CN108517567B - Adaptor, primer group, kit and library construction method for cfDNA library construction - Google Patents
Adaptor, primer group, kit and library construction method for cfDNA library construction Download PDFInfo
- Publication number
- CN108517567B CN108517567B CN201810359753.XA CN201810359753A CN108517567B CN 108517567 B CN108517567 B CN 108517567B CN 201810359753 A CN201810359753 A CN 201810359753A CN 108517567 B CN108517567 B CN 108517567B
- Authority
- CN
- China
- Prior art keywords
- cfdna
- sequence
- primer
- library
- seq
- 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
Images
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a connector, a primer group, a kit and a library construction method for cfDNA library construction, wherein the connector comprises a top connector and a bottom connector which are respectively shown as SEQ ID NO 1 and SEQ ID NO 2; the primer group comprises sequences shown as SEQ ID NO. 4-6; the kit comprises the joint and the primer group; the method for cfDNA library construction comprises the following steps: (1) performing end repair on the cfDNA, and adding an A base at the 3' end of the cfDNA; (2) connecting the end of the cfDNA with a linker to obtain a ligation product; (3) performing first round amplification on the connection product, and performing magnetic bead purification on the product; (4) and (4) performing library building amplification on the first round amplification product after magnetic bead purification, and obtaining a high-throughput sequencing library after magnetic bead purification of the product. The library obtained by the adaptor and the cfDNA library construction method is suitable for an Illumina sequencing platform and can improve the conversion efficiency of the library.
Description
Technical Field
The invention relates to the technical field of molecular genetics, in particular to a connector, a primer group, a kit and a library construction method for cfDNA library construction.
Background
Plasma cell-free DNA (cfDNA) is extracellular DNA existing in human blood in a cell-free state, and a certain amount of cfDNA can be detected in body fluids such as urine, cerebrospinal fluid, bronchoalveolar lavage fluid, amniotic fluid, semen and the like of a human body and in vitro cell culture fluid. Since the discovery of cfDNA by mantel and Metais in 1947, with the rapid development of molecular detection technology, more than 300 kinds of plasma free DNA have been detected, and the same gene mutation as tumor cell DNA is observed, so that cfDNA as a marker has potential value in clinical disease diagnosis, prognosis, detection and the like. In recent years cfDNA detection has been used in the diagnosis and prognostic evaluation of a variety of acute or chronic diseases.
Today, high-throughput analysis of cfDNA is commonly performed using Next Generation Sequencing (NGS) technology to see which genes have mutations and to determine the next step in the diagnosis based on the mutation information. The NGS technology takes Hiseq and Nextseq sequencers of Illumina as representative second generation sequencing technologies, and has the advantages of high detection flux (hundreds of G of gene data can be generated in each operation), wide coverage (the sequencing analysis of human whole genome can be carried out), high cost performance (the average cost per G is only dozens of money), and the like, so the NGS technology is particularly suitable for the parallel detection of multiple mutation types of multiple genes. However, the NGS technology firstly needs to construct a DNA library, and in the existing library construction process, the steps are complicated, and multiple purification processes are needed, which may cause DNA loss, and at the same time, a linker self-ligation phenomenon may occur in the linker ligation process, resulting in low library construction efficiency and poor quality. Or the method comprises the steps of end repair, purification after adding A, purification after connecting a connector, amplification, purification and the like. Although the purification steps are reduced and the loss of DNA is reduced, the problem of self-connection of the joint is still difficult to solve.
Disclosure of Invention
In view of the above, the invention aims to provide a linker, a primer group, a kit and a library construction method for cfDNA library construction, and the method can effectively eliminate random mutation introduced by PCR reaction in the library construction process, eliminate false positive and greatly improve the accuracy of NGS data analysis.
In view of the above objects, the present invention provides an adaptor for cfDNA library construction, wherein the adaptor is a double-stranded adaptor comprising a top adaptor and a bottom adaptor, and the sequence of the top adaptor is: 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGCCGCCGCCGCCG T-3' which is represented by SEQ ID NO: 1; the sequence of the bottom linker is: 5 ' -P-CGGCGGCGGCGGC-3 ' which is shown in SEQ ID NO. 2, and 5 ' end is phosphorylated; wherein the 12 bases "NNNNNNNNNNNN" in the top linker are the tag sequence.
The present invention designed a special adaptor (as shown in fig. 1), the 3' end of which (green) is a fixed fragment with high GC content, and the prominent T is used to ligate with the repaired cfDNA with a; the middle of the joint is a random single molecular label sequence of 12 basic groups (yellow), and the label can realize the labeling of all initial library building template nucleic acid fragments so as to effectively eliminate random mutation introduced by PCR reaction in the library building process, eliminate false positive and greatly improve the accuracy of NGS data analysis; the 5' end of the adaptor is a partial sequence (purple) of a primer for building the library P5, after the cfDNA is connected with the adaptor, the sequence can be directly used as an upstream non-specific primer and a specific downstream primer of fusion gene containing a P7 sequence for carrying out multiple PCR, and the obtained target fragment is amplified to build the library.
Where N in the top linker is a random base (i.e., a random nucleotide), each N independently can be any one of A, G, C, T. Since N is a random base, different sequences may be formed when N is different bases, and the mixture of different double stranded sequences formed by annealing these different sequences constitutes the adaptor of the present invention. Therefore, in the present invention, the random adaptor used is a mixture containing a plurality of different double-stranded sequences annealed by different sequences formed when N is different bases.
The preparation method of the joint comprises the following steps: diluting the two synthesized sequences to a concentration of 30 mu M respectively; then respectively taking 20 mul of the mixture to be uniformly mixed, and transferring the mixture into a PCR tube; placing the mixed solution in a PCR instrument, setting the temperature at 95 ℃, and incubating for 5 min; after the incubation is finished, the PCR instrument is directly closed, and the incubation is continued for 2 hours to obtain the prepared joint.
The invention also provides a primer group for cfDNA library construction, wherein the primer group comprises a sequence shown in SEQ ID NO. 4-6.
Further, the invention also provides a cfDNA library construction kit, which comprises the adaptor and the primer group.
Further, the present invention also provides a method for pooling cfDNA, comprising the steps of:
(1) performing end repair on the cfDNA, and adding an A base at the 3' end of the cfDNA;
(2) connecting the end of the cfDNA obtained after the treatment in the step (1) with a linker containing a random tag sequence to obtain a ligation product, and performing magnetic bead purification on the ligation product;
(3) performing first round amplification on the ligation product by using a universal front primer and a specific rear primer, and performing magnetic bead purification on the first round amplification product;
(4) and performing library construction and amplification on the first round of amplified products after magnetic bead purification by using primers P7 and P5, and performing magnetic bead purification on the library construction and amplification products to obtain a high-throughput sequencing library.
In some embodiments of the invention, in step (2), the linker is a double-stranded linker comprising a top linker and a bottom linker, the sequence of the top linker being: 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGCCGCCGCCGCCG T-3' which is represented by SEQ ID NO: 1; the sequence of the bottom linker is: 5 ' -P-CGGCGGCGGCGGC-3 ' which is shown in SEQ ID NO. 2, and 5 ' end is phosphorylated; wherein the 12 bases "NNNNNNNNNNNN" in the top linker are the tag sequence.
In some embodiments of the invention, in step (3), the sequence of the universal pre-primer is: 5'-ACACTCTTTCCCTACACGACGCTCTTC CGATCT-3', which is represented by SEQ ID NO. 3; the sequence of the specific rear primer is as follows: 5 '-CAGACGTGTGCTTCCGATCTxxxxxxxxxxxxx-3', shown in SEQ ID NO: 4. The sequence of the universal front primer is a partial sequence of an illiminia pool P5 primer, the sequence of 'CAGACGTGTGCTCTTCCGATCT' in the specific rear primer is a partial sequence of an illiminia pool P7 primer, and 'xxxxxxxxxxxxxxxxx' can be designed according to different target sequences. After the cfDNA is connected with the adaptor, multiple PCR can be directly carried out by taking a universal front primer as an upstream non-specific primer and taking fusion gene containing a P7 sequence as a specific rear primer, and the obtained target fragment is amplified and subjected to library construction.
In some embodiments of the invention, in step (4), the sequence of the P5 primer: 5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC T-3' which is represented by SEQ ID NO: 5; sequence of the P7 primer: 5'-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', which is represented by SEQ ID NO. 6.
As can be seen from the above, the present invention has the following advantageous effects:
the library obtained by the adaptor and cfDNA library construction method is suitable for an Illumina sequencing platform and can improve the conversion efficiency of the library. The database construction method provided by the invention is simple to operate, the sequencing result is stable, and the sequencing cost is low. After adding a molecular tag sequence to a joint, the invention can identify and classify the sequencing result of the amplification product belonging to the same original template through the molecular tag after PCR and sequencing. Theoretically, certain DNA molecules derived from the same molecular tag should carry the same sequence, so that real gene mutation and systematic errors can be identified through the molecular tag, the systematic errors introduced in the PCR and sequencing processes are removed, the final sequencing result is prevented from being interfered by the systematic errors, and the result of improving the sensitivity of cfDNA sequencing is finally achieved.
Drawings
FIG. 1 is a schematic view of a joint of the present invention;
FIG. 2 is a flow chart of cfDNA pooling of the present invention;
FIG. 3 is a capillary electrophoresis of a library;
FIG. 4 is a diagram showing the results of analysis of the fusion gene.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and accompanying drawings.
In the present invention, the cfDNA library building process is shown in fig. 2, and includes: (1) performing end repair on the cfDNA, and adding an A base at the 3' end of the cfDNA; (2) connecting the end of the cfDNA obtained after the treatment in the step (1) with a linker containing a random tag sequence to obtain a ligation product, and performing magnetic bead purification on the ligation product; (3) amplifying the connecting product by using a universal front primer and a specific rear primer, and purifying the amplified product by using magnetic beads; (4) and amplifying the products after magnetic bead purification by using primers P7 and P5, and purifying the amplified products by using magnetic beads to obtain a high-throughput sequencing library.
Example 1 construction method of cfDNA library
(1) DNA end repair reaction
To a 200. mu.l PCR tube (placed on ice, immediately following the reaction) were added the following reagents:
the total volume of the solution in the tube at this time was 65. mu.l.
This end repair reaction included the addition of an "A" to the 3' end.
According to the instruction of QIAamp Circulating nucleic Acid Kit (50) provided by QIAGEN company, the Kit is used for extracting free cfDNA in plasma;
and secondly, lightly blowing and sucking the solution by using a gun head, uniformly mixing, and centrifuging for a short time to collect all components to the bottom of the tube.
Putting the PCR tube into a PCR instrument, wherein the reaction procedure is as follows:
37℃ 10min
72℃ 10min
Hold on 4℃
(2) adaptor connection
Adding the following reagents directly into the reaction solution after the DNA end repair is completed:
the total volume of the solution in the tube at this time was 83.5. mu.l.
The joint is a double-link joint and comprises a top joint and a bottom joint, and the sequence of the top joint is as follows: 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGCCGCCGCCGCCG T-3' which is represented by SEQ ID NO: 1; the sequence of the bottom linker is: 5 ' -P-CGGCGGCGGCGGC-3 ' as shown in SEQ ID NO 2, and 5 ' end is phosphorylated.
The preparation method of the joint comprises the following steps: the two sequences (SEQ ID NO:1 sequence and SEQ ID NO:2 sequence) synthesized are respectively diluted to the concentration of 30 mu M; then respectively taking 20 mul of the mixture to be uniformly mixed, and transferring the mixture into a PCR tube; placing the mixed solution in a PCR instrument, setting the temperature at 95 ℃, and incubating for 5 min; after the incubation is finished, the PCR instrument is directly closed, and the incubation is continued for 2 hours to obtain the prepared joint.
Secondly, the reagent is evenly blown and sucked by a gun head and is centrifuged for a short time, so that the solution is collected at the bottom of the tube.
③ 20 ℃ for 15 minutes, if the operation uses the PCR instrument, please close the hot lid.
(3) Recovery of DNA fragments
1. CMpure was vortexed for 20 seconds to mix thoroughly as a homogeneous solution.
2. The adaptor ligation reaction was transferred to a new 1.5ml centrifuge tube.
5. Add 120. mu.l of well mixed CMPure to the tube, vortex for 5 seconds and then allow to stand at room temperature for 5 minutes.
6. Briefly, the tube is placed on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (about 5 minutes), and the supernatant is carefully aspirated and discarded, while avoiding contact with the target DNA-bound beads.
Note that: the beads are not discarded.
7. The tube was kept on the magnetic frame, 250. mu.l of freshly prepared 80% ethanol was added to the tube, left at room temperature for 30 seconds, and after the suspended beads were completely adsorbed, the supernatant was carefully discarded.
8. And 7, repeating the step.
9. Keeping the centrifuge tube fixed on the magnetic frame, standing for 10 minutes at room temperature to dry the magnetic beads in the air.
10. The centrifuge tube was removed from the magnetic frame, 25. mu.l EB (Ready-made) or deionized water was added, the beads were completely resuspended in deionized water by vortexing, and the mixture was allowed to stand at room temperature for 5 minutes.
11. Briefly, centrifuge the tube on a magnetic rack until the solution is clear (about 5 minutes), and transfer 23. mu.l of clear solution to a new PCR tube.
(4) First round PCR amplification
Adding the following reagents into a PCR tube and uniformly mixing.
In the first round of PCR amplification, a universal pre-primer and a specific post-primer are used, wherein the sequence of the universal pre-primer is as follows: 5'-ACACTCTTTCCCTACACGACGCTCTTC CGATCT-3', which is shown in SEQ ID NO. 3; the sequence of the specific rear primer is as follows: 5 '-CAGACGTGTGCTTCCGATCTxxxxxxxxxxxxx-3', shown in SEQ ID NO: 4.
② PCR reaction conditions
Purification of first round PCR product
1. CMpure was vortexed for 20 seconds to mix thoroughly as a homogeneous solution.
2. The PCR reaction was transferred to a new 1.5ml centrifuge tube.
3. CMpure of 1.5 sample volumes was added, vortexed for 5 seconds and allowed to stand at room temperature for 5 minutes.
4. Briefly, the tube is placed on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (about 5 minutes), and the supernatant is carefully aspirated and discarded, while avoiding contact with the target DNA-bound beads.
Note that: the beads are not discarded.
5. The tube was kept on the magnetic frame, 250. mu.l of freshly prepared 80% ethanol was added to the tube, left at room temperature for 30 seconds, and after the suspended beads were completely adsorbed, the supernatant was carefully discarded.
6. And (5) repeating the step.
7. Keeping the centrifuge tube fixed on the magnetic frame, standing for 10 minutes at room temperature to dry the magnetic beads in the air.
8. The centrifuge tube was removed from the magnetic frame, 25. mu.l EB (Ready-made) or deionized water was added, the beads were completely resuspended in deionized water by vortexing, and the mixture was allowed to stand at room temperature for 5 minutes.
9. Briefly, centrifuge the tube on a magnetic rack until the solution cleared (approximately 5 minutes), and transfer 22. mu.l of the cleared solution to a new PCR tube. The concentration was measured using nanodrop.
(5) PCR library construction and amplification
Adding the following reagents into a PCR tube and uniformly mixing.
The universal pre-primer and the specific post-primer are used in the first round of PCR amplification,
sequence of the P5 primer: 5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC T-3' which is represented by SEQ ID NO: 5; sequence of the P7 primer: 5'-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', which is shown in SEQ ID NO. 6.
② PCR reaction conditions
③ purification of PCR products
1. CMpure was vortexed for 20 seconds to mix thoroughly as a homogeneous solution.
2. The PCR reaction was transferred to a new 1.5ml centrifuge tube.
3. CMpure was added at 1 sample volume, vortexed for 5 seconds and allowed to stand at room temperature for 5 minutes.
4. Briefly, the tube is placed on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (about 5 minutes), and the supernatant is carefully aspirated and discarded, while avoiding contact with the target DNA-bound beads.
Note that: the beads are not discarded.
5. The tube was kept on the magnetic frame, 250. mu.l of freshly prepared 80% ethanol was added to the tube, left at room temperature for 30 seconds, and after the suspended beads were completely adsorbed, the supernatant was carefully discarded.
6. And (5) repeating the step.
7. Keeping the centrifuge tube fixed on the magnetic frame, standing for 10 minutes at room temperature to dry the magnetic beads in the air.
8. The centrifuge tube was removed from the magnetic frame, 30. mu.l EB (Ready-made) or deionized water was added, the beads were completely resuspended in deionized water by vortexing, and the mixture was allowed to stand at room temperature for 5 minutes.
9. Briefly, centrifuge the tube on a magnetic rack until the solution is clear (about 5 minutes), and transfer 25. mu.l of clear solution to a new PCR tube. The DNA library was stored at-20 ℃.
(6) Capillary electrophoresis of library: the purified library was diluted to 1ng/ul and analyzed by bioAnalyzer, the results are shown in FIG. 3. As only the downstream primer is a specific primer, the size of the library is wider, the main peak is positioned at 300bp, and the positions of the main peaks of different samples slightly deviate.
The result of fusion gene analysis is schematically shown in FIG. 4, and we successfully detected the fusion of ROS1 gene and SLC34A2 gene. The breakpoint of the ROS1 gene is at intron 31, the chromosomal location is chr6:117658326, the breakpoint of the SLC34A2 gene is at intron 4, the chromosomal location is chr4:25666631, and specific gene fusion sequences are given. The reference genome for data analysis was hg 19.
As can be seen from the above, the present invention has the following advantageous effects:
the library obtained by the adaptor and cfDNA library construction method is suitable for an Illumina sequencing platform and can improve the conversion efficiency of the library. The database construction method provided by the invention is simple to operate, the sequencing result is stable, and the sequencing cost is low. After adding a molecular tag sequence to a joint, the invention can identify and classify the sequencing result of the amplification product belonging to the same original template through the molecular tag after PCR and sequencing. Theoretically, certain DNA molecules derived from the same molecular tag should carry the same sequence, so that real gene mutation and systematic errors can be identified through the molecular tag, the systematic errors introduced in the PCR and sequencing processes are removed, the final sequencing result is prevented from being interfered by the systematic errors, and the result of improving the sensitivity of cfDNA sequencing is finally achieved.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> Jiangsukang was a century Biotechnology Co., Ltd
<120> adaptor, primer set, kit and banking method for cfDNA banking
<130>FI180129
<160>6
<170>PatentIn version 3.5
<210>1
<211>59
<212> Artificial sequence
<223> Top Joint
<400>1
acactctttc cctacacgac gctcttccga tctnnnnnnn nnnnngccgc cgccgccg*t 59
<210>2
<211>13
<212> Artificial sequence
<223> bottom joint
<400>2
cggcggcggc ggc 13
<210>3
<211>33
<212> Artificial sequence
<223> Universal Pre-primer
<400>3
acactctttc cctacacgac gctcttccga tct 33
<210>4
<211>39
<212> Artificial sequence
<223> specific rear primer
<400>4
cagacgtgtg ctcttccgat ctxxxxxxxx xxxxxxxxx 39
<210>5
<211>58
<212> Artificial sequence
<223> P5 primer
<400>5
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatc*t 58
<210>6
<211>58
<212> Artificial sequence
<223> P7 primer
<400>6
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatct 58
Claims (3)
1. A method for constructing a cfDNA library is characterized by comprising the following steps:
(1) performing end repair on the cfDNA, and adding an A base at the 3' end of the cfDNA;
(2) connecting the end of the cfDNA obtained after the treatment in the step (1) with a linker containing a random tag sequence to obtain a ligation product, and performing magnetic bead purification on the ligation product;
the joint is a double-link joint and comprises a top joint and a bottom joint, and the sequence of the top joint is as follows: 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGCCGCCGCCGCCG T-3' which is represented by SEQ ID NO: 1; the sequence of the bottom linker is: 5 '-P-CGGCGGCGGCGGC-3' which is shown in SEQ ID NO: 2; wherein the 12 bases "NNNNNNNNNNNN" in the top linker are the tag sequence;
(3) performing first round amplification on the ligation product by using a universal front primer and a specific rear primer, and performing magnetic bead purification on the first round amplification product;
(4) and performing library construction and amplification on the first round of amplified products after magnetic bead purification by using primers P7 and P5, and performing magnetic bead purification on the library construction and amplification products to obtain a high-throughput sequencing library.
2. The method of pooling cfDNA of claim 1, wherein in step (3), the sequence of the universal pre-primer is: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', which is represented by SEQ ID NO. 3; the sequence of the specific rear primer is as follows: 5 '-CAGACGTGTGCTTCCGATCTxxxxxxxxxxxxx-3', shown in SEQ ID NO: 4.
3. The method of pooling cfDNA of claim 1, wherein in step (4), the sequence of the P5 primer: 5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC T-3' which is shown in SEQ ID NO. 5; sequence of the P7 primer: 5'-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', which is shown in SEQ ID NO. 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810359753.XA CN108517567B (en) | 2018-04-20 | 2018-04-20 | Adaptor, primer group, kit and library construction method for cfDNA library construction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810359753.XA CN108517567B (en) | 2018-04-20 | 2018-04-20 | Adaptor, primer group, kit and library construction method for cfDNA library construction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108517567A CN108517567A (en) | 2018-09-11 |
| CN108517567B true CN108517567B (en) | 2020-08-11 |
Family
ID=63429894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810359753.XA Active CN108517567B (en) | 2018-04-20 | 2018-04-20 | Adaptor, primer group, kit and library construction method for cfDNA library construction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108517567B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295050A (en) * | 2018-09-26 | 2019-02-01 | 刘强 | Both-end label specific linkers, kit and the banking process in the library Blood Trace cfDNA |
| CN111005073A (en) * | 2019-09-29 | 2020-04-14 | 深兰科技（上海）有限公司 | Method and device for constructing multi-sample library |
| CN110791814A (en) * | 2019-10-07 | 2020-02-14 | 深圳易倍科华生物科技有限公司 | Rapid single-chain library building method |
| CN113046835A (en) * | 2019-12-27 | 2021-06-29 | 深圳华大生命科学研究院 | Sequencing library construction method for detecting lentivirus insertion site and lentivirus insertion site detection method |
| CN113502287A (en) * | 2021-06-28 | 2021-10-15 | 深圳市核子基因科技有限公司 | Molecular tag joint and construction method of sequencing library |
| CN117568450A (en) * | 2023-11-17 | 2024-02-20 | 厦门飞朔生物技术有限公司 | Improved construction method and application of amplicon library carrying specificity molecular tag |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016028316A1 (en) * | 2014-08-22 | 2016-02-25 | Resolution Bioscience, Inc. | Methods for quantitative genetic analysis of cell free dna |
| CN105483267A (en) * | 2016-01-15 | 2016-04-13 | 古博 | Plasma cfDNA (cell-free deoxyribonucleic acid) bi-molecular marker, method for marking and detecting plasma cfDNA and application of plasma cfDNA bi-molecular marker |
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN107447021A (en) * | 2017-09-07 | 2017-12-08 | 上海交通大学 | A kind of method and its application of the accurate identification genotype based on high-flux sequence |
| CN107794256A (en) * | 2016-09-01 | 2018-03-13 | 埃提斯生物技术（上海）有限公司 | CfDNA banking process and kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2951683T3 (en) * | 2015-04-17 | 2023-10-24 | Translational Genomics Res Inst | Evaluation of the quality of circulating acellular DNA using multiplexed nanodroplet digital PCR |
-
2018
- 2018-04-20 CN CN201810359753.XA patent/CN108517567B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016028316A1 (en) * | 2014-08-22 | 2016-02-25 | Resolution Bioscience, Inc. | Methods for quantitative genetic analysis of cell free dna |
| CN105483267A (en) * | 2016-01-15 | 2016-04-13 | 古博 | Plasma cfDNA (cell-free deoxyribonucleic acid) bi-molecular marker, method for marking and detecting plasma cfDNA and application of plasma cfDNA bi-molecular marker |
| CN107794256A (en) * | 2016-09-01 | 2018-03-13 | 埃提斯生物技术（上海）有限公司 | CfDNA banking process and kit |
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN107447021A (en) * | 2017-09-07 | 2017-12-08 | 上海交通大学 | A kind of method and its application of the accurate identification genotype based on high-flux sequence |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108517567A (en) | 2018-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108517567B (en) | Adaptor, primer group, kit and library construction method for cfDNA library construction | |
| CN108893466B (en) | Sequencing joint, sequencing joint group and detection method of ultralow frequency mutation | |
| CN111748551B (en) | Blocking sequence, capture kit, library hybridization capture method and library construction method | |
| CN110218781B (en) | Composite amplification system of 21 micro haplotype sites, next generation sequencing and typing kit and typing method | |
| CN111808854B (en) | Balanced joint with molecular bar code and method for quickly constructing transcriptome library | |
| WO2012068919A1 (en) | Dna library and preparation method thereof, and method and device for detecting snps | |
| CN108866174B (en) | Method for detecting low-frequency mutation of circulating tumor DNA | |
| CN109593757B (en) | Probe and method for enriching target region by using same and applicable to high-throughput sequencing | |
| CN108103164B (en) | Method for detecting copy number variation by using multiple fluorescent competitive PCR | |
| CN113373524B (en) | ctDNA sequencing tag joint, library, detection method and kit | |
| WO2018184495A1 (en) | Method for constructing amplicon library through one-step process | |
| CN111321208A (en) | Database building method based on high-throughput sequencing | |
| CN110004225B (en) | Tumor chemotherapeutic drug individualized gene detection kit, primers and method | |
| CN113136422A (en) | Method for detecting high-throughput sequencing sample contamination by grouping SNP sites | |
| CN109853047A (en) | A kind of genomic DNA sequencing library fast construction method and matched reagent box | |
| CN107354207B (en) | liquid phase hybridization capture kit based on double-stranded probe, washing kit and application thereof | |
| TW201321520A (en) | Method and system for virus detection | |
| CN110951827B (en) | Rapid construction method and application of transcriptome sequencing library | |
| CN111979353A (en) | Library construction method for sequencing novel coronavirus SARS-CoV-2 full-length genome | |
| CN112259165A (en) | Method and system for detecting microsatellite instability state | |
| CN110651050A (en) | Targeted enrichment method and kit for detecting low-frequency mutation | |
| CN113444769B (en) | Construction method and application of DNA tag sequence | |
| CN107904297B (en) | Primer group, joint group and sequencing method for microbial diversity research | |
| CN111793623A (en) | Typing genetic marker composition, kit, identification system and typing method of 62 multi-allelic SNP-NGS | |
| CN109517819A (en) | A kind of detection probe, method and kit modified for detecting multiple target point gene mutation, methylation modification and/or methylolation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 225300 4 floor, building TQB, No. 1, Yao Cheng Avenue, Taizhou, Jiangsu. Patentee after: Jiangsu Kangwei century Biotechnology Co.,Ltd. Address before: 225300 4 floor, building TQB, No. 1, Yao Cheng Avenue, Taizhou, Jiangsu. Patentee before: CoWin Biosciences Co.,Ltd. |