CN110241462A - A kind of method and apparatus of two generations sequencing library targeting fast enriching - Google Patents
A kind of method and apparatus of two generations sequencing library targeting fast enriching Download PDFInfo
- Publication number
- CN110241462A CN110241462A CN201910568971.9A CN201910568971A CN110241462A CN 110241462 A CN110241462 A CN 110241462A CN 201910568971 A CN201910568971 A CN 201910568971A CN 110241462 A CN110241462 A CN 110241462A
- Authority
- CN
- China
- Prior art keywords
- primer
- target gene
- specific primer
- enrichment
- specific
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Abstract
This application discloses a kind of method and apparatus of two generation sequencing libraries targeting fast enriching.The two generation sequencing libraries of the application target fast enriching method, including primer hybridization and extension step, are annealed, are extended to DNA sample using primer mixture；Primer mixture designs a specific primer at interval of 50-200bp on target gene by forming for the specific primer of target gene design, and 5 ' ends of specific primer have biotin modification；Beads enrichment and washing step obtain target gene including capturing using the magnetic bead of Streptavidin modification to extension products.The present processes extend target gene using specific primer, replace long probe, at low cost, high-efficient, shorten the enrichment reaction time.Specific primer is spaced setting on target gene, and relatively few number of primer can be used and cover longer target gene region, compared to probe hybrid method, the application method is simpler, easy to operate.
Description
Technical field
This application involves high throughput sequencing technologies fields, more particularly to a kind of two generation sequencing libraries targeting fast enriching
Method and apparatus.
Background technique
High throughput sequencing technologies are also known as two generation sequencing technologies (Next Generation Sequencing, NGS), and are recognized
Traditional Sanger Capillary Electrophoresis order-checking method to be first generation sequencing technologies is compared, and two generation sequencing technologies are with lower cost
More high-throughput data are provided, and realize large-scale genome research, single operation data amount is big, can be simultaneously to millions of
A DNA molecular implements sequencing.With the progress of contemporary science and technology, two generation microarray datasets are gradually introduced into exist including oncology
In interior various fields, such as clinical diagnosis, agriculture genomics and medical jurisprudence.
The cost of genome sequencing is very high, the complicated time-consuming of operating process.Specifically in the application in a certain field, technology people
Member usually requires that interested target gene selective enrichment is only sequenced and is analyzed to interested gene region,
Such as full exon sequencing, to achieve the purpose that reduce cost and reduce data analysis workload.
The targeting enrichment method of sequencing of most widely used two generation at present is probe hybrid capture, and specific embodiment is to treat
It surveys DNA and carries out library construction, then carry out hybrid capture using DNA probe or rna probe.Probe is the key component of hybridization,
It is simultaneously also the major part of cost.When probe hybrid method designs probe, in order to ensure the complete capture to target gene, usually
It needs to carry out all standing design to target gene, so often quantity is various for probe, and segment is longer.The numerous long probes of quantity
Not only synthesis cost is high, but also reduces the specificity of probe, causes capture time-consuming very long, it usually needs 12 hours to more than 70
The time of hour prepares the sample for sequencing, and takes more time to complete entire sequencing procedure.
Therefore, it needs to research and develop a kind of new quick and save the cost two generation sequencing library targeting beneficiation technologies, to meet
Increasingly developed high-flux sequence demand.
Summary of the invention
The purpose of the application is to provide a kind of method and apparatus of new two generation sequencing libraries targeting fast enriching.
To achieve the goals above, the application uses following technical scheme:
The one side of the application discloses a kind of method of two generation sequencing libraries targeting fast enriching, includes the following steps,
Primer hybridization and extension step, including annealed, extended to DNA sample using primer mixture；Primer mixture is by being directed to
The specific primer composition of target gene design, designs a specific primer at interval of 50-200bp on target gene, makes
Entire target gene can be covered by obtaining primer mixture, and 5 ' ends of specific primer have biotin modification；Beads enrichment
And washing step catches primer hybridization and the extension products for extending step including the magnetic bead using Streptavidin modification
It obtains, and is separated the specific primer of biotin modification and corresponding target gene by magnetic bead, washing removes non-target base
Cause obtains the target gene of targeting enrichment.
It should be noted that the targeting enrichment method of the application, principle still using the specific primer being pre-designed,
Hybrid capture is carried out to target gene；But it is different from existing probe hybrid capture, the application design is that specificity is drawn
Object.On the one hand, the specific primer of the application is the design spaced apart in target gene region, i.e. interval 50-
200bp, for the target gene of same size, the quantity of specific primer used by the present processes is significantly smaller than probe
The number of probes of hybrid capture method；On the other hand, the application utilizes the extension of specific primer, can be using shorter special
Property primer capture longer targeting regions, without designing long probe, greatly reduce cost, such as a kind of reality of the application
The length of specific primer is only 15-25 base in existing mode, and the length of the long probe of general cross capture is 60-120
Base；In addition, the present processes realize the specific hybrid capture of target gene by annealing, the extension of specific primer,
List is for hybrid capture, in a kind of implementation of the application, it is thus only necessary to can be completed within 8 minutes, improve hybridization and catch
The efficiency obtained.
It is appreciated that the primer mixture of the application was made of a plurality of specific primer, these specific primers are
Spaced design is in the primer in the same amplification direction on target gene, for example, being all upstream primer or being all downstream primer.This
In the method for application, primer hybridization and the purpose for extending step are not the PCR amplification that index times is carried out to target gene, mesh
Be only can more preferably more complete hybrid capture target gene, therefore, the amplification direction of all specific primers is identical, energy
The coverage after each specific primer extends to target gene is enough ensured to greatest extent.In the application method to DNA sample
Product are annealed, are extended, and actually at an annealing temperature, held for some time makes specific primer hybridize to target base
Because upper, then the held for some time under elongating temperature, extends specific primer along target gene；Wherein, it is used
Reagent, such as enzyme, buffer and reaction condition can carry out with reference to Standard PCR, such as elongating temperature is usually 72
℃；The difference is that there is no the design of cyclic amplification in the primer hybridization of the application and extension step.
Preferably, the present processes further include sequencing library amplification step, wherein sequencing library amplification step includes adopting
PCR amplification is carried out with the target gene of library construction primer pair targeting enrichment, obtains the sequencing library for being used for high-flux sequence.
It should be noted that sequencing library amplification step is for high-flux sequence, in fact, the side of the application
In method, by primer hybridization and extend step and Beads enrichment and washing step, the targeting enrichment to target gene can be completed；
But continue subsequent high-flux sequence if necessary, then it is generally necessary to sequencing library amplification step.The present processes
In, the final purpose of sequencing library amplification step is to carry out PCR amplification to the target gene of enrichment using library construction primer,
Obtain the library for being used directly for high-flux sequence.In general, if the DNA sample of primer hybridization and extension step process
It is pre- library sample, then sequencing library amplification step, directlys adopt universal primer amplification enriched product；Wherein, pre- library
Sample refers to by conventional segment processing, end is repaired and the DNA sample of the first-class processing of adjunction.If primer hybridization and prolonged
The DNA sample for stretching step process is not pre- library sample, then sequencing library amplification step also needs to include the target base to enrichment
The DNA fragmentation of cause carries out the first-class operation of adjunction, then carries out PCR amplification again and obtains sequencing library.As for DNA fragmentation processing, end
The specific method that end is repaired, adjunction is first-class can refer to existing library constructing method, be not specifically limited herein.
Preferably, the length of specific primer is not less than 10 bases.
Preferably, the length of specific primer is 15-25 base.
It should be noted that on the one hand the length of specific primer needs to consider its specificity, on the other hand need to consider
Synthesis cost.It is appreciated that its longer specificity of the length of specific primer is better, still, corresponding synthesis cost is high, it is difficult to
Embody the difference with long probe.Therefore, in the case where ensureing specificity, the application proposes that the length of specific primer is not less than
10 bases, the i.e. length of specific primer can accomplish minimum 10 bases, can either ensure specificity in this way, and can reduce into
This.
Preferably, annealed, extended to DNA sample, actual conditions are, 98 DEG C be denaturalized 5 minutes, 65 DEG C of prehybridizations 1 divide
Clock, 58 DEG C anneal 1 minute, 72 DEG C extend 1 minute, after reaction 4 DEG C it is standby.
It should be noted that the primer hybridization of the application and extend step in, annealed to DNA sample, extend it is anti-
Answer system can be with reference to conventional PCR amplification, only reaction condition is different.For example, only carrying out a step after denaturation
Annealing and extension, without circulation step；Also, introduce pre-hybridization step before the anneal, i.e., 65 DEG C prehybridization 1 minute so that
All specific primers can be preferably integrated on the target gene of design.It is appreciated that the above reaction condition is
Optimal reference, the temperature and time of each step can carry out the adjustment of adaptability according to test situation, such as denaturation temperature is led to
Often at 90 DEG C or more, denaturation time is usually -10 minutes 30 seconds；In another example the temperature of prehybridization is usually 60-70 DEG C, the time is
- 10 minutes 30 seconds；The temperature of annealing is usually 50-60 DEG C, and the time is -2 minutes 30 seconds；Extending usually all is 65-75 DEG C of extension
1-10 minutes.Standby temperature after reaction is intended merely to preferably save reaction product, usually 4 DEG C, or can not
It is directly after the completion of reaction taken out immediately spare in the refrigerator for deposit in 4 DEG C or so with the standby temperature.
The another side of the application discloses a kind of device of two generation sequencing libraries targeting fast enriching, including primer hybridization and
Extension of module and Beads enrichment and wash module；Primer hybridization and extension of module, including for using primer mixture to DNA sample
Product are annealed, are extended；Primer mixture for the specific primer of target gene design by forming, every on target gene
A specific primer is designed every 50-200bp, enables primer mixture to cover entire target gene, and specificity is drawn
5 ' ends of object have biotin modification；Beads enrichment and wash module are right including the magnetic bead for being modified using Streptavidin
The extension products of primer hybridization and extension of module are captured, and pass through magnetic bead for the specific primer and phase of biotin modification
The target gene separation answered, washing remove non-target gene, that is, obtain the target gene of targeting enrichment.
Preferably, the device of the application further includes sequencing library amplification module, and it includes for adopting that sequencing library, which expands module,
PCR amplification is carried out with the target gene of library construction primer pair targeting enrichment, obtains the sequencing library for being used for high-flux sequence.
It should be noted that the device of the application, actually realizes that text is sequenced in two generation of the application by modules
Library targets each step in fast enriching method, to realize the automation enrichment of target gene.Wherein, primer hybridization and extension
Module and sequencing library expand module, core be that controlled by temperature the annealing realized primer hybridization and extend step,
Extend and the PCR amplification of sequencing library amplification step, structure are similar to thermostat or PCR instrument；Beads enrichment and washing mould
Block, core is that magnetic bead adsorption precipitation is made to its separation by magnetic frame, matched to be arranged, such as oscillation, stirring
It mixes or the components such as ultrasonic wave mixes, resuspension for realizing magnetic bead automatically etc. operates.The behaviour such as addition as sample or reagent
Make that existing automatic sample-adding system can be referred to, the movement of test tube can be not specifically limited herein using conventional mechanical arm.
It is appreciated that the method for the application two generations sequencing library targeting fast enriching, all or part of function can lead to
The mode for crossing hardware is realized, can also be realized by way of computer program.When being realized by way of computer program,
The program can be stored in a computer readable storage medium, and storage medium may include: read-only memory, random storage
Device, disk, CD, hard disk etc. execute the program by computer to realize the present processes.For example, program is stored in
In the memory of equipment, when executing program in memory by processor, the present processes can be realized.When the side of the application
When all or part of function is realized by way of computer program in method, which also be can store in server, Ling Yiji
In the storage mediums such as calculation machine, disk, CD, flash disk or mobile hard disk, pass through downloading or copying and saving depositing to local device
In reservoir, or version updating is carried out to the system of local device, then when processor executes the program in memory, Ji Keshi
Existing the application corrects all or part of function of the method for high-flux sequence data.
Due to using the technology described above, the beneficial effects of the present application are as follows:
The two generation sequencing libraries of the application target fast enriching method, are prolonged using specific primer to target gene
It stretches, replaces long probe, it is not only at low cost but also high-efficient, substantially reduce the enrichment reaction time.Also, specific primer exists
It is interval setting on target gene, longer target gene region can be covered using relatively small number of specific primer quantity,
Compared to the numerous probes of quantity, the present processes are simpler, easy to operate, and the reduction of specific primer quantity is also further
Reduce costs.The present processes provide a kind of new quick and save the cost targeting enrichment skill for high-flux sequence
Art.
Detailed description of the invention
Fig. 1 is the flow diagram of two generation sequencing libraries targeting fast enriching method in the embodiment of the present application；
Fig. 2 is the part flow diagram of two generation sequencing libraries targeting fast enriching method in the embodiment of the present application；
Fig. 3 is the structural block diagram of two generation sequencing libraries targeting fast enriching device in the embodiment of the present application.
Specific embodiment
Sequencing library enrichment method more conventional at present is probe hybrid method, and still, probe hybrid method is in order to ensure pair
The coverage of target gene is needed using the numerous probes of quantity, also, probe length is longer, and every probe usually requires 60-
The length of 120 bases or even longer.
The it is proposed of the application creativeness, it is spaced apart on target gene, such as interval 50-200bp, design is specifically
Property primer, utilize primer extend to substitute long probe, wherein the length of specific primer can accomplish minimum 10 bases, the application
A kind of implementation in be specially 15-25 base, it is not only more shorter than long probe, but also using interval setting, for identical big
Small target gene, in the case where ensureing the coverage of target gene, the quantity of specific primer is also more than the quantity of probe
It is few, synthesis cost is greatly saved.Moreover, the application extends replacement long probe using specific primer, entire enrichment reaction is only
It needs can be completed within about 8 minutes, i.e. primer hybridization in the application method and extension step, improves the enrichment effect of target gene
Rate.
It more than gene studies and recognizes, present applicant proposes a kind of methods of two generation sequencing libraries targeting fast enriching, such as
Shown in Fig. 1 and Fig. 2, including primer hybridization and extension step 11, Beads enrichment and washing step 12, and particular for sequencing text
The sequencing library amplification step 13 of library building.In Fig. 2, DNA sample is pre- library, and therefore, the both ends of DNA fragmentation are library connector
Sequence, wherein solid black lines are target gene, remaining is non-target gene.
Wherein, primer hybridization and extension step 11, including annealed, extended to DNA sample using primer mixture；This
The primer mixture of application for the specific primer of target gene design by forming, at interval of 50-200bp on target gene
A specific primer is designed, primer mixture is enabled to cover entire target gene, and 5 ' end tools of specific primer
There is biotin modification.
In a kind of implementation of the application, annealing extends specifically using purchased from Thermo Fisher Scientific Inc.
AmpliTaqDNTP Mix and MgCl are added in reaction system by DNAPolymerase and PCR Buffer II2, and
Benefit is filled with water to 50 μ L, and the specific dosage of each component refers to existing Standard PCR.Reaction condition is, 98 DEG C be denaturalized 5 minutes, 65 DEG C it is pre-
Hybridization 1 minute, 58 DEG C anneal 1 minute, 72 DEG C extend 1 minute, after reaction 4 DEG C it is standby.The application is every on target gene
It is spaced 50-200bp and designs a specific primer, every specific primer includes at least 10 bases, generally 15-25 alkali
Base, relative to the numerous long probe hybrid captures of existing quantity, the present processes can be using short and few quantity special
Property primer completely covers target gene, ensures the enrichment quality and integrality of target gene.
Beads enrichment and washing step 12 to primer hybridization and extend step including the magnetic bead using Streptavidin modification
Rapid extension products are captured, and are divided the specific primer of biotin modification and corresponding target gene by magnetic bead
From washing removes non-target gene, that is, obtains the target gene of targeting enrichment.
In a kind of implementation of the application, the specific DynabeadsTMM- for using Invitrogen
270Streptavidin magnetic bead carries out the separation of target gene, the knot that magnetic bead uses biotin and Streptavidin to react in advance
Buffer washing is closed, then magnetic bead is resuspended in the combination buffer, is incubated with primer hybridization and the product room temperature for extending step
It educates about 30 minutes, magnetic bead is rinsed in the washing reagent and method of the offer according to magnetic bead kit, finally uses ddH2O weight
Outstanding magnetic bead, i.e. acquisition target gene.
Sequencing library amplification step 13, including carrying out PCR expansion using the target gene of library construction primer pair targeting enrichment
Increase, obtains the sequencing library for being used for high-flux sequence.
In a kind of implementation of the application, specifically using purchased from the primer of KAPABiosystems company, enzyme and
Reaction solution is constructed for sequencing library.Specifically, buffer is 2 × KAPAHiFi HotStart ReadyMix, library construction
Primer is 10 × LibraryAmplification PrimerMix.Reaction condition is, 98 DEG C initial denaturation 45 seconds, subsequently into 15
A circulation: it anneals 30 seconds, 72 DEG C within denaturation 15 seconds, 60 DEG C for 98 DEG C and extends 30 seconds, extend 1 minute, last 4 DEG C for 72 DEG C after circulation terminates
It is standby.Reaction terminates to obtain sequencing library, can directly upper machine sequencing.
The two generation sequencing libraries based on the application target fast enriching method, and the application further provides a kind of two generations survey
Preface library targets the device of fast enriching, as shown in figure 3, the device include primer hybridization and extension of module 31, Beads enrichment and
Wash module 32 and sequencing library expand module 33.Primer hybridization and extension of module 31, including for using primer mixture pair
DNA sample is annealed, is extended；Primer mixture for the specific primer of target gene design by forming, in target gene
On at interval of 50-200bp design a specific primer, enable primer mixture to cover entire target gene, and special
5 ' ends of specific primer have biotin modification；Beads enrichment and wash module 32, including for being modified using Streptavidin
Magnetic bead, the extension products of primer hybridization and extension of module 31 are captured, and by magnetic bead by the special of biotin modification
Property primer and the separation of corresponding target gene, washing remove non-target gene, that is, obtain the target gene of targeting enrichment；Sequencing
Amplified library module 33 includes being used for carrying out PCR amplification using the target gene of library construction primer pair targeting enrichment
In the sequencing library of high-flux sequence.
The application is described in further detail below by specific embodiments and the drawings.Following embodiment is only to the application
It is further described, should not be construed as the limitation to the application.
Embodiment
This example designs specific primer according to the sequence information of target gene.In specific primer and two generations to be enriched with, survey
The pre- library of sequence carries out enrichment reaction, adsorbs hybrid product with affine strepavidin magnetic beads after reaction, and clean by rinsing liquid
Remove the nucleic acid of non-specific adsorption.The magnetic bead for being adsorbed with hybrid product carries out POST-PCR, then upper machine sequencing.Wherein, two generation
Be sequenced pre- library refer to handled by fragmentation, end is repaired, the DNA sample of the first-class step of adjunction.This example is made using pre- library
For process object, the method for two generation sequencing libraries targeting fast enriching is illustrated, as follows in detail:
One, specific primer design and synthesis
The specific primer of this example is according to the sequence design of target gene, and target gene complementary pairing, specifically, in target
It marks and designs a specific primer at interval of 50-200bp on gene, every specific primer includes at least 10 bases, and
5 ' ends of primer have biotin modification.
All specific primers of this example are blended together as primer mixture to use, all specificity are drawn in principle
The annealing temperature of object will be consistent, such as in the range of 58 DEG C or so 1-2 DEG C, all special to ensure in annealing steps
Property primer can be integrated on target gene.
Two, enrichment reaction
It annealed, extended to DNA sample using primer mixture, the AmpliTaq that this example specifically uses
DNAPolymerase, PCR Buffer II are purchased from Thermo Fisher Scientific Inc..The reaction system of 50 μ L includes:
AmpliTaq5 μ of primer mixture of 0.25 μ L of DNAPolymerase, pre- library DNA sample 1ng-3 μ g, 10pmol
L, the MgCl of 10 × PCR Buffer II, 5 μ L, dNTP Mix1 μ L of 10mM, 25mM23 μ L supplement ddH2O to 50 μ L.
Prepared reaction system is reacted in the thermal cycler, specific reaction condition are as follows: 98 DEG C of denaturation 5 minutes, 65
Anneal 1 minute, 72 DEG C within DEG C prehybridization 1 minute, 58 DEG C and extend 1 minute, after reaction 4 DEG C it is standby.
Three, Beads enrichment purifies
1. magnetic bead pre-processes
This example uses the DynabeadsTMM-270Streptavidin magnetic bead purchased from Invitrogen.Magnetic bead pretreatment packet
Include following steps:
A. M-270 magnetic bead is resuspended on vortex blending instrument, 50 μ L magnetic beads are added into new centrifuge tube in each response sample.
B. 200 μ L combination buffers are respectively added, mixes magnetic bead with pipettor piping and druming or vortex instrument, centrifuge tube is placed on magnetic
On power frame, after waiting 1 minute, draws and discard supernatant liquid.
C. it is primary to repeat step b.
D. 50 μ L combination buffers are added, magnetic bead is resuspended.
2. Beads enrichment purifies
Hybridize the specific primer of absorption biotin modification using Streptavidin MagneSphere, and matched with Primers complementary
Target gene.Specifically, the product of " two, enrichment reaction " is added in pretreated magnetic bead, 30 points are incubated at room temperature after mixing
Clock.Then magnetic bead rinsing is carried out, the target gene of enrichment is obtained, specific as follows:
A. by magnetic frame on the sample cell containing magnetic bead and DNA, supernatant is removed after waiting 1 minute；
B. 200 μ L wash buffers are added, 65 DEG C of metal baths are incubated for 2 minutes, and upper magnetic frame removes after waiting 1 minute
Clear liquid；
C. step b is repeated twice；
D. the ddH of 20 μ L is added2Magnetic bead is resuspended in O, that is, obtains the target gene of targeting enrichment.
Four, sequencing library constructs
The target gene of targeting enrichment is expanded using POST-PCR, obtain can directly upper machine sequencing sequencing library, this example
The primer and enzyme reaction solution that PCR is used are purchased from KAPABiosystems company.The reaction system of 50 μ L includes: 2 × KAPA
25 μ L of HiFi HotStart ReadyMix, 10 × Library Amplification PrimerMix, 5 μ L, it is adsorbed with target
Mark the 20 μ L of magnetic bead of gene.
Reaction condition are as follows: 98 DEG C initial denaturation 45 seconds, subsequently into 15 recycle: 98 DEG C be denaturalized 15 seconds, 60 DEG C anneal 30 seconds,
72 DEG C extend 30 seconds, extend 1 minute for 72 DEG C after circulation terminates, last 4 DEG C standby.
PCR after reaction, recycles pcr amplification product, i.e. acquisition sequencing library.This example is specifically using nucleic acid purification
Magnetic bead recovery purifying PCR reaction product, magnetic bead usage amount be 1 ×, sequencing library after purification is dissolved in the ddH of 30 μ L2In O.Most
Afterwards, the directly upper machine sequencing of sequencing library after purification.
Test example
This example tests KEAP1 gene according to above method and step, specifically devises 82 primers, every is drawn
The long 15-25 base of object is used for KEAP1 genetic enrichment.As a comparison, using conventional probe hybrid method, 85 spies are devised
Needle is used for the enrichment of identical target gene.This example uses Novaseq microarray dataset, quickly rich to 5 two generation sequencing libraries targetings
The sequencing library of set method preparation and the sequencing library of identical 5 probe hybrid methods preparation are sequenced and are analyzed.Statistics
Different enrichment methods are to the capture rate of KEAP1 gene, and the results are shown in Table 1.
The capture rate statistical result of the different enrichment methods of table 1
Table 1 the results show that using this example enrichment method, to the capture rate of target gene all 90% or more, and
The capture rate of sonde method is only 50-60%.As it can be seen that the primer of this example design and the probe of conventional method design be to target gene,
Although covered to target gene 100% in design；But using the enrichment method of this example, it is clear that than traditional probe
Method capture rate is much higher.Also, for KEAP1 gene, the quantity of primer used by the enrichment method of this example,
Also fewer than the quantity of probe.Therefore, the enrichment method of this example, the length of primer replacement routine that can be less, shorter using quantity
Probe carries out capture enrichment to target gene；Also, the enrichment method of this example capture rate with higher, can be improved target
The efficiency and quality of gene trap enrichment.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen
Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off
Under the premise of from the application design, a number of simple deductions or replacements can also be made.
Claims (8)
1. a kind of method of two generation sequencing libraries targeting fast enriching, it is characterised in that: include the following steps,
Primer hybridization and extension step, including annealed, extended to DNA sample using primer mixture；The primer mixing
Object designs a specificity at interval of 50-200bp on target gene by forming for the specific primer of target gene design
Primer enables primer mixture to cover entire target gene, and 5 ' ends of specific primer have biotin modification；
Beads enrichment and washing step to the primer hybridization and extend step including the magnetic bead using Streptavidin modification
Extension products captured, and the specific primer of biotin modification and corresponding target gene are separated by magnetic bead,
Washing removes non-target gene, that is, obtains the target gene of targeting enrichment.
2. according to the method described in claim 1, it is characterized by also including sequencing library amplification step, the sequencing library
Amplification step includes carrying out PCR amplification using the target gene of library construction primer pair targeting enrichment, obtains and measures for high pass
The sequencing library of sequence.
3. method according to claim 1 or 2, it is characterised in that: the length of the specific primer is not less than 10 bases；
Preferably, the length of the specific primer is 15-25 base.
4. method according to claim 1 or 2, it is characterised in that: described to be annealed, extended to DNA sample, specific item
Part is that 98 DEG C are denaturalized anneal within prehybridization 1 minute, 58 DEG C 1 minute, 72 DEG C of extensions 1 minute, after reaction 4 DEG C in 5 minutes, 65 DEG C
It is standby.
5. a kind of device of two generation sequencing libraries targeting fast enriching, it is characterised in that: including primer hybridization and extension of module and
Beads enrichment and wash module；
The primer hybridization and extension of module, including for being annealed, being extended to DNA sample using primer mixture；It is described
Primer mixture for the specific primer of target gene design by forming, at interval of 50-200bp design one on target gene
Specific primer enables primer mixture to cover entire target gene, and 5 ' ends of specific primer have biology
Element modification；
The Beads enrichment and wash module, including for the magnetic bead using Streptavidin modification, to the primer hybridization and
The extension products of extension of module are captured, and pass through magnetic bead for the specific primer of biotin modification and corresponding target base
Because of separation, washing removes non-target gene, that is, obtains the target gene of targeting enrichment.
6. device according to claim 5, it is characterised in that: further include sequencing library amplification module, the sequencing library
Amplification module includes obtaining for carrying out PCR amplification using the target gene of library construction primer pair targeting enrichment and being used for high pass
Measure the sequencing library of sequence.
7. device according to claim 5 or 6, it is characterised in that: the length of the specific primer is not less than 10 bases；
Preferably, the length of the specific primer is 15-25 base.
8. device according to claim 5 or 6, it is characterised in that: described to be annealed, extended to DNA sample, specific item
Part is that 98 DEG C are denaturalized anneal within prehybridization 1 minute, 58 DEG C 1 minute, 72 DEG C of extensions 1 minute, after reaction 4 DEG C in 5 minutes, 65 DEG C
It is standby.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910568971.9A CN110241462A (en) | 2019-06-27 | 2019-06-27 | A kind of method and apparatus of two generations sequencing library targeting fast enriching |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910568971.9A CN110241462A (en) | 2019-06-27 | 2019-06-27 | A kind of method and apparatus of two generations sequencing library targeting fast enriching |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110241462A true CN110241462A (en) | 2019-09-17 |
Family
ID=67889884
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910568971.9A Pending CN110241462A (en) | 2019-06-27 | 2019-06-27 | A kind of method and apparatus of two generations sequencing library targeting fast enriching |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110241462A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680796A (en) * | 2021-01-18 | 2021-04-20 | 深圳市睿法生物科技有限公司 | Target gene enrichment and library construction method |
| CN113981041A (en) * | 2021-11-25 | 2022-01-28 | 首都医科大学附属北京安贞医院 | Targeted enrichment sequencing reagent and targeted enrichment method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212879A (en) * | 2013-06-05 | 2014-12-17 | 上海派森诺生物科技有限公司 | Magnetic bead enrichment technique based method for high-flux development of genome SSR markers |
-
2019
- 2019-06-27 CN CN201910568971.9A patent/CN110241462A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212879A (en) * | 2013-06-05 | 2014-12-17 | 上海派森诺生物科技有限公司 | Magnetic bead enrichment technique based method for high-flux development of genome SSR markers |
Non-Patent Citations (2)
| Title |
|---|
| BRIGGS AW: "Rapid retrieval of DNA target sequences by primer extension capture.", 《METHODS MOL BIOL. 》 * |
| MAYRA EDUARDOFF等: "Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin", 《GENES (BASEL).》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680796A (en) * | 2021-01-18 | 2021-04-20 | 深圳市睿法生物科技有限公司 | Target gene enrichment and library construction method |
| CN113981041A (en) * | 2021-11-25 | 2022-01-28 | 首都医科大学附属北京安贞医院 | Targeted enrichment sequencing reagent and targeted enrichment method |
| CN113981041B (en) * | 2021-11-25 | 2024-04-30 | 首都医科大学附属北京安贞医院 | Targeting enrichment sequencing reagent and targeting enrichment method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fan et al. | Single-cell RNA-seq analysis of mouse preimplantation embryos by third-generation sequencing | |
| CN105714383B (en) | A kind of sequencing library construction method and reagent based on the reverse probe of molecule | |
| CN105219766B (en) | A kind of multiple PCR method of three-wheel amplification | |
| Blumenstiel et al. | Targeted exon sequencing by in‐solution hybrid selection | |
| CN107075513A (en) | The oligonucleotides of separation and its purposes in nucleic acid sequencing | |
| CN106574286A (en) | Selective amplification of nucleic acid sequences | |
| CN105647907B (en) | It is a kind of for targeting the preparation method of the modified DNA hybridization probe of hybrid capture | |
| CN109234356A (en) | A kind of method and application constructing hybrid capture sequencing library | |
| CN106282353A (en) | A kind of method utilizing clamp primers to carry out multiplex PCR | |
| Bevilacqua et al. | Technique development for probing RNA structure in vivo and genome-wide | |
| CN109486923B (en) | Primer system for sequencing multiple amplicons, application thereof and method for constructing sequencing library | |
| CN109971846A (en) | Use the method for the diallele SNP antenatal measurement aneuploid of Noninvasive for targeting next-generation sequencing | |
| CN110241462A (en) | A kind of method and apparatus of two generations sequencing library targeting fast enriching | |
| WO2016095736A1 (en) | Target region enrichment method based on multiplex pcr, and reagent | |
| WO2016023431A1 (en) | Wash solution and washing method for hybrid-enrichment-capture dna sequencing library | |
| Jameson et al. | DNA-, RNA-, and protein-based stable-isotope probing for high-throughput biomarker analysis of active microorganisms | |
| Garosi et al. | Defining best practice for microarray analyses in nutrigenomic studies | |
| CN105506747B (en) | It is enriched with construction method and its application in the libraries RNA of primary transcript information | |
| JP2021536255A (en) | Methods and kits for building RNA libraries | |
| CN106795650B (en) | PF quick database building method and application thereof | |
| CN103882532B (en) | A kind of synthesis of lead compound and screening method and test kit | |
| CN104404154B (en) | Zooplankter rrnL gene magnifications primer and its screening technique and application and application process | |
| CN110273028A (en) | Enrichment method, sequencing data analysis method and the device of viral integrase type DNA | |
| CN113493932A (en) | Method and kit for constructing capture library with high detection performance | |
| Gazestani et al. | circTAIL-seq, a targeted method for deep analysis of RNA 3′ tails, reveals transcript-specific differences by multiple metrics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |