CN107557450A - A kind of reagent and method and its application of rapid build high-throughput sequencing library - Google Patents
A kind of reagent and method and its application of rapid build high-throughput sequencing library Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biology, and in particular to a kind of reagent and method and its application of rapid build high-throughput sequencing library.The high flux library construction reagent of the present invention includes nucleic acid releasing agent, and nucleic acid releasing agent effectively can discharge as buffer system, nucleic acid from sample, and is directly used in high-throughput sequencing library structure without purifying.Thus, it is possible to simplify purification step, storehouse efficiency is built in raising；And reaction terminating reagent both can effectively terminate swivel base reaction, and without purifying, moreover it is possible to be efficiently used for subsequent treatment, magnetic bead activating reagent can effective activated magnetic beads, the ability for making magnetic bead regain absorption nucleic acid fragment.High-throughput sequencing library construction method provided by the present invention, it is tubular type operation truly, quickly, step is easy, easily operated, as a result reproducible, while can avoid pollution and sample sample mixing between sample.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of reagent of rapid build high-throughput sequencing library
And method and its application.
Background technology
Nucleic acid is the carrier of life hereditary information, and the ways and means understood to hereditary information is not quite similar.In recent years
The second generation sequencing technologies of rise, it is the Basics of Biology such as gene expression and regulation the characteristics of with its high flux and high accuracy
Research provides significant data, while applies the methods of also be medical diagnosis on disease analysis and gene therapy and provide important support.
However, currently used high-flux sequence platform, is both needed to carry out sequencing library structure.And in library construction flow,
Both needed to carry out nucleic acid extraction purifying to sample, and needed high concentration high-purity nucleic acid-templated again, and all refer to isolate and purify.Therefore,
Whole process very complicated, intermediate product are excessive, time-consuming, cost is high.
The content of the invention
The problem of existing for prior art, the present invention provide reagent and the side of a kind of rapid build high-throughput sequencing library
Method and its application, it is therefore an objective to which easy rapidly progress nucleic acid extraction and library construction are integrated.
The reagent of the rapid build high-throughput sequencing library of the present invention includes nucleic acid releasing agent, reaction terminating liquid and magnetic bead and lived
Agent；
Wherein described nucleic acid releasing agent includes：5~500mM (Tris-HCl, 50~1000mM of pH7.0~9.0
The dodecyl sodium sulfate (SDS) of NaCl, 0.1~10mM EDTA, mass/volume than 0.01%~2%, mass/volume ratio
0.04%~3% glycine betaine and matter of lithium dodecyl sulfate (LLS), 0.01%~2% mass/volume than 0.01~2%
Amount/volume ratio 0.2-1.5% BSA, percent by volume therein is using sterile water volume as calculating benchmark；
Described reaction terminating liquid includes：0.1~100mM EDTA, mass/volume than the SDS for 0.1~5% and
15~500mM Tris-HCl of pH7.0~9.0；
Described magnetic bead activator includes 100~5000mM NH4OH, 25mM~500mM KCl, pH5.0-9.0.
The present invention also provides a kind of method of fast high-flux sequencing library structure, and this method follows the steps below：
(1) sample nucleic acid is discharged, and sample is handled using nucleic acid releasing agent, obtains the template of library construction；
(2) nucleic acid absorption magnetic bead is directly added into after step (1), the nucleic acid of release is enriched with；
(3) swivel base enzyme system is directly added into after step (2), fragmentation and special is carried out to the nucleic acid that is enriched on magnetic bead
Property joint is added to nucleic acid fragment both ends；
(4) reaction terminating liquid is directly added into after step (3), swivel base reaction is terminated, is prepared for follow-up amplified library；
(5) amplification in amplification system progress library is directly added into step (4)；
(6) magnetic bead activator is directly added into after step (5), the ability for making magnetic bead regain absorption nucleic acid, and meanwhile it is right
Library after amplification is purified, and product after purification is upper machine library.
The present invention high flux library constructing method, can be used for Roche, Illumina, ThermoFisher,
Pacific Biosciences, Hua Da gene, Oxford Nanopore Technologies, Hua Yinkang, big desert gene are contour
Flux microarray dataset.
Whole blood of the described sample type including the mankind, flesh tissue, paraffin-embedded tissue (FFPE), oral cavity come off carefully
Born of the same parents, cell line, excrement, urine and other body fluid, and other animal species, plant, bacterium and virus.
Compared with prior art, the features of the present invention and beneficial effect are：
The high flux library construction reagent of the present invention includes nucleic acid releasing agent, and nucleic acid releasing agent is as buffer system, core
Acid effectively can discharge from sample, and be directly used in high-throughput sequencing library structure without purifying.Thus, it is possible to simplify purifying
Storehouse efficiency is built in step, raising；And reaction terminating reagent both can effectively terminate swivel base reaction, and without purifying, moreover it is possible to effectively
Ground is used for subsequent treatment, magnetic bead activating reagent can effective activated magnetic beads, magnetic bead is regained the energy of absorption nucleic acid fragment
Power.
High-throughput sequencing library construction method provided by the present invention, it is tubular type operation truly, quickly, step
It is rapid easy, it is easily operated, it is as a result reproducible, while pollution and sample sample mixing between sample can be avoided.
The present invention high flux library constructing method, can be used for Roche, Illumina, ThermoFisher,
Pacific Biosciences, Hua Da gene, Oxford Nanopore Technologies, Hua Yinkang, big desert gene are contour
Flux microarray dataset.
Whole blood of the applicable sample type of the present invention including the mankind, flesh tissue, paraffin-embedded tissue (FFPE), oral cavity take off
Fall cell, cell line, excrement, urine and other body fluid.It can be used for other animal species, plant, bacterium and virus simultaneously
Deng.
The library that the present invention is built, both can directly go up machine sequencing, be used for the experiment such as subsequent captured but also as pre- library.
The nucleic acid releasing agent of the present invention, can complete effective release of nucleic acid, better than needed for traditional library constructing method
The sample of nucleic acid of High Purity, applicability is wide, easy to spread；
Present invention introduces transposase to build storehouse scheme, enormously simplify Library development flow；
The swivel base reaction terminating liquid of the present invention, can save the purification step after swivel base, greatly save experimental period, carry
Height builds storehouse efficiency；
The magnetic bead activator of the present invention, it is possible to achieve magnetic bead is reused, and Kucheng's sheet is built in attenuating, simplifies purification step；
The banking process of offer, it can be used for different microarray datasets, applicability is wide, easy to spread.
Brief description of the drawings
Fig. 1 is the flow chart that the present invention establishes rapid build high-throughput sequencing library；
Fig. 2 is the agarose gel electrophoresis of the embodiment of the present invention 2% detection library result；
Fig. 3 is 2% agarose gel electrophoresis detection library knot of the embodiment of the present invention 2 using conventional method structure library
Fruit.
Embodiment
Now by taking Illumina platforms as an example, the present invention is further described in conjunction with the embodiments.
Embodiment 1：
The present embodiment carries out human whole blood sample high flux library rapid build and sequencing analysis.
The reagent of rapid build high-throughput sequencing library includes nucleic acid releasing agent, reaction terminating liquid and magnetic bead in the present embodiment
Activator；
Wherein described nucleic acid releasing agent includes：250mM (Tris-HCl of pH7.0~9.0,50mM NaCl,
0.1EDTA, 0.01%SDS, 0.04%LLS, 0.01% glycine betaine and 0.2%BSA；
Described reaction terminating liquid includes：50mM EDTA, 0.1%SDS and the 15mM of pH7.0~9.0 Tris-HCl；
Described magnetic bead activator includes 5000mM NH4OH, 500mM KCl, pH5.0-9.0.
The method of the present embodiment fast high-flux sequencing library structure, as shown in figure 1, following the steps below：
(1) sample nucleic acid discharges：By a whole blood sample, centrifugation behaviour is carried out according to the sample preprocessing method of respective type
Make, after centrifugation, draw 200 μ L in 1.5mL centrifuge tubes, add 200 μ L nucleic acid releasing agents, be vortexed and mix, brief centrifugation,
5~30min is incubated at 80~100 DEG C, 5min is stored at room temperature, obtains the template of library construction；
(2) 50 μ L nucleic acid absorption magnetic beads are directly added into after step (1), the nucleic acid of release is enriched with, is vortexed and mixes,
After being stored at room temperature 10min, it is placed on magnetic frame, abandons supernatant, magnetic bead is gone in new 0.2mL PCR pipes, preserved with standby；
(3) swivel base enzyme system is directly added into after step (2), fragmentation and special is carried out to the nucleic acid that is enriched on magnetic bead
Property joint be added to nucleic acid fragment both ends, the swivel base reaction concussion that is vortexed mixes, and brief centrifugation, 55 DEG C of 15~30min of incubation, is incubated
5 μ L reaction terminating liquids are added after end, the concussion that is vortexed mixes, brief centrifugation；
Wherein swivel base multienzyme complex assembling is according to following table：
| Composition | Concentration/volume |
| Transposase | 8μL |
| Adaptor1-Core | 5μL |
| Adaptor2-Core | 5μL |
| Assemble buffer solution | 2μL |
| Cumulative volume | 20μL |
Finger tip flicks mixing, and brief centrifugation, 25 DEG C of incubation 90min, compound is in 4 DEG C of preservations；
Adaptor1-Core and Adaptor2-Core assembling：
Adaptor1 and Core and Adaptor2 and Core is completed to anneal in annealing buffer respectively, completes annealing
Adaptor1-Core and Adaptor2-Core afterwards is used for the assembling of transposase；
Wherein：Adaptor1, Adaptor2, Core design nucleotide sequence are as follows:
Adaptor1:
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'
Adaptor 2:
5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'
Core:
5’-P-CTGTCTCTTATACACATCT-3’；
According to following table, swivel base reaction system is prepared：
| Component | Volume |
| Swivel base buffer solution | 5μL |
| Swivel base multienzyme complex | 5μL |
| Step 1.2.1 nucleic acid (magnetic bead) | --- |
| ddH2O | Complement to 45 μ L |
(4) reaction terminating liquid is directly added into after step (3), swivel base reaction is terminated, is prepared for follow-up amplified library；
(5) amplification in amplification system progress library is directly added into step (4)；
The preparation of PCR reaction systems is carried out according to the following table：
| Component | Volume |
| 5xPCR reaction solutions | 10μL |
| dNTPs(2.5mM each) | 4μL |
| Universal Primer | 1μL |
| Index Primer | 1μL |
| Archaeal dna polymerase (5U/ μ L) | 0.2μL |
| Step 1.2.2 sample (magnetic bead) | 25μL |
| BSA(40ng/μL) | 5μL |
| ddH2O | Complement to 50 μ L |
Wherein：
Universal Primer：
5'-AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC-3'；
Index Primer：
5'-AAGCAGAAGACGGCATACGAGATATTAAGGGTCTCGTGGGCTCGG-3', wherein underlined sequences are
Index, for distinguishing different samples.
According to following table；Perform PCR amplification programs：
72℃30min；
95℃2min；
95℃30s；Then 62 DEG C of 30s, 6~15 circulations (period needed for different sample types is slightly different)
72℃3min；
4℃forever。
(6) 100 μ L magnetic bead activators are directly added into after step (5), 50 DEG C of incubation 30min, magnetic bead is regained suction
The ability of attached nucleic acid, reaction tube are placed on magnetic frame, are stood 3-5min, are abandoned supernatant, add the ethanol of 200 μ L 80% to be eluted, weight
It is multiple 2~3 times, eluted with 35 μ L ultra-pure waters, purified product is detected with 2% agarose gel electrophoresis.Product after purification is
For upper machine library, product is preserved in case upper machine in -20 DEG C.
QPCR quality inspections：
According to KAPA Library Quantifcation Kit (Kapabiosystems, Cat No.KK4854) explanation
Book is operated, and is detected using the real-time fluorescence quantitative PCR instrument of Roche Light Cycler 480, with reference to the standard in kit
Product carry out the absolute quantitation of library concentration.
Upper machine sequencing：
Library denaturation, dilution and the sequencing of 75bp both-ends are carried out according to NextSeq sequenators operating process.
Experimental result：
1. library electrophoresis detection analysis result
According to electrophoretic analysis result (Fig. 2), this electrophoretic analysis result is provided by precious bioengineering (Dalian) Co., Ltd, text
Storehouse master tape understands and is mainly distributed on 200bp or so, and band brightness becomes clear, consistent with expected results.
2.qPCR quantitative analysis results：
QPCR testing results are as follows：
| Sample | 1 |
| QPCR concentration (nM) | 127 |
Illustrate to build library text storehouse and reach confidential and seek concentration, available for examination with computer.
Machine Quality Control data on 3.
| Library title | Total sequence number | Compare genome sequence columns | Compare genome sequence ratio | Redundancy |
| 1 | 9011625 | 8948543 | 99.3% | 1.37% |
Embodiment 2
The present embodiment contrasts quick library constructing method and traditional library constructing method, comprises the following steps that：
(1) quick library constructing method：
Whole blood library rapid build is carried out according to method in embodiment 1；
(2) traditional library constructing method：
2.1 extracting genome DNA：
Quick library constructing method identical whole blood sample is taken, (Tiangeng is biochemical according to poba gene group DNA extraction kit
Scientific and technological (Beijing) Co., Ltd, article No.：DP318) specification carries out Whole Blood Genomic DNA extraction, comprises the following steps：
A) sample cracks；
B) DNA and adsorption column combine；
C) DNA cleanings (altogether 3 times)；
D) centrifugal drying of adsorption column；
E) DNA elution.
2.2 library construction：
Said according to KAPA LTP Library Preparation Kit (Kapabiosystems, Cat No.KK8232)
Bright book carries out library construction, comprises the following steps：
A) genomic DNA that step 2.2.1 has been extracted carries out Physical fragmentation；
B) purify；
C) end is repaired；
D) purify；
E) 3 ' ends add base dA；
F) purify；
G) joint connects；
H) purify；
I) PCR is expanded.
2.3qPCR quality inspections and the sequencing of upper machine：
QPCR Quality Controls are carried out to the library of flow 2.1 and 2.2 according to method in embodiment 1 and upper machine is sequenced.
2.4 experimental result：
2.4.1 library electrophoresis detection analysis result：
According to electrophoretic analysis result (Fig. 3), this electrophoretic analysis result is provided by precious bioengineering (Dalian) Co., Ltd, this
The quick banking process of invention and the library master tape difference in distribution that traditional banking process is obtained are little, and band brightness is without obvious poor
Not.
2.4.2qPCR library quantitative analysis results：
QPCR testing results are as follows：
| Sample | Fast run-up storehouse method | Tradition builds storehouse method |
| QPCR concentration (nM) | 132 | 114 |
As seen from the above table, the library of two kinds of banking process meets machine sequencing and required, fast run-up storehouse method slightly better than tradition
Method.
2.4.3 upper machine Quality Control data：
| Library title | Total sequence number | Compare genome sequence columns | Compare genome sequence ratio | Redundancy |
| Fast run-up storehouse method | 7374636 | 7330388 | 99.4% | 1.28% |
| Tradition builds storehouse method | 7358332 | 7292107 | 99.1% | 2.31% |
In terms of upper table comparing result, in the case of suitable data quantum of output, fast run-up storehouse method will be slightly better than tradition and build storehouse method.
2.4.4 fast run-up storehouse method is built storehouse method with tradition and contrasted：
| Banking process | Tradition builds storehouse method | Fast run-up storehouse method |
| Uncap number | 29 | 6 |
| Tube number | 10 | 1 |
| Experimental procedure | 103 | 18 |
| Test the used time (h) | 56 | 3.5 |
| Experimental cost | It is high | It is low |
In terms of upper table comparing result, fast run-up storehouse method had both simplified experimental implementation flow and step, reduced tube of uncapping again
Number, it is tubular type operation truly, while can also avoids pollution and sample sample mixing between sample, reduces because of Library development flow
Complexity triggers faulty operation risk, improves the repeatability and accuracy of experimental result.
Claims (4)
- A kind of 1. reagent of rapid build high-throughput sequencing library, it is characterised in that including nucleic acid releasing agent, reaction terminating liquid and Magnetic bead activator；Wherein described nucleic acid releasing agent includes：5~500mM (Tris-HCl of pH7.0~9.0,50~1000mM NaCl, The SDS of 0.1~10mM EDTA, mass/volume than 0.01%~2%, LLS of the mass/volume than 0.04%~3%, 0.01% The BSA of glycine betaine of~2% mass/volume than 0.01~2% and mass/volume than 0.2-1.5%, percent by volume therein Using sterile water volume as calculating benchmark；Described reaction terminating liquid includes：0.1~100mM EDTA, mass/volume than the SDS and pH7.0 for 0.1~5%~ 9.0 15~500mM Tris-HCl；Described magnetic bead activator includes 100~5000mM NH4OH, 25mM~500mM KCl, pH5.0-9.0.
- A kind of 2. method of fast high-flux sequencing library structure, it is characterised in that follow the steps below：(1) sample nucleic acid is discharged, and sample is handled using nucleic acid releasing agent, obtains the template of library construction；(2) nucleic acid absorption magnetic bead is directly added into after step (1), the nucleic acid of release is enriched with；(3) swivel base enzyme system is directly added into after step (2), fragmentation is carried out to the nucleic acid being enriched on magnetic bead and specificity connects Head is added to nucleic acid fragment both ends；(4) reaction terminating liquid is directly added into after step (3), swivel base reaction is terminated, is prepared for follow-up amplified library；(5) amplification in amplification system progress library is directly added into step (4)；(6) magnetic bead activator is directly added into after step (5), the ability for making magnetic bead regain absorption nucleic acid, while to amplification Library afterwards is purified, and product after purification is upper machine library.
- A kind of 3. method of fast high-flux sequencing library structure according to claim 2, it is characterised in that described sample This type includes whole blood, flesh tissue, FFPE, mouth desquamated cells, cell line, excrement, urine and the other body fluid of the mankind, And other animal species, plant, bacterium and virus.
- A kind of 4. application of the method for fast high-flux sequencing library structure, it is characterised in that available for Roche, Illumina, ThermoFisher, Pacific Biosciences, Hua Da gene, Oxford Nanopore Technologies, Hua Yinkang With big desert gene high-flux sequence platform.
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| CN108330546A (en) * | 2018-03-21 | 2018-07-27 | 东莞博奥木华基因科技有限公司 | A kind of library constructing method and reagent of simplification |
| CN108588200A (en) * | 2018-05-06 | 2018-09-28 | 湖南大地同年生物科技有限公司 | A kind of R-Loop high-throughput sequencing libraries construction method |
| CN108611346A (en) * | 2018-05-06 | 2018-10-02 | 湖南大地同年生物科技有限公司 | A kind of construction method in unicellular gene expression amount detection library |
| CN108753924A (en) * | 2018-06-08 | 2018-11-06 | 安徽鼎晶生物科技有限公司 | A kind of lung cancer high-throughput sequencing library and its construction method and application |
| CN109207572A (en) * | 2018-09-29 | 2019-01-15 | 苏州贝康医疗器械有限公司 | Unicellular high-throughput sequencing library construction method and its kit |
| CN109610011A (en) * | 2018-12-28 | 2019-04-12 | 厦门胜芨科技有限公司 | A kind of NanoDNA overlength is adjoint to build library kit and its application method |
| CN109778321A (en) * | 2019-02-28 | 2019-05-21 | 北京先声医学检验实验室有限公司 | A kind of banking process, kit and sequencing approach for macro gene order-checking |
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| CN110129414A (en) * | 2019-04-28 | 2019-08-16 | 安徽鼎晶生物科技有限公司 | A kind of BRCA high-throughput sequencing library and its construction method and application |
| CN111575348A (en) * | 2020-05-19 | 2020-08-25 | 广州微远基因科技有限公司 | Metagenome library, and library construction method and application thereof |
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| CN108330546B (en) * | 2018-03-21 | 2019-02-01 | 东莞博奥木华基因科技有限公司 | A kind of library constructing method and reagent of simplification |
| CN108588200A (en) * | 2018-05-06 | 2018-09-28 | 湖南大地同年生物科技有限公司 | A kind of R-Loop high-throughput sequencing libraries construction method |
| CN108611346A (en) * | 2018-05-06 | 2018-10-02 | 湖南大地同年生物科技有限公司 | A kind of construction method in unicellular gene expression amount detection library |
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| CN110129414A (en) * | 2019-04-28 | 2019-08-16 | 安徽鼎晶生物科技有限公司 | A kind of BRCA high-throughput sequencing library and its construction method and application |
| CN111575348A (en) * | 2020-05-19 | 2020-08-25 | 广州微远基因科技有限公司 | Metagenome library, and library construction method and application thereof |
| CN111575348B (en) * | 2020-05-19 | 2024-01-09 | 广州微远医疗器械有限公司 | Metagenomic library, library building method and application |
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Application publication date: 20180109 |