WO2024056012A1 - Automatic ngs library preparation and sub-packaging kit and library construction method - Google Patents

Automatic ngs library preparation and sub-packaging kit and library construction method Download PDF

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WO2024056012A1
WO2024056012A1 PCT/CN2023/118715 CN2023118715W WO2024056012A1 WO 2024056012 A1 WO2024056012 A1 WO 2024056012A1 CN 2023118715 W CN2023118715 W CN 2023118715W WO 2024056012 A1 WO2024056012 A1 WO 2024056012A1
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final concentration
well
solution
reaction
take
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PCT/CN2023/118715
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French (fr)
Chinese (zh)
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张华�
秦资
徐安然
李芳敏
祝君
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上海思路迪生物医学科技有限公司
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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of biological high-throughput sequencing, and in particular to an automated NGS library preparation and packaging kit and a library construction method.
  • NGS High-throughput sequencing
  • This application provides an automated NGS library preparation and packaging kit and a library construction method, which solves the problem of incompatible kits in manual detection and automated detection processes in the existing technology, and avoids the inability of existing commercial kits to be directly used in automated testing. In the process of building the database.
  • the present application provides an automated NGS library preparation dispensing kit, which includes: a first dispensing reagent combination, a second dispensing reagent combination, and a second dispensing reagent combination, wherein the first dispensing reagent combination
  • the reagent combination includes a first hot washing liquid, a cleaning liquid, and an eluent
  • the second packaging reagent combination includes a second hot washing liquid, a normal temperature washing liquid, a library building reagent, and a capture reagent
  • the third packaging reagent The combination includes magnetic bead reagents.
  • the first hot washing liquid in the first aliquoted reagent combination includes washing liquid 1; the washing liquid includes ethanol and water; and the eluent includes water;
  • the second hot washing liquid in the second packaged reagent combination includes enhanced washing liquid;
  • the normal temperature washing liquid It includes magnetic bead cleaning solution, washing solution 1, washing solution 2 and washing solution 3;
  • the library construction reagents include end repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution ;
  • the capture reagent includes blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer and PCR reaction solution;
  • the magnetic bead reagents in the third packaging reagent combination include Ampure XP beads and streptavidin magnetic beads.
  • the end repair buffer includes: Tris-HCl, MgCl 2 , DTT, ATP, dATP, dCTP, dGTP and dTTP;
  • the end repair enzyme includes: T4 polynucleotide kinase, T4 DNA Polymerase, Taq DNA polymerase, Klenow fragment and glycerol;
  • the ligation reaction solution includes: Tris-HCl, MgCl2, DTT, ATP, dNTP, PEG8000 and water;
  • the joint includes an adapter
  • the ligase includes T4 ligase
  • the PCR reaction solution includes KAPA HiFi hotstart ready mix
  • the PCR amplification primers include P5 and P7 primer;
  • the blocking solution includes Human Cot DNA and Blocker Oligo TS Mix
  • the hybridization reaction solution includes sodium citrate, dextran sulfate, betaine, formamide, tetramethylammonium chloride, NaCl, Probe, water, glycerol and polysorbate (Tween);
  • the magnetic bead cleaning solution includes NaCl, Tris-HCl, EDTA, Tween and water;
  • the lotion 1 includes sodium citrate, NaCl and SDS;
  • the enhanced lotion includes sodium citrate, NaCl and Tween;
  • the lotion 2 includes sodium citrate, NaCl and water;
  • the lotion 3 includes sodium citrate, NaCl and water;
  • the magnetic bead resuspension includes sodium citrate, dextran sulfate, betaine, formamide, tetramethylammonium chloride, NaCl, water, glycerol and Tween.
  • the volume of water in the hot washing solution is 2000 ⁇ L, and the volume of the washing solution 1 is 150 ⁇ L;
  • Equal volumes of ethanol in the first cleaning solution are divided into three parts, and the volume of each part of ethanol is 500 ⁇ L, and the concentration of ethanol is 80%;
  • the eluent is divided into two parts of water, and the volumes of the two parts of water are 50 ⁇ L and 60 ⁇ L respectively;
  • the enhanced lotion in the second hot lotion is divided into two parts, each with a volume of 200 ⁇ L;
  • the volume of the magnetic bead cleaning solution in the normal temperature washing solution is 320 ⁇ L, and the volumes of the washing solutions 1, 2, and 3 are all 175 ⁇ L;
  • the volumes of end repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution in the library construction reagent are 10 ⁇ L, 4.3 ⁇ L, 40 ⁇ L, 5 ⁇ L, 15 ⁇ L, 30 ⁇ L, and 30 ⁇ L respectively. ;
  • the volumes of blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer, and PCR reaction solution in the capture reagent are 25 ⁇ L, 20 ⁇ L, 20 ⁇ L, 30 ⁇ L, and 30 ⁇ L respectively;
  • the Ampure XP beads in the magnetic bead reagent are divided into three parts, with volumes of 120 ⁇ L, 150 ⁇ L, and 100 ⁇ L respectively; the volume of the streptavidin magnetic beads is 60 ⁇ L.
  • the final concentration of Tris-HCl is 150-250mM
  • the final concentration of MgCl2 is 35-45mM
  • the final concentration of DTT is 35-45mM
  • the final concentration of ATP is 3.5- 4.5mM
  • the final concentration of dATP is 10-20mM
  • the final concentration of dCTP is 1.0-2.0mM
  • the final concentration of dGTP is 1.0-2.0mM
  • the final concentration of dTTP is 1.0-2.0mM
  • the final concentration of Tris-HCl is 150-250mM
  • the final concentration of MgCl2 is 35-45mM
  • the final concentration of DTT is 35-45mM
  • the final concentration of ATP is 3.5- 4.5mM
  • the final concentration of dATP is 10-20mM
  • the final concentration of dCTP is 1.0-2.0mM
  • the final concentration of dGTP is 1.0-2.0mM
  • the final concentration of dTTP is 1.0-2.0
  • the final concentration of T4 polynucleotide kinase in the end repair enzyme is 3.5-4.5U/ ⁇ L
  • the final concentration of T4 DNA Polymerase is 0.8-1.2U/ ⁇ L
  • the final concentration of Taq DNA polymerase is 0.8-1.2U.
  • the final concentration of Klenow fragment is 0.8-1.2U/ ⁇ L
  • the final concentration of glycerol is 5-30%;
  • the final concentration of Tris-HCl in the ligation reaction solution is 100-150nM, the final concentration of MgCl2 is 25-30mM, the final concentration of DTT is 25-30mM, the final concentration of ATP is 2-3mM, and the final concentration of dNTP is 2-3mM, the final concentration of PEG8000 is 20%-30%;
  • the final concentration of the adapter in the adapter is 0.5-0.8 ⁇ M
  • the final concentration of T4 ligase in the ligase is 250-350U/ ⁇ L;
  • the final concentration of P5 and P7 primer in the PCR amplification primer is 5-15 ⁇ M
  • the final concentration of Human Cot DNA in the blocking solution is 0.25-0.5mg/ ⁇ L;
  • the final concentration of sodium citrate in the hybridization reaction solution is 10-20mM, the final concentration of dextran sulfate is 4.5-5.5%, the final concentration of betaine is 0.8-1.2M, and the final concentration of formamide is 15-25 %, the final concentration of tetramethylammonium chloride is 0.8-1.2M, the final concentration of NaCl is 135-165mM, the final concentration of Probe is 0.1-0.2nM, the final concentration of glycerol is 5%-30%, and the final concentration of Tween The final concentration is 0.05%-0.5%;
  • the final concentration of NaCl in the magnetic bead cleaning solution is 0.8-1.2M, and the final concentration of Tris-HCl is 5-15
  • the final concentration of mM and EDTA is 0.8-1.2mM, and the final concentration of Tween is 0.05%-0.5%;
  • the final concentration of sodium citrate in the lotion 1 is 13.5-16.5mM, the final concentration of NaCl is 135-165mM, and the final concentration of SDS is 0.05%-0.3%;
  • the final concentration of sodium citrate in the enhanced lotion is 13.5-16.5mM, the final concentration of NaCl is 50-100mM, and the final concentration of Tween is 0.05%-0.5%;
  • the final concentration of sodium citrate in the lotion 2 is 5-10mM, and the final concentration of NaCl is 50-100mM;
  • the final concentration of sodium citrate in the lotion 3 is 2-5mM, and the final concentration of NaCl is 25-35mM;
  • the final concentration of sodium citrate in the magnetic bead resuspension is 10-20mM, the final concentration of dextran sulfate is 4.5-5.5%, the final concentration of betaine is 0.8-1.2M, and the final concentration of formamide is 15 -25%, the final concentration of tetramethylammonium chloride is 0.8-1.2M, the final concentration of NaCl is 135-165mM, the final concentration of glycerol is 5%-30%, the final concentration of Tween is 0.05%-0.5% .
  • this application also provides an automatic library construction method using an automated NGS library preparation and packaging kit, which includes the following steps:
  • Reagents and sample preparation Take out the automated library construction kit, balance it to room temperature, shake and mix well, take 50 ⁇ L of the fragmented sample into the first reaction well and centrifuge it for later use, with a rotation speed of 1000 rpm/min;
  • End repair Take the end repair buffer and pipet and mix into the end repair enzyme well containing the end repair enzyme; take 10 ⁇ L of the mixture in the end repair enzyme well into the centrifuged sample and pipet and mix;
  • PCR amplification Take 25 ⁇ L of PCR amplification primer into the first reaction well to elute the dried magnetic beads, and take the elution supernatant into the second reaction well, and wash the first reaction well with water at the same time; Pour 25 ⁇ L of PCR reaction solution into the second reaction well and mix by pipetting;
  • Hybridization capture Take 17 ⁇ L of hybridization reaction solution from the hybridization reaction solution well to the first reaction well, mix by pipetting; after incubation, take all the elution supernatant into the second reaction well, and run the following hybridization program;
  • the washing and enrichment in step S90 includes the following steps:
  • streptavidin magnetic beads Take 50 ⁇ L of streptavidin magnetic beads from the streptavidin magnetic bead well and pipet and mix the streptavidin magnetic beads to the well to be used, and use the magnetic bead cleaning solution in the magnetic bead cleaning solution well to wash the streptavidin magnetic beads three times;
  • Capture PCR amplification Take 25 ⁇ L of PCR amplification primer from the PCR amplification primer well and 25 ⁇ L of PCR reaction solution from the PCR reaction solution well to the second reaction well, mix by pipetting, and run the PCR amplification program:
  • the PCR product is purified with 75 ⁇ L AMpure XP Beads (1.5X) in the third Ampure XP beads well and 80% ethanol in the third ethanol well, and 30 ⁇ L water is taken from the third water well to elute the library.
  • the program run in S20 the reaction temperature is 20°C, the reaction time is 30min; the reaction temperature is 65°C, the reaction time is 30min;
  • reaction temperature is 95°C, reaction time is 30s; reaction temperature is 65°C, reaction time is 12h.
  • the PCR amplification runs as follows:
  • the PCR program for the capture PCR amplification run is as follows:
  • the kit described in this application is an automated NGS library preparation kit. It uses pre-packaged kits and automated instruments to prepare libraries. It can automatically prepare libraries with one click.
  • the instrument operation is stable and reproducible; only one person is needed. There is no need for personnel to participate in the whole process after adding the sample, and the manual operation time is 5 minutes. At the same time, it can be run unattended at night, which improves the preparation efficiency.
  • a fully enclosed cartridge is used to perform the reaction in a sealed space, achieving zero contamination in library preparation.
  • Figure 1 is a comparison chart of target accuracy between manual library construction and instrument-automated library construction in this application.
  • Figure 2 is a comparison of the CV values of the on-target rate between manual library construction and instrument-automated library construction in this application.
  • Figure 3 is a comparison chart of the uniformity of manual library construction and instrument-automated library construction in this application.
  • Figure 4 is a comparison chart of uniformity CV values between manual library construction and instrument-automated library construction in this application.
  • Figure 5 is a diagram showing the well position distribution of each component of the automated library construction kit of the present application.
  • This embodiment provides an automated NGS library preparation and packaging kit, which includes a first packaging reagent combination, a second packaging reagent combination, and a second packaging reagent combination, wherein the first packaging reagent combination includes a third packaging reagent combination.
  • the third packaged reagent combination includes a magnetic bead reagent .
  • the first hot washing liquid in the first packaged reagent combination in the kit includes washing liquid 1; the washing liquid includes ethanol and water; the eluent includes water;
  • the second hot washing liquid in the second packaging reagent combination includes enhanced washing liquid;
  • the normal temperature washing liquid includes magnetic bead cleaning liquid, washing liquid 1, washing liquid 2 and washing liquid 3;
  • the library building reagent includes terminal Repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution;
  • the capture reagent includes blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer and PCR The reaction solution;
  • the magnetic bead reagents in the third packaging reagent combination include Ampure XP beads and streptavidin magnetic beads.
  • the automated NGS library preparation kit uses pre-packaged reagents onto the cartridge base as shown in Figure 5, and divides the hole locations into Reaction well combination, hot washing solution well combination, room temperature washing solution well combination, cleaning solution well combination, eluent well combination, library building reagent well combination, capture reagent well combination and magnetic bead reagent well combination and other different areas, reagent well positions
  • the distribution and loading are shown in Table 1.
  • the required reagent components of the second aliquot reagent set, their final concentration, optimal concentration, and the volume of each reagent in the second aliquot reagent set are as follows: As shown in Table 2 below.
  • Blocker Oligo TS Mix in the third reagent set Company
  • KAPA HiFi hotstart ready mix KAPA Biosysterms
  • streptavidin magnetic beads Invitrogen
  • AMpure XP Beads Bosset Group
  • water Invitrogen
  • This embodiment adopts the above-mentioned automated library construction kit to be packed into the cartridge base and used with the automated library construction equipment in conjunction with the automated library construction instrument.
  • the specific internal operation process of the instrument is as follows:
  • This embodiment also provides an application of automated NGS library preparation and packaging
  • the automatic library construction method of the test kit includes the following steps:
  • Adapter connection After the end repair program is completed, take 35 ⁇ L of the connection buffer in well 30 and add 5 ⁇ L of adapter to well 31. After pipetting and mixing, take 40 ⁇ L into reaction well 1;
  • reaction well 2 Take 20 ⁇ L of the eluent supernatant from reaction well 2 to reaction well 1, and wash reaction well 2 (use water in well 9);
  • sample was purified and washed using an automated library construction instrument (using 80% ethanol in well 13).
  • wash once with 100 ⁇ L of Wash Solution 1 preheated to 65°C in well 10 then wash twice with 150 ⁇ L of Enhanced Wash Solution preheated to 65°C in wells 19 and 20; then wash once with 150 ⁇ L of Wash Solution 1, Wash Solution 2, and Wash Solution 3 in wells 23, 24, and 25, respectively;
  • the PCR product was purified with 75 ⁇ L AMpure XP Beads (1.5X) in well 47 (using 80% ethanol in well 15), and 30 ⁇ L water was taken from well 17 to elute the library.
  • connection reaction solution After the program is finished, add the connection reaction solution and connector connection component formula according to the following table:
  • Step 3 Purification of ligation reaction products
  • Step 4 PCR Amplification
  • Step 5 PCR product recovery
  • the PCR product was purified with 75 ⁇ L AMpure XP Beads (1.5X), and the captured library was subjected to Qubit quantification and fragment size analysis.
  • This embodiment provides an automated library construction kit for use in conjunction with an automated library construction instrument.
  • the operation process is as follows:
  • the target-on-target rate of manual library construction by different personnel varies greatly, with large fluctuations.
  • the CV value of the target-on-target rate is larger than that of instrument library construction, and the results are not as stable as those of instrument library construction.
  • the uniformity is good regardless of whether the instrument or manual library construction is performed.
  • the instrument library construction is uniform.
  • the one-dimensional CV value is smaller and more stable.
  • the components of the commercial manual kits currently on the market are more complicated than this application, with more components and smaller volumes.

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Abstract

An automatic NGS library preparation and sub-packaging kit and a library construction method. The kit is an automatic NGS library preparation kit. Library preparation is carried out using a pre-sub-packaged kit with an automatic instrument, and the library is automatically prepared in a one-click mode. The instrument operation is stable, and the repeatability is high. Only one person is needed to add the sample, after which no personnel involvement is required throughout the process. The manual operation time is 5 minutes, and unattended operation at night can be achieved, improving the preparation efficiency. In addition, a totally enclosed cassette is adopted, such that the reaction is carried out in a sealed space, achieving zero contamination in the library preparation.

Description

一种自动化NGS文库制备分装试剂盒及建库方法An automated NGS library preparation and packaging kit and library construction method 技术领域Technical field
本申请涉及生物高通量测序领域，尤其涉及一种自动化NGS文库制备分装试剂盒及建库方法。The present application relates to the field of biological high-throughput sequencing, and in particular to an automated NGS library preparation and packaging kit and a library construction method.
背景技术Background technique
高通量测序(NGS)技术凭借其高通量、精确度高和信息量丰富等优势，在基础研究、产前诊断、遗传病诊断、肿瘤诊断与精准医疗等领域应用广泛。然而，相比于传统分子检测技术，NGS操作十分复杂和繁琐，实验流程较长，对实验人员要求较高，需要大量专业技术人员较长时间的培训才能顺利操作。这些情况严重制约了NGS技术的大规模推广和应用，为此开发自动化NGS建库产品成为打破束缚，大规模推广NGS技术的必由之路。High-throughput sequencing (NGS) technology has been widely used in basic research, prenatal diagnosis, genetic disease diagnosis, tumor diagnosis and precision medicine due to its advantages of high throughput, high accuracy and rich information. However, compared with traditional molecular detection technology, NGS operations are very complex and cumbersome, with long experimental procedures and high requirements for experimental personnel. It requires a large number of professional and technical personnel and a long period of training to operate smoothly. These situations have seriously restricted the large-scale promotion and application of NGS technology. Therefore, the development of automated NGS library building products has become the only way to break the constraints and promote NGS technology on a large scale.
现有文库制备都是采用手工法，手工制备文库的方法十分繁琐，对操作人员专业水平要求较高；且手工制备步骤繁多，出错概率高，且不同人员间不稳定，波动较大；同时文库制备工作量大，需要3-4名专业操作人员，至少两天时间的操作。另外，多管操作，多次开盖移液，不可避免的会产生污染。Existing library preparation methods are all manual. The method of manual library preparation is very cumbersome and requires a high professional level of operators. Moreover, manual preparation steps are numerous, the probability of errors is high, and it is unstable and fluctuates greatly among different personnel. At the same time, the library The preparation workload is large and requires 3-4 professional operators and at least two days of operation. In addition, multi-tube operation and multiple opening and pipetting will inevitably cause contamination.
发明内容Contents of the invention
本申请提供一种自动化NGS文库制备分装试剂盒及建库方法，解决现有技术中手动检测以及自动化检测过程中的试剂盒不配套的问题，避免现有的商用试剂盒无法直接使用到自动化建库的过程中。This application provides an automated NGS library preparation and packaging kit and a library construction method, which solves the problem of incompatible kits in manual detection and automated detection processes in the existing technology, and avoids the inability of existing commercial kits to be directly used in automated testing. In the process of building the database.
第一方面，本申请提供一种自动化NGS文库制备分装试剂盒，其包括：第一分装试剂组合和第二分装试剂组合以及第二分装试剂组合，其中，所述第一分装试剂组合包括第一热洗液、清洗液、洗脱液；所述第二分装试剂组合包括第二热洗液、常温洗液、建库试剂、捕获试剂；以及所述第三分装试剂组合包括磁珠试剂。In a first aspect, the present application provides an automated NGS library preparation dispensing kit, which includes: a first dispensing reagent combination, a second dispensing reagent combination, and a second dispensing reagent combination, wherein the first dispensing reagent combination The reagent combination includes a first hot washing liquid, a cleaning liquid, and an eluent; the second packaging reagent combination includes a second hot washing liquid, a normal temperature washing liquid, a library building reagent, and a capture reagent; and the third packaging reagent The combination includes magnetic bead reagents.
在一个实施方式中，所述第一分装试剂组合中所述的第一热洗液包括洗液1；所述清洗液包括乙醇、水；以及所述洗脱液包括水；In one embodiment, the first hot washing liquid in the first aliquoted reagent combination includes washing liquid 1; the washing liquid includes ethanol and water; and the eluent includes water;
所述第二分装试剂组合中所述的第二热洗液包括增强洗液；所述常温洗液 包括磁珠清洗液、洗液1、洗液2和洗液3；所述建库试剂包括末端修复缓冲液、末端修复酶、连接缓冲液、接头、连接酶、PCR扩增引物和PCR反应液；以及所述捕获试剂包括封闭液、杂交反应液、磁珠重悬液、PCR扩增引物和PCR反应液；以及The second hot washing liquid in the second packaged reagent combination includes enhanced washing liquid; the normal temperature washing liquid It includes magnetic bead cleaning solution, washing solution 1, washing solution 2 and washing solution 3; the library construction reagents include end repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution ; And the capture reagent includes blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer and PCR reaction solution; and
所述第三分装试剂组合中所述的磁珠试剂包括Ampure XP beads、链霉亲和素磁珠。The magnetic bead reagents in the third packaging reagent combination include Ampure XP beads and streptavidin magnetic beads.
在一个实施方式中，所述末端修复缓冲液包括：Tris-HCl、MgCl2、DTT、ATP、dATP、dCTP、dGTP和dTTP；In one embodiment, the end repair buffer includes: Tris-HCl, MgCl 2 , DTT, ATP, dATP, dCTP, dGTP and dTTP;
所述末端修复酶包括：T4多聚核苷酸激酶、T4 DNA Polymerase、Taq DNA聚合酶、Klenow片段和甘油；The end repair enzyme includes: T4 polynucleotide kinase, T4 DNA Polymerase, Taq DNA polymerase, Klenow fragment and glycerol;
所述连接反应液包括：Tris-HCl、MgCl2、DTT、ATP、dNTP、PEG8000和水；The ligation reaction solution includes: Tris-HCl, MgCl2, DTT, ATP, dNTP, PEG8000 and water;
所述接头包括adapter；The joint includes an adapter;
所述连接酶包括T4连接酶；The ligase includes T4 ligase;
所述PCR反应液包括KAPA HiFi hotstart ready mix；The PCR reaction solution includes KAPA HiFi hotstart ready mix;
所述PCR扩增引物包括P5、P7 primer；The PCR amplification primers include P5 and P7 primer;
所述封闭液包括Human Cot DNA和Blocker Oligo TS Mix；The blocking solution includes Human Cot DNA and Blocker Oligo TS Mix;
所述杂交反应液包括柠檬酸钠、硫酸葡聚糖、甜菜碱、甲酰胺、四甲基氯化铵、NaCl、Probe、水、甘油和聚山梨酯(吐温)；The hybridization reaction solution includes sodium citrate, dextran sulfate, betaine, formamide, tetramethylammonium chloride, NaCl, Probe, water, glycerol and polysorbate (Tween);
所述磁珠清洗液包括NaCl、Tris-HCl、EDTA、吐温和水；The magnetic bead cleaning solution includes NaCl, Tris-HCl, EDTA, Tween and water;
所述洗液1包括柠檬酸钠、NaCl和SDS；The lotion 1 includes sodium citrate, NaCl and SDS;
所述增强洗液包括柠檬酸钠、NaCl和吐温；The enhanced lotion includes sodium citrate, NaCl and Tween;
所述洗液2包括柠檬酸钠、NaCl和水；The lotion 2 includes sodium citrate, NaCl and water;
所述洗液3包括柠檬酸钠、NaCl和水；以及The lotion 3 includes sodium citrate, NaCl and water; and
所述磁珠重悬液包括柠檬酸钠、硫酸葡聚糖、甜菜碱、甲酰胺、四甲基氯化铵、NaCl、水、甘油和吐温。The magnetic bead resuspension includes sodium citrate, dextran sulfate, betaine, formamide, tetramethylammonium chloride, NaCl, water, glycerol and Tween.
在一个实施方式中，所述热洗液中水的体积为2000μL，所述洗液1的体积为150μL；In one embodiment, the volume of water in the hot washing solution is 2000 μL, and the volume of the washing solution 1 is 150 μL;
所述第一清洗液中乙醇等体积分为三份，且每份乙醇的体积为500μL，所述乙醇的浓度为80％； Equal volumes of ethanol in the first cleaning solution are divided into three parts, and the volume of each part of ethanol is 500 μL, and the concentration of ethanol is 80%;
所述洗脱液中分装为两份水，所述两份水的体积分别为50μL和60μL；The eluent is divided into two parts of water, and the volumes of the two parts of water are 50 μL and 60 μL respectively;
所述第二热洗液中的增强洗液分装成两份，体积均为200μL；The enhanced lotion in the second hot lotion is divided into two parts, each with a volume of 200 μL;
所述常温洗液中的磁珠清洗液的体积为320μL，所述洗液1、洗液2、洗液3的体积均为175μL；The volume of the magnetic bead cleaning solution in the normal temperature washing solution is 320 μL, and the volumes of the washing solutions 1, 2, and 3 are all 175 μL;
所述建库试剂中末端修复缓冲液、末端修复酶、连接缓冲液、接头、连接酶、PCR扩增引物以及PCR反应液的体积分别为10μL、4.3μL、40μL、5μL、15μL、30μL、30μL；The volumes of end repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution in the library construction reagent are 10 μL, 4.3 μL, 40 μL, 5 μL, 15 μL, 30 μL, and 30 μL respectively. ;
所述捕获试剂中的封闭液、杂交反应液、磁珠重悬液、PCR扩增引物、PCR反应液的体积分别为25μL、20μL、20μL、30μL、30μL；The volumes of blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer, and PCR reaction solution in the capture reagent are 25 μL, 20 μL, 20 μL, 30 μL, and 30 μL respectively;
所述磁珠试剂中的Ampure XP beads分为三份，体积分别为120μL、150μL、100μL；所述链霉亲和素磁珠的体积为60μL。The Ampure XP beads in the magnetic bead reagent are divided into three parts, with volumes of 120 μL, 150 μL, and 100 μL respectively; the volume of the streptavidin magnetic beads is 60 μL.
在一个实施方式中，所述末端修复缓冲液中：Tris-HCl的终浓度为150-250mM、MgCl2的终浓度为35-45mM、DTT的终浓度为35-45mM、ATP的终浓度为3.5-4.5mM、dATP终浓度为10-20mM、dCTP终浓度为1.0-2.0mM、dGTP的终浓度为1.0-2.0mM、dTTP的终浓度为1.0-2.0mM；In one embodiment, in the end repair buffer: the final concentration of Tris-HCl is 150-250mM, the final concentration of MgCl2 is 35-45mM, the final concentration of DTT is 35-45mM, and the final concentration of ATP is 3.5- 4.5mM, the final concentration of dATP is 10-20mM, the final concentration of dCTP is 1.0-2.0mM, the final concentration of dGTP is 1.0-2.0mM, the final concentration of dTTP is 1.0-2.0mM;
所述末端修复酶中T4多聚核苷酸激酶的终浓度为3.5-4.5U/μL、T4 DNA Polymerase的终浓度为0.8-1.2U/μL、Taq DNA聚合酶的终浓度为0.8-1.2U/μL、Klenow片段的终浓度为0.8-1.2U/μL、甘油的终浓度为5-30％；The final concentration of T4 polynucleotide kinase in the end repair enzyme is 3.5-4.5U/μL, the final concentration of T4 DNA Polymerase is 0.8-1.2U/μL, and the final concentration of Taq DNA polymerase is 0.8-1.2U. /μL, the final concentration of Klenow fragment is 0.8-1.2U/μL, and the final concentration of glycerol is 5-30%;
所述连接反应液中Tris-HCl的终浓度为100-150nM、MgCl2的终浓度为25-30mM、DTT的终浓度为25-30mM、ATP的终浓度为2-3mM、dNTP的终浓度为2-3mM、PEG8000的终浓度为20％-30％；The final concentration of Tris-HCl in the ligation reaction solution is 100-150nM, the final concentration of MgCl2 is 25-30mM, the final concentration of DTT is 25-30mM, the final concentration of ATP is 2-3mM, and the final concentration of dNTP is 2-3mM, the final concentration of PEG8000 is 20%-30%;
所述接头中adapter的终浓度为0.5-0.8μM；The final concentration of the adapter in the adapter is 0.5-0.8 μM;
所述连接酶中T4连接酶的终浓度为250-350U/μL；The final concentration of T4 ligase in the ligase is 250-350U/μL;
所述PCR扩增引物中P5、P7 primer的终浓度为5-15μM；The final concentration of P5 and P7 primer in the PCR amplification primer is 5-15 μM;
所述封闭液中Human Cot DNA的终浓度为0.25-0.5mg/μL；The final concentration of Human Cot DNA in the blocking solution is 0.25-0.5mg/μL;
所述杂交反应液中柠檬酸钠的终浓度为10-20mM、硫酸葡聚糖的终浓度为4.5-5.5％、甜菜碱的终浓度为0.8-1.2M、甲酰胺的终浓度为15-25％、四甲基氯化铵的终浓度为0.8-1.2M、NaCl的终浓度为135-165mM、Probe的终浓度为0.1-0.2nM、甘油的终浓度为5％-30％、吐温的终浓度为0.05％-0.5％；The final concentration of sodium citrate in the hybridization reaction solution is 10-20mM, the final concentration of dextran sulfate is 4.5-5.5%, the final concentration of betaine is 0.8-1.2M, and the final concentration of formamide is 15-25 %, the final concentration of tetramethylammonium chloride is 0.8-1.2M, the final concentration of NaCl is 135-165mM, the final concentration of Probe is 0.1-0.2nM, the final concentration of glycerol is 5%-30%, and the final concentration of Tween The final concentration is 0.05%-0.5%;
所述磁珠清洗液中NaCl的终浓度为0.8-1.2M、Tris-HCl的终浓度为5-15 mM、EDTA的终浓度为0.8-1.2mM、吐温的终浓度为0.05％-0.5％；The final concentration of NaCl in the magnetic bead cleaning solution is 0.8-1.2M, and the final concentration of Tris-HCl is 5-15 The final concentration of mM and EDTA is 0.8-1.2mM, and the final concentration of Tween is 0.05%-0.5%;
所述洗液1中柠檬酸钠的终浓度为13.5-16.5mM、NaCl的终浓度为135-165mM、SDS的终浓度为0.05％-0.3％；The final concentration of sodium citrate in the lotion 1 is 13.5-16.5mM, the final concentration of NaCl is 135-165mM, and the final concentration of SDS is 0.05%-0.3%;
所述增强洗液中柠檬酸钠的终浓度为13.5-16.5mM、NaCl的终浓度为50-100mM、吐温的终浓度为0.05％-0.5％；The final concentration of sodium citrate in the enhanced lotion is 13.5-16.5mM, the final concentration of NaCl is 50-100mM, and the final concentration of Tween is 0.05%-0.5%;
所述洗液2中柠檬酸钠的终浓度为5-10mM、NaCl的终浓度为50-100mM；The final concentration of sodium citrate in the lotion 2 is 5-10mM, and the final concentration of NaCl is 50-100mM;
所述洗液3中柠檬酸钠的终浓度为2-5mM、NaCl的终浓度为25-35mM；The final concentration of sodium citrate in the lotion 3 is 2-5mM, and the final concentration of NaCl is 25-35mM;
所述磁珠重悬液中柠檬酸钠的终浓度为10-20mM、硫酸葡聚糖的终浓度为4.5-5.5％、甜菜碱的终浓度为0.8-1.2M、甲酰胺的终浓度为15-25％、四甲基氯化铵的终浓度为0.8-1.2M、NaCl的终浓度为135-165mM、甘油的终浓度为5％-30％、吐温的终浓度为0.05％-0.5％。The final concentration of sodium citrate in the magnetic bead resuspension is 10-20mM, the final concentration of dextran sulfate is 4.5-5.5%, the final concentration of betaine is 0.8-1.2M, and the final concentration of formamide is 15 -25%, the final concentration of tetramethylammonium chloride is 0.8-1.2M, the final concentration of NaCl is 135-165mM, the final concentration of glycerol is 5%-30%, the final concentration of Tween is 0.05%-0.5% .
第二方面，本申请还提供一种应用自动化NGS文库制备分装试剂盒的自动建库方法，其包括如下步骤：In a second aspect, this application also provides an automatic library construction method using an automated NGS library preparation and packaging kit, which includes the following steps:
S10.试剂和样本准备：将自动化建库试剂盒取出，平衡至室温后振荡混匀，取50μL片段化后的样本至第一反应孔中并离心后备用，转速为1000rpm/min；S10. Reagents and sample preparation: Take out the automated library construction kit, balance it to room temperature, shake and mix well, take 50 μL of the fragmented sample into the first reaction well and centrifuge it for later use, with a rotation speed of 1000 rpm/min;
S20.末端修复：取末端修复缓冲液至含有末端修复酶的末端修复酶孔中吹打混匀；取末端修复酶孔中的混合液10μL至离心样本中吹打混匀；S20. End repair: Take the end repair buffer and pipet and mix into the end repair enzyme well containing the end repair enzyme; take 10 μL of the mixture in the end repair enzyme well into the centrifuged sample and pipet and mix;
S30.接头连接：将连接缓冲液孔中与接头吹打混匀后，取40μL至第一反应孔中；取连接酶10μL至第一反应孔中，吹打混匀；S30. Adapter connection: After pipetting and mixing the ligation buffer well and the adapter, take 40 μL into the first reaction well; take 10 μL of ligase into the first reaction well, pipe and mix;
S40.连接产物纯化：连接程序运行结束后，取99μL吹打混匀的Ampure XP beads至第一反应孔中并吹打混匀；并进行样本纯化和80％乙醇洗涤；S40. Purification of the ligation product: After the ligation program is completed, take 99 μL of Ampure
S50.PCR扩增：取25μL PCR扩增引物至第一反应孔中洗脱晾干的磁珠，并取洗脱上清液至第二反应孔中，同时使用水清洗第一反应孔；取25μL PCR反应液至第二反应孔中，吹打混匀；S50. PCR amplification: Take 25 μL of PCR amplification primer into the first reaction well to elute the dried magnetic beads, and take the elution supernatant into the second reaction well, and wash the first reaction well with water at the same time; Pour 25 μL of PCR reaction solution into the second reaction well and mix by pipetting;
S60.PCR产物纯化：PCR运行结束后，取45μL吹打混匀的Ampure XP beads至第二反应孔吹打混匀；并对样本进行纯化和80％乙醇洗涤；取40μL水至第二反应孔中，吹打混匀；S60. PCR product purification: After the PCR run, take 45 μL of Ampure Whisk to mix;
S70.文库封闭与浓缩：取20μL洗脱液上清至第一反应孔，并清洗第二反应孔并用第一水孔中的水；取20μL封闭液至第一反应孔，吹打混匀；然后取72μL吹打混匀的Ampure XP beads至第一反应孔吹打混匀；孵育后进行纯化和 并使用80％乙醇进行洗涤；S70. Library blocking and concentration: Take 20 μL of eluent supernatant to the first reaction well, and wash the second reaction well with water from the first water well; take 20 μL of blocking solution to the first reaction well, pipet and mix; then Take 72μL of Ampure And use 80% ethanol for washing;
S80.杂交捕获：从杂交反应液孔取17μL杂交反应液至第一反应孔，吹打混匀；孵育后取全部洗脱上清液至第二反应孔中，运行如下杂交程序；
S80. Hybridization capture: Take 17 μL of hybridization reaction solution from the hybridization reaction solution well to the first reaction well, mix by pipetting; after incubation, take all the elution supernatant into the second reaction well, and run the following hybridization program;
S90.洗涤富集。S90. Washing and enrichment.
在一个实施方式中，所述步骤S90中的洗涤富集包括如下步骤：In one embodiment, the washing and enrichment in step S90 includes the following steps:
从链霉亲和素磁珠孔取50μL吹打混匀的链霉亲和素磁珠至待用孔，并使用磁珠清洗液孔中的磁珠清洗液清洗链霉亲和素磁珠三次；Take 50 μL of streptavidin magnetic beads from the streptavidin magnetic bead well and pipet and mix the streptavidin magnetic beads to the well to be used, and use the magnetic bead cleaning solution in the magnetic bead cleaning solution well to wash the streptavidin magnetic beads three times;
取磁珠重悬液孔中17μL磁珠重悬液加入待用孔重悬链霉亲和素磁珠；Take 17 μL of the magnetic bead resuspension solution from the magnetic bead resuspension solution well and add it to the well to be used to resuspend the streptavidin magnetic beads;
待杂交程序结束后，将待用孔中的重悬链霉亲和素磁珠全部加入到第二反应孔的杂交液中，吹打混匀，65℃条件下继续孵育45min，期间每15min吹打混匀一次；After the hybridization program is completed, add all the resuspended streptavidin magnetic beads in the well to be used to the hybridization solution in the second reaction well, mix by pipetting, and continue to incubate at 65°C for 45 minutes, pipetting and mixing every 15 minutes. Even once;
孵育结束后，使用第一热洗液孔组合中洗液1孔的100μL预热65℃的洗液1清洗一次，然后使用第二热洗液孔组合中两个增强洗液孔中150μL预热65℃的增强洗液清洗两次；之后分别使用常温洗液孔组合中洗液1孔、洗液2孔以及洗液3孔中150μL洗液1、洗液2和洗液3清洗一次；After the incubation, wash once with 100 μL of wash solution 1 in well 1 of the first hot wash solution well combination that is preheated at 65°C, and then use 150 μL of preheated wash solution 1 in the two enhanced wash solution wells of the second hot wash solution well combination. Wash twice with enhanced washing solution at 65°C; then wash once with 150 μL washing liquid 1, washing liquid 2 and washing liquid 3 in washing liquid 1, washing liquid 2 and washing liquid 3 in the room temperature washing liquid well combination;
捕获PCR扩增：取PCR扩增引物孔中25μL PCR扩增引物和PCR反应液孔中25μL PCR反应液至第二反应孔中，吹打混匀，运行PCR扩增程序：
Capture PCR amplification: Take 25 μL of PCR amplification primer from the PCR amplification primer well and 25 μL of PCR reaction solution from the PCR reaction solution well to the second reaction well, mix by pipetting, and run the PCR amplification program:
运行结束后，PCR产物用第三Ampure XP beads孔中75μL AMpure XP Beads(1.5X)和第三乙醇孔中80％乙醇进行纯化，取第三水孔中30μL水洗脱文库。After the run is completed, the PCR product is purified with 75 μL AMpure XP Beads (1.5X) in the third Ampure XP beads well and 80% ethanol in the third ethanol well, and 30 μL water is taken from the third water well to elute the library.
在一个实施方式中，所述S20中运行的程序：反应温度为20℃，反应时间为30min；反应温度为65℃，反应时间为30min； In one embodiment, the program run in S20: the reaction temperature is 20°C, the reaction time is 30min; the reaction temperature is 65°C, the reaction time is 30min;
所述接头连接中运行的程序：反应温度为20℃；反应时间为25min；The program run during the joint connection: reaction temperature is 20°C; reaction time is 25 minutes;
所述杂交捕获中运行的程序：反应温度为95℃，反应时间为30s；反应温度为65℃，反应时间为12h。The program run in the hybridization capture: reaction temperature is 95°C, reaction time is 30s; reaction temperature is 65°C, reaction time is 12h.
在一个实施方式中，所述PCR扩增运行的程序如下：
In one embodiment, the PCR amplification runs as follows:
在一个实施方式中，所述捕获PCR扩增运行的PCR程序为如下：
In one embodiment, the PCR program for the capture PCR amplification run is as follows:
与现有技术相比，本申请提供的一种自动化NGS文库制备分装试剂盒及建库方法至少具有以下有益效果：Compared with the existing technology, the automated NGS library preparation and packaging kit and library construction method provided by this application have at least the following beneficial effects:
本申请所述的试剂盒为自动化NGS文库制备试剂盒，采用预分装的试剂盒与自动化仪器配合进行文库制备，一键式自动化制备出文库，仪器操作稳定，重复性高；只需1人加入样本后全程无需人员参与，手动操作时间5分钟，同时夜间无人值守也可运行，提高了制备效率；此外，采用全封闭式卡盒，在密封空间内进行反应，实现文库制备零污染。The kit described in this application is an automated NGS library preparation kit. It uses pre-packaged kits and automated instruments to prepare libraries. It can automatically prepare libraries with one click. The instrument operation is stable and reproducible; only one person is needed. There is no need for personnel to participate in the whole process after adding the sample, and the manual operation time is 5 minutes. At the same time, it can be run unattended at night, which improves the preparation efficiency. In addition, a fully enclosed cartridge is used to perform the reaction in a sealed space, achieving zero contamination in library preparation.
附图说明Description of drawings
图1为本申请手动建库和仪器自动化建库上靶率比较图。Figure 1 is a comparison chart of target accuracy between manual library construction and instrument-automated library construction in this application.
图2为本申请手动建库和仪器自动化建库上靶率CV值比较。Figure 2 is a comparison of the CV values of the on-target rate between manual library construction and instrument-automated library construction in this application.
图3为本申请手动建库和仪器自动化建库均一性比较图。 Figure 3 is a comparison chart of the uniformity of manual library construction and instrument-automated library construction in this application.
图4为本申请手动建库和仪器自动化建库均一性CV值比较图。Figure 4 is a comparison chart of uniformity CV values between manual library construction and instrument-automated library construction in this application.
图5为本申请自动化建库试剂盒各组分孔位分布图。Figure 5 is a diagram showing the well position distribution of each component of the automated library construction kit of the present application.
具体实施方式Detailed ways
现有商业化建库试剂均为手动，与自动化相比，手动无需考虑体积大小，而自动化试剂首先就要考虑组分体积是否适配于自动化仪器，而日常使用的手动建库试剂盒，其组分较复杂、部分体积较小，无法适配到自动化建库过程中的。Existing commercial library construction reagents are all manual. Compared with automation, manual does not need to consider the volume. For automated reagents, you must first consider whether the component volume is suitable for automated instruments. However, manual library construction kits used daily have The components are more complex and the parts are smaller in volume, which cannot be adapted to the automated library construction process.
实施例1Example 1
本实施例提供一种自动化NGS文库制备分装试剂盒，其包括第一分装试剂组合和第二分装试剂组合以及第二分装试剂组合，其中，所述第一分装试剂组合包括第一热洗液、清洗液和洗脱液，所述第二分装试剂组合包括第二热洗液、常温洗液、建库试剂和捕获试剂；所述第三分装试剂组合包括磁珠试剂。This embodiment provides an automated NGS library preparation and packaging kit, which includes a first packaging reagent combination, a second packaging reagent combination, and a second packaging reagent combination, wherein the first packaging reagent combination includes a third packaging reagent combination. A hot washing liquid, cleaning liquid and eluent, the second packaged reagent combination includes a second hot washing liquid, a normal temperature washing liquid, a library building reagent and a capture reagent; the third packaged reagent combination includes a magnetic bead reagent .
所述试剂盒中第一分装试剂组合中的第一热洗液包括洗液1；所述清洗液包括乙醇和水；所述洗脱液包括水；The first hot washing liquid in the first packaged reagent combination in the kit includes washing liquid 1; the washing liquid includes ethanol and water; the eluent includes water;
所述第二分装试剂组合中的第二热洗液包括增强洗液；所述常温洗液包括磁珠清洗液、洗液1、洗液2和洗液3；所述建库试剂包括末端修复缓冲液、末端修复酶、连接缓冲液、接头、连接酶、PCR扩增引物和PCR反应液；所述捕获试剂包括封闭液、杂交反应液、磁珠重悬液、PCR扩增引物和PCR反应液；The second hot washing liquid in the second packaging reagent combination includes enhanced washing liquid; the normal temperature washing liquid includes magnetic bead cleaning liquid, washing liquid 1, washing liquid 2 and washing liquid 3; the library building reagent includes terminal Repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution; the capture reagent includes blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer and PCR The reaction solution;
所述第三分装试剂组合中的磁珠试剂包括Ampure XP beads、链霉亲和素磁珠。The magnetic bead reagents in the third packaging reagent combination include Ampure XP beads and streptavidin magnetic beads.
所述自动化NGS文库制备试剂盒采用提前将试剂预分装至如图5所示的卡盒底座上，根据不同组分性质以及卡盒底座孔容量、位置、形状等差异，将孔位划分为反应孔组合、热洗液孔组合、常温洗液孔组合、清洗液孔组合、洗脱液孔组合、建库试剂孔组合、捕获试剂孔组合和磁珠试剂孔组合等不同区域，试剂孔位分布和装量如表1所示。The automated NGS library preparation kit uses pre-packaged reagents onto the cartridge base as shown in Figure 5, and divides the hole locations into Reaction well combination, hot washing solution well combination, room temperature washing solution well combination, cleaning solution well combination, eluent well combination, library building reagent well combination, capture reagent well combination and magnetic bead reagent well combination and other different areas, reagent well positions The distribution and loading are shown in Table 1.
表1


Table 1


在使用自动化NGS文库制备分装试剂盒自动建库时，所需要的第二分装试剂组合的试剂组分及其终浓度、最佳浓度以及第二分装试剂组合中的各试剂的体积比如下表2所示。When using the automated NGS library preparation aliquot kit to automatically build a library, the required reagent components of the second aliquot reagent set, their final concentration, optimal concentration, and the volume of each reagent in the second aliquot reagent set are as follows: As shown in Table 2 below.
表2



Table 2



除上述自配试剂外，第三分装试剂组合中的Blocker Oligo TS Mix(IDT公 司)、KAPA HiFi hotstart ready mix(KAPA Biosysterms)、链霉亲和素磁珠(Invitrogen)、AMpure XP Beads(贝克曼公司)、无水乙醇(国药集团)和水(Invitrogen)等均购自商业化试剂。In addition to the above self-prepared reagents, Blocker Oligo TS Mix (IDT) in the third reagent set Company), KAPA HiFi hotstart ready mix (KAPA Biosysterms), streptavidin magnetic beads (Invitrogen), AMpure XP Beads (Beckman Company), absolute ethanol (Sinopharm Group) and water (Invitrogen) were purchased from commercial chemical reagents.
实施例2Example 2
本实施例采用上述自动化建库试剂盒分装至卡盒底座上与自动化建库设备配合自动化建库仪器使用，具体仪器内部运行流程如下：本实施例还提供一种应用自动化NGS文库制备分装试剂盒的自动建库方法，其包括如下步骤：This embodiment adopts the above-mentioned automated library construction kit to be packed into the cartridge base and used with the automated library construction equipment in conjunction with the automated library construction instrument. The specific internal operation process of the instrument is as follows: This embodiment also provides an application of automated NGS library preparation and packaging The automatic library construction method of the test kit includes the following steps:
S10.试剂和样本准备S10. Reagents and sample preparation
将自动化建库试剂盒取出，平衡至室温后振荡混匀，取50μL片段化后的样本并加入到反应孔1。将自动化建库试剂盒放入离心机中离心，转速为1000rpm/min，然后将自动化建库试剂盒放入配套一键式自动化建库仪中，选择对应程序后开始运行，仪器具体运行流程如下。Take out the automated library construction kit, equilibrate to room temperature, shake and mix well, take 50 μL of the fragmented sample and add it to reaction well 1. Put the automated library construction kit into the centrifuge and centrifuge at a speed of 1000rpm/min. Then put the automated library construction kit into the matching one-click automated library construction instrument. Select the corresponding program and start running. The specific operation process of the instrument is as follows .
S20.末端修复S20. End repair
取28号孔中末端修复缓冲液10μL至含有4.3μL末端修复酶的29号孔中，吹打混匀；Add 10 μL of the end repair buffer in well No. 28 to well No. 29 containing 4.3 μL of end repair enzyme, pipet and mix;
取29号孔中末端修复混合液10μL至加入了50μL打断后样本的反应孔1中，吹打混匀；Take 10 μL of the end repair mixture in well 29 and add 50 μL of the interrupted sample to reaction well 1, and pipet to mix;
运行如下末端修复程序：
Run the following terminal fix:
S30.接头连接：末端修复程序运行结束后，取30号孔中35μL连接缓冲液至含有5μL的接头的31号孔中，吹打混匀后，取40μL至反应孔1中；S30. Adapter connection: After the end repair program is completed, take 35 μL of the connection buffer in well 30 and add 5 μL of adapter to well 31. After pipetting and mixing, take 40 μL into reaction well 1;
取32号孔中连接酶10μL至反应孔1中，吹打混匀；Add 10 μL of ligase from well 32 to reaction well 1, mix by pipetting;
运行如下连接程序：
Run the following connection program:
S40.连接产物纯化S40. Purification of ligation products
连接程序运行结束后，从44号孔取99μL吹打混匀的Ampure XP beads至 1号反应孔，并吹打混匀；After the connection program is completed, take 99 μL of Ampure XP beads from well 44 and pipet to mix them. Reaction well No. 1, and pipet to mix;
使用自动化建库仪器对样本进行纯化和洗涤(使用12号孔中80％乙醇)；Use automated library construction equipment to purify and wash the samples (use 80% ethanol in well 12);
S50.PCR扩增S50.PCR amplification
取33号孔中25μL PCR扩增引物至1号孔中洗脱晾干的磁珠，并取洗脱上清液至反应孔2，同时清洗反应孔1(使用9号孔中的水)；Take 25 μL of PCR amplification primer from well No. 33 to elute the dried magnetic beads in well No. 1, and take the elution supernatant to reaction well 2, and wash reaction well 1 at the same time (use the water in well 9);
取34号孔中25μL PCR反应液至反应孔2，吹打混匀；Take 25 μL of PCR reaction solution from well 34 to reaction well 2, pipet and mix;
运行PCR扩增程序：
Run the PCR amplification program:
S60.PCR产物纯化S60. PCR product purification
PCR运行结束后，从45号孔取45μL吹打混匀的Ampure XP beads至反应孔2，吹打混匀；After the PCR run is completed, take 45 μL of Ampure XP beads from well 45 and mix by pipetting, to reaction well 2, and mix by pipetting;
使用自动化建库仪器对样本进行纯化和洗涤(使用12号孔中80％乙醇)；Use automated library construction equipment to purify and wash the samples (use 80% ethanol in well 12);
从16号孔取40μL水至反应孔2，吹打混匀。Take 40 μL of water from well 16 to reaction well 2, pipet and mix.
S70.文库封闭与浓缩S70. Library blocking and concentration
从反应孔2取20μL洗脱液上清至反应孔1，并清洗反应孔2(用9号孔中的水)；Take 20 μL of the eluent supernatant from reaction well 2 to reaction well 1, and wash reaction well 2 (use water in well 9);
从37号孔取20μL封闭液至反应孔1，吹打混匀；Take 20 μL of blocking solution from well 37 to reaction well 1, pipet and mix;
然后从45号孔取72μL吹打混匀的Ampure XP beads至反应孔1，吹打混匀；Then take 72μL of Ampure XP beads from well 45 and pipet to mix evenly, to reaction well 1, pipet to mix;
孵育后使用自动化建库仪器对样本进行纯化和洗涤(使用13号孔中80％乙醇)。After incubation, the sample was purified and washed using an automated library construction instrument (using 80% ethanol in well 13).
S80.杂交捕获S80. Hybrid capture
从38号孔取17μL杂交反应液至反应孔1，吹打混匀；Take 17 μL of hybridization reaction solution from well 38 to reaction well 1, pipet and mix;
孵育后取全部洗脱上清液至2号反应孔中，运行如下杂交程序：

After incubation, transfer all the elution supernatant to reaction well No. 2, and run the following hybridization program:

S90.洗涤富集S90. Washing and enrichment
从46号孔取50μL吹打混匀的链霉亲和素磁珠至11号孔，并使用22号孔磁珠清洗液，清洗链霉亲和素磁珠三次；Take 50 μL of streptavidin magnetic beads from well No. 46 and mix well by pipetting to well No. 11, and use the magnetic bead cleaning solution in well No. 22 to wash the streptavidin magnetic beads three times;
取40号孔中17μL磁珠重悬液加入11号孔重悬链霉亲和素磁珠；Take 17 μL of the magnetic bead resuspension solution in well No. 40 and add the resuspended streptavidin magnetic beads in well No. 11;
待杂交程序结束后，将11号孔中的重悬链霉亲和素磁珠全部加入到2号反应孔的杂交液中，吹打混匀，65℃条件下继续孵育45min，期间每15min吹打混匀一次；After the hybridization procedure is completed, add all the resuspended streptavidin magnetic beads in well No. 11 to the hybridization solution in reaction well No. 2, mix by pipetting, and continue to incubate at 65°C for 45 minutes, pipetting and mixing every 15 minutes. Even once;
孵育结束后，使用10号孔中100μL预热65℃的洗液1清洗一次，然后使用19、20号孔中150μL预热65℃的增强洗液清洗两次；之后分别使用23、24和25号孔中150μL洗液1、洗液2和洗液3清洗一次；After the incubation, wash once with 100 μL of Wash Solution 1 preheated to 65°C in well 10, then wash twice with 150 μL of Enhanced Wash Solution preheated to 65°C in wells 19 and 20; then wash once with 150 μL of Wash Solution 1, Wash Solution 2, and Wash Solution 3 in wells 23, 24, and 25, respectively;
捕获PCR扩增：Capture PCR amplification:
取42号孔中25μL PCR扩增引物和43号孔25μL PCR反应液至反应孔2，吹打混匀，运行PCR扩增程序：
Take 25 μL of PCR amplification primer from well 42 and 25 μL of PCR reaction solution from well 43 to reaction well 2, mix by pipetting, and run the PCR amplification program:
运行结束后，PCR产物用47号孔中75μL AMpure XP Beads(1.5X)进行纯化(使用15号孔中80％乙醇)，取17号孔中30μL水洗脱文库。After the run, the PCR product was purified with 75 μL AMpure XP Beads (1.5X) in well 47 (using 80% ethanol in well 15), and 30 μL water was taken from well 17 to elute the library.
实施例3Example 3
对比实验：按照第二分装试剂组合中的组分采用手动建库捕获流程如下：Comparative experiment: According to the components in the second aliquot of reagent combination, the manual library construction and capture process is as follows:
步骤1：末端修复Step 1: End Repair
在PCR管中加入50μL片段化后DNA，根据以下体系配制反应液；Add 50 μL of fragmented DNA to the PCR tube and prepare the reaction solution according to the following system;
末端修复组分配方：

End repair component recipe:

震荡混匀，短暂离心，置于PCR仪上，在PCR仪上运行末端修复程序：Vortex and mix, centrifuge briefly, place on the PCR machine, and run the end repair program on the PCR machine:
末端修复程序：
End fix:
步骤2：连接Step 2: Connect
程序运行结束后，按照下表加入连接反应液，接头连接组分配方：
After the program is finished, add the connection reaction solution and connector connection component formula according to the following table:
加入连接混合液后，涡旋混匀，短暂离心后在PCR仪上运行连接程序：After adding the ligation mixture, vortex to mix, centrifuge briefly and then run the ligation program on the PCR machine:
接头连接程序：
Joint connection procedure:
步骤3：连接反应产物纯化Step 3: Purification of ligation reaction products
反应结束后取出PCR反应管，加入99μL(0.9X)已室温平衡30min的XP 磁珠对连接反应产物进行纯化；并使用21μL无酶水进行洗脱，取20μL上清至新的PCR管中。After the reaction, take out the PCR reaction tube and add 99 μL (0.9X) of XP that has been equilibrated at room temperature for 30 minutes. Use magnetic beads to purify the ligation reaction product; use 21 μL of enzyme-free water for elution, and take 20 μL of the supernatant into a new PCR tube.
步骤4：PCR扩增Step 4: PCR Amplification
按照下表配制PCR反应体系，加入PCR管充分混匀，短暂离心，此时管内总体积为50μL。
Prepare the PCR reaction system according to the table below, add it to the PCR tube, mix thoroughly, and centrifuge briefly. At this time, the total volume in the tube is 50 μL.
在PCR仪上运行Pre-PCR程序：Run the Pre-PCR program on the PCR machine:
Pre-PCR程序：
Pre-PCR procedure:
步骤5：PCR产物回收Step 5: PCR product recovery
PCR结束后，取出PCR反应管，加入45μL(0.9X)AMpure Beads进行纯化，加入41μL无酶水洗脱，并取40μL上清至新的1.5mL离心管中；After the PCR is completed, take out the PCR reaction tube, add 45μL (0.9X) AMpure Beads for purification, add 41μL enzyme-free water for elution, and take 40μL supernatant into a new 1.5mL centrifuge tube;
对文库进行Qubit定量并记录文库浓度。文库制备完成后可用于捕获。Quantify the library using Qubit and record the library concentration. Once library preparation is complete, it is ready for capture.
步骤6：文库封闭与浓缩Step 6: Library blocking and concentration
在500ng待捕获文库中加入6μL封闭液，混匀后用真空浓缩仪抽干。Add 6 μL of blocking solution to 500 ng of the library to be captured, mix well, and drain using a vacuum concentrator.
步骤7：文库杂交 Step 7: Library Hybridization
取17μL杂交反应液加入到上述抽干管中，混匀后静置10min，置于PCR仪上运行杂交程序；Add 17 μL of the hybridization reaction solution into the above-mentioned drain tube, mix well, let it stand for 10 minutes, and place it on the PCR machine to run the hybridization program;
杂交程序：
Hybridization procedure:
步骤8：洗涤富集Step 8: Wash Enrichment
(1)取50μL平衡至室温的链霉亲和素磁珠，加入100μL磁珠清洗液，混匀后置于磁力架，磁珠完全分离后，去掉上清，同样的方式再清洗所述磁珠两次。(1) Take 50 μL of streptavidin magnetic beads that have been equilibrated to room temperature, add 100 μL of magnetic bead cleaning solution, mix well and place on a magnetic stand. After the magnetic beads are completely separated, remove the supernatant and clean the magnetic beads in the same way. beads twice.
(2)用17μL磁珠重悬液重悬所述磁珠，待杂交程序结束后，将重悬磁珠全部加入杂交结束后的杂交液中，涡旋震荡混匀然后放回PCR仪上在65℃条件下继续孵育45min，期间每15min涡旋混匀一次。(2) Resuspend the magnetic beads with 17 μL of magnetic bead resuspension solution. After the hybridization procedure is completed, add all the resuspended magnetic beads into the hybridization solution after hybridization, vortex to mix and then put them back on the PCR machine. Continue to incubate at 65°C for 45 minutes, vortexing every 15 minutes.
(3)孵育结束后，将100μL预热65℃的洗液1加入到上述杂交液中，混匀后置于磁力架。所述磁珠完全分离后，立即去掉上清，然后加入150μL预热65℃的增强洗液，混匀后置于磁力架，所述磁珠完全分离，立即去掉上清，同样的方式用150μL预热65℃的所述增强洗液重复清洗一次。(3) After the incubation, add 100 μL of washing solution 1 preheated at 65°C to the above hybridization solution, mix well, and place on the magnetic stand. After the magnetic beads are completely separated, immediately remove the supernatant, then add 150 μL of enhanced wash solution preheated to 65°C, mix well, and place on a magnetic stand. When the magnetic beads are completely separated, immediately remove the supernatant, and use 150 μL of the enhanced wash solution in the same manner. Repeat washing once with the enhanced wash solution preheated to 65°C.
(4)加入室温保存的150μL洗液1，涡旋震荡30s，静置30s，2min后短暂离心，然后静置于磁力架上，所述磁珠完全分离后去掉上清，然后以同样的方式依次用室温保存的150μL的洗液2、150μL的洗液3清洗所述磁珠。(4) Add 150 μL of Washing Solution 1 stored at room temperature, vortex for 30 seconds, let stand for 30 seconds, centrifuge briefly after 2 minutes, and then place on a magnetic stand. After the magnetic beads are completely separated, remove the supernatant, and then proceed in the same way. The magnetic beads were washed sequentially with 150 μL of washing solution 2 and 150 μL of washing solution 3 stored at room temperature.
(5)加入21μL无酶水重悬所述磁珠，取20μL上清至新的PCR管中，然后加入25μL PCR反应液和5μL PCR扩增引物，混匀后PCR仪上运行Post-PCR程序：(5) Add 21 μL of enzyme-free water to resuspend the magnetic beads, take 20 μL of supernatant into a new PCR tube, then add 25 μL of PCR reaction solution and 5 μL of PCR amplification primer, mix and run the Post-PCR program on the PCR machine :
Post-PCR反应程序：

Post-PCR reaction procedure:

所述PCR产物用75μL AMpure XP Beads(1.5X)进行纯化，对捕获文库进行Qubit定量及片段大小分析。The PCR product was purified with 75 μL AMpure XP Beads (1.5X), and the captured library was subjected to Qubit quantification and fragment size analysis.
实施例4Example 4
手动建库和自动化建库比较Comparison between manual database construction and automated database construction
为了比较手动建库和自动化建库之间的准确性和稳定性，分别使用三个人员，三台仪器，对四个样本进行建库，并对测序结果进行分析，考察手动和仪器之间的差异。手动操作流程见实施例3。In order to compare the accuracy and stability between manual and automated library construction, three people and three instruments were used to construct libraries for four samples, and the sequencing results were analyzed to examine the differences between manual and automated library construction. difference. See Example 3 for the manual operation process.
本实施例提供一种自动化建库试剂盒与自动化建库仪配套使用，其操作流程如下：This embodiment provides an automated library construction kit for use in conjunction with an automated library construction instrument. The operation process is as follows:
将自动化建库试剂盒底座从-20±5℃冰箱中取出，平衡至室温后振荡混匀；将磁珠部分从2-8℃取出，使用震荡仪充分混匀后放入试剂底座中。取25μL打断后的样本并加入到1号反应孔，将自动化建库试剂盒放入离心机中离心，转速为1000rpm/min，然后将自动化建库试剂盒放入配套一键式自动化建库仪中，选择对应程序后开始运行。具体运行流程见实施例1.Take out the automated library construction kit base from the -20±5°C refrigerator, balance to room temperature and shake to mix; take out the magnetic bead part from 2-8°C, use a shaker to mix thoroughly and put it into the reagent base. Take 25 μL of the interrupted sample and add it to reaction well No. 1. Put the automated library construction kit into a centrifuge and centrifuge at a speed of 1000 rpm/min. Then put the automated library construction kit into the supporting one-click automated library construction. In the instrument, select the corresponding program and start running it. See Example 1 for the specific operating process.
仪器运行结束后，弹出卡盒，将2号反应孔中所有组分全部转移至新的1.5mL EP管中，做好标记，然后将EP管置于磁力架上，待溶液澄清后小心吸取上清至与新的样本收集管中，即为纯化好的文库，保存于-20±5℃。对捕获文库进行生信分析，上靶率分析结果如图1、图2所示，均一性分析结果如图3、图4所示。After the instrument is running, eject the cartridge, transfer all the components in reaction well No. 2 to a new 1.5mL EP tube, mark it, and then place the EP tube on the magnetic stand. After the solution is clarified, carefully absorb it. The purified library is then placed in a new sample collection tube and stored at -20±5°C. The capture library was subjected to bioinformatics analysis. The on-target rate analysis results are shown in Figures 1 and 2, and the homogeneity analysis results are shown in Figures 3 and 4.
从图1和图2中可以看出，不同人员手动建库上靶率有高有低，波动较大，上靶率CV值较仪器建库大，结果不如仪器建库稳定。从图3和图4中可以看出，无论仪器还是手动建库，均一性都较好，但是从稳定性方面来看，仪器建库均 一性CV值更小，更加稳定。相较于手动，仪器能把NGS文库制备做好很不容易，目前市面上商品化的手动试剂盒组分比本申请要复杂，组分更多，体积要更小，还没有自动化试剂盒，而且并非所有的试剂都是适合自动化建库的试剂。本申请对试剂盒进行预分装并对试剂体系进行调整，以使试剂盒更加适应仪器进行检测，且建库均一性CV值更小，更加稳定。As can be seen from Figures 1 and 2, the target-on-target rate of manual library construction by different personnel varies greatly, with large fluctuations. The CV value of the target-on-target rate is larger than that of instrument library construction, and the results are not as stable as those of instrument library construction. It can be seen from Figure 3 and Figure 4 that the uniformity is good regardless of whether the instrument or manual library construction is performed. However, from the perspective of stability, the instrument library construction is uniform. The one-dimensional CV value is smaller and more stable. Compared with manual preparation, it is not easy to prepare NGS libraries with instruments. The components of the commercial manual kits currently on the market are more complicated than this application, with more components and smaller volumes. There is no automated kit yet. And not all reagents are suitable for automated library construction. This application prepackages the kit and adjusts the reagent system so that the kit is more suitable for instrument detection, and the uniformity CV value of library construction is smaller and more stable.
应当说明的是，上述实施例均可根据需要自由组合。以上所述仅是本申请的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。 It should be noted that the above embodiments can be freely combined as needed. The above are only the preferred embodiments of the present application. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications can also be made. should be regarded as the scope of protection of this application.

Claims (10)

  1. 一种自动化NGS文库制备分装试剂盒，其包括：第一分装试剂组合和第二分装试剂组合以及第三分装试剂组合；An automated NGS library preparation and packaging kit, which includes: a first packaging reagent combination, a second packaging reagent combination, and a third packaging reagent combination;
    其中，所述第一分装试剂组合包括第一热洗液、清洗液和洗脱液；Wherein, the first packaged reagent combination includes a first hot washing liquid, a cleaning liquid and an eluent;
    所述第二分装试剂组合包括第二热洗液、常温洗液、建库试剂和捕获试剂；以及The second packaged reagent combination includes a second hot washing solution, a normal temperature washing solution, a library building reagent and a capture reagent; and
    所述第三分装试剂组合包括磁珠试剂。The third packaged reagent combination includes magnetic bead reagents.
  2. 根据权利要求1所述的自动化NGS文库制备分装试剂盒，其中，所述第一分装试剂组合中所述的第一热洗液包括洗液1；所述清洗液包括乙醇和水；所述洗脱液包括水；The automated NGS library preparation dispensing kit according to claim 1, wherein the first hot washing liquid in the first dispensing reagent combination includes washing liquid 1; the washing liquid includes ethanol and water; The eluent includes water;
    所述第二分装试剂组合中所述的第二热洗液包括增强洗液；所述常温洗液包括磁珠清洗液、洗液1、洗液2和洗液3；所述建库试剂包括末端修复缓冲液、末端修复酶、连接缓冲液、接头、连接酶、PCR扩增引物和PCR反应液；所述捕获试剂包括封闭液、杂交反应液、磁珠重悬液、PCR扩增引物和PCR反应液；以及The second hot washing liquid in the second packaging reagent combination includes enhanced washing liquid; the normal temperature washing liquid includes magnetic bead cleaning liquid, washing liquid 1, washing liquid 2 and washing liquid 3; the library building reagent Including end repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution; the capture reagent includes blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer and PCR reaction solution; and
    所述第三分装试剂组合中所述的磁珠试剂包括Ampure XP beads、链霉亲和素磁珠。The magnetic bead reagents in the third packaging reagent combination include Ampure XP beads and streptavidin magnetic beads.
  3. 根据权利要求2所述的自动化NGS文库制备分装试剂盒，其中，所述末端修复缓冲液包括：Tris-HCl、MgCl2、DTT、ATP、dATP、dCTP、dGTP和dTTP；The automated NGS library preparation and packaging kit according to claim 2, wherein the end repair buffer includes: Tris-HCl, MgCl2, DTT, ATP, dATP, dCTP, dGTP and dTTP;
    所述末端修复酶包括：T4多聚核苷酸激酶、T4 DNA Polymerase、Taq DNA聚合酶、Klenow片段和甘油；The end repair enzyme includes: T4 polynucleotide kinase, T4 DNA Polymerase, Taq DNA polymerase, Klenow fragment and glycerol;
    所述连接反应液包括：Tris-HCl、MgCl2、DTT、ATP、dNTP、PEG8000和水；The ligation reaction solution includes: Tris-HCl, MgCl 2 , DTT, ATP, dNTP, PEG8000 and water;
    所述接头包括adapter；The joint includes an adapter;
    所述连接酶包括T4连接酶；The ligase includes T4 ligase;
    所述PCR反应液包括KAPA HiFi hotstart ready mix；The PCR reaction solution includes KAPA HiFi hotstart ready mix;
    所述PCR扩增引物包括P5、P7 primer；The PCR amplification primers include P5 and P7 primer;
    所述封闭液包括Human Cot DNA和Blocker Oligo TS Mix；The blocking solution includes Human Cot DNA and Blocker Oligo TS Mix;
    所述杂交反应液包括柠檬酸钠、硫酸葡聚糖、甜菜碱、甲酰胺、四甲基氯化铵、NaCl、Probe、水、甘油和聚山梨酯；The hybridization reaction solution includes sodium citrate, dextran sulfate, betaine, formamide, tetramethylammonium chloride, NaCl, Probe, water, glycerin and polysorbate;
    所述磁珠清洗液包括NaCl、Tris-HCl、EDTA、聚山梨酯和水； The magnetic bead cleaning solution includes NaCl, Tris-HCl, EDTA, polysorbate and water;
    所述洗液1包括柠檬酸钠、NaCl和SDS；The lotion 1 includes sodium citrate, NaCl and SDS;
    所述增强洗液包括柠檬酸钠、NaCl和聚山梨酯；The enhanced lotion includes sodium citrate, NaCl and polysorbate;
    所述洗液2包括柠檬酸钠、NaCl和水；The lotion 2 includes sodium citrate, NaCl and water;
    所述洗液3包括柠檬酸钠、NaCl和水；以及The lotion 3 includes sodium citrate, NaCl and water; and
    所述磁珠重悬液包括柠檬酸钠、硫酸葡聚糖、甜菜碱、甲酰胺、四甲基氯化铵、NaCl、水、甘油和聚山梨酯。The magnetic bead resuspension includes sodium citrate, dextran sulfate, betaine, formamide, tetramethylammonium chloride, NaCl, water, glycerol and polysorbate.
  4. 根据权利要求2所述的自动化NGS文库制备分装试剂盒，其中，所述热洗液中洗液1的体积为150μL；The automated NGS library preparation and packaging kit according to claim 2, wherein the volume of washing liquid 1 in the hot washing liquid is 150 μL;
    所述第一清洗液中乙醇等体积分为三份，且每份乙醇的体积为500μL，所述乙醇的浓度为80％；Equal volumes of ethanol in the first cleaning solution are divided into three parts, and the volume of each part of ethanol is 500 μL, and the concentration of ethanol is 80%;
    所述洗脱液中分装为两份水，所述两份水的体积分别为50μL和60μL；The eluent is divided into two parts of water, and the volumes of the two parts of water are 50 μL and 60 μL respectively;
    所述第二热洗液中的增强洗液分装成两份，体积均为200μL；The enhanced lotion in the second hot lotion is divided into two parts, each with a volume of 200 μL;
    所述常温洗液中的磁珠清洗液的体积为320μL，所述洗液1、洗液2、洗液3的体积均为175μL；The volume of the magnetic bead cleaning solution in the normal temperature washing solution is 320 μL, and the volumes of the washing solutions 1, 2, and 3 are all 175 μL;
    所述建库试剂中末端修复缓冲液、末端修复酶、连接缓冲液、接头、连接酶、PCR扩增引物以及PCR反应液的体积分别为10μL、4.3μL、40μL、5μL、15μL、30μL、30μL；The volumes of end repair buffer, end repair enzyme, ligation buffer, adapter, ligase, PCR amplification primer and PCR reaction solution in the library construction reagent are 10 μL, 4.3 μL, 40 μL, 5 μL, 15 μL, 30 μL, and 30 μL respectively. ;
    所述捕获试剂中的封闭液、杂交反应液、磁珠重悬液、PCR扩增引物、PCR反应液的体积分别为25μL、20μL、20μL、30μL、30μL；The volumes of blocking solution, hybridization reaction solution, magnetic bead resuspension solution, PCR amplification primer, and PCR reaction solution in the capture reagent are 25 μL, 20 μL, 20 μL, 30 μL, and 30 μL respectively;
    所述磁珠试剂中的Ampure XP beads分为三份，体积分别为120μL、150μL、100μL；所述链霉亲和素磁珠的体积为60μL。The Ampure XP beads in the magnetic bead reagent are divided into three parts, with volumes of 120 μL, 150 μL, and 100 μL respectively; the volume of the streptavidin magnetic beads is 60 μL.
  5. 根据权利要求2-4中任一项所述的自动化NGS文库制备分装试剂盒，其中，所述末端修复缓冲液中Tris-HCl的终浓度为150-250mM、MgCl2的终浓度为35-45mM、DTT的终浓度为35-45mM、ATP的终浓度为3.5-4.5mM、dATP终浓度为10-20mM、dCTP终浓度为1.0-2.0mM、dGTP的终浓度为1.0-2.0mM、dTTP的终浓度为1.0-2.0mM；The automated NGS library preparation and packaging kit according to any one of claims 2-4, wherein the final concentration of Tris-HCl in the end repair buffer is 150-250mM, and the final concentration of MgCl is 35-35 45mM, the final concentration of DTT is 35-45mM, the final concentration of ATP is 3.5-4.5mM, the final concentration of dATP is 10-20mM, the final concentration of dCTP is 1.0-2.0mM, the final concentration of dGTP is 1.0-2.0mM, the final concentration of dTTP The final concentration is 1.0-2.0mM;
    所述末端修复酶中T4多聚核苷酸激酶的终浓度为3.5-4.5U/μL、T4 DNA Polymerase的终浓度为0.8-1.2U/μL、Taq DNA聚合酶的终浓度为0.8-1.2U/μL、Klenow片段的终浓度为0.8-1.2U/μL、甘油的终浓度为5-30％；The final concentration of T4 polynucleotide kinase in the end repair enzyme is 3.5-4.5U/μL, the final concentration of T4 DNA Polymerase is 0.8-1.2U/μL, and the final concentration of Taq DNA polymerase is 0.8-1.2U. /μL, the final concentration of Klenow fragment is 0.8-1.2U/μL, and the final concentration of glycerol is 5-30%;
    所述连接反应液中Tris-HCl的终浓度为100-150nM、MgCl2的终浓度为 25-30mM、DTT的终浓度为25-30mM、ATP的终浓度为2-3mM、dNTP的终浓度为2-3mM、PEG8000的终浓度为20％-30％；The final concentration of Tris-HCl in the ligation reaction solution is 100-150nM, and the final concentration of MgCl2 is 25-30mM, the final concentration of DTT is 25-30mM, the final concentration of ATP is 2-3mM, the final concentration of dNTP is 2-3mM, the final concentration of PEG8000 is 20%-30%;
    所述接头中adapter的终浓度为0.5-0.8μM；The final concentration of the adapter in the adapter is 0.5-0.8 μM;
    所述连接酶中T4连接酶的终浓度为250-350U/μL；The final concentration of T4 ligase in the ligase is 250-350U/μL;
    所述PCR扩增引物中P5、P7primer的终浓度为5-15μM；The final concentration of P5 and P7 primer in the PCR amplification primer is 5-15 μM;
    所述封闭液中Human Cot DNA的终浓度为0.25-0.5mg/μL；The final concentration of Human Cot DNA in the blocking solution is 0.25-0.5mg/μL;
    所述杂交反应液中柠檬酸钠的终浓度为10-20mM、硫酸葡聚糖的终浓度为4.5-5.5％、甜菜碱的终浓度为0.8-1.2M、甲酰胺的终浓度为15-25％、四甲基氯化铵的终浓度为0.8-1.2M、NaCl的终浓度为135-165mM、Probe的终浓度为0.1-0.2nM、甘油的终浓度为5％-30％、聚山梨酯的终浓度为0.05％-0.5％；The final concentration of sodium citrate in the hybridization reaction solution is 10-20mM, the final concentration of dextran sulfate is 4.5-5.5%, the final concentration of betaine is 0.8-1.2M, and the final concentration of formamide is 15-25 %, the final concentration of tetramethylammonium chloride is 0.8-1.2M, the final concentration of NaCl is 135-165mM, the final concentration of Probe is 0.1-0.2nM, the final concentration of glycerol is 5%-30%, polysorbate The final concentration is 0.05%-0.5%;
    所述磁珠清洗液中NaCl的终浓度为0.8-1.2M、Tris-HCl的终浓度为5-15mM、EDTA的终浓度为0.8-1.2mM、聚山梨酯的终浓度为0.05％-0.5％；The final concentration of NaCl in the magnetic bead cleaning solution is 0.8-1.2M, the final concentration of Tris-HCl is 5-15mM, the final concentration of EDTA is 0.8-1.2mM, and the final concentration of polysorbate is 0.05%-0.5% ;
    所述洗液1中柠檬酸钠的终浓度为13.5-16.5mM、NaCl的终浓度为135-165mM、SDS的终浓度为0.05％-0.3％；The final concentration of sodium citrate in the lotion 1 is 13.5-16.5mM, the final concentration of NaCl is 135-165mM, and the final concentration of SDS is 0.05%-0.3%;
    所述增强洗液中柠檬酸钠的终浓度为13.5-16.5mM、NaCl的终浓度为50-100mM、吐温的终浓度为0.05％-0.5％；The final concentration of sodium citrate in the enhanced lotion is 13.5-16.5mM, the final concentration of NaCl is 50-100mM, and the final concentration of Tween is 0.05%-0.5%;
    所述洗液2中柠檬酸钠的终浓度为5-10mM、NaCl的终浓度为50-100mM；The final concentration of sodium citrate in the lotion 2 is 5-10mM, and the final concentration of NaCl is 50-100mM;
    所述洗液3中柠檬酸钠的终浓度为2-5mM、NaCl的终浓度为25-35mM；The final concentration of sodium citrate in the lotion 3 is 2-5mM, and the final concentration of NaCl is 25-35mM;
    所述磁珠重悬液中柠檬酸钠的终浓度为10-20mM、硫酸葡聚糖的终浓度为4.5-5.5％、甜菜碱的终浓度为0.8-1.2M、甲酰胺的终浓度为15-25％、四甲基氯化铵的终浓度为0.8-1.2M、NaCl的终浓度为135-165mM、甘油的终浓度为5％-30％、聚山梨酯的终浓度为0.05％-0.5％。The final concentration of sodium citrate in the magnetic bead resuspension is 10-20mM, the final concentration of dextran sulfate is 4.5-5.5%, the final concentration of betaine is 0.8-1.2M, and the final concentration of formamide is 15 -25%, the final concentration of tetramethylammonium chloride is 0.8-1.2M, the final concentration of NaCl is 135-165mM, the final concentration of glycerol is 5%-30%, the final concentration of polysorbate is 0.05%-0.5 %.
  6. 一种应用自动化NGS文库制备分装试剂盒的自动建库方法，其包括如下步骤：An automatic library construction method using an automated NGS library preparation and packaging kit, which includes the following steps:
    S10.试剂和样本准备：将自动化建库试剂盒取出，平衡至室温后振荡混匀，取50μL片段化后的样本至第一反应孔中并离心后备用，转速为1000rpm/min；S10. Reagents and sample preparation: Take out the automated library construction kit, balance it to room temperature, shake and mix well, take 50 μL of the fragmented sample into the first reaction well and centrifuge it for later use, with a rotation speed of 1000 rpm/min;
    S20.末端修复：取末端修复缓冲液至含有末端修复酶的末端修复酶孔中吹打混匀；取末端修复酶孔中的混合液10μL至离心样本中吹打混匀；S20. End repair: Take the end repair buffer and pipet and mix into the end repair enzyme well containing the end repair enzyme; take 10 μL of the mixture in the end repair enzyme well into the centrifuged sample and pipet and mix;
    S30.接头连接：将连接缓冲液孔中与接头吹打混匀后，取40μL至第一反应孔中；取连接酶10μL至第一反应孔中，吹打混匀； S30. Adapter connection: After pipetting and mixing the ligation buffer well and the adapter, take 40 μL into the first reaction well; take 10 μL of ligase into the first reaction well, pipe and mix;
    S40.连接产物纯化：连接程序运行结束后，取99μL吹打混匀的Ampure XP beads至第一反应孔中并吹打混匀；并进行样本纯化和80％乙醇洗涤；S40. Purification of the ligation product: After the ligation program is completed, take 99 μL of Ampure
    S50.PCR扩增：取25μL PCR扩增引物至第一反应孔中洗脱晾干的磁珠，并取洗脱上清液至第二反应孔，同时使用水清洗第一反应孔；取25μL PCR反应液至第二反应孔中，吹打混匀；S50. PCR amplification: Take 25 μL of PCR amplification primer into the first reaction well to elute the dried magnetic beads, and take the elution supernatant to the second reaction well. At the same time, use water to wash the first reaction well; take 25 μL Transfer the PCR reaction solution to the second reaction well and mix by pipetting;
    S60.PCR产物纯化：PCR运行结束后，取45μL吹打混匀的Ampure XP beads至第二反应孔吹打混匀；并对样本进行纯化和80％乙醇洗涤；取40μL水至第二反应孔，吹打混匀；S60. PCR product purification: After the PCR run, take 45 μL of Ampure mix;
    S70.文库封闭与浓缩：取20μL洗脱液上清至第一反应孔，并清洗第二反应孔并用第一水孔中的水；取20μL封闭液至第一反应孔，吹打混匀；然后取72μL吹打混匀的Ampure XP beads至第一反应孔吹打混匀；孵育后进行纯化和并使用80％乙醇进行洗涤；S70. Library blocking and concentration: Take 20 μL of eluent supernatant to the first reaction well, and wash the second reaction well with water from the first water well; take 20 μL of blocking solution to the first reaction well, pipet and mix; then Take 72μL of Ampure XP beads that are pipetted and mixed to the first reaction well and pipetted to mix; after incubation, purify and wash with 80% ethanol;
    S80.杂交捕获：从杂交反应液孔取17μL杂交反应液至第一反应孔，吹打混匀；孵育后取全部洗脱上清液至第二反应孔中，运行如下杂交程序；
    S80. Hybridization capture: Take 17 μL of hybridization reaction solution from the hybridization reaction solution well to the first reaction well, mix by pipetting; after incubation, take all the elution supernatant into the second reaction well, and run the following hybridization program;
    S90.洗涤富集。S90. Washing and enrichment.
  7. 根据权利要求6所述的应用自动化NGS文库制备分装试剂盒的自动建库方法，其中，所述S90中的洗涤富集包括如下步骤：The automatic library construction method using automated NGS library preparation and packaging kits according to claim 6, wherein the washing and enrichment in S90 includes the following steps:
    从链霉亲和素磁珠孔取50μL吹打混匀的链霉亲和素磁珠至待用孔，并使用磁珠清洗液孔中的磁珠清洗液清洗链霉亲和素磁珠三次；Take 50 μL of streptavidin magnetic beads from the streptavidin magnetic bead well and pipet and mix the streptavidin magnetic beads to the well to be used, and use the magnetic bead cleaning solution in the magnetic bead cleaning solution well to wash the streptavidin magnetic beads three times;
    取磁珠重悬液孔中17μL磁珠重悬液加入待用孔重悬链霉亲和素磁珠；Take 17 μL of the magnetic bead resuspension solution from the magnetic bead resuspension solution well and add it to the well to be used to resuspend the streptavidin magnetic beads;
    待杂交程序结束后，将待用孔中的重悬链霉亲和素磁珠全部加入到第二反应孔的杂交液中，吹打混匀，65℃条件下继续孵育45min，期间每15min吹打混匀一次；After the hybridization program is completed, add all the resuspended streptavidin magnetic beads in the well to be used to the hybridization solution in the second reaction well, mix by pipetting, and continue to incubate at 65°C for 45 minutes, pipetting and mixing every 15 minutes. Even once;
    孵育结束后，使用第一热洗液孔组合中洗液1孔的100μL预热65℃的洗液1清洗一次，然后使用第二热洗液孔组合中两个增强洗液孔中150μL预热65℃的增强洗液清洗两次；之后分别使用常温洗液孔组合中洗液1孔、洗液2孔以及洗液3孔中150μL洗液1、洗液2和洗液3清洗一次； After the incubation, wash once with 100 μL of wash solution 1 in well 1 of the first hot wash solution well combination that is preheated at 65°C, and then use 150 μL of preheated wash solution 1 in the two enhanced wash solution wells of the second hot wash solution well combination. Wash twice with enhanced washing solution at 65°C; then wash once with 150 μL washing liquid 1, washing liquid 2 and washing liquid 3 in washing liquid 1, washing liquid 2 and washing liquid 3 in the room temperature washing liquid well combination;
    捕获PCR扩增：取PCR扩增引物孔中25μL PCR扩增引物和PCR反应液孔中25μL PCR反应液至第二反应孔中，吹打混匀，运行PCR扩增程序：
    Capture PCR amplification: Take 25 μL of PCR amplification primer from the PCR amplification primer well and 25 μL of PCR reaction solution from the PCR reaction solution well to the second reaction well, mix by pipetting, and run the PCR amplification program:
    运行结束后，PCR产物用第三Ampure XP beads孔中75μL 1.5X的AMpure XP Beads和第三乙醇孔中80％乙醇进行纯化，取第三水孔中30μL水洗脱文库。After the run is completed, the PCR product is purified with 75 μL of 1.5X AMpure XP Beads in the third Ampure XP beads well and 80% ethanol in the third ethanol well, and 30 μL of water is taken from the third water well to elute the library.
  8. 根据权利要求6所述的应用自动化NGS文库制备分装试剂盒的自动建库方法，其中，所述S20中运行的程序：反应温度为20℃，反应时间为30min；反应温度为65℃，反应时间为30min；The automatic library construction method using automated NGS library preparation and packaging kits according to claim 6, wherein the program run in S20: the reaction temperature is 20°C, the reaction time is 30min; the reaction temperature is 65°C, the reaction Time is 30min;
    所述接头连接中运行的程序：反应温度为20℃；反应时间为25min；The program run during the joint connection: reaction temperature is 20°C; reaction time is 25min;
    所述杂交捕获运行的程序：反应温度为95℃，反应时间为30s；反应温度为65℃，反应时间为12h。The program of the hybridization capture operation: the reaction temperature is 95°C, the reaction time is 30s; the reaction temperature is 65°C, and the reaction time is 12h.
  9. 根据权利要求6所述的应用自动化NGS文库制备分装试剂盒的自动建库方法，其中，所述PCR扩增运行的程序如下：
    The automatic library construction method using automated NGS library preparation and packaging kits according to claim 6, wherein the procedure of the PCR amplification operation is as follows:
  10. 根据权利要求9所述的应用自动化NGS文库制备分装试剂盒的自动建库方法，其中，所述捕获PCR扩增运行的PCR程序如下：

    The automatic library construction method using automated NGS library preparation and packaging kits according to claim 9, wherein the PCR program for the capture PCR amplification run is as follows:

PCT/CN2023/118715 2022-09-15 2023-09-14 Automatic ngs library preparation and sub-packaging kit and library construction method WO2024056012A1 (en)

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