CN102061335A - Asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using second-generation high-throughput sequencing technology and application thereof - Google Patents
Asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using second-generation high-throughput sequencing technology and application thereof Download PDFInfo
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Abstract
The invention provides asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using a second-generation high-throughput sequencing technology, which is composed of two DNA oligonucleotide single strands. The preparation method comprises the following steps: dissolving the two DNA oligonucleotide single strands into a quenching solution; regulating the final concentration to 2mM and volume to 20microlitre; carrying out a quenching reaction to form the DNA artificial adapters which are locally and mutually complemented at a temperature of 95 DEG C for 5 minutes; reducing the temperature of 95 DEG C to 12 DEG C at the speed of 0.1 DEG C per second; keeping the temperature of 12 DEG C, and diluting an adapter solution after quenching to 500 mu M at a ratio of 1:4; and storing at the temperature of minus 20 DEG C. An application of the asymmetric DNA artificial adapters by using the second-generation high-throughput sequencing technology is as follows: A, asymmetric DNA adapters are in ligation with DNA samples; B, non-connected DNA artificial adapters are purified and isolated by using electrophoresis tapping; C, judgment is carried out; and D, a polymerase chain reaction (PCR) amplification reaction is performed based on an asymmetric sequence. The asymmetric DNA artificial adapters by using the second-generation high-throughput sequencing technology has the obvious advantages that: 1) the usage of original DNA samples for establishing a library is reduced to 50 nanogram and the sensitivity of the original DNA samples is improved by 100 times; and 2) 100% of effective sequencing samples are produced in the process of adapter connection for establishing the library.
Description
Technical field:
The present invention relates to a kind of two generation high-flux sequence double-stranded joint of asymmetric D NA and application thereof.
Background technology:
In the end of the year 2005,454 Corp. has released Genome Sequencer 20 System of ultra-high throughput gene order-checking system based on the tetra-sodium sequencing of novelty.Released the more excellent s-generation gene order-checking system of performance in 2007 again: Genome Sequencer FLX System.454 high throughput sequencing technologies when order-checking, has used " Pico TiterPlate " flat board (PTP) of a kind of being called, and it contains more than 160 ten thousand holes of being made up of optical fiber, is loaded with chemiluminescence reaction required various enzymes and substrate in the hole.When beginning order-checking is placed on four kinds of bases in four independent reagent bottles, and circulating successively according to the order of T, A, C, G enters the PTP plate, only enters a base at every turn.If the generation base pairing will discharge a tetra-sodium.This tetra-sodium is under the effect of various enzymes, and through a building-up reactions and a chemiluminescence reaction, fluorescein is oxidized to oxyluciferin the most at last, discharges optical signal simultaneously.The optical signal that this reaction discharges is captured by the high-sensitive CCD of instrument configuration in real time.There are a base and sequencing template to match, will capture the optical signal of a part; Corresponding one by one thus, just can determine the base sequence of template to be measured accurately and rapidly.
The committed step of order-checking experiment flow is the library preparation of DNA sample.The standard method of the library of 454 platforms preparation is: with genomic dna or transcribe the fragment that cDNA that the group reverse transcription becomes interrupted and selected 400-800bp.After end was repaired, (the B joint has vitamin H (Biotin) to carry out tack (blunt-end) and is connected (ligation), and the result forms the fragment of three kinds of different splice combinations: AA, AB, BB for the joint A different with two kinds and B.To connect (ligation) product through with the magnetic bead adhesion of streptavidin (Streptavidin), wash-out, alkaline denaturation, treatment step such as wash-out again, being recovered to two ends at last is single stranded DNAs (sstDNA) of A and B joint sequence, is used for next step latex PCR (emulsion-PCR) and tetra-sodium sequencing reaction.
The library preparation of 454 sequenced dna samples, bigger to the sample size requirement of DNA/cDNA, at least 5 microgram samples.Simultaneously, the efficient of the technology of the library of DNA sample preparation is lower, and is low as the efficient of tack (blunt-end) connection own; 50% connection (ligation) product (AA, BB) is invalid; And step is more loaded down with trivial details, need carry out the detection of a plurality of steps to the DNA amount, not only needs expensive instrument, and the 2100Bioanalyzer (instrument name) as Agilent (u s company) also will consume a lot of consumptive materials and reagent.
Summary of the invention:
In order to solve the problems of the technologies described above, the invention provides a kind of two generation high-flux sequence double-stranded joint of asymmetric D NA and application thereof.
The technical solution adopted for the present invention to solve the technical problems is: a kind of two generation high-flux sequence the double-stranded joint of asymmetric D NA, described two generation high-flux sequence the double-stranded joint of asymmetric D NA form by two DNA oligonucleotide strands, its preparation method is as follows:
Described DNA oligonucleotide strand is dissolved in the quenching solution, adjusts ultimate density and reach 2mM, volume 20 microlitres utilize the reaction of quenching of following condition then, form local complementary dna double chain joint: in 95 ℃ 5 minutes; 95 ℃ drop to 12 ℃, 0.1 ℃ of per second; Remain on 12 ℃, the joint solution after the annealing is diluted to 500 μ M at 1: 4, and-20 ℃ of preservations.
Above-mentioned two DNA oligonucleotide strands can adopt following flag sequence, thereby form 3 different, local complementary dna double chain joints, Y1-ACGAGTGCGT, Y2-AGCGTCGACT, Y3-TGACGCACCT.
Simultaneously, the present invention also provides the banking process of the double-stranded joint of asymmetric D NA of a kind of application this high-flux sequence in two generation, and it adopts following flow process:
The ligation of A, asymmetric D NA joint and dna sample:
After the dna sample fragment uses atomising method to interrupt, behind Qiagen MinElute PCR purification kit purifying, after terminal reparation and 5 ' phosphorylation, utilize Klenow archaeal dna polymerase and dATP to add 3 ' A-tail end, then 3 ' the A-tail end DNA sample that forms is connected with the double-stranded joint of aforesaid asymmetric D NA;
The dna double chain joint that B, electrophoresis rubber tapping purifies and separates do not connect:
To the dna sample after the ligation, earlier behind Qiagen MinElute PCR purification kit purifying, carry out electrophoretic separation with 2.0% agaropectin again, extract the agar blob of viscose that comprises DNA of 400-800bp position, utilize QIAquick Gel Extraction Kit that DNA is reclaimed;
C, judgement
, can directly enter 454 emPCR reactions steps, and then check order quantitatively greater than 10 nanograms as the DNA that reclaims; , also can carry out PCR by following step D and amplify in the emPCR step that directly enters 454 as the DNA quantity not sufficient that reclaims, electrophoresis rubber tapping purifying is removed the unreacted dna primer of PCR then, enters 454 emPCR reactions steps at last;
D, amplify reaction based on the PCR of asymmetric sequence:
Recovery DNA with the B step is a template, with following two oligonucleotide strand primers
PA：5’-CCATCTCATCCCTGCGTGTCTC-3’70C
PB：5’-CCTATCCCCTGTGTGCCTTGGCA-3’66C
Dna profiling is carried out 10-30 cycle P CR amplify reaction.
Further:
In above-mentioned flow process A, 3 ' A-tail end DNA sample with the condition that the double-stranded joint of asymmetric D NA is connected is: 0.1 μ g DNA/ μ l; The asymmetric joint of 20 μ M; 2mM ATP; 1/20 T4 dna ligase; Insulation at 14 ° above 6 hours.
In above-mentioned flow process D, described PCR amplifies the condition of reacting the solution that is adopted and is: 2 μ M PA primers; 2 μ M PB primers; 1x Phusion Master Mix.
In above-mentioned flow process D, it is as follows that PCR amplifies institute's temperature condition that adopts of reaction: the 1st goes on foot: in 98 ℃ 30 seconds; The 2nd step: in 98 ℃ 10 seconds; The 3rd step: in 65 ℃ 45 seconds; The 4th step: in 72 ℃ 30 seconds; The 5th step: repeat 2-4 and go on foot 10-30 time; The 6th step: in 72 ℃ 5 seconds; The 7th step: remain in 4 ℃.
The present invention is with respect to building the storehouse for 454 platforms of high-flux sequence several once significant advantages are arranged when the first two: 1) build storehouse initiate dna amount of samples and be low to moderate 50 nanograms, sensitivity and improve and reach 100 times, 454 platforms are built the initiate dna sample that the storehouse needs the 3-5 microgram before this; 2) the joint connection procedure of building the storehouse can produce effective order-checking sample of 100%, and 454 platforms are built the storehouse sample that effectively checks order and only accounted for connection and connected 50% of product before this; The joint efficiency of joint is higher when 3) building the storehouse, adopt in the art of this patent be 3 ' A-tail end DNA with stick terminal the connection with the outstanding joint of 5 ' T, and 454 platforms adopt flat end connection before this; 4) simplified and build the storehouse flow process, shortened the stability of building the storehouse time, having improved technology, 454 platforms are built the storehouse and need be adopted various step to handle the DNA sample before this, finally to obtain effective single-stranded DNA banks; 4) the DNA library that builds is stable, easy long-time the preservation, and the DNA library that utilizes the art of this patent finally to build is two strands, and before this 454 platforms be strand.
Description of drawings:
Fig. 1 interrupts working drawing for the dna sample fragment by atomization.
Fig. 2 builds quality monitoring figure in the process of storehouse for the DNA sample.
Fig. 3 is the DNA staple diagram.
Among the figure: 1, blank; 2, the standard DNA sample of Agilent; 3, the dna sample of smashing with atomization; 4, connect the dna library of 3 ' A-tail end before the double-stranded joint; 5, connect the dna library behind the double-stranded joint; 6, use the dna sample of ultrasonic grinding.
Embodiment:
The present invention is further described below in conjunction with drawings and Examples.
A kind of two generation high-flux sequence the double-stranded joint of asymmetric D NA, constitute by two DNA oligonucleotide strands, its preparation method is as follows:
Described DNA oligonucleotide strand is dissolved in the quenching solution, adjusts ultimate density and reach 2mM, volume 20 microlitres utilize following condition to carry out double-stranded joint annealing: 5min in 95 ℃ then; 95 ℃ drop to 12 ℃, 0.1 ℃ of per second; Remain on 12 ℃, the double-stranded joint solution after the annealing is diluted to 500 μ M at 1: 4, and-20 ℃ of preservations.
Described two DNA oligonucleotide strands can adopt following flag sequence:
Be the banking process of the double-stranded joint of asymmetric D NA of using this high-flux sequence below, it adopts following flow process in two generation: (Fig. 2 builds quality monitoring figure in the process of storehouse for the DNA sample, and in conjunction with shown in Figure 1)
1) the interrupting of long segment gDNA or cDNA (atomization):
Prepare atomization damping fluid: 10ml (total amount)
Glycerine 5.31ml
1M?Tris-HCl(pH?7.5)0.37ml
0.5M?EDTA(pH?8.0)0.11ml
The super hydrostatic of VWR (product of the VWR of u s company) 4.21ml
Dna sample (0.05-1 μ g) dissolved in atomization damping fluid (200 μ l) and put into the cup of spraying gun, feed nitrogen, pressure 30psi, time 1min.Reclaim the dna sample that atomizing is smashed.((also can use hyperacoustic method to DNA, but the method that the rate of recovery is smashed not as atomizing, with reference to figure 3).
2) DNA mends gentle cleaning:
Prepare dna filling-in liquid: 20 μ l (total amount)
Polysaccharase holds liquid (10X NEB T4 DNA: the 6 μ l product of U.S. New England Biolab company)
The super hydrostatic of VWR (product of the VWR of u s company) 4.5 μ l
The bovine serum albumin 0.5 μ l of 100X U.S. New England Biolab company
25mM dNTP mother liquor 2.5 μ l
100mM ATP (mononucleotide) 0.5 μ l
3U/ μ l T4 DNA Polymerase (archaeal dna polymerase) 3 μ l
10U/ μ l T4 Polynucleotide Kinase (oligonucleotide kinases) 3 μ l
Add the dna sample 40 μ l after atomizing is smashed during the DNA filling-in, follow these steps to react:
In 12 ℃ 20 minutes
In 25 ℃ 20 minutes
In 75 ℃ 20 minutes
After finishing, reaction clears up and recovery DNA (32 μ l) with QIAquick PCR Purification Kit (U.S. Qiagen company product).
3) add 3 ' A tail end to dna fragmentation:
Prepare dna adds the reaction solution of 3 ' A-tail end: 50 μ l (total amount)
Dna sample 32 μ L
Klenow solution 5 μ L
dATP?10μL
Klenow excision enzyme (3 ' to, 5 ' the circumscribed shortage) 3 μ L
Then reaction solution was handled 30 minutes at 37 ℃.After finishing, reaction clears up and recovery DNA (10 μ L) with Qiagen MinElute PCR Purification kit (U.S. Qiagen company product).
4) dna fragmentation of 3 ' A-tail end connects the double-stranded joint of asymmetric D NA:
Prepare dna ligation liquid: 50 μ l (total amount)
Nuclease free water (Nuclease-free water) 4 μ L
Ligase enzyme solution (DNA ligase buffer) 25 μ L
The double-stranded joint of asymmetric D NA: 6 μ L
Ligase enzyme (DNA ligase) 5 μ L
Reaction was finished in about 6 hours at 14 ° reaction solution insulation.After finishing, reaction clears up and recovery DNA (20 μ l) with QIAquick PCR Purification Kit (U.S. Qiagen company product).
5) electrophoresis rubber tapping purifying extracts the dna fragmentation that connects double-stranded joint
Prepare 2.0% the Agarose glue and the electrophoresis chamber of packing into, pack into DNA sample fragment and low molecular dna mark with the TAE damping fluid.Run the adhesive tape part: the about 45min of 120V cuts out the adhesive tape of 400-800bp position under UV light with sterile blade.Utilize Qiagen Gel Extraction Kit (U.S. Qiagen company product) that dna sample is reclaimed.The DNA that reclaims can directly enter 454 emPCR reactions steps, and then check order (if directly carry out emPCR after this step, need carry out effectively the quantitative of (being the dna fragmentation that two ends all connect joint) to the DNA that reclaims, carry out qPCR with the amplification of the PCR in this programme primer and can carry out accurately quantitatively).
6) based on asymmetric sequence dna sample is carried out PCR and amplify reaction
(can carry out this step working the lower situation of storehouse sample size of establishing, not enough 500ng can consider as sample size)
The DNA that step 4 reclaims can directly enter 454 emPCR reactions steps.If the DNA quantity not sufficient that reclaims can carry out PCR by this step and amplify, reclaim enough DNA after, in the emPCR reactions steps that enters 454.
Reclaiming DNA with step 4 is template, is primer with two sequences that describe below,
PA：5’-CCATCTCATCCCTGCGTGTCTC
PB：5’-CCTATCCCCTGTGTGCCTTGGCA
Carry out 10-30 cycle P CR according to the amount of template and amplify reaction.PCR amplifies the final condition of reacting the solution that is adopted: μ M PA primer; 2 μ M PB primers; 1x Phusion Master Mix (product of U.S. New England Biolab company) [see handbook: " Phusion High Fidelity Master Mix User ' s Guide (Finnzymes) "]; The DNA that connects double-stranded joint.
The temperature condition that PCR amplification reaction is adopted is as follows:
The 1st step: in 98 ℃ 30 seconds
The 2nd step: in 98 ℃ 10 seconds
The 3rd step: in 65 ℃ 45 seconds
The 4th step: in 72 ℃ 30 seconds
The 5th step: repeat the 2-4 step, 10-30 time
The 6th step: in 72 ℃ 5 minutes
The 7th step: in 4 ℃, keep
PCR utilizes the method identical with step B after amplifying reaction, and electrophoresis rubber tapping purifying is removed the unreacted dna primer of PCR.The DNA that reclaims can directly enter 454 emPCR reactions steps, and then checks order.
Claims (6)
- One kind two generation high-flux sequence the double-stranded joint of asymmetric D NA, it is characterized in that: described two generation high-flux sequence the double-stranded joint of asymmetric D NA form by two DNA oligonucleotide strands, its preparation method is as follows:Described DNA oligonucleotide strand is dissolved in the quenching solution, adjusts ultimate density and reach 2mM, volume 20 microlitres utilize the reaction of quenching of following condition then, form local complementary dna double chain joint: in 95 ℃ 5 minutes; 95 ℃ drop to 12 ℃, 0.1 ℃ of per second; Remain on 12 ℃, the joint solution after the annealing is diluted to 500 μ M at 1: 4, and-20 ℃ of preservations.
- 2. according to claim 1 two generation high-flux sequence the double-stranded joint of asymmetric D NA, it is characterized in that: described two DNA oligonucleotide strands adopt following flag sequence:
- An application rights require 1 described two generation high-flux sequence the banking process of the double-stranded joint of asymmetric D NA, it is characterized in that: described banking process adopts following flow process:The ligation of A, asymmetric D NA joint and dna sample:After the dna sample fragment uses atomising method to interrupt, behind Qiagen MinElute PCR purification kit purifying, after terminal reparation and 5 ' phosphorylation, utilize Klenow archaeal dna polymerase and dATP to add 3 ' A-tail end, then 3 ' the A-tail end DNA sample that forms is connected with the double-stranded joint of aforesaid asymmetric D NA;The dna double chain joint that B, electrophoresis rubber tapping purifies and separates do not connect:To the dna sample after the ligation, earlier behind Qiagen MinElute PCR purification kit purifying, carry out electrophoretic separation with 2.0% agaropectin again, extract the agar blob of viscose that comprises DNA of 400-800bp position, utilize QIAquick Gel Extraction Kit that DNA is reclaimed;C, judgement, can directly enter 454 emPCR reactions steps, and then check order quantitatively greater than 10 nanograms as the DNA that reclaims; , also can carry out PCR by following step D and amplify in the emPCR step that directly enters 454 as the DNA quantity not sufficient that reclaims, electrophoresis rubber tapping purifying is removed the unreacted dna primer of PCR then, enters 454 emPCR reactions steps at last;D, amplify reaction based on the PCR of asymmetric sequence:Recovery DNA with the B step is a template, with following two oligonucleotide strand primersPA：5’-CCATCTCATCCCTGCGTGTCTC-3’70CPB：5’-CCTATCCCCTGTGTGCCTTGGCA-3’66CDna profiling is carried out 10-30 cycle P CR amplify reaction.
- 4. the banking process of the double-stranded joint of asymmetric D NA of a kind of application two generations high-flux sequence according to claim 3, it is characterized in that: in described flow process A, 3 ' A-tail end DNA sample with the condition that the double-stranded joint of asymmetric D NA is connected is: 0.1 μ g DNA/ μ l; The asymmetric joint of 20 μ M; 2mMATP; 1/20 T4 dna ligase; Insulation at 14 ° above 6 hours.
- 5. the banking process of the double-stranded joint of asymmetric D NA of a kind of application two generations high-flux sequence according to claim 3, it is characterized in that: in described flow process D, the solution that described PCR amplification reaction is adopted is: 2 μ M PA primer 2 μ M PB primer 1x Phusion Master Mix.
- 6. according to claim 3 a kind of use two generation high-flux sequence the banking process of the double-stranded joint of asymmetric D NA, it is characterized in that: in described flow process D, it is as follows that PCR amplifies institute's temperature condition that adopts of reaction: the 1st goes on foot: in 98 ℃ 30 seconds; The 2nd step: in 98 ℃ 10 seconds; The 3rd step: in 65 ℃ 45 seconds; The 4th step: in 72 ℃ 30 seconds; The 5th step: repeat 2-4 and go on foot 10-30 time; The 6th step: in 72 ℃ 5 seconds; The 7th step: remain in 4 ℃.
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Cited By (13)
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| CN102296065A (en) * | 2011-08-04 | 2011-12-28 | 盛司潼 | System and method for constructing sequencing library |
| CN102978205A (en) * | 2012-11-19 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing junction applied to marker development and application method thereof |
| CN104862302A (en) * | 2015-05-05 | 2015-08-26 | 华南师范大学 | DNA (Deoxyribose Nucleic Acid) fragmentation method and device for implementing same |
| CN105154444A (en) * | 2015-10-15 | 2015-12-16 | 南京普东兴生物科技有限公司 | Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers |
| CN105567681A (en) * | 2015-12-31 | 2016-05-11 | 广州赛哲生物科技股份有限公司 | Noninvasive biopsy virus detection method based on high throughput gene sequencing and tagged connector |
| WO2018006567A1 (en) * | 2016-07-08 | 2018-01-11 | 北京全式金生物技术有限公司 | High-efficiency method for linking dna connector |
| CN108034705A (en) * | 2018-01-15 | 2018-05-15 | 武汉爱基百客生物科技有限公司 | A kind of full-length genome methylates high-flux sequence method |
| CN108148899A (en) * | 2018-01-15 | 2018-06-12 | 武汉爱基百客生物科技有限公司 | A kind of genome simplifies and two generations sequencing SNP compound detections system and detection method |
| CN108300716A (en) * | 2018-01-05 | 2018-07-20 | 武汉康测科技有限公司 | Joint component, its application and the method that targeting sequencing library structure is carried out based on asymmetric multiplex PCR |
| CN109971826A (en) * | 2014-01-31 | 2019-07-05 | 斯威夫特生物科学股份有限公司 | Improved method for processing DNA substrate |
| CN110791813A (en) * | 2018-08-01 | 2020-02-14 | 广州华大基因医学检验所有限公司 | Method for processing single-stranded DNA and application |
| WO2020043174A1 (en) * | 2018-08-31 | 2020-03-05 | 成都先导药物开发股份有限公司 | High-throughput next-generation sequencing method for double-stranded nucleotide sequencing |
| CN111041069A (en) * | 2019-12-26 | 2020-04-21 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1618962A (en) * | 2004-11-12 | 2005-05-25 | 南京大学 | Method of preparing DNA linker |
| CN101503733A (en) * | 2008-12-17 | 2009-08-12 | 上海人类基因组研究中心 | DNA cohesive end joint facilitating high throughput gene sequence label sequencing and use |
| CN101802223A (en) * | 2007-08-15 | 2010-08-11 | 香港大学 | methods and compositions for high-throughput bisulphite dna-sequencing and utilities |
| CN101845500A (en) * | 2010-05-18 | 2010-09-29 | 苏州众信生物技术有限公司 | Method for correcting sequence abundance deviation of secondary high-flux sequence test by DNA sequence bar codes |
-
2010
- 2010-11-15 CN CN201010544306.5A patent/CN102061335B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1618962A (en) * | 2004-11-12 | 2005-05-25 | 南京大学 | Method of preparing DNA linker |
| CN101802223A (en) * | 2007-08-15 | 2010-08-11 | 香港大学 | methods and compositions for high-throughput bisulphite dna-sequencing and utilities |
| CN101503733A (en) * | 2008-12-17 | 2009-08-12 | 上海人类基因组研究中心 | DNA cohesive end joint facilitating high throughput gene sequence label sequencing and use |
| CN101845500A (en) * | 2010-05-18 | 2010-09-29 | 苏州众信生物技术有限公司 | Method for correcting sequence abundance deviation of secondary high-flux sequence test by DNA sequence bar codes |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296065B (en) * | 2011-08-04 | 2013-05-15 | 盛司潼 | System and method for constructing sequencing library |
| CN102296065A (en) * | 2011-08-04 | 2011-12-28 | 盛司潼 | System and method for constructing sequencing library |
| CN102978205A (en) * | 2012-11-19 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing junction applied to marker development and application method thereof |
| CN102978205B (en) * | 2012-11-19 | 2014-08-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing junction applied to marker development and application method thereof |
| CN109971826A (en) * | 2014-01-31 | 2019-07-05 | 斯威夫特生物科学股份有限公司 | Improved method for processing DNA substrate |
| CN104862302A (en) * | 2015-05-05 | 2015-08-26 | 华南师范大学 | DNA (Deoxyribose Nucleic Acid) fragmentation method and device for implementing same |
| CN104862302B (en) * | 2015-05-05 | 2020-12-15 | 华南师范大学 | DNA fragmentation method and device for implementing same |
| CN105154444A (en) * | 2015-10-15 | 2015-12-16 | 南京普东兴生物科技有限公司 | Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers |
| CN105567681B (en) * | 2015-12-31 | 2018-08-31 | 广州赛哲生物科技股份有限公司 | A kind of method and label connector based on the noninvasive biopsy virus of high-throughput gene sequencing |
| CN105567681A (en) * | 2015-12-31 | 2016-05-11 | 广州赛哲生物科技股份有限公司 | Noninvasive biopsy virus detection method based on high throughput gene sequencing and tagged connector |
| WO2018006567A1 (en) * | 2016-07-08 | 2018-01-11 | 北京全式金生物技术有限公司 | High-efficiency method for linking dna connector |
| CN108300716A (en) * | 2018-01-05 | 2018-07-20 | 武汉康测科技有限公司 | Joint component, its application and the method that targeting sequencing library structure is carried out based on asymmetric multiplex PCR |
| CN108300716B (en) * | 2018-01-05 | 2020-06-30 | 武汉康测科技有限公司 | Linker element, application thereof and method for constructing targeted sequencing library based on asymmetric multiplex PCR |
| CN108148899A (en) * | 2018-01-15 | 2018-06-12 | 武汉爱基百客生物科技有限公司 | A kind of genome simplifies and two generations sequencing SNP compound detections system and detection method |
| CN108034705A (en) * | 2018-01-15 | 2018-05-15 | 武汉爱基百客生物科技有限公司 | A kind of full-length genome methylates high-flux sequence method |
| CN110791813A (en) * | 2018-08-01 | 2020-02-14 | 广州华大基因医学检验所有限公司 | Method for processing single-stranded DNA and application |
| CN110791813B (en) * | 2018-08-01 | 2023-06-16 | 广州华大基因医学检验所有限公司 | Method for processing single-stranded DNA and application thereof |
| WO2020043174A1 (en) * | 2018-08-31 | 2020-03-05 | 成都先导药物开发股份有限公司 | High-throughput next-generation sequencing method for double-stranded nucleotide sequencing |
| CN111041069A (en) * | 2019-12-26 | 2020-04-21 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
| CN111041069B (en) * | 2019-12-26 | 2021-01-19 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
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