CN108330546B - A kind of library constructing method and reagent of simplification - Google Patents
A kind of library constructing method and reagent of simplification Download PDFInfo
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Abstract
The invention discloses a kind of library constructing method of simplification and reagents.Banking process and reagent of the invention can be realized fragmentation and end is repaired a step and completed, product after fragmentation and end reparation, which can choose, not to be purified, and entire banking process eliminates library enriching step, the sequencing library finally obtained, which is not only able to satisfy, is sequenced above confidential ask, more purification bring DNA loss is also reduced, and does not influence the testing result of sample.
Description
Technical field
The invention belongs to gene sequencing fields, are more particularly to the library constructing method and reagent of a kind of simplification.
Background technique
With the development of sequencing technologies, gene order-checking has become a kind of universal technological means.Previous genome
Sequencing mostly is carried out based on many cells level, and in recent years, such as circulating tumor cell, screening cell is unicellular before embryo implantation
Horizontal gene order-checking has become hot spot, and for these single celled gene order-checkings, existing sequencing approach is mainly pair
Unicellular carry out whole genome amplification meets the needs of high-flux sequence platform to improve template quantity, then obtains to amplification
Complete genome DNA carries out building library, and library is finally carried out high-flux sequence and analysis.
For building library step, the process of the prior art is main including the following steps: 1. to complete genome DNA into
Row fragmentation；2. carrying out end reparation to fragmentation products；3. repairing product to end carries out connector connection；4. butt joint connects
Product is enriched with；Above four steps are required to after each step carry out magnetic beads for purifying.
As it can be seen that existing build library complex steps, time-consuming on multiple magnetic beads for purifying, and fragmentation and end reparation step can not
Integration, connector connection product need further enrichment, and not only operating cost is high for these processes, and time-consuming, and more purification
The loss of DNA sample can inevitably be increased.Therefore, for the prior art, a kind of banking process of simplification is developed, especially
It, will be very with practical value suitable for the banking process of unicellular gene order-checking.
Summary of the invention
The purpose of the present invention is to provide a kind of library constructing method of simplification and related reagents.
The technical solution used in the present invention is:
A kind of library constructing method, includes the following steps:
(1) fragmentation is carried out to DNA to be measured by single step reaction and end is repaired, obtained fragmentation and end is repaired and produced
Object；
(2) fragmentation is repaired product progress connector with end to connect, purifies, obtain sequencing library, is measured for high pass
Sequence；
Wherein, it before fragmentation is connected with end reparation product progress connector, can carry out purifying or not purifying.
Preferred as the above method, the step of single step reaction includes: by DNA to be measured and reaction solution A, enzyme mixation
B is reacted, wherein the reaction solution A includes following final concentration substance in the system of single step reaction: 30~50mM buffering is molten
Liquid, 15~25mM MgCl2, 4~6mM DTT, 0.15%~0.25% (V/V) Triton X-100, the sub- essence of 0.5~1.5mM
Amine, 0.5~1.5mM ATP, 0.3~0.7mM dATP, 0.3~0.7mM dGTP, 0.3~0.7mM dCTP, 0.3~0.7mM
dTTP；Enzyme mixation B includes fragmentation enzyme and end repair enzyme.
Preferred as the above method, in reaction solution A, buffer solution is selected from Tris-HCl, phosphate buffer solution, buffering
PH value of solution is 7.5~8.5.
It is preferred as the above method, the reaction condition of the single step reaction are as follows: (3~5) DEG C reaction (50~70) s；(30
~35) DEG C reaction (15~25) min；(60~70) DEG C reaction (25~35) min.
Preferred as the above method, the purifying is magnetic beads for purifying.
Preferred as the above method, the DNA to be measured is the full genome for directly extracting or obtaining through whole genome amplification
Group DNA.
Preferred as the above method, the high-flux sequence includes proton sequencing.
The system of reaction solution A, the reaction solution A in single step reaction of single step reaction is repaired for DNA fragmentation and end
In include following final concentration substance: 30~50mM buffer solution, 15~25mM MgCl2, 4~6mM DTT, 0.15%~
0.25% (V/V) Triton X-100,0.5~1.5mM spermidine, 0.5~1.5mM ATP, 0.3~0.7mM dATP, 0.3~
0.7mM dGTP, 0.3~0.7mM dCTP, 0.3~0.7mM dTTP.
Preferred as above-mentioned reaction solution A, buffer solution is selected from Tris-HCl, phosphate buffer solution, buffer solution pH
It is 7.5~8.5.
Application of the above-mentioned reaction solution A in high-throughput sequencing library building.
The beneficial effects of the present invention are:
Banking process and reagent of the invention can be realized fragmentation and end is repaired a step and completed, and fragmentation and end are repaired
Product after multiple, which can choose, not to be purified, and entire banking process eliminates library enriching step, the sequencing text finally obtained
Library be not only able to satisfy sequencing it is upper it is confidential ask, also reduce more purification bring DNA loss, and do not influence the detection knot of sample
Fruit.
Detailed description of the invention
Fig. 1 is 2100 maps of the sequencing library that embodiment 1 obtains；
Fig. 2 is 2100 maps of the sequencing library that comparative example 1 obtains.
Specific embodiment
On the basis of existing banking process, inventor provides a kind of library constructing method of simplification, includes the following steps:
(1) fragmentation is carried out to DNA to be measured by single step reaction and end is repaired, obtained fragmentation and end is repaired and produced
Object；
(2) fragmentation is repaired product progress connector with end to connect, purifies, obtain sequencing library, is measured for high pass
Sequence；
Wherein, it before fragmentation is connected with end reparation product progress connector, can carry out purifying or not purifying.
Preferred as the above method, the step of single step reaction includes: by DNA to be measured and reaction solution A, enzyme mixation
B is reacted, wherein the reaction solution A includes following final concentration substance in the system of single step reaction: 30~50mM buffering is molten
Liquid, 15~25mM MgCl2, 4~6mM DTT, 0.15%~0.25% (V/V) Triton X-100, the sub- essence of 0.5~1.5mM
Amine, 0.5~1.5mM ATP, 0.3~0.7mM dATP, 0.3~0.7mM dGTP, 0.3~0.7mM dCTP, 0.3~0.7mM
dTTP；Enzyme mixation B includes fragmentation enzyme and end repair enzyme.
Preferred as the above method, in reaction solution A, buffer solution is selected from Tris-HCl, phosphate buffer solution, buffering
PH value of solution is 7.5~8.5.
It is preferred as the above method, the reaction condition of the single step reaction are as follows: (3~5) DEG C reaction (50~70) s；(30
~35) DEG C reaction (15~25) min；(60~70) DEG C reaction (25~35) min.
Preferred as the above method, the purifying is magnetic beads for purifying.
Preferred as the above method, the DNA to be measured is the full genome for directly extracting or obtaining through whole genome amplification
Group DNA.
Preferred as the above method, the high-flux sequence includes proton sequencing.
Based on the above method, the present invention also provides the reaction solution A that single step reaction is repaired for DNA fragmentation and end, institutes
Reaction solution A is stated in the system of single step reaction comprising following final concentration substance: 30~50mM buffer solution, 15~25mM
MgCl2, 4~6mM DTT, 0.15%~0.25% (V/V) Triton X-100,0.5~1.5mM spermidine, 0.5~1.5mM
ATP, 0.3~0.7mM dATP, 0.3~0.7mM dGTP, 0.3~0.7mM dCTP, 0.3~0.7mM dTTP.
Preferred as above-mentioned reaction solution A, buffer solution is selected from Tris-HCl, phosphate buffer solution, buffer solution pH
It is 7.5~8.5.
Application of the above-mentioned reaction solution A in high-throughput sequencing library building.
The present invention is explained further below by way of specific embodiment.
Not specified experimental method is carried out with reference to conventional method in that art or reagent specification in embodiment, not especially
The reagent and instrument of explanation can be obtained by commercial channel, be not intended to limit the scope of the invention.What embodiment was related to
Cell to be measured is purchased from oriell institute, there is positive reference product, negative reference product, chimera reference material and dyeing respectively
The micro-deleted micro- duplicate sample of body.
Embodiment 1, a kind of library constructing method of simplification
A kind of library constructing method of simplification of the invention, detailed process are as follows:
(1) fragmentation and end are repaired
It is formulated for fragmentation and the reaction solution A (10X) of single step reaction is repaired in end, be formulated as follows:
Configure following system:
Wherein, enzyme mixation B includes fragmentation enzyme (NEB company) and end repair enzyme (Life company), and the two presses volume
It is mixed than 1:1.
It after mixing, is reacted as follows, reaction is placed on 4 DEG C of preservations:
(2) sequence measuring joints are connected
Connector connection is carried out to the product after previous step reaction, following reagent is added:
Wherein, P1 connector is the P1-Adapter being made of following two positive and negative sequences:
5'-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3'(SEQ ID NO:1),
3'-T*T*GGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA-5'(SEQ IDNO:2)。
It after mixing, is reacted as follows, reaction is placed on 4 DEG C of preservations:
(3) it purifies
Using magnetic beads for purifying: the XP magnetic beads for purifying 5min of 60 μ L of previous step reaction product, after placing 3min on magnetic frame
Liquid is sucked, with 500 μ L75% ethanol washing 2 times, residual liquid is blotted, dries, 20 μ L Low TE solution are added, is mixed, it is quiet
5min is set, drawing liquid is sequencing library.
Embodiment 2
A kind of library constructing method of simplification supplements " magnetic beads for purifying " step, other steps after 1 step of embodiment (1)
It is constant.Wherein, steps are as follows for the magnetic beads for purifying of supplement: the XP magnetic beads for purifying of the 36 μ L of product after fragmentation and end are repaired
5min sucks liquid after placing 3min on magnetic frame, with 500 μ L, 75% ethanol washing 2 times, blots residual liquid, dry, add
Enter 20 μ L Low TE solution, mix, stand 5min, drawing liquid is that fragmentation after purification repairs product with end, is used for
Connect sequence measuring joints.
Embodiment 3
A kind of library constructing method of simplification carries out the formula of reaction solution A (10X) excellent in 1 step of embodiment (1)
Choosing, each group of formula is as follows, other steps are constant:
Comparative example 1, conventional libraries construction method
A kind of conventional libraries construction method, includes the following steps:
(1) fragmentation of DNA: preparing following reaction system, after mixing, 37 DEG C of reaction 20min:
(2) magnetic beads for purifying is carried out to fragmentation products, and is dissolved with 40 μ L low TE；
(3) end is repaired, and prepares following reaction system, after mixing, reacts at room temperature 20min:
(4) product is repaired to end and carries out magnetic beads for purifying, and dissolved with 40 μ L low TE；
(5) connector connects: preparing following reaction system, after mixing, reacts at room temperature 30min, wherein the same embodiment of P1 connector
1:
(6) butt joint connection product carries out magnetic beads for purifying, and is dissolved with 19 μ L low TE；
(7) library is enriched with: prepare following reaction system:
It is executed by following response procedures, reaction is placed on 4 DEG C of preservations:
(8) magnetic beads for purifying, low TE dissolution are carried out to library enriched product.
Comparative example 2
Fragmentation in conventional banking process and end are repaired and simply merge into step progress by this comparative example, it may be assumed that will compare
The reagent that the step of example 1 (1) and step (3) are related to is mixed by dosage, and 20min is reacted at 37 DEG C, is deleted step (2), step
(4)~(7) are constant.
Test example 1
Using the banking process of Examples 1 to 3 and comparative example 1~2, whole genome amplification product is carried out to build library, wherein
Whole genome amplification, which is related to following existing steps, (can refer to SIGMA-ALDRICH GenomePlex single cell whole genome amplification
Kit).
(1) cell cracking
2.0 μ L Proteinase Ks are mixed with 32.0 μ L buffers, fresh lysate liquid is configured to, takes 1 μ L fresh lysate liquid and 9 μ
L cell to be measured (supplying 9.0 μ L with PBS, contain 5 cells to be measured) is uniformly mixed, and reaction: 50 DEG C of reactions is carried out as follows
1h, subsequent 99 DEG C of reactions 4min, 4 DEG C of preservations after reaction.
(2) pre- amplification
2 μ L of buffer, 1 μ L of stabilizing solution are sequentially added in the product that the reaction of upper step terminates, 95 DEG C of reaction 2min after mixing,
4℃Forever；Then library is added and prepares 1 μ L of enzyme mixing, reaction condition are as follows: 16 DEG C of 20min, 24 DEG C of 20min, 37 DEG C
20min, 75 DEG C of 5min；4 DEG C of preservations after reaction.
(3) exponential amplification
Amplification 7.5 μ L of premixed liquid, 48.5 μ L of molecule rank water, archaeal dna polymerase 5 are added in the product that the reaction of upper step terminates
μ L is carried out amplification reaction, reaction condition after mixing well are as follows: 95 DEG C of 3min, 25 circulations (94 DEG C of 30s, 65 DEG C of 5min), reaction
4 DEG C of preservations afterwards.
(4) it purifies
Using magnetic beads for purifying: the XP magnetic beads for purifying 5min of 135 μ L of previous step reaction product, after placing 5min on magnetic frame
Liquid is sucked, with 500 μ L, 75% ethanol washing 2 times, residual liquid is blotted, dries, 50 μ L Low TE solution are added, is mixed,
5min is stood, drawing liquid is whole genome amplification product, is used for library construction.
It takes whole genome amplification product to carry out library construction, the sequencing library for building library acquisition is quantified, using fluorescence
Dye method carries out molecular number using absolute quantification method and quantifies, can also be by 2100 biological analyser of Agilent to library
Size and molecular number analyzed and detected；Upper machine is carried out using semiconductor sequencing system Ion Proton and its matched reagent
Sequencing, upper machine concentration recommends according to official, and each library as a result, equivalent is mixed, takes 20pM to carry out later according to quantitative
Upper machine sequencing.
Table 1, embodiment 1 and comparative example 2 obtain the qPCR concentration in library
It is carried out together as it can be seen from table 1 fragmentation is merely repaired step with end and is merged, can not achieve library
Building, it is necessary to creatively the reaction solution of single step reaction is repaired in exploitation for fragmentation and end, be just able to achieve fragmentation with
It repairs a step and completes in end.
Table 2, embodiment 1 and comparative example 1 obtain the qPCR concentration in library
From table 2 it can be seen that the conventional method that the method for the present invention and comparative example 1 that embodiment 1 represents represent, builds library acquisition
Library content it is suitable, and refer to Fig. 1 and Fig. 2, it can be seen that the library fragments size of the two is all very close, and library size is equal
Meet the requirement of Proton microarray dataset, therefore, the method for the present invention does not interfere with Library Quality.
Table 3, embodiment 1 and embodiment 2 obtain the qPCR concentration in library
From table 3 it can be seen that after banking process of the invention is repaired in fragmentation and end, product can be carried out purifying or
It does not purify, the product not purified will not influence library construction, and compare with the product purified, the concentration phase after building library
To higher, this may to reduce loss of product related with reaction step is reduced.
Influence of each group buffer formulation to sequencing library qPCR concentration in table 4, embodiment 3
It is improved from table 4, it can be seen that repairing the spermidine in the reaction solution A of single step reaction for fragmentation and end and having
The effect of library concentration.
Test example 2
Choosing positive reference product, negative reference product, chimera reference material and the micro- duplicate sample of microdeletion is
Example, by the banking process of embodiment 1 and comparative example 1, carries out building library, sequencing, analysis, wherein analysis includes: by sequencing result benefit
It is compared to human genome with bioinformatic analysis method with reference on map, comparison result is filtered and optimized, and carries out matter
Control obtains sample chromosomes letter by altered fragments result to guarantee that reading long sequence using reliable comparison carries out downstream analysis
Breath.
The testing result of table 5, positive reference product
The testing result of table 6, negative reference product
The testing result of table 7, chimera reference material
Table 8, micro-deleted micro- duplicate pattern detection result
Positive reference product, negative reference product, chimera reference material and micro-deleted micro- repetition has been selected to join in this experimental example
Examining product, conventionally (comparative example 1) and banking process of the present invention (embodiment 1) carries out library construction, sequencing and analysis respectively
The two (5~table of reference table 8) completely the same to the testing result of reference material as the result is shown.Illustrate the banking process that the present invention simplifies
The sequencing library of acquisition has no effect on subsequent upper machine sequencing and information analysis process.
To sum up, banking process of the invention and reagent, which can be realized fragmentation and end and repair a step, completes, fragmentation and
Product after the reparation of end, which can choose, not to be purified, and entire banking process eliminates library enriching step, finally obtains
Sequencing library be not only able to satisfy sequencing it is upper it is confidential ask, also reduce more purification bring DNA loss, and do not influence sample
Testing result.
Sequence table
<110>Dongguan Bo Aomuhua Gene Tech. Company Limited
<120>library constructing method and reagent of a kind of simplification
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213>manual splice ()
<400> 1
ccactacgcc tccgctttcc tctctatggg cagtcggtga t 41
<210> 2
<211> 43
<212> DNA
<213>manual splice ()
<220>
<221>thio-modification
<222> (42)..(43)
<400> 2
atcaccgact gcccatagag aggaaagcgg aggcgtagtg gtt 43
Claims (9)
1. a kind of library constructing method, includes the following steps:
(1) fragmentation is carried out to DNA to be measured by single step reaction and end is repaired, obtained fragmentation and product is repaired in end；
(2) fragmentation is repaired product progress connector with end to connect, purifies, obtain sequencing library, is used for high-flux sequence；
Wherein, it before fragmentation is connected with end reparation product progress connector, can carry out purifying or not purifying；
The step of single step reaction includes: to react DNA to be measured with reaction solution A, enzyme mixation B, wherein the reaction
Liquid A includes following final concentration substance: 30~50mM buffer solution, 15~25 mM MgCl in the system of single step reaction2, 4~
6 mM DTT, 0.15%~0.25% V/V Triton X-100,0.5~1.5mM spermidine, 0.5~1.5mM ATP, 0.3~
0.7 mM dATP, 0.3~0.7mM dGTP, 0.3~0.7mM dCTP, 0.3~0.7mM dTTP；Enzyme mixation B includes segment
Change enzyme and end repair enzyme.
2. according to the method described in claim 1, it is characterized by: buffer solution is selected from Tris-HCl, phosphoric acid in reaction solution A
Salt buffer solution, buffer solution pH are 7.5~8.5.
3. according to the method described in claim 1, it is characterized by: the reaction condition of the single step reaction are as follows: 3~5 DEG C of reactions
50~70s；30~35 DEG C of 15~25min of reaction；60~70 DEG C of 25~35min of reaction.
4. according to the method described in claim 1, it is characterized by: the purifying is magnetic beads for purifying.
5. according to the method described in claim 1, it is characterized by: the DNA to be measured is directly to extract or expand through full-length genome
Increase the complete genome DNA obtained.
6. according to the method described in claim 1, it is characterized by: the high-flux sequence includes proton sequencing.
7. repairing the reaction solution A of single step reaction for DNA fragmentation and end, it is characterised in that: the reaction solution A is anti-in a step
It include following final concentration substance: 30~50mM buffer solution, 15~25 mM MgCl in the system answered2, 4~6 mM DTT,
0.15%~0.25% V/V Triton X-100,0.5~1.5mM spermidine, 0.5~1.5mM ATP, 0.3~0.7 mM
DATP, 0.3~0.7mM dGTP, 0.3~0.7mM dCTP, 0.3~0.7mM dTTP.
8. reaction solution A according to claim 7, it is characterised in that: it is molten that buffer solution is selected from Tris-HCl, phosphate-buffered
Liquid, buffer solution pH are 7.5~8.5.
9. application of the reaction solution A described in claim 7 or 8 in high-throughput sequencing library building.
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| CN111020018B (en) * | 2019-11-28 | 2021-09-14 | 天津金匙医学科技有限公司 | Macrogenomics-based pathogenic microorganism detection method and kit |
| CN111394799A (en) * | 2020-03-11 | 2020-07-10 | 广州赛哲生物科技股份有限公司 | Method for constructing meningitis pathogen metagenome second-generation sequencing library and kit thereof |
| CN111500704B (en) * | 2020-04-28 | 2023-10-27 | 广州市金圻睿生物科技有限责任公司 | Kit and method for detecting human fetal chromosome aneuploidy |
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