段化后，从磁珠存储区L7分出10～20微升的磁珠并将其与打断的产物混合并常温孵育，待目标片段结合到磁珠上后将混合物移动到磁珠吸附区1，利用外加磁铁捕获磁珠并去除废液到W1。在芯片上将L6内的洗涤液运送到磁珠吸附区洗涤后，运输L5内的洗脱液到磁珠吸附区，放开磁珠进行洗脱。
(4)带有接头序列的基因组DNA片段的制备方法：
在上步反应液中加入1～3微升末端修复缓冲液(0.1～1M dATP，0.03～0.2M dNTPs，0.01～0.1％吐温20)及1～3微升末端修复酶混合液(0.05～0.5U/μL T4 PNK，0.05～0.5U/μL T4 DNA聚合酶，0.005～0.05U/μL Klenow聚合酶，0.005～0.05U/μL rTaq聚合酶，0.01～0.1％吐温20)通过电润湿原理移动到加热板1(H1)的位置混合并混匀，利用37℃及65℃的梯度反应45分钟完成末端修复。再移动1～3微升接头连接缓冲液(0.1～1μM接头，0.1～10mM三磷酸腺苷，3％～20％聚乙二醇8000，0.1～10mM三氯化六氨合钴，0.01～0.1％吐温20)及1～3微升接头连接酶 混合液(1～20U/μL T4连接酶，0.01～0.1％吐温20)到上一步混合液中混合并混匀，23℃反应5～60分钟完成接头连接。
(5)连接产物纯化方法：
接头连接完成后，从磁珠存储区L7分出3～10微升的磁珠并将其与接头连接产物混合并常温孵育，待目标片段结合到磁珠上后将混合物移动到磁珠吸附区1，利用外加磁铁捕获磁珠并去除废液到W1。在芯片上将L6内的洗涤液运送到磁珠吸附区洗涤后，运输L5内的洗脱液到磁珠吸附区，放开磁珠进行洗脱。
(6)两种单链环制备方法：
方案一：将步骤(5)纯化后的接头连接产物中加入1～3微升单链环化缓冲液(0.1～5μM锚定核苷酸，0.1～10mM三磷酸腺苷，0.01～0.1％吐温20)，移动到加热板3(H3)的位置混合混匀，加热到95℃高温变性1～10分钟，快速冷却复性后加入1～3微升单链环化酶混合液(0.1～5U/μL T4连接酶，0.01～0.1％吐温20)，37℃反应5～60分钟，反应结束后加入1～3微升消化酶混合液(0.1～1U/μL外切酶I，0.1～10U/μL外切酶III，0.01～0.1％吐温20)混合均匀，37℃反应5～60分钟，最后加入1～3微升消化终止液(0.1～1mM乙二胺四乙酸，0.01～0.1％吐温20)混合均匀。单链环制备完成后，从磁珠存储区L7分出10～20微升的磁珠并将其与单链环化的产物混合并常温孵育，待目标片段结合到磁珠上后将混合物移动到磁珠吸附区2，利用外加磁铁捕获磁珠并去除废液到W2。在芯片上将L6内的洗涤液运送到磁珠吸附区洗涤后，运输L5内的洗脱液到磁珠吸附区，放开磁珠进行洗脱。
方案二：将步骤(5)纯化后的接头连接产物中加入1～3微升单链环化缓冲液(0.1～5μM锚定核苷酸，0.1～10mM三磷酸腺苷，0.01～0.1％吐温20)，移动到加热板3(H3)的位置混合混匀，加热到95℃高温变性1～10分钟，快速冷却复性后加入1～3微升单链环化酶混合液(0.1～5U/μL T4连接酶，0.01～0.1％吐温20)，37℃反应5～60分钟。
(7)DNA纳米球的制备方法：
向步骤(6)制备的单链环中加入1～5微升M7试剂槽中的DNA纳米球制备缓冲液，进行95℃ 1分钟，65℃ 1分钟，40℃ 1分钟的孵育，然后加入1～3微升M2试剂槽中的DNA纳米球制备酶混合液(0.1～1U/μL phi29聚合酶，0.01～0.1％ 吐温20)，在加热板3(H3)的位置常温混合均匀后30℃孵育10～60分钟后加入1～3微升M6试剂槽中的DNA纳米球制备终止液来终止滚环线性扩增。
(8)DNA纳米球定量质检方法：
分别将S7试剂槽中的荧光定量标准液1，S9试剂槽中的单链荧光定量标准液2与L1试剂槽中的单链荧光定量试剂移动到加热板4(H4)的位置常温混合混匀，利用外加荧光镜头获取荧光强度并做标准曲线。取1微升DNA纳米球溶液到加热板4(H4)的位置与双链荧光定量试剂常温混合混匀并利用荧光镜头读取相应的荧光强度，计算所得DNA纳米球的浓度。
以上实施例1和实施例2中，实施例1为低起始量低成本快速便携式全基因组PCR全流程自动化建库，实施例2为免PCR(PCR free)全流程自动化建库，所得DNA纳米球质检浓度结果，如下表1所示。
表1
Figure PCTCN2019095868-appb-000001
手动建库与数字化微流控自动化建库所得文库测序质量对比结果，如下表2所示。
表2
Figure PCTCN2019095868-appb-000002
本发明实施例中，利用宏基因组测序方案实现低丰度病原文库的构建及测序， 图4示出了手动建库与数字化微流控建库检测效果对比结果，结果显示：(1)针对样本中混入的八种微生物物种，数字化微流控建库及手工建库均可检测出相当的各菌种丰度值，表明数字化微流控全自动化建库可得到与手工建库一致的文库质量；(2)利用微流控小体积方案得到的文库比常规大体积所建文库有更少的样本污染。
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 (18)

  1. 一种全基因组全流程微流控自动化建库方法，其特征在于，所述方法包括：在数字化微流控芯片上，以基因组DNA作为建库起始原料，通过电润湿原理控制液滴在所述数字化微流控芯片上的移动，依次进行基因组DNA打断、末端修复、接头连接、单链环化和DNA纳米球制备的反应，其中每一步反应的反应体系不超过10微升，所述反应体系包含反应组分和用于降低表面张力的表面活性剂成分。
  2. 根据权利要求1所述的方法，其特征在于，所述方法还包括在所述接头连接和单链环化之间进行PCR扩增。
  3. 根据权利要求1所述的方法，其特征在于，以10～500纳克基因组DNA作为建库起始原料。
  4. 根据权利要求1所述的方法，其特征在于，所述表面活性剂成分是吐温20。
  5. 根据权利要求4所述的方法，其特征在于，所述吐温20在所述反应体系中的浓度是0.01～0.1％。
  6. 根据权利要求1所述的方法，其特征在于，所述数字化微流控芯片上具有帕尔帖板，以实现试剂反应区的温度控制。
  7. 根据权利要求1所述的方法，其特征在于，所述基因组DNA打断的反应体系包括1～5微升基因组打断缓冲液和1～3微升基因组打断酶。
  8. 根据权利要求7所述的方法，其特征在于，所述方法还包括：在所述基因组DNA打断之后，通过电润湿原理移动1微升0.1～1M乙二胺四乙酸到上一步混合液中混匀以终止打断反应。
  9. 根据权利要求1所述的方法，其特征在于，所述基因组DNA打断之后，取10～20微升磁珠与打断产物混合并孵育进行产物纯化。
  10. 根据权利要求1所述的方法，其特征在于，所述末端修复的反应体系包括1～3微升末端修复缓冲液和1～3微升末端修复酶混合液，其中所述末端修复缓冲液包括0.1～1M dATP，0.03～0.2M dNTPs，0.01～0.1％吐温20；所述末端修复酶混合液包括0.05～0.5U/μL T4 PNK，0.05～0.5U/μL T4 DNA聚合酶，0.005～0.05U/μL Klenow聚合酶，0.005～0.05U/μL rTaq聚合酶，0.01～0.1％吐温20。
  11. 根据权利要求1所述的方法，其特征在于，所述接头连接的反应体系包括1～3微升接头连接缓冲液和1～3微升接头连接酶混合液，其中所述接头连接缓冲液包括0.1～1μM接头，0.1～10mM三磷酸腺苷，3％～20％聚乙二醇8000，0.1～10mM三氯化六氨合钴，0.01～0.1％吐温20；所述接头连接酶混合液包括1～20U/μL T4连接酶，0.01～0.1％吐温20。
  12. 根据权利要求1所述的方法，其特征在于，所述接头连接之后，取3～10微升磁珠与接头连接产物混合并孵育进行产物纯化。
  13. 根据权利要求1所述的方法，其特征在于，所述单链环化的反应体系包括上一步的纯化产物，以及1～3微升单链环化缓冲液和1～3微升单链环化酶混合液，其中所述单链环化缓冲液包括0.1～5μM锚定核苷酸，0.1～10mM三磷酸腺苷，0.01～0.1％吐温20；所述单链环化酶混合液包括0.1～5U/μL T4连接酶，0.01～0.1％吐温20。
  14. 根据权利要求13所述的方法，其特征在于，所述方法还包括：在所述单链环化之后，加入1～3微升消化酶混合液进行消化，然后加入1～3微升消化终止液终止反应，其中所述消化酶混合液包括0.1～1U/μL外切酶I，0.1～10U/μL外切酶III，0.01～0.1％吐温20；所述消化终止液包括0.1～1mM乙二胺四乙酸，0.01～0.1％吐温20。
  15. 根据权利要求13或14所述的方法，其特征在于，所述单链环化之后，取10～20微升磁珠与单链环化产物混合并孵育进行产物纯化。
  16. 根据权利要求1所述的方法，其特征在于，所述DNA纳米球制备的反应体系包括1～5微升DNA纳米球制备缓冲液以及1～3微升DNA纳米球制备酶混合液，其中所述DNA纳米球制备酶混合液包括0.1～1U/μL phi29聚合酶，0.01～0.1％吐温20；
    所述DNA纳米球制备反应之后，加入1～3微升DNA纳米球制备终止液来终止滚环线性扩增。
  17. 根据权利要求2所述的方法，其特征在于，所述PCR扩增的反应体系包括3～5微升PCR扩增酶混合液，所述PCR扩增酶混合液包括PCR酶，0.1～2μM引物，0.01～0.1％吐温20。
  18. 一种全基因组全流程微流控自动化建库装置，其特征在于，所述装置包括：微流控芯片，该微流控芯片包括芯片基底和设置在所述芯片基底上的电极分布及电极开关的组合控制，所述微流控芯片用于实现权利要求1-17中任一项所述的方法。
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