CN105200530A - Method for establishing multi-sample hybrid library suitable for high-flux whole-genome sequencing - Google Patents
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Abstract
The invention provides a method for establishing a multi-sample hybrid library suitable for high-flux whole-genome sequencing. The method comprises the following steps: breaking genome DNA of a plurality of samples, distributing a concentration zone at 500bp, simultaneously carrying out the 5'-end restoration and adding a basic group A to a 3' end after the sample is broken, connecting DNA fragments having 5'phosphoration and 3' viscous end A of different samples and connectors containing different barcode sequences, carrying out low-circulation PCR for a connection product, purifying and mixing an amplified product to obtain a multi-sample hybrid sequencing library suitable for the high-flux whole-genome sequencing. The method has the advantages of high flux, low cost and short time, the generated library sequencing quality is high, the method is suitable for establishing a large-scale multi-sample sequencing library, and the application prospect is very wide in the research such as high-flux SNP detection and identifier development, genetic map drawing, whole-genome correlation analysis and the like.
Description
Technical field
The present invention relates to field of bioinformatics, specifically, relate to a kind of construction process being applicable to the Multi-example mixing sequencing library of high-flux sequence.
Background technology
In the order-checking of two generations, usually Illumina, NEB or Vazyme test kit Library development flow used is mature and stable, after sample has interrupted, the follow-up DNA chain end carried out is repaired, 3 ' end adds base step A does not all need purifying, next step reaction can be carried out, therefore build storehouse process simple and efficient, and it is high to build Kucheng's power; But build storehouse for multiple sample, test kit conveniently builds storehouse, then complex operation, especially the library fragments in later stage is selected, need single sample to be carried out respectively electrophoresis cut glue screening fragment or utilize magnetic bead to screen fragment, cause building storehouse cost intensive, need consume a large amount of time and build storehouse expense.The high-throughput of prior art simplifies gene order-checking library constructing method mainly for not with reference to genomic sample in addition, cut prediction by enzyme to carry out enzyme and cut and interrupt, build library, randomness is good not, again by removing tumor-necrosis factor glycoproteins and other redundant sequences, by remaining data analysis, obtain relevant associated region, accuracy is not high enough.
Summary of the invention
The object of this invention is to provide a kind of construction process being applicable to the Multi-example mixing sequencing library of high-throughput genome sequencing, to make up in prior art the deficiency building such library longer, complex operation consuming time.
The construction process being applicable to the Multi-example mixing sequencing library of high-throughput genome sequencing provided by the invention, comprises the following steps:
(1) genomic dna of different sample interrupts, and obtains the DNA fragmentation concentrating band to be distributed in 500bp;
(2) sample DNA of having no progeny of simultaneously fighting each other carries out the flat end reparation of 5 ' phosphorylation and 3 ' end interpolation base A, obtains the DNA fragmentation with 5 ' phosphorylation and 3 ' sticky end A;
(3) respectively 5 ' the phosphorylation that has of different sample is connected with the joint containing the unique barcode sequence can distinguishing sample with the DNA fragmentation of 3 ' sticky end A, obtains the connection product can distinguishing sample source;
(4) respectively pcr amplification is carried out to the connection product containing different barcode, obtain the amplified production containing different bar code sequence, purifying, quantitatively, mixed sample; Mixed PCR primer carries out sepharose separation and purification, and purified product forms the multiple sample mixing sequencing libraries being suitable for high-throughput genome sequencing.
In the inventive method, the genomic dna concentration described in step (1) is 20 ~ 100ng/ μ L.Described genomic dna be through agarose gel electrophoresis detect and ultraviolet spectrophotometer method detection.
Wherein, the sample gene group DNA of step (1) also comprises magnetic beads for purifying step after interrupting.
In the inventive method, the reaction system of step (2) is:
Wherein, the reaction conditions of step (2) is: 20 DEG C of 30min, 65 DEG C of 30min.
In the inventive method, the sequence of step (3) joint contains the normal chain of 8 base bar code sequences and the minus strand of 8 base bar code sequences.
Step (3) described joint, its sequence is:
Normal chain 5 '-3 ':
AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCTTTCCCTACACGACGCTCTTCCGATCT
Minus strand 5 '-3 ':
CAAGCAGAAGACGGCATACGAGATYYYYYYYYGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
Wherein: the XXXXXXXX in normal chain represents normal chain bar code sequence, the YYYYYYYY in minus strand represents minus strand bar code sequence.
The bar code sequence of above-mentioned positive and negative chain is the random combine of A, T, C, G tetra-kinds of bases, can design according to the actual needs of those skilled in the art; The bar code sequence of positive and negative chain need make for distinguishing sample simultaneously.
In step (4), pcr amplification primer sequence used is:
P1:5’-3’：AATGATACGGCGACCGAGATCTACAC
P2:5’-3’:CAAGCAGAAGACGGCATACGAGAT。
In the inventive method, the PCR response procedures of step (4) is: 95-98 DEG C of 3min; 95-98 DEG C of 10sec, 65 DEG C of 30sec, 72 DEG C of 30-40sec, totally 10 circulations; 72 DEG C of 5min.
It is that the fragment cutting 650bp lower edges carries out purifying that step (4) mixed PCR primer carries out sepharose separation and purification.
The invention provides the application of above-mentioned construction process in Multi-example genome association is analyzed.
In the step (4) of the inventive method, after purifying, the quantitative detecting method of amplified production can be ultraviolet spectrophotometer method, nucleic acid fluorescent quantitative method, agarose gel electrophoresis detection.
The inventive method is the improvement on Vazyme test kit banking process, the method of building storehouse is carried out for batch sample, magnetic beads for purifying is carried out after sample interrupts, DNA end reparation afterwards, 3 ' end add base step A and all operate on 96 orifice plates, greatly improve flux and efficiency, when jointing, add the both-end Index that can identify different sample simultaneously, PCR carries out enrichment, is then mixed in the library after different pcr amplification, cuts glue carry out library fragments selection finally by electrophoresis.The method is compared with traditional method, and 96 orifice plate batch operations can improve flux greatly, becomes a library after final multiple sample mixes sample, selects fragment step saves a large amount of reagent cost, human cost and time at follow-up glue of cutting.The method is specially adapted to resurvey sequence and the low genome of the order-checking degree of depth of the little biological genome of genome and resurveys sequence.
Technical scheme of the present invention can bring following beneficial effect:
(1) sample initial amount requires to reduce.In the prior art, conventional single library construction needs at least 5 μ g samples, therefore higher to the demand of sample, cause the rare sample of part because less not the reaching of total amount builds library standard, the minimizing of purification step in Library development flow of the present invention, initial amount can be reduced to 2.5 μ g by 5 original μ g, the DNA initial amount of this level, both reduced the requirement to sample, also meet the output to follow-up data and analysis, and the reduction of initial amount had made to build storehouse reagent cost reduction simultaneously simultaneously;
(2) for the sample had with reference to genome sequence, carry out machinery and interrupt at random, build library, final literature data covers full-length genome, and randomness is better; By order-checking gained all data by with compare with reference to genome, therefore higher to the SNP site of interpretation of result, Indel site accuracy;
(3) before mixed sample, the advanced performing PCR amplification of each sample, namely PCR enriched product is obtained, carry out quantitative and mixed sample again, if first carry out mixed sample, first mixed sample can be caused irregular due to the content difference of different sample, and the difference of this sample room can be amplified further by the PCR process after mixed sample, therefore larger on the output impact of later stage different sample data amount, and the present invention first carries out pcr amplification to each sample, and then carry out mixed sample, this operation can avoid mixed sample error to be amplified further after pcr amplification, makes the homogeneity of sample better;
(4) pcr amplification has Preference, in pcr amplification process, primer may be partial in conjunction with genomic certain section of region, a large amount of amplification this section of region, therefore the product obtained there will be genomic covering uneven, and then redundancy is high to make sequencing data occur, so, in experimentation, in order to avoid too high and the reducing genomic coverage of causing of later data redundancy, the present invention by the design of the cycle number of pcr amplification lower than 10 circulations, like this, effectively can reduce the Preference that pcr amplification brings, ensure that the validity of data analysis;
(5) in conventional Library development flow, single sample needs to cut glue screening fragment respectively, each sample runs glue from point sample, electrophoresis, contaminate glue, cut glue to expend time in length to purifying, waste time and energy especially for Multi-example, and present design carries out cutting glue after being mixed by sample, such as, 100 samples cut the glue time, the present invention can be utilized completely required time to be down to the time of 1 sample, therefore to greatly reduce time and cost that single sample electrophoresis cuts glue.
For the library construction of 100 samples, conventionally build storehouse, building the storehouse cycle reaches 10 working dayss, and utilizes the inventive method 100 samples can be built storehouse cycle time to 4 working days, greatly saves and builds the storehouse time.On the other hand, to resurvey sequence for resurvey sequence and the low genome of the order-checking degree of depth of the biological genome that genome is little, library is built separately according to existing routine techniques, not only require higher to genomic dna initial amount, and it is higher to build storehouse reagent cost, utilizing the inventive method, reduce the DNA initial amount building storehouse, making to build this reduction of Kucheng by reducing using amount of reagent simultaneously.
Accompanying drawing explanation
Fig. 1 is the distribution plan of data analysis Insert Fragment after the embodiment of the present invention 1 checks order.
Fig. 2 is the distribution plan of data analysis Insert Fragment after the embodiment of the present invention 2 checks order.
Fig. 3 is the distribution plan of data analysis Insert Fragment after the embodiment of the present invention 3 checks order.
Fig. 4 is the schema that the Multi-example mixing sequencing library of high-flux sequence of the present invention builds.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.Biochemical reagents used in following examples are commercially available.
In following embodiment, its sequence of the joint of use is:
Normal chain 5 '-3 ':
AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCTTTCCCTACACGACGCTCTTCCGATCT
Minus strand 5 '-3 ':
CAAGCAGAAGACGGCATACGAGATYYYYYYYYGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
Wherein: the XXXXXXXX in normal chain represents normal chain bar code sequence, the YYYYYYYY in minus strand represents minus strand bar code sequence.
In following embodiment, pcr amplification primer sequence used is:
P1:5’-3’：AATGATACGGCGACCGAGATCTACAC
P2:5’-3’:CAAGCAGAAGACGGCATACGAGAT。
Embodiment 120 paddy rice samples mix the construction process (1) in sample library
Select 20 paddy rice samples, library construction comprises the steps:
1, genomic DNA fragment and purifying are got 20 paddy rice sample gene group DNA5 μ g respectively and are moved into the low absorption centrifuge tube of 1.5mL, use ddH
2o mends to cumulative volume 55 μ L, and utilize Ultrasonic Cell Disruptor to carry out machinery to sample and interrupt, the time of interrupting is 5min, and electrophoresis detection concentrates band to be positioned at 500bp.Residue interrupts product and is determining that getting half after segment ranges correctly interrupts product (25 μ L), carries out purifying with the AMpure magnetic bead (45 μ L) of 1.8 times of volumes, finally with 18 μ LddH
2o carries out back dissolving.
2, end reparation, add reagent to interrupting in product after the 1st step purifying, reaction system is as follows:
Reaction system is placed in constant-temperature metal bath 20 DEG C and hatches 30min, carry out purifying with the AMpure magnetic bead (45 μ L) of 1.8 times of volumes, finally with 17 μ LddH
2o carries out back dissolving.
3,3 '-end adds A reaction, and in the product after the 2nd step purifying, add reagent, reaction system is:
Reaction system is placed in constant-temperature metal bath 37 DEG C and hatches 30min, carries out purifying with the AMpure magnetic bead (45 μ L) of 1.8 times of volumes, finally with 30 μ LddH
2o carries out back dissolving.
4, jointing reaction, in the product after the 3rd step purifying, add reagent, wherein C4-N is the joint with Index, synthesizes in Takara) reaction system is as follows:
Reaction system is placed in constant-temperature metal bath 20 DEG C and hatches 30min, carries out purifying with the AMpure magnetic bead (72 μ L) of 1.8 times of volumes, finally with 30 μ LddH
2o carries out back dissolving.
5, pcr amplification PCR reaction system:
Pcr amplification is carried out: 98 DEG C of 3min according to the program of following settings; 98 DEG C of 10sec, 65 DEG C of 30sec, 72 DEG C of 30sec, 10 circulations; 72 DEG C of 5min.
Get 5 μ LPCR products, carry out electrophoresis (1.8% sepharose) with 100bpDNALadderMarker, detect between 500bp-700bp whether have disperse.Purifying is carried out, finally with 30 μ LddH with the AMpure magnetic bead (40 μ L) of 1 times of volume
2o carries out back dissolving.
6, Concentration Testing and mixed sample
The PCR primer of getting after 2 μ L above-mentioned steps purifying carries out Nanodrop detection.Mixed sample is carried out according to detected result.By unified for the sample Cmin value be diluted in sample, each sample is got 150ng and is carried out mixed sample.
7, cut glue to reclaim and get the mixed PCR purified product of 600ng and carry out sepharose (2%) electrophoretic separation, cut the fragment of 650bp lower edges.QIAquickGelExtractionKit purifying is utilized to cut film segment, 35 μ LElutionBuffer wash-outs.
8, corresponding reagent is added: Q5E is that Q5 surpasses fidelity dna polysaccharase.
Pcr amplification is carried out: 98 DEG C of 3min according to the program of following settings; 98 DEG C of 10sec, 65 DEG C of 30sec, 72 DEG C of 30sec, 10 circulations; 72 DEG C of 5min.
Get 5 μ LPCR products, carry out electrophoresis (1.8% sepharose) with 100bpDNALadderMarker, detect whether clip size is about 650bp, carry out purifying with the AMpure magnetic bead (40 μ L) of 1 times of volume, finally with 30 μ LddH
2o carries out back dissolving.
9, the sequencing library that Library Quality controls the 8th step obtains carries out quality examination on Agilent2100Bioanalyzer, and segment ranges meets substantially cuts glue scope.
10, check order and to check order being diluted to proper concn by the library of quality inspection in the 9th step, order-checking platform is IlluminaHighSeq2500, and order-checking length is both-end 125bp, and the Reads Insert Fragment size distribution that order-checking produces is shown in Fig. 1.Insert Fragment main peak is distributed between 400-500bp as seen from Figure 1, but between 150-400bp, there is a large amount of small segment, reason may be that the global cycle number of twice PCR is too much, small segment excessive amplification caused, in addition, pcr amplification cycle number too much can cause data redudancy high, genome coverage low (see table 1), the redundancy of 1X data is up to 70%, genome coverage is on average only 20%, be not enough to carry out genetic map analysis, therefore, carry out embodiment 2, flow process is optimized, save second step PCR process, reduce total PCR cycle number.
Table 1 single sample data measured amount, data redudancy and genome coverage
In the present embodiment, because pcr amplification cycle number is too much, cause later data to analyze Insert Fragment abnormal, data redudancy is high, and coverage is low, is not enough to carry out genetic map analysis.
Embodiment 220 paddy rice samples mix sample sequencing library construction process (2)
The present embodiment selects 20 paddy rice samples, on the basis of embodiment 1 method, is optimized flow process, saves second step PCR process, thus reduces total PCR cycle number.Library construction comprises the steps:
1, genomic DNA fragment and purifying
Get genomic dna 5 μ g and move into the low absorption centrifuge tube of 1.5mL, use ddH
2o mends to cumulative volume 55 μ L, utilize Ultrasonic Cell Disruptor to carry out machinery to sample to interrupt, the time of interrupting is 5min, electrophoresis detection concentrates band to be positioned at 500bp, residue interrupts product and is determining that getting half after segment ranges correctly interrupts product (25 μ L), purifying is carried out, finally with 18 μ LddH with the AMpure magnetic bead (45 μ L) of 1.8 times of volumes
2o carries out back dissolving.
2, end reparation, add reagent to interrupting in product after the 1st step purifying, reaction system is as follows:
Reaction system is placed in constant-temperature metal bath 20 DEG C and hatches 30min, carry out purifying with the AMpure magnetic bead (45 μ L) of 1.8 times of volumes, finally with 17 μ LddH
2o carries out back dissolving.
3,3 '-end adds A reaction, and in the product after the 2nd step purifying, add reagent, reaction system is:
Reaction system is placed in constant-temperature metal bath 37 DEG C and hatches 30min, carries out purifying with the AMpure magnetic bead (45 μ L) of 1.8 times of volumes, finally with 30 μ LddH
2o carries out back dissolving.
4, jointing reaction, in the product after the 3rd step purifying, add reagent, reaction system is as follows:
Reaction system is placed in constant-temperature metal bath 20 DEG C and hatches 30min, carries out purifying with the AMpure magnetic bead (72 μ L) of 1.8 times of volumes, finally with 30 μ LddH
2o carries out back dissolving.
5, pcr amplification adds corresponding reagent:
Pcr amplification is carried out: 98 DEG C of 3min according to the program of following settings; 98 DEG C of 10sec, 65 DEG C of 30sec, 72 DEG C of 30sec, 10 circulations; 72 DEG C of 5min.
Get 5 μ LPCR products, carry out electrophoresis (1.8% sepharose) with 100bpDNALadderMarker, detect between 500bp-700bp whether have disperse, carry out purifying with the AMpure magnetic bead (40 μ L) of 1 times of volume, finally with 30 μ LddH
2o carries out back dissolving.
6, Concentration Testing and mixed sample
The PCR primer of getting after 2 μ L above-mentioned steps purifying carries out Nanodrop detection.Mixed sample is carried out according to detected result.By unified for the sample Cmin value be diluted in sample, each sample is got 150ng and is carried out mixed sample.
7, cut glue to reclaim and get the mixed PCR purified product of 600ng and carry out sepharose (2%) electrophoretic separation, cut the fragment of 650bp lower edges.QIAquickGelExtractionKit purifying is utilized to cut film segment, 35 μ LElutionBuffer wash-outs.
8, Library Quality controls
The sequencing library that 7th step obtains is carried out quality examination on Agilent2100Bioanalyzer, and segment ranges meets 650bp size substantially.
9, check order and to check order being diluted to proper concn by the library of quality inspection in the 8th step, order-checking platform is IlluminaHighSeq2500, and order-checking length is both-end 125bp.In the present embodiment, the main peak of Insert Fragment is positioned at about 400-500bp, coincidence theory value, see Fig. 2, meanwhile, conclusion as can be drawn from Table 2, the redundancy of data is only 1%, illustrate same area on genome repeat lower, therefore surveyed data distribution uniform on genome, genome coverage is high
Meet the structure of genetic map.But, in the present embodiment library construction process, end reparation, add A reaction and add joint reaction carry out respectively, all need to carry out purifying after each step simultaneously, not only expend the operating time, also make DNA further loss in each step purification step, therefore for above-mentioned deficiency, carry out embodiment 3, the flow process of mixed sample being built to storehouse is optimized further.
Table 2 single sample data measured amount, data redudancy and genome coverage
The construction process of embodiment 3 20 paddy rice sample mix sequencing libraries of the present invention
Select 20 the paddy rice samples identical with the first two embodiment, library construction comprises the steps:
1, genomic DNA fragment and purifying
Get genomic dna 2.5 μ g and move into the low absorption centrifuge tube of 1.5mL, use ddH
2o mends to cumulative volume 55 μ L, utilize Ultrasonic Cell Disruptor to carry out machinery to sample to interrupt, the time of interrupting is 5min, and electrophoresis detection determination fragment concentrates scope to be 500bp, the AMpure magnetic bead (45 μ L) that residue interrupts product 1.8 times of volumes carries out purifying, finally with 27 μ LddH
2o carries out back dissolving.
2, end reparation, add reagent respectively to interrupting in product after the 1st step purifying, reaction system is as follows:
Pressure-vaccum mixing also brief centrifugation, reacts in PCR instrument according to following program.After reaction terminates, carry out next step joint connection immediately.20℃30min；65℃30min。
3, jointing reaction, in the 2nd step reaction product, add reagent, reaction system is as follows:
Reaction system is placed in constant-temperature metal bath 20 DEG C and hatches 15min, carries out purifying with the AMpure magnetic bead (96 μ L) of 1.8 times of volumes, finally with 30 μ LddH
2o carries out back dissolving.
4, pcr amplification adds corresponding reagent according to following table: Q5E is that Q5 surpasses fidelity dna polysaccharase.
Pcr amplification is carried out: 98 DEG C of 3min according to the program of following settings; 98 DEG C of 10sec, 65 DEG C of 30sec, 72 DEG C of 30sec, 10 circulations; 72 DEG C of 5min.
Get 5 μ LPCR products, carry out electrophoresis (1.8% sepharose) with 100bpDNALadderMarker, detect between 500bp-700bp whether have disperse, carry out purifying with the AMpure magnetic bead (40 μ L) of 1 times of volume, finally with 30 μ LddH
2o carries out back dissolving.
5, the PCR primer after 2 μ L above-mentioned steps purifying got by Concentration Testing and mixed sample carries out Nanodrop detection.Mixed sample is carried out according to detected result.By unified for the sample Cmin value be diluted in sample, each sample is got 150ng and is carried out mixed sample.
6, cut glue recovery and mixed PCR purified product is carried out sepharose (2%) electrophoretic separation, cut the fragment of 650bp lower edges.QIAquickGelExtractionKit purifying is utilized to cut film segment, 35 μ LElutionBuffer wash-outs.
7, the sequencing library that Library Quality controls the 6th step obtains carries out quality examination on Agilent2100Bioanalyzer, and segment ranges meets 650bp size substantially.
8, check order and to check order being diluted to proper concn by the library of quality inspection in the 7th step, order-checking platform is IlluminaHighSeq2500, and order-checking length is both-end 125bp.
The present invention is through the optimization gradually of embodiment 1-3, and finally obtain the method for Multi-example genome sequencing library construction in embodiment 3, schema is shown in Fig. 4.The inventive method (embodiment 3) is relative to embodiment 1 and 2, in step prepared by library, more save the operating time, data aspect, Insert Fragment normal in size, is shown in Fig. 3, and data redudancy reduces, genome coverage higher (see table 3), meets the structure carrying out genetic map.
Table 3 single sample data measured amount, data redudancy and genome coverage
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. be applicable to a construction process for multiple sample mixing sequencing libraries of high-throughput genome sequencing, comprise the following steps:
(1) genomic dna of different sample interrupts, and obtains the DNA fragmentation concentrating band to be distributed in 500bp;
(2) sample DNA of having no progeny of simultaneously fighting each other carries out the flat end reparation of 5 ' phosphorylation and 3 ' end interpolation base A, obtains the DNA fragmentation with 5 ' phosphorylation and 3 ' sticky end A;
(3) respectively 5 ' the phosphorylation that has of different sample is connected with the joint containing the unique barcode sequence can distinguishing sample with the DNA fragmentation of 3 ' sticky end A, obtains the connection product can distinguishing sample source;
(4) respectively pcr amplification is carried out to the connection product containing different barcode, obtain the amplified production containing different bar code sequence, purifying, quantitatively, mixed sample; Mixed PCR primer carries out sepharose separation and purification, and purified product forms the multiple sample mixing sequencing libraries being suitable for high-flux sequence.
2. construction process according to claim 1, is characterized in that, the genomic dna concentration described in step (1) is 50 ~ 100ng/ μ L.
3. construction process according to claim 1, is characterized in that, the reaction system of step (2) is:
4. construction process according to claim 1, is characterized in that, the reaction conditions of step (2) is: 20 DEG C of 30min, 65 DEG C of 30min.
5. construction process according to claim 1, is characterized in that, the sequence of step (3) joint contains the normal chain of 8 base bar code sequences and the minus strand of 8 base bar code sequences.
6. construction process according to claim 1, is characterized in that, step (3) described joint, and its sequence is:
Normal chain 5 '-3 ':
AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCTTTCCCTACACGACGCTCTTCCGATCT
Minus strand 5 '-3 ':
CAAGCAGAAGACGGCATACGAGATYYYYYYYYGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
Wherein: the XXXXXXXX in normal chain represents normal chain bar code sequence, the YYYYYYYY in minus strand represents minus strand bar code sequence.
7. according to the arbitrary described construction process of claim 1-6, it is characterized in that, in step (4), pcr amplification primer sequence used is:
P1:5’-3’：AATGATACGGCGACCGAGATCTACAC
P2:5’-3’:CAAGCAGAAGACGGCATACGAGAT。
8., according to the arbitrary described construction process of claim 1-6, it is characterized in that, the PCR response procedures of step (4) is: 95 DEG C of-98 DEG C of 3min; 95 DEG C of-98 DEG C of 10sec, 65 DEG C of 30sec, 72 DEG C of 30-40sec, totally 10 circulations; 72 DEG C of 5min.
9., according to the arbitrary described construction process of claim 1-6, it is characterized in that, it is that the fragment cutting 650bp lower edges carries out purifying that step (4) mixed PCR primer carries out sepharose separation and purification.
10. the application of the arbitrary described construction process of claim 1-9 in Multi-example genome association is analyzed.
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| CN108504651A (en) * | 2017-02-27 | 2018-09-07 | 深圳市乐土精准医疗科技有限公司 | The library constructing method and reagent in library are built in PCR product large sample size mixing based on high-flux sequence |
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| CN110872615A (en) * | 2018-08-31 | 2020-03-10 | 成都先导药物开发股份有限公司 | A high-throughput next-generation sequencing method for sequencing nucleotide duplexes |
| WO2022199242A1 (en) * | 2021-03-25 | 2022-09-29 | 南方医科大学 | Set of barcode linkers and medium-flux multi-single-cell representative dna methylation library construction and sequencing method |
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