CN105332063B - Construction method of single-tube and high-flux sequencing library - Google Patents
Construction method of single-tube and high-flux sequencing library Download PDFInfo
- Publication number
- CN105332063B CN105332063B CN201510496049.5A CN201510496049A CN105332063B CN 105332063 B CN105332063 B CN 105332063B CN 201510496049 A CN201510496049 A CN 201510496049A CN 105332063 B CN105332063 B CN 105332063B
- Authority
- CN
- China
- Prior art keywords
- probe
- stage
- sequencing library
- construction method
- annealing
- 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.)
- Active
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
The invention discloses a construction method of a single-tube and high-flux sequencing library. The method comprises the following steps: 1, respectively designing multiple pairs of basic amplimers for multiple target genes; 2, designing a pair of asymmetrically connected probes and a general primer not complementary to a mankind genome; 3, amplifying a template, the multiple pairs of basic amplimers, the asymmetric probes and the general primer in a PCR reaction system containing a DNA ligase and a DNA end modification enzyme to obtain an amplification product, wherein the amplification program sequentially comprises initial denaturation, first stage amplification and second stage amplification; and 4, purifying the amplification product to obtain the high-flux sequencing library. The construction method of the single-tube and high-flux sequencing library realizes single-tube rapid library completion of multiple target sequences can effectively solve the difficulty of polygene and multi-target detection of somatic cells of present clinic tumors, genetic diseases and other diseases on the basis of a small amount of clinic samples through combining a high-flux sequencing platform, and has low cost.
Description
Technical field
The invention belongs to detect biological technical field, and in particular to a kind of construction method of single tube high-throughput sequencing library.
Background technology
The crystallization that diagnostic technique in molecular biology is modern molecular biology to be made great progress with molecular genetics, be
People are produced on the basis of increasingly deepening to the understanding of life quintessence's problems such as the expression and regulation and control of the structure and gene of gene
's.In recent years, the methodological study of diagnostic technique in molecular biology has made great progress, and successively establishes restricted enzyme
The methods such as enzyme spectrum analysis, making nucleic acid molecular hybridization, restriction fragment length polymorphism linkage analysises.1985 public by U.S. Cetus
DNA Amplification Technologies (the Polymerase that department human geneticss research department Mullis etc. is founded and subsequently developed rapidly
Chain Reaction, PCR), and the DNA chip technology (DNA Chip) for growing up the nineties, and by molecular biology
Diagnostic techniquess bring up to a brand-new stage.
The core technology of most of molecular diagnostic laboratories be all concentrated mainly on detection specificity, relatively short DNA or
In RNA fragments.With going deep into for medical research, the mechanism of more and more diseases is relevant with the variation of related gene, such as loses
Hereditary diseases β-thalassemia is related to gene delection, hereditary hearing impairment and GJB2, SLC26A4 and mitochondrial DNA A1555G
Related to C1494T mutation, phenylketonuria has found more than 70 kinds gene mutation, Familial Occurrence breast in Chinese population
Adenocarcinoma is related to BRCA1/2 gene mutation etc..Therefore the gene mutation related to disease is presented polygenes, various variation types
(point mutation, insertion, disappearance).Clinic is in the urgent need to a kind of method that can detect polygenes, changeable ectopic sites simultaneously.
The development of molecular diagnostic techniques is paid much attention in countries in the world, and high-flux sequence becomes molecular diagnosis agents of new generation and opens
The main flow sent out.High-flux sequence is the crystallization of the combine multidisciplinarilies such as molecular biology, microelectronics, computer, is combined various existing
For high-quality precision and sophisticated technology.High-flux sequence has the function that can detect multiple target spots simultaneously, with fast and effectively feature.Thus
High-flux sequence becomes the main developing way of molecular diagnosis agents of new generation.
Existing high-throughput sequencing library construction method is broadly divided into two kinds：One kind is Ampliseq technologies, i.e., using multiple
The many target sequences of round pcr are expanded, and particular sequence is connected to amplified production two by DNA ligase by amplified production purification
End, purification is expanded again.This has the disadvantage that operating procedure is more, and library construction is of long duration, about needs 8 hours.It is another kind of
Be by probe capture technique, will sample DNA enter Break Row, filter out target length size, using with markd probe
Hybridized, combined with the probe of labelling using the magnetic bead of tape label, eluting is captured all target sequences, after carry out distinguished sequence
Connection and expand.The method has the disadvantage that hybridization efficiency is low, and library construction is of long duration about to need 24 hours.
The content of the invention
It is an object of the invention to overcome prior art defect, there is provided a kind of structure side of single tube high-throughput sequencing library
Method.
The concrete technical scheme of the present invention is as follows：
A kind of construction method of single tube high-throughput sequencing library, comprises the steps：
(1) separately design for some genes of interest some to basic amplimer, draw in the forward direction of basic amplimer
5 ' ends of thing and reverse primer are all connected with 2～5 T, and carry out PNA in 2～5 T on 3 ' first T for holding and repair
Decorations, it is unrelated with genes of interest, the efficiency of initial pcr amplification reaction is neither affected, is not also resulted in non-specific between primer pair
Property amplification so that amplified production can produce sticky end, and some Tm values differences to basic amplimer are less than 1 DEG C,
Corresponding annealing temperature is 56～64 DEG C；
(2) a pair asymmetric linking probes and are designed the universal primer of complementation is not formed with human genome, this pair is not
But symmetrical linking probe include a positive probe and the reverse probe for itself forming ring-type, the positive probe and reverse probe from
3 ' hold to 5 ' ends successively including one can with the complementary seriess of the 5 ' of the asymmetric linking probe some continuous base pairings in end, one
With the extension increasing sequence of the universal primer complementary pairing and a sticky end recognition sequence corresponding with above-mentioned 2～5 T-phase, on
The Tm values difference of positive probe and reverse probe is stated less than 1 DEG C, and above-mentioned positive probe and reverse probe itself form ring-type
Annealing temperature differ with the annealing temperature of basic amplimer less than 1 DEG C；
(3) by template, it is above-mentioned it is some basic amplimer, asymmetric probe, universal primer are placed in one containing DNA connect
Being expanded in the PCR reaction systems of enzyme and the end modified enzymes of DNA, obtained amplified production, time of the program of the amplification is 2～
3h, and include successively：Denaturation, first stage amplification and second stage amplification, wherein first stage amplification include first successively
Denaturation stage, the first annealing stage and first extend the cycle stage, and the first period for extending the cycle stage is 5～15, first
The annealing temperature of annealing stage be 60～65 DEG C, second stage amplification successively include the second denaturation stage, the second annealing stage and
Second extends the cycle stage, and the second period for extending the cycle stage is 15～30 (preferably 20)；First annealing stage is moved back
Fiery temperature is higher than the annealing temperature of the second annealing stage 4～8 DEG C；As the product of each circulation can be used as next circulation
Template, after first stage amplification, the product expanded by the basic amplimer of present invention design obtains substantial amounts of accumulation,
The product of these accumulation template again as second stage amplification, now universal primer can be with complete of these templates
Match somebody with somebody, joint efficiency is significantly lifted；
(4) by amplified production after purification, obtain final product the high-throughput sequencing library.
In a preferred embodiment of the invention, a labelling sequence is additionally provided between the complementary seriess and extension increasing sequence
Row, to be identified to different samples.
In a preferred embodiment of the invention, the length of the labelled sequence is 10～15bp.
In a preferred embodiment of the invention, the length of the complementary seriess is 3～5bp.
In a preferred embodiment of the invention, the length of the universal primer is 10～15bp.
In a preferred embodiment of the invention, the positive probe and reverse probe of the asymmetric linking probe
Length is no more than 50bp.
In a preferred embodiment of the invention, the DNA ligase is T4DNA ligases.
The invention has the beneficial effects as follows：
(1) construction method of single tube high-throughput sequencing library of the invention carries out single tube for multiple target sequences, quick complete
Into the structure in library, whole library construction process only needs 2～3 hours, needs only to manufal operation time 15～30 minutes, knot
Closing high-flux sequence platform can be with highly effective solution at present for facing in a small amount of in the clinically disease such as tumor, heredopathia
Need to detect this difficult point to somatic cell polygenes, Mutiple Targets on bed sample basis, and it is with low cost；
(2) library sequence prepared by construction method of the invention by current high-flux sequence system identification and can be examined
Survey, so as to realize that library construction carries out the application of the detection of nucleotide sequence, the detection of nucleic acids can be applicable to current various high fluxs
Microarray dataset, gene chip platform, hybridization check platform.
Description of the drawings
Fig. 1 is the schematic diagram of the first stage amplification and second stage amplification of the present invention；
Fig. 2 is the total Reads quantity of the high-flux sequence chip of the embodiment of the present invention 1
3 sample difference Reads quantity and average length of the Fig. 3 for the embodiment of the present invention 1
Matching degrees of the Fig. 4 for the sample data and target sequence of the embodiment of the present invention 1
Matching degree and homogeneity of the Fig. 5 for each sample of the embodiment of the present invention 1
Specific embodiment
Accompanying drawing is combined below by way of specific embodiment to be further detailed technical scheme and describe.
As shown in figure 1, the technical scheme is that：
A kind of construction method of single tube high-throughput sequencing library, comprises the steps：
(1) separately design for some genes of interest some to basic amplimer, draw in the forward direction of basic amplimer
5 ' the ends of thing F1 and reverse primer R1 are all connected with 2～5 T 10, and in 2～5 T 10 on 3 ' first T for holding
PNA modifications are carried out, less than 1 DEG C, corresponding annealing temperature is 56～64 DEG C to some Tm values differences to basic amplimer；
(2) a pair asymmetric linking probes and are designed the universal primer F3 of complementation, this pair is not formed with human genome
But asymmetric linking probe includes the forward direction probe F2 and a reverse probe R2, the positive probe F2 for itself forming ring-type and instead
Holding to probe R2 from 3 ' successively can be with some continuous base pairing in the 5 ' of asymmetric linking probe ends including one to 5 ' ends
The extension increasing sequence 13 of complementary seriess 11, a labelled sequence (barcode) 12, one and the universal primer F3 complementary pairings and one with
The corresponding sticky end recognition sequence 14 of above-mentioned 2～5 T-phase, the Tm values of above-mentioned positive probe F2 and reverse probe R2 are differed not
More than 1 DEG C, and above-mentioned positive probe F2 and reverse probe itself form the annealing temperature of ring-type and the annealing of basic amplimer
Temperature difference is less than 1 DEG C；
(3) by template, above-mentioned some basic amplimer F1 and R1, asymmetric probe F2 and R2, universal primer F3 are put
Expanded in the PCR reaction systems containing DNA ligase and the end modified enzymes of DNA, obtained amplified production, the journey of the amplification
The time of sequence is 2～3h, and is included successively：Denaturation, first stage amplification and second stage amplification, wherein first stage amplification
Include that the first denaturation stage, the first annealing stage and first extend the cycle stage successively, first extends the period of cycle stage
For 5～15, the annealing temperature of the first annealing stage is 60～65 DEG C, second stage amplification include successively the second denaturation stage, the
Two annealing stages and second extend the cycle stage, and the second period for extending the cycle stage is 15～30；First annealing stage
Annealing temperature is higher than the annealing temperature of the second annealing stage 4～8 DEG C；
(4) by amplified production after purification, obtain final product the high-throughput sequencing library.
Skilled person will appreciate that, the parameter of the present invention still is able to obtain and following enforcements when changing in the following ranges
The same or like technique effect of example：
The length of the labelled sequence is 10～15bp.
The length of the complementary seriess is 3～5bp.
The length of the universal primer F3 is 10～15bp.
The length of the positive probe F2 and reverse probe R2 of the asymmetric linking probe is no more than 50bp.
Embodiment 1
In the present embodiment, design is carried out for 5 gene common mutations sites of current tumor area correlation in the process of the present invention
Library construction system design in combination with high-flux sequence instrument Ion torrent PGM detection of platform EGFR, KRAS, BRAF,
The gene mutation such as PIK3CA, HER2, TP53.
Material：
1st, the preparation of sample and reference substance
Receive from clinical paraffin organization sample, sample is cut into 5-10 μm, add the dewaxing of 1ml dimethylbenzene, be collected by centrifugation
Precipitation, adds 1ml dehydrated alcohol in precipitation, and room temperature or 37 DEG C dry, and adds E.C. 3.4.21.64 and Buffer ATL, 56 DEG C of digestion
Cracking 1 hour, 90 DEG C of hatching 1h add 200ml Buffer AL mixings to add 200 μ L dehydrated alcohol and fully mix, will be upper
Carefully it is transferred in QIA 2ml centrifugal columns clearly, 6000 × g (8000rpm) is from 1min, 500 μ L Buffer AW1 of addition, 6000
From 1min, careful lid of opening adds 500 μ L Buffer AW2 to × g (8000rpm), and 6000 × g (8000rpm) is from 1min, empty
Pipe is centrifuged 20000 × g (14000rpm) from 3min, adds 100 μ L Buffer ATE central in film, incubation 5 minutes, 20000 ×
G (14000rpm) is from 1min.Take 3 μ L and survey OD values, the sample DNA of extraction is diluted to into 2ng/ μ L, take 5 μ L and enter performing PCR reaction.
2nd, the design and preparation of library primer
Show outside EGFR gene exons 1 8～21, KRAS gene extrons 2, BRAF gene according to the announcement of Cosmic data
Son 16, PIK3CA exon16s, HER2 exons 8,2 gene mutation gene sequencing of TP53 exons 1s, separately design a pair
Basic amplimer, it is as shown in the table, while be attached two T at 5 ' ends of the primer of following table, and to hold near 3 ' the
PNA modifications are carried out on one T：
Positive probe F2, the reverse probe R2 and universal primer F3 for designing asymmetric probe is as follows：
By more than, the primer and probe of design is sent to the synthesis of DNA Synesis Company, dilute with Tris-HCl (pH8.0) after having synthesized
Release.
3rd, library construction PCR amplifications
PCR reaction systems are：
1 × PCR buffer
The program of real time PCR amplification is：
Denaturation：95 DEG C, 5 minutes, 1 circulation；
First stage expands：95 DEG C of degeneration 30 seconds, 64 DEG C are annealed 4 minutes, and 72 DEG C extend 30 seconds, 13 circulations；
Second stage is expanded：95 DEG C of degeneration 30 seconds, 58 DEG C are annealed 4 minutes, and 72 DEG C extend 30 seconds, 20 circulations.
Obtain initial libraries product
4th, library purification
UseXP reagent 1.5x sample volumes
4.1 carefully throw off shrouding film and add 45 μ L's (1.5x sample volumes) in the initial libraries of above-mentioned preparationXP reagents.Being blown and beaten 5 times with rifle makes DNA fully mix with bead suspension, obtains mixed liquor.
Above-mentioned mixed liquor is placed in room temperature 5 minutes by 4.2.
96 orifice plates are placed in DynaMag by 4.3TMOn -96Side Magnet (Cat.no.12331D) magnetic frame, 2 points are stood
Clock waits solution turned clear.Supernatant is carefully siphoned away and discarded, magnetic bead should not be disturbed.
4.4 add freshly prepared 70% ethanol of 150 μ L in hole, move back and forth 96 orifice plates to wash magnetic on magnetic force
Pearl, then carefully supernatant discarded, should not disturb magnetic bead.
4.5 repeat the 4th step, carry out second washing.
4.6 guarantee that ethanol drop is all siphoned away from hole.Plate is positioned on magnetic frame, air at room temperature is dried 5 minutes,
It is careful not to over-drying.
96 orifice plates are taken away from magnetic frame by 4.7, add 50 μ L Low TE fully to infiltrate magnetic bead in every hole.Use96 orifice plates are tamping by adhesive film films, and fully vibration is mixed, and liquid is collected ttom of pipe by mild centrifugation.
(can also select the liquid of more than half is drawn with rifle blow and beat at least 5 times up and down to mix)
96 orifice plates are placed in magnetic frame upper 2 minute by 4.8.Containing library after purification in supernatant.5 μ L of supernatant are taken out, is tied
The seedless sour waters of 495 μ L are closed, it is quantitative for library.
5th, detect
Using libraries after purification of Ion torrent PGM high-flux sequence instrument (ThermoFisher companies) to acquisition
Carry out subsequent detection,
6th, interpretation of result
The total Reads quantity of 6.1 library constructions as shown in Figures 2 to 5, the ISP in the library that the embodiment builds
Loading reaches 85%, and average to read long in 116bp, total reads quantity has more than 410,000.
The above, only presently preferred embodiments of the present invention, therefore the scope of present invention enforcement can not be limited according to this, i.e.,
The equivalence changes made according to the scope of the claims of the present invention and description and modification, all still should belong in the range of the present invention covers.
Claims (7)
1. a kind of construction method of single tube high-throughput sequencing library, it is characterised in that：Comprise the steps：
(1) separately design for some genes of interest it is some to basic amplimer, basic amplimer forward primer and
5 ' ends of reverse primer are all connected with 2～5 T, and carry out PNA modifications in 2～5 T on 3 ' first T for holding, should
Less than 1 DEG C, corresponding annealing temperature is 56～64 DEG C to some Tm values differences to basic amplimer；
(2) a pair asymmetric linking probes and a universal primer that complementation is not formed with human genome are designed, this pair is or not
But claim linking probe to include a positive probe and a reverse probe, the positive probe and reverse probe of itself formation ring-type
Holding from 3 ' successively can be with the complementary sequence of some continuous base pairings in the 5 ' of asymmetric linking probe ends including one section to 5 ' ends
Row, one section know with the extension increasing sequence of the universal primer complementary pairing and one section of corresponding with above-mentioned 2～5 T-phase sticky end
The Tm values difference of other sequence, above-mentioned positive probe and reverse probe is less than 1 DEG C, and above-mentioned positive probe and reverse probe itself
The annealing temperature for forming ring-type is differed with the annealing temperature of basic amplimer less than 1 DEG C；
(3) by template, it is above-mentioned it is some one is placed in basic amplimer, asymmetric probe, universal primer containing DNA ligase and
Expanded in the PCR reaction systems of the end modified enzymes of DNA, obtained amplified production, the time of the program of the amplification is 2～3h, and
Include successively：Denaturation, first stage amplification and second stage amplification, wherein first stage amplification include the first degeneration rank successively
Section, the first annealing stage and first extend the cycle stage, and the first period for extending the cycle stage is 5～15, the first annealing rank
The annealing temperature of section is 60～65 DEG C, and second stage amplification includes that the second denaturation stage, the second annealing stage and second prolong successively
Stretch the cycle stage, the second period for extending the cycle stage is 15～30；The annealing temperature of the first annealing stage is than the second annealing
The annealing temperature in stage is high 4～8 DEG C；
(4) by amplified production after purification, obtain final product the high-throughput sequencing library.
2. a kind of construction method of single tube high-throughput sequencing library as claimed in claim 1, it is characterised in that：The complementary sequence
A segment mark sequence is additionally provided between row and extension increasing sequence, to be identified to different samples.
3. a kind of construction method of single tube high-throughput sequencing library as claimed in claim 2, it is characterised in that：The labelling sequence
The length of row is 10～15bp.
4. a kind of construction method of single tube high-throughput sequencing library as claimed in claim 1, it is characterised in that：The complementary sequence
The length of row is 3～5bp.
5. a kind of construction method of single tube high-throughput sequencing library as claimed in claim 1, it is characterised in that：It is described general to draw
The length of thing is 10～15bp.
6. a kind of construction method of single tube high-throughput sequencing library as claimed in claim 1, it is characterised in that：It is described asymmetric
The length of the positive probe and reverse probe of linking probe is no more than 50bp.
7. a kind of construction method of single tube high-throughput sequencing library as claimed in claim 1, it is characterised in that：The DNA connects
Enzyme is connect for T4DNA ligases.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510496049.5A CN105332063B (en) | 2015-08-13 | 2015-08-13 | Construction method of single-tube and high-flux sequencing library |
| PCT/CN2015/095393 WO2017024690A1 (en) | 2015-08-13 | 2015-11-24 | Construction method for a single tube high-throughput sequencing library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510496049.5A CN105332063B (en) | 2015-08-13 | 2015-08-13 | Construction method of single-tube and high-flux sequencing library |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105332063A CN105332063A (en) | 2016-02-17 |
| CN105332063B true CN105332063B (en) | 2017-04-12 |
Family
ID=55282816
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510496049.5A Active CN105332063B (en) | 2015-08-13 | 2015-08-13 | Construction method of single-tube and high-flux sequencing library |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN105332063B (en) |
| WO (1) | WO2017024690A1 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312822A (en) * | 2016-04-26 | 2017-11-03 | 厦门飞朔生物技术有限公司 | A kind of construction method in oncogene variation library detected for high-flux sequence and its application |
| CN107312770A (en) * | 2016-04-26 | 2017-11-03 | 厦门飞朔生物技术有限公司 | A kind of construction method in tumour BRCA1/2 genetic mutations library detected for high-flux sequence and its application |
| CN106498035A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for detecting chemotherapeutics gene SNP variation library for high-flux sequence |
| CN106497916A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method in the NK cell polygenic variations library for high-flux sequence detection and its application |
| CN106498036A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method in the Drug Discovery SNP variations library for high-flux sequence detection and its application |
| CN106591957A (en) * | 2016-11-10 | 2017-04-26 | 复旦大学附属中山医院 | Construction method and application of gastrointestinal stromal tumor polygenic variation library |
| CN106987906A (en) * | 2017-05-15 | 2017-07-28 | 重庆市肿瘤研究所 | The construction method in oncotherapy cardiac toxic predicted gene abrupt climatic change library |
| CN107475429A (en) * | 2017-09-28 | 2017-12-15 | 上海思路迪生物医学科技有限公司 | The primer and kit of more target library constructions are uniformed based on high-flux sequence |
| CN110714071B (en) * | 2018-07-12 | 2023-06-20 | 深圳华大智造科技股份有限公司 | High-throughput detection kit for human BRCA1/2 gene mutation |
| CN109897898A (en) * | 2019-02-14 | 2019-06-18 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for resetting library for high-flux sequence detection lymthoma IG and TCR |
| CN112064122B (en) * | 2020-09-30 | 2021-06-04 | 厦门飞朔生物技术有限公司 | Library construction method for detecting endometrial cancer related gene mutation based on high-throughput sequencing |
| CN112144125B (en) * | 2020-09-30 | 2021-06-15 | 厦门飞朔生物技术有限公司 | Library construction method for detecting bile duct cancer FGFR1/2/3 fusion gene based on high-throughput sequencing |
| CN113136435A (en) * | 2021-05-25 | 2021-07-20 | 上海锐品生物科技有限公司 | PCR technology-based detection kit for detecting PAX1 gene methylation and application thereof |
| CN114015749A (en) * | 2021-10-18 | 2022-02-08 | 浙江博圣生物技术股份有限公司 | Construction method of mitochondrial genome sequencing library based on high-throughput sequencing and amplification primer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796808A (en) * | 2011-05-23 | 2012-11-28 | 深圳华大基因科技有限公司 | Methylation high-flux detection method |
| CN103060924A (en) * | 2011-10-18 | 2013-04-24 | 深圳华大基因科技有限公司 | Library preparation method of trace nucleic acid sample and application thereof |
| CN104293940A (en) * | 2014-09-30 | 2015-01-21 | 天津华大基因科技有限公司 | Method for constructing sequencing library and application of sequencing library |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967684B (en) * | 2010-09-01 | 2013-02-27 | 深圳华大基因科技有限公司 | Sequencing library, preparation method thereof, and terminal sequencing method and device |
| US10017761B2 (en) * | 2013-01-28 | 2018-07-10 | Yale University | Methods for preparing cDNA from low quantities of cells |
| WO2014182574A1 (en) * | 2013-05-04 | 2014-11-13 | The Board Of Trustees Of The Leland Stanford Junior University | Enrichment of dna sequencing libraries from samples containing small amounts of target dna |
-
2015
- 2015-08-13 CN CN201510496049.5A patent/CN105332063B/en active Active
- 2015-11-24 WO PCT/CN2015/095393 patent/WO2017024690A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796808A (en) * | 2011-05-23 | 2012-11-28 | 深圳华大基因科技有限公司 | Methylation high-flux detection method |
| CN103060924A (en) * | 2011-10-18 | 2013-04-24 | 深圳华大基因科技有限公司 | Library preparation method of trace nucleic acid sample and application thereof |
| CN104293940A (en) * | 2014-09-30 | 2015-01-21 | 天津华大基因科技有限公司 | Method for constructing sequencing library and application of sequencing library |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105332063A (en) | 2016-02-17 |
| WO2017024690A1 (en) | 2017-02-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105332063B (en) | Construction method of single-tube and high-flux sequencing library | |
| JP6722179B2 (en) | Universal blocking oligo system for multiple capture reactions and improved hybridization capture method | |
| JP6783590B2 (en) | Amplified nucleic acid detection method and detection device | |
| JP6404714B2 (en) | Multivariate diagnostic assay and method for using the same | |
| CN104254617B (en) | Method of dna detection and quantification by single-molecule hybridization and manipulation | |
| CN107849607A (en) | The single-molecule sequencing of plasma dna | |
| CN105899680A (en) | Nucleic acid probe and method of detecting genomic fragments | |
| CN108611399A (en) | The multiple labelling of length dna segment | |
| CN107636166A (en) | The method of highly-parallel accurate measurement nucleic acid | |
| CN107922966B (en) | Sample preparation for nucleic acid amplification | |
| CN106834515A (en) | A kind of probe library of the exons mutation of detection MET genes 14, detection method and kit | |
| CN110036117A (en) | Increase the method for the treating capacity of single-molecule sequencing by multi-joint short dna segment | |
| CN109477245A (en) | Generate the method and kit of the DNA library for large-scale parallel sequencing | |
| CN106536735A (en) | Probe set for analyzing a dna sample and method for using the same | |
| CN106566876A (en) | Oligonucleotide probe and acquisition method thereof | |
| CN107475403A (en) | The analysis method of the method for detection Circulating tumor DNA, kit and its sequencing result from peripheral blood dissociative DNA | |
| US11359193B2 (en) | Preparation method for in-situ hybridization probe | |
| CN107312770A (en) | A kind of construction method in tumour BRCA1/2 genetic mutations library detected for high-flux sequence and its application | |
| CN108517567A (en) | Connector, primer sets, kit and the banking process in library are built for cfDNA | |
| CN114958997A (en) | Method for detecting chaperone gene | |
| KR101955080B1 (en) | Circulating cancer biomarker and its use | |
| CN111690748A (en) | Probe set and kit for detecting instability of microsatellite by using high-throughput sequencing and detection method for instability of microsatellite | |
| CN102943107A (en) | Method of analyzing target nucleic acid of biological samples | |
| CN108374047B (en) | Kit for detecting bladder cancer based on high-throughput sequencing technology | |
| CN106497916A (en) | A kind of construction method in the NK cell polygenic variations library for high-flux sequence detection and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Xi Ke Zhen Xi Zhou Lu, Tong'an District, Xiamen City, Fujian Province, No. 2041 361000 Applicant after: XIAMEN SPACEGEN BIOTECHNOLOGY CO., LTD. Address before: 361000 Fujian Province, Haicang District of Xiamen City No. 11 2602A clock Etna Applicant before: XIAMEN SPACEGEN BIOTECHNOLOGY CO., LTD. |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |