CN109853047A - A kind of genomic DNA sequencing library fast construction method and matched reagent box - Google Patents
A kind of genomic DNA sequencing library fast construction method and matched reagent box Download PDFInfo
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Abstract
The invention discloses a kind of genomic DNA sequencing library fast construction method and matched reagent boxes.Method includes: one, selects I deoxyribonuclease I of DNase as DNA fragmentation enzyme, carries out enzyme cutting method fragmentation to sample DNA, obtains fragmentation DNA；Two, fragmentation DNA is directly carried out adding dA tail, obtains the DNA for adding dA tail；Three, the DNA of dA tail will be added to connect with connector, obtains connector connection product；Four, butt joint connection product carries out magnetic beads for purifying；Five, butt joint connection product carries out PCR amplification, obtains amplified production；Six, magnetic beads for purifying is carried out to amplified production, obtains genomic DNA sequencing library product；Seven, segment distribution inspection is carried out using agarose gel electrophoresis detection method to library production and library is evaluated.Banking process of the invention selects DNase I to be used as DNA fragmentation enzyme, reduces end and repairs and phosphatising step, makes to build that the library time is shorter, and cost is lower.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of genomic DNA sequencing library fast construction method and mating examination
Agent box.
Background technique
The acquisition of life entity hereditary information is of great significance to the research of life science, has pushed biologic medical
The fast development in field.In recent years, including more and more genomes such as people, arabidopsis and rice are sequenced, full-length genome
Sequencing becomes the important means of life medical research.The research of various bio-science fields or clinical detection field are directed to
The building and high-flux sequence service of sequencing library.
Conventional sequencing library preparation is related to the fragmentation of sample DNA, end-filling, 5 ' terminal phosphates, 3 ' ends add
The plurality of step such as dA tail, connector connection and PCR amplification, and multiple nucleic acid purification is also needed between step, not only time-consuming but also appearance
Easily cause sample losses.There is part to be commercialized matched reagent box, such as the production that KAPA Biosystems, NEB company are provided at present
End is repaired and the library step of building of dA tail is added to merge by product, be can achieve and is saved time, the low-loss purpose of drop.But needle
The processing of some daily requirements is largely built for the staff that library sample and first contacts build library, traditional banking process is still
It is too cumbersome.The especially fragmentation of DNA need to generally be completed using special ultrasonic fragmentation instrument, and consumables cost is larger, nothing
Method meets the needs of all users.
To sum up, existing genomic DNA sequencing library construction method has the following deficiencies: that 1, device dependence is strong,
It is at high cost；2, cumbersome, time-consuming, builds library low efficiency；3, it can not achieve integrated operation；4, single sample fragmentation, cannot
Realize that high throughput builds library.Therefore it provides a kind of quicker, convenient and fast genome dna library construction method, raising is built on a large scale
The efficiency in library reduces and builds the operation difficulty in library, realizes that high throughput builds library, gets rid of equipment limit, meet the needs of different clients at
For the task of top priority.
Summary of the invention
It is an object of the present invention to overcome the deficiencies of the prior art and provide a kind of genomic DNA sequencing library rapid build
Method and matched reagent box.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of genomic DNA sequencing library fast construction method of the present invention, technical concept and technology path are as follows:
(1) DNA fragmentation enzyme DNase I is chosen: according to the demand and feature of fragmentation DNA in library construction process
Random cutting DNA segment, Preference is low, strong operability.DNase I (deoxyribonuclease I) is chosen in the present invention is used as DNA
Single-stranded or double-stranded DNA equally Stochastic Decomposition can be generated the deoxidation core with 5 '-P terminal oligos by fragmentation enzyme
Ribosomal ribonucleic acid restriction endonuclease.DNase I can cut off simultaneously double-stranded DNA in the presence of Mn2+, make DNA fragmentation, can be used for subsequent DNA
Library production.Compared with traditional banking process, banking process of the invention chooses DNase I and is used as DNA fragmentation enzyme, can reduce
End is repaired and phosphatising step, makes to build that the library time is shorter, and cost is lower.
(2) test enzyme amount, buffer and digestion time: by adjusting buffer, gradient tests enzyme amount and digestion used time
Between, filter out the suitable buffer component that library is continued after being suitable for, enzyme amount and digestion time.
(3) completely build library test optimization: endonuclease bamhi product continues the progress of library process and completely builds library test after combining, from
Library is built as a result, i.e. yield, library size etc. are started with, further does corresponding adjusting and optimizing.
(4) high-flux sequence: the genome sample of the microbial genome sample and people of choosing different G/C contents carries out
High throughput builds library sequencing analysis, by analysis coverage rate, GC Preference, the data such as Q20, Q30 come comprehensive assessment this to build library mating
Kit.
A kind of genomic DNA sequencing library fast construction method provided by the invention, comprising the following steps:
Step 1: DNase I (deoxyribonuclease I) is selected to be used as DNA fragmentation enzyme, enzyme cutting method is carried out to sample DNA
Fragmentation obtains fragmentation DNA；
Step 2: directly carrying out adding dA tail to the fragmentation DNA that step 1 obtains, the DNA for adding dA tail is obtained；
Step 3: DNA that step 2 is obtained plus dA tail is attached with connector, connector connection product is obtained；
Step 4: butt joint connection product carries out magnetic beads for purifying；
Step 5: butt joint connection product carries out PCR amplification, amplified production is obtained；
Step 6: carrying out magnetic beads for purifying to amplified production, genomic DNA sequencing library product is obtained；
Step 7: library production is carried out segment distribution using agarose gel electrophoresis detection method and is checked and library evaluation.
Further, the genomic DNA sequencing library fast construction method, further comprising the steps of:
Step 8: completely building library test optimization: endonuclease bamhi product continues library process and carries out completely building library survey after combining
Examination, Cong Jianku further do corresponding adjusting and optimizing as a result, i.e. yield, library size etc. are started with；
Step 9: carrying out high-flux sequence to genomic DNA sequencing library product；Also that is, choosing the micro- of different G/C contents
Biological genome sample and the genome sample of people carry out high throughput and build library sequencing analysis, by analyzing coverage rate, GC preference
The data such as property, Q20, Q30 carry out comprehensive assessment, and this builds library matched reagent box.
Further, the genomic DNA sequencing library fast construction method, sequentially includes the following steps:
1), sample DNA fragmentation
It takes sample DNA as fragmentation template, enzyme cutting buffering liquid is added, DNA fragmentation enzyme DNase I is deoxyribose core
Sour enzyme I is configured to reaction system and carries out fragmentation reaction, obtains the gDNA of fragmentation；
2), dA tail adds
Da- tail addition buffer, da- tail addition enzyme are added into the gDNA of fragmentation, is configured to reaction system and carries out dA
Tail addition reaction obtains dA- tail addition product；
3), connector connects
T4 DNA ligase, connection promotor, DNA connector, ligase buffer solution are added into dA- tail addition product, matches
Reaction system is made and carries out connector connection reaction, obtains connector connection product；
4), connector connection product magnetic beads for purifying
By connector connection product obtained in the previous step, magnetic bead is sorted by DNA, is purified back with the magnetic bead ratio of 0.8x
It receives；
5), PCR amplification
High-fidelity DNA polymerase, polymerization enzyme buffer are added in the connector connection product of the purification and recovery obtained one step up
Liquid is configured to reaction system and carries out pcr amplification reaction, obtains pcr amplification product:
6), amplified production magnetic beads for purifying
By pcr amplification product obtained in the previous step, magnetic bead is sorted by DNA, is purified back with the magnetic bead ratio of 0.9x
It receives, obtains genomic DNA sequencing library product；
7), to library production obtained in the previous step, library size is detected with agarose gel electrophoresis, carries out segment distribution inspection
It looks into and is evaluated with library.
Further, the genomic DNA sequencing library fast construction method, further comprising the steps of:
8), completely build library test optimization: endonuclease bamhi product continues the progress of library process and completely builds library test after combining, from
Library is built as a result, i.e. yield, library size etc. are started with, further does corresponding adjusting and optimizing；
9), to genomic DNA sequencing library product, high-flux sequence is carried out；Also that is, choosing the microorganism of different G/C contents
Genome sample and the genome sample of people carry out high throughput and build library sequencing analysis, by analysis coverage rate, GC Preference,
The data such as Q20, Q30 carry out comprehensive assessment, and this builds library matched reagent box.
Further, genomic DNA sequencing library fast construction method of the present invention, specifically according to the following steps into
Row:
1, sample genomic dna fragmentation
It takes 50ng sample genomic dna as fragmentation template, 10x Fragment buffer (digestion buffering is added
Liquid), 1ul DNase I (deoxyribonuclease I, fragmentation enzyme) is configured to the reaction system of 50ul, by following response procedures:
25 DEG C of 6min, 85 DEG C of 10min carry out fragmentation, the gDNA of fragmentation can be obtained.
2, dA tail adds
To addition 6ul dA-Tailing Buffer in Fragmented DNA (gDNA of fragmentation), (addition of da- tail is slow
Fliud flushing), 2ul dA-Tailing Enzyme (da- tail adds enzyme) is configured to the reaction system of 60ul, by following response procedures
DA tail addition: 72 DEG C of 15min is carried out, dA- tail addition product is obtained.
3, connector connects
The T4 DNA ligase of 5ul high concentration is added into dA- tail addition product, (connection increases the connection promotor of 30ul
Strong agent), the Illumina DNA connector of 5ul 10uM, the 10X ligase buffer solution of 5ul is configured to the system of 100ul, by such as
Lower response procedures carry out connector connection: 25 DEG C of 15min obtain connector connection product.
4, connector connection product magnetic beads for purifying
By connector connection product obtained in the previous step, pass through Hieff NGSTMDNA Selection Beads magnetic bead
(Hieff NGSTMDNA sorts magnetic bead), illustrate by reagent, purification and recovery is carried out with the magnetic bead ratio of 0.8x.
5, PCR amplification
2 × Super Canace of 25ul is added into the connector connection product by purification and recovery obtained in the previous stepTM
High-Fidelity Mix (high-fidelity DNA polymerase premixed liquid), 5ul Primer Mix (primary premixed liquid), is configured to
The reaction system of 50ul carries out PCR amplification (polymerase chain reaction) by following procedure:
6, amplified production magnetic beads for purifying
By pcr amplification product obtained in the previous step, pass through Hieff NGSTMDNA Selection Beads magnetic bead
(Hieff NGSTMDNA sorts magnetic bead), illustrate by reagent, purification and recovery is carried out with the magnetic bead ratio of 0.9x, gene can be obtained
Group DNA sequencing library production.
7, library size is detected with agarose gel electrophoresis, carries out segment distribution and checks and library evaluation；
To library production obtained in the previous step, library size is detected with agarose gel electrophoresis.
Further, sample genomic dna described in the above method is human cel gene group DNA；Previous step is obtained
The library production arrived, the library size detected with agarose gel electrophoresis, obtained testing result are shown in Fig. 1, from the electricity of Fig. 1
Swimming testing result figure, it can be clearly seen that, the stripe size distribution of gradient, this article is presented in human cel gene group DNA library
DNA fragmentation size in library is between 100-700bp.
Further, the genomic DNA sequencing library fast construction method, further comprising the steps of:
8, completely build library test optimization: endonuclease bamhi product continues the progress of library process and completely builds library test after combining, from
Library is built as a result, i.e. yield, library size etc. are started with, further does corresponding adjusting and optimizing；
9, to genomic DNA sequencing library product, high-flux sequence is carried out；Also that is, choosing the microorganism of different G/C contents
Genome sample and the genome sample of people carry out high throughput and build library sequencing analysis, by analysis coverage rate, GC Preference,
The data such as Q20, Q30 carry out comprehensive assessment, and this builds library matched reagent box.
The present invention also provides a kind of matched reagent box for rapid build genomic DNA sequencing library according to the above method,
The matched reagent box includes: DNase I (deoxyribonuclease I), enzyme cutting buffering liquid, da- tail addition buffer, the addition of da- tail
Enzyme, T4 DNA ligase, connection promotor, DNA connector, high-fidelity DNA polymerase (high-fidelity amplified library premixed liquid), primary
Premixed liquid, DNA sort magnetic bead.And operated using the matched reagent box by such as above method, supper-fast building may be implemented
Genomic DNA sequencing library.
Further, the matched reagent box includes: 1ul DNase I (deoxyribonuclease I), 10ul 10X enzyme
The connection for cutting buffer, 6ul da- tail addition buffer, the addition of 2ul da- tail enzyme, 5ul T4 DNA ligase, 30ul promotes
Agent, the Illumina DNA connector of 5ul 10uM, 5ul 10X ligase buffer solution, 2 × high-fidelity DNA polymerase of 25ul are (high
Fidelity amplified library premixed liquid), 5ul primary premixed liquid, 0.8x DNA sorting magnetic bead, 0.9x DNA sort magnetic bead.
Beneficial effects of the present invention:
The present invention provides a kind of more simple and quick DNA sequencing library constructing methods, it may be assumed that chooses DNase I and is used as DNA
Fragmentation enzyme gets rid of cumbersome ultrasonic step, reduces costs using enzyme cutting method by DNA fragmentation, while by DNA fragmentation
Change, the addition of dA tail, connector connection, merge into single step reaction, shortens and build the library time, improve and build library efficiency.Of the invention builds
Library method can be applied in DNA fragmentation and DNA sequencing library construction.
Banking process of the invention has the advantages that compared with existing traditional banking process
1, the present invention selects DNase I to be used as DNA fragmentation enzyme, reduces end reparation and phosphatising step, when making to build library
Between it is shorter, cost is lower.
2, the present invention uses enzyme cutting method fragmentation DNA, at low cost without using large-scale instrument and equipment；
3, method of the invention is easy to operate, and step is simple, and it is high-efficient to build library；
4, integrated Library development flow can be achieved in the present invention, is suitable for automation platform；
5, the present invention can carry out fragmentation processing, it can be achieved that high throughput builds library to multisample simultaneously.
Detailed description of the invention
Fig. 1 is the testing result figure obtained in the embodiment of the present invention 1 with agarose gel electrophoresis detection library size.
Specific embodiment
Below in conjunction with drawings and examples, the present invention is further illustrated.
Embodiment 1
A kind of fast construction method in human cel gene group DNA sequencing library of the present invention, carries out in the steps below:
1, human cel gene group DNA fragmentation
It takes 50ng human cel gene group DNA as fragmentation template in this example, 10ul 10x Fragment is added
Buffer (enzyme cutting buffering liquid), 1ul DNase I (deoxyribonuclease I), is configured to the reaction system of 50ul, by following anti-
Program: 25 DEG C of 6min is answered, 85 DEG C of 10min carry out fragmentation, the gDNA of fragmentation can be obtained.
2, dA tail adds
6ul dA-Tailing Buffer (da- is added in this example into Fragmented DNA (gDNA of fragmentation)
Tail adds buffer), 2ul dA-Tailing Enzyme (da- tail adds enzyme) is configured to the reaction system of 60ul, by as follows
Response procedures carry out dA tail addition: 72 DEG C of 15min, obtain dA- tail addition product.
3, connector connects
The T4 DNA ligase of 5ul high concentration, the connection promotor of 30ul, 5ul are added into dA- tail addition product
The Illumina DNA connector of 10uM, the 10X ligase buffer solution of 5ul are configured to the system of 100ul, by following response procedures
Carry out connector connection: 25 DEG C of 15min obtain connector connection product.
4, connector connection product magnetic beads for purifying
By connector connection product obtained in the previous step, pass through Hieff NGSTMDNA Selection Beads magnetic bead
(Hieff NGSTMDNA sorts magnetic bead), illustrate by reagent, purification and recovery is carried out with the magnetic bead ratio of 0.8x.
5, amplified library
2 × Super CanaceTM of 25ul is added into the connector connection product by purification and recovery obtained in the previous step
High-Fidelity Mix (high-fidelity amplified library premixed liquid), 5ul Primer Mix (primary premixed liquid) are configured to 50ul
Reaction system, by following procedure carry out PCR amplification (polymerase chain reaction), obtain amplified production:
6, amplified production magnetic beads for purifying
By amplified library product obtained in the previous step, pass through Hieff NGSTMDNA Selection Beads magnetic bead
(Hieff NGSTMDNA sorts magnetic bead), illustrate by reagent, purification and recovery is carried out with the magnetic bead ratio of 0.9x, gene can be obtained
Group DNA sequencing library production.
7, library size is detected with agarose gel electrophoresis, carries out segment distribution and checks and library evaluation；
To library production obtained in the previous step, library size is detected with agarose gel electrophoresis, obtained testing result is shown in
Fig. 1.From the electrophoresis detection of Fig. 1, it can be clearly seen that, the stripe size point of gradient is presented in human cel gene group DNA library
Cloth, the DNA fragmentation size in the library is between 100-700bp.
8, completely build library test optimization: endonuclease bamhi product continues the progress of library process and completely builds library test after combining, from
Library is built as a result, i.e. yield, library size etc. are started with, further does corresponding adjusting and optimizing；
9, to genomic DNA sequencing library product, high-flux sequence is carried out；Also that is, choosing the microorganism of different G/C contents
Genome sample and the genome sample of people carry out high throughput and build library sequencing analysis, by analysis coverage rate, GC Preference,
The data such as Q20, Q30 carry out comprehensive assessment, and this builds library matched reagent box.
It is from embodiment 1 as can be seen that of the present invention a kind of for the survey of rapid build genomic DNA according to the above method
The matched reagent box in preface library, comprising: DNase I (deoxyribonuclease I), enzyme cutting buffering liquid, da- tail addition buffer,
Da- tail addition enzyme, T4 DNA ligase, connection promotor, DNA connector, (high-fidelity amplified library is pre- for high-fidelity DNA polymerase
Mixed liquid), primary premixed liquid, DNA sort magnetic bead.
The matched reagent box specifically includes: 1ul DNase I (deoxyribonuclease I), 10ul 10X enzyme cutting buffering liquid,
6ul da- tail adds connection promotor, the 5ul of buffer, 2ul da- tail addition enzyme, 5ul T4 DNA ligase, 30ul
The Illumina DNA connector of 10uM, 5ul 10X ligase buffer solution, 25ul 2 × high-fidelity DNA polymerase (high-fidelity text
Library expand premixed liquid), 5ul primary premixed liquid, 0.8x DNA sorting magnetic bead, 0.9x DNA sort magnetic bead.
It is operated using the matched reagent box by method described in embodiment 1, supper-fast building genome may be implemented
DNA sequencing library.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of genomic DNA sequencing library fast construction method, which comprises the following steps:
Step 1: selecting DNase I is deoxyribonuclease I as DNA fragmentation enzyme, enzyme cutting method piece is carried out to sample DNA
Duan Hua obtains fragmentation DNA；
Step 2: directly carrying out adding dA tail to the fragmentation DNA that step 1 obtains, the DNA for adding dA tail is obtained；
Step 3: DNA that step 2 is obtained plus dA tail is attached with connector, connector connection product is obtained；
Step 4: butt joint connection product carries out magnetic beads for purifying；
Step 5: butt joint connection product carries out PCR amplification, amplified production is obtained；
Step 6: carrying out magnetic beads for purifying to amplified production, genomic DNA sequencing library product is obtained；
Step 7: library production using agarose gel electrophoresis detection method, is carried out segment distribution and is checked and library evaluation.
2. genomic DNA sequencing library fast construction method as described in claim 1, which is characterized in that further include following step
It is rapid:
Step 8: completely building library test optimization: endonuclease bamhi product continues the progress of library process and completely builds library test after combining, from
Library is built as a result, i.e. yield, library size etc. are started with, further does corresponding adjusting and optimizing；
Step 9: carrying out high-flux sequence to genomic DNA sequencing library product；Also that is, choosing the microorganism of different G/C contents
Genome sample and the genome sample of people carry out high throughput and build library sequencing analysis, by analysis coverage rate, GC Preference,
The data such as Q20, Q30 carry out comprehensive assessment, and this builds library matched reagent box.
3. genomic DNA sequencing library fast construction method as described in claim 1, which is characterized in that according to the following steps into
Row:
1), sample DNA fragmentation
It takes sample DNA as fragmentation template, enzyme cutting buffering liquid is added, DNA fragmentation enzyme DNase I is deoxyribonuclease
I is configured to reaction system and carries out fragmentation reaction, obtains the gDNA of fragmentation；
2), dA tail adds
Da- tail addition buffer, da- tail addition enzyme are added into the gDNA of fragmentation, is configured to reaction system progress dA tail and adds
Add reaction, obtains dA- tail addition product；
3), connector connects
T4 DNA ligase, connection promotor, DNA connector are added into dA- tail addition product, is configured to reaction system and is connect
Head connection reaction, obtains connector connection product；
4), connector connection product magnetic beads for purifying
By connector connection product obtained in the previous step, magnetic bead is sorted by DNA, purification and recovery is carried out with the magnetic bead ratio of 0.8x；
5), PCR amplification
High-fidelity DNA polymerase, polymerase buffer are added in the connector connection product of the purification and recovery obtained one step up, matches
Reaction system is made and carries out pcr amplification reaction, obtains pcr amplification product:
6), amplified production magnetic beads for purifying
By pcr amplification product obtained in the previous step, magnetic bead is sorted by DNA, purification and recovery is carried out with the magnetic bead ratio of 0.9x, is obtained
To genomic DNA sequencing library product；
7), to library production obtained in the previous step, detect library size with agarose gel electrophoresis, carry out segment distribution check and
Library evaluation.
4. genomic DNA sequencing library fast construction method as claimed in claim 3, which is characterized in that further include following step
It is rapid:
8), completely build library test optimization: endonuclease bamhi product continues library process and carries out completely building library test, Cong Jianku after combining
As a result, i.e. yield, library size etc. are started with, corresponding adjusting and optimizing is further done；
9), to genomic DNA sequencing library product, high-flux sequence is carried out；Also that is, choosing the microbial gene of different G/C contents
Group sample and people genome sample carry out high throughput build library sequencing analysis, by analysis coverage rate, GC Preference, Q20,
The data such as Q30 carry out comprehensive assessment, and this builds library matched reagent box.
5. genomic DNA sequencing library fast construction method as claimed in claim 3, which is characterized in that specifically press following step
It is rapid to carry out:
1), sample genomic dna fragmentation
It takes 50ng sample genomic dna as fragmentation template, 10x enzyme cutting buffering liquid is added, 1ul fragmentation enzyme DNase I is
Deoxyribonuclease I is configured to the reaction system of 50ul, by following response procedures: 25 DEG C of 6min, and 85 DEG C of 10min carry out piece
Sectionization reaction, obtains the gDNA of fragmentation；
2), dA tail adds
6ul da- tail is added into the gDNA of fragmentation and adds buffer, and 2ul da- tail adds enzyme, is configured to the reaction of 60ul
System carries out dA tail addition: 72 DEG C of 15min by following response procedures, obtains dA- tail addition product；
3), connector connects
The T4 DNA ligase of addition 5ul high concentration into dA- tail addition product, the connection promotor of 30ul, 5ul10uM's
Illumina DNA connector, the 10X ligase buffer solution of 5ul, is configured to the system of 100ul, is connect by following response procedures
Head connection: 25 DEG C of 15min obtain connector connection product；
4), connector connection product magnetic beads for purifying
By connector connection product obtained in the previous step, pass through Hieff NGSTMDNA sort magnetic bead, with the magnetic bead ratio of 0.8x into
Row purification and recovery；
5), PCR amplification
2 × Super Canace of 25ul is added into the connector connection product by purification and recovery obtained in the previous stepTMHigh-fidelity
Archaeal dna polymerase premixed liquid, 5ul primary premixed liquid, is configured to the reaction system of 50ul, carries out PCR amplification by following procedure:
6), amplified production magnetic beads for purifying
By pcr amplification product obtained in the previous step, pass through Hieff NGSTMDNA sorts magnetic bead, is carried out with the magnetic bead ratio of 0.9x
Purification and recovery obtains genomic DNA sequencing library product；
7), to library production obtained in the previous step, detect library size with agarose gel electrophoresis, carry out segment distribution check and
Library evaluation.
6. genomic DNA sequencing library fast construction method as claimed in claim 5, which is characterized in that the sample gene
Group DNA is human cel gene group DNA；To library production obtained in the previous step, library size is detected with agarose gel electrophoresis
Obtain: the stripe size distribution of gradient is presented in human cel gene group DNA library, and the DNA fragmentation size in the library is located at
Between 100-700bp.
7. such as genomic DNA sequencing library fast construction method described in claim 5 or 6, which is characterized in that further include following
Step:
8), completely build library test optimization: endonuclease bamhi product continues library process and carries out completely building library test, Cong Jianku after combining
As a result start in terms of yield, library size, further do corresponding adjusting and optimizing；
9), to genomic DNA sequencing library product, high-flux sequence is carried out；Also that is, choosing the microbial gene of different G/C contents
Group sample and people genome sample carry out high throughput build library sequencing analysis, by analysis include coverage rate, GC Preference,
Data including Q20, Q30 carry out comprehensive assessment, and this builds library matched reagent box.
8. a kind of mating examination for by the method rapid build genomic DNA sequencing library as described in claim 2-7 is any
Agent box, which is characterized in that the matched reagent box includes: that DNase I i.e. deoxyribonuclease I, enzyme cutting buffering liquid, da- tail add
Add buffer, da- tail addition enzyme, T4 DNA ligase, connection promotor, DNA connector, high-fidelity DNA polymerase, polymerase slow
Fliud flushing, DNA sort magnetic bead.
9. matched reagent box as claimed in claim 8, which is characterized in that the high-fidelity DNA polymerase is high-fidelity DNA
Polymerase premixed liquid is Super CanaceTMHigh-Fidelity Mix；The polymerase buffer is primary premix
Liquid is Primer Mix.
10. matched reagent box as claimed in claim 9, which is characterized in that the matched reagent box includes: 1ul DNase
I i.e. deoxyribonuclease I, 10ul10X enzyme cutting buffering liquid, 6ul da- tail addition buffer, 2ul da- tail add enzyme, 5ul
The 10X connection of the T4 DNA ligase, the connection promotor, the Illumina DNA connector of 5ul10uM, 5ul of 30ul of high concentration
2 × Super Canace of enzyme buffer liquid, 25ulTMHigh-Fidelity Mix, 5ul Primer Mix, 0.8x DNA sorting
Magnetic bead, 0.9x DNA sort magnetic bead.
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| CN111269980A (en) * | 2020-02-11 | 2020-06-12 | 圣湘生物科技股份有限公司 | BRCA1/2 mutation detection composition, kit and library construction method |
| CN113073133A (en) * | 2021-04-01 | 2021-07-06 | 深圳易倍科华生物科技有限公司 | Method for amplifying trace amount of DNA and detecting multiple nucleic acids, and nucleic acid detecting apparatus |
| CN114736950A (en) * | 2022-02-24 | 2022-07-12 | 北京组学生物科技有限公司 | DNA next-generation sequencing library, and construction method and construction kit thereof |
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