CN112375809A - Hybridization capture kit and method for performing hybridization capture by using same - Google Patents
Hybridization capture kit and method for performing hybridization capture by using same Download PDFInfo
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- 238000009396 hybridization Methods 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 49
- 238000010828 elution Methods 0.000 claims abstract description 36
- 230000000903 blocking effect Effects 0.000 claims abstract description 13
- 238000012408 PCR amplification Methods 0.000 claims abstract description 5
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 239000003112 inhibitor Substances 0.000 claims abstract description 3
- 239000011324 bead Substances 0.000 claims description 18
- 108020004414 DNA Proteins 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000000523 sample Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 108020004518 RNA Probes Proteins 0.000 claims description 5
- 239000003391 RNA probe Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 230000027455 binding Effects 0.000 claims description 2
- 230000009870 specific binding Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 abstract description 15
- 238000005516 engineering process Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
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- 108010090804 Streptavidin Proteins 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
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- 238000007664 blowing Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
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- 239000007788 liquid Substances 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
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- 239000012452 mother liquor Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 108091035233 repetitive DNA sequence Proteins 0.000 description 1
- 102000053632 repetitive DNA sequence Human genes 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a hybridization capture kit and a method for performing hybridization capture by using the kit, and belongs to the technical field of target area hybridization capture. The kit comprises a blocking reagent, an RNA Inhibitor, a hybridization buffer solution, an elution reagent and a PCR amplification reagent; the hybrid capture kit obtained by the invention has low cost and high capture efficiency, can meet the use requirement of targeted sequencing, and has good market application prospect.
Description
Technical Field
The invention relates to a hybridization capture kit and a method for performing hybridization capture by using the kit, belonging to the technical field of target area hybridization capture.
Background
At present, the second generation sequencing is widely applied in the fields of identifying genetic change related to cancer, researching genetic susceptibility of diseases and the like. The hybridization capture technology based on the second generation sequencing is mature, and mainly comprises a multiple PCR capture technology, a solid phase chip technology, a liquid phase probe capture technology and a molecular inversion capture technology. Wherein, the liquid phase hybridization capture technology is based on the improvement of a solid phase probe synthesis method. The liquid phase hybridization capture technology is characterized in that a target DNA fragment is directly hybridized with a probe with a biotin label in a solution, and the target DNA fragment is effectively enriched through the reaction of biotin and streptavidin on magnetic beads. The target DNA fragment obtained by the liquid phase hybridization capture technology has good uniformity and repeatability, thereby being widely applied.
Currently, commercial kits using liquid phase hybridization capture technology are various in types, but still have many defects. For example, current commercially available kits are expensive, capture efficiency is generally low, which reduces the effective data volume of samples, virtually increases reagent cost of single samples, and is difficult to meet the current requirements of targeted sequencing. Therefore, there is a need for a new hybrid capture kit with lower cost and higher capture efficiency.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a hybridization capture kit and a method for performing hybridization capture by using the kit, which have the advantages of low cost and high capture efficiency.
The technical scheme of the invention is as follows:
the invention provides a kit for hybridization capture of a target area, which comprises a sealing reagent, an RNA Inhibitor, a hybridization buffer solution, an elution reagent and a PCR amplification reagent; wherein the blocking reagent comprises commercially available Human Cot-1DNA, Salmonon DNA and a linker sequence blocking reagent.
Further, the elution reagent comprises an elution reagent I, an elution reagent II and an elution reagent III, wherein the elution reagent I consists of NaCl, Tris-Cl and EDTA, and the elution reagent II and the elution reagent III both consist of SSC and SDS.
Further, the linker sequence blocking reagent is TS Mix.
Further, the kit also comprises a nucleic-free water.
The invention also provides a method for carrying out hybridization capture by using the kit, which comprises the following steps:
(1) blocking linker and repeat sequence: adding TS Mix to the library to be hybridized to block the linker sequence, and adding Human Cot-1DNA and Salmonon DNA to block the repeat DNA sequence; (2) binding of the RNA probe to the target region; (3) capturing the hybrid library;
(4) elution of the captured library;
(5) and (4) carrying out PCR enrichment and magnetic bead purification on the eluted library.
Further, in the step 1, the closed reaction system is as follows:
the following reaction procedure was performed in a PCR instrument:
further, the step 2 specifically includes: when the reaction program is carried out to the third step of 65 ℃ and PAUSE, adding Hybridization Mix containing an RNA probe into the library mixed liquor, controlling the fifth step of 65 ℃ and HOLD time to be 8-12h, and realizing the specific binding of the probe and the target region; the Hybridization Mix was formulated as follows:
further, the step (3) includes: washing the Dynabeads Myone Sreptavidin T1 magnetic beads with an elution reagent I; resuspending the beads with elution reagent I; adding the hybrid product, placing on a four-dimensional mixing instrument, and mixing at room temperature for 30 min; the objective was to allow the streptavidin-bearing T1 magnetic beads to bind well to the biotin-labeled hybridization product.
In step (4), the captured library is eluted with an elution reagent II and an elution reagent III to remove nucleic acid fragments and impurities that are not bound to the probes, thereby obtaining T1 magnetic beads to which the probes and the target sequences are bound.
The beneficial technical effects of the invention are as follows:
compared with the library obtained by using the Agilent target capture kit, the library obtained by using the method has much higher repetition rate, but the capture efficiency after repetition removal is obviously improved by about 1 time; meanwhile, the effective sequencing depth is also obviously increased. The method provided by the invention can meet the use requirement of the current targeted sequencing.
Detailed Description
As described in the background, the conventional commercial hybrid capture kit is not only expensive, but also has poor capture efficiency when the capture target region is small in size. In order to solve the above problems, the present inventors have developed a novel kit for hybrid capture of a target region.
In the following examples, Invitrogen Human Cot-1DNA and Ultrapure Salmonon Sperm DNA Solution were used for blocking the repetitive DNA sequence, and IDT Co was used for blocking the linker sequence
Universal Blockers-TS Mix, RNase Inhibitor used SUPERAse-In RNase Inhibitor from Invitrogen, PCR amplification reagents used Herculase II Fusion DNA polymers from Agilent.
In the following examples, the hybridization buffer consisted of the following components: wherein CaCl2The working concentration was 1080mM, the working concentration of sodium carboxymethyl starch (CMS) was 0.085% (v/v), and the working concentration of Tris (pH 8.0) was 5 mM.
In the following examples, elution reagent I consisted of a working concentration of 1M NaCl, 10mM Tris-HCl and 1mM EDTA; elution reagent II was organized by a consensus concentration of SSC of 1X and a working concentration of SDS of 0.1% (v/v); the elution reagent III consists of SSC at a working concentration of 0.1X and SDS at a working concentration of 0.1% (v/v). The three elution reagents can be diluted by mother liquor with different concentrations for use.
The following examples relate to an example of plasma-free DNA and an example of leukocyte genomic DNA, and are extracted using QIAamp Circulating Nucleic Acid Kit (ref:55114) and Tiangen blood genomic DNA extraction Kit (cat # DP304-02) from QIAGEN, respectively, and library construction is performed using KAPA Hyper Prep Kit (ref: KK 8504). After the library construction was completed, hybrid capture was performed simultaneously using the Agilent SureSelect XT Target entity Kit (cat #5190-7334) as a control group.
Example 1
1. Concentration of hybridization libraries
The library to be hybridized, 550-600ng, was concentrated on a concentrator at a temperature below 45 ℃ in a maximum volume of no more than 5. mu.l.
2. Blocking linkers and repeat sequences
The following reagents were added to the concentrated library in order:
the mixture was mixed by pipetting, centrifuged briefly, placed in a PCR instrument and the following procedure was started:
binding of RNA probes to target regions
Hybridization reagent Hybridization Mix was prepared as follows
When the PCR reaction program is carried out to the third step (65 ℃, PAUSE), the reaction program is suspended, 13 mu l of Hybridization Mix is added into the library mixed liquid under the condition of keeping the library mixed liquid at 65 ℃, the mixture is evenly blown and sucked up and down by a gun for 8 to 10 times, the mixture is placed on a PCR instrument after short centrifugation, the next step of reaction is continued, and the reaction time of the last step (65 ℃, Hold) is ensured to be 8 to 12 hours.
4. Post-hybridization library Capture
(1) Placing streptavidin magnetic beads Dynabeads Myone Sreptavidin T1beads at room temperature for balancing 30min in advance;
(2) 50 μ l of Dynabeads Myone Sreptavidin T1 was taken for each hybridization reaction in a 1.5ml PCR tube;
(3) adding 500 μ l of elution reagent I, shaking, mixing, centrifuging for a short time, placing on a magnetic frame, and sucking supernatant after the solution is clarified;
(4) repeating the steps twice, and resuspending the magnetic beads with 200. mu.l of elution reagent I;
(5) adding the hybridization product into the solution, reversing and mixing uniformly for 3-5 times, placing on a four-dimensional mixing machine, and mixing uniformly for 30min at room temperature.
5. Elution of the library
(1) Placing the centrifugal tube on a magnetic frame, standing until the solution is clear, and sucking the supernatant;
(2) adding 500 mul of elution reagent II, shaking and mixing uniformly; after short-time centrifugation, placing the centrifuge tube on a magnetic frame, standing until the solution is clear, and absorbing and removing the supernatant;
(3) adding 500 μ l of 65 deg.C preheated elution reagent III, shaking, mixing, and performing 65 deg.C warm bath on PCR instrument for 5 min; placing the centrifugal tube on a magnetic frame, standing until the solution is clear, and sucking the supernatant;
(4) after repeating the above procedure twice, 37. mu.l of nucleic-Free Water was added and the beads were resuspended by pipetting the mixture with a gun.
Enrichment by PCR
The PCR amplification reaction system was prepared as follows:
the reaction system was run on a PCR instrument with the following program:
6. magnetic bead purification
(1) Placing AMPure XP beads at room temperature for at least 30min, and fully and uniformly mixing an AMPure XP bead suspension;
(2) adding a 1.8-time volume (90 mu l) of the uniformly mixed AMPure XP beads suspension and an amplified DNA sample into a 1.5ml EP tube, blowing and sucking the mixture uniformly for more than 10 times, and standing the mixture at room temperature for 5 min;
(3) centrifuging for a short time, placing the centrifuge tube on a magnetic frame, and standing for about 3-5min until the solution becomes clear; carefully sucking the supernatant in the tube on a magnetic frame, wherein the gun head cannot touch the magnetic beads;
(5) on a magnetic frame, adding 500 μ l of 80% ethanol into each tube, standing for 30s, shaking upside down, and then removing ethanol by suction;
(6) repeating the step (5) once, and completely evaporating the residual ethanol in the tube, wherein the surfaces of the magnetic beads are not reflective and are frosted;
(7) adding 21 μ l of clean-free water, blowing and sucking, mixing well for more than 10 times, standing at room temperature for 5 min;
(8) centrifuging for a short time, placing the centrifuge tube on a magnetic frame, and standing for about 2-3min until the solution becomes clear; approximately 20. mu.l of the supernatant was taken into a new 1.5ml centrifuge tube to obtain the post-hybridization library.
7. Quantitative computer for library
The above library was quantitatively detected with Agilent 2100 and Roche 480 and subjected to in-machine sequencing on the Illumina NextSeq500 sequencing platform.
8. Analysis of sequencing results
The method simultaneously adopts a SureSelect XT Target entity Kit of Agilent company to carry out Target region hybridization capture on the two samples, and is used for evaluating the Target region hybridization capture method provided by the invention.
The four obtained libraries were subjected to on-machine sequencing using Illumina NextSeq500 sequencing platform, the data size of each sample was 2G, and the capture efficiency (rmdup), the repetition rate and the average effective sequencing depth of the four libraries were analyzed. The four library sequencing results (table 1) show that, compared with the library obtained by using the Agilent targeted capture kit, the library obtained by using the method of the invention has much higher repetition rate, but the capture efficiency after repetition removal is obviously improved by about 1 time; meanwhile, the effective sequencing depth is also obviously increased. The method provided by the invention can meet the current use requirement and can obtain higher capture efficiency.
TABLE 1 library sequencing results
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A kit for target region hybridization capture is characterized by comprising a blocking reagent, an RNA Inhibitor, a hybridization buffer solution, an elution reagent and a PCR amplification reagent; wherein the blocking reagent comprises commercially available Human Cot-1DNA, Salmonon DNA and a linker sequence blocking reagent.
2. The kit according to claim 1, wherein the elution reagent comprises elution reagent I, elution reagent II and elution reagent III, wherein elution reagent I consists of NaCl, Tris-Cl and EDTA, and wherein elution reagent II and elution reagent III both consist of SSC and SDS.
3. The kit of claim 1, wherein the linker sequence blocking reagent is TS Mix.
4. The kit of claim 1, wherein said kit further comprises a nucleic-free water.
5. A method for performing hybrid capture using the kit of any one of claims 1 to 4, comprising the steps of:
(1) blocking linker and repeat sequence: adding TS Mix to the library to be hybridized to block the linker sequence, and adding Human Cot-1DNA and Salmonon DNA to block the repeat DNA sequence;
(2) binding of the RNA probe to the target region;
(3) capturing the hybrid library;
(4) elution of the captured library;
(5) and (4) carrying out PCR enrichment and magnetic bead purification on the eluted library.
6. The method according to claim 5, wherein in the step (1), the closed reaction system is as follows:
the blocked reaction procedure was as follows:
7. the method according to claim 6, characterized in that said step (2) is in particular: when the reaction program is carried out to the third step of 65 ℃ and PAUSE, adding Hybridization Mix containing an RNA probe into the library mixed liquor, controlling the fifth step of 65 ℃ and HOLD time to be 8-12h, and realizing the specific binding of the probe and the target region; the Hybridization Mix was formulated as follows:
8. the method of claim 5, wherein step (3) comprises: washing the Dynabeads Myone Sreptavidin T1 magnetic beads with an elution reagent I; resuspending the beads with elution reagent I; adding the hybridization product, placing on a four-dimensional mixing machine, and mixing at room temperature for 30 min.
9. The method of claim 5 or 8, wherein step 4 is performed by eluting the captured library with an elution reagent II and an elution reagent III.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480579A (en) * | 2021-12-28 | 2022-05-13 | 上海英基生物科技有限公司 | Hybridization capture kit for sequencing genome target region, capture method and application |
| CN114480579B (en) * | 2021-12-28 | 2024-05-17 | 上海英基生物科技有限公司 | Hybridization capture kit, capture method and application for genome target region sequencing |
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| US20100029498A1 (en) * | 2008-02-04 | 2010-02-04 | Andreas Gnirke | Selection of nucleic acids by solution hybridization to oligonucleotide baits |
| US20150211047A1 (en) * | 2014-01-29 | 2015-07-30 | Agilent Technologies, Inc. | Fast hybridization for next generation sequencing target enrichment |
| CN108456713A (en) * | 2017-11-27 | 2018-08-28 | 天津诺禾致源生物信息科技有限公司 | The construction method of tab closure sequence, library construction Kit and sequencing library |
| CN110564831A (en) * | 2019-08-30 | 2019-12-13 | 北京优迅医学检验实验室有限公司 | Blocking reagent for sequencing library and method for improving targeted capture efficiency |
| CN110600082A (en) * | 2019-11-13 | 2019-12-20 | 上海仁东医学检验所有限公司 | Nucleic acid capture probe for HLA typing and design method thereof |
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- 2020-11-19 CN CN202011299951.5A patent/CN112375809A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100029498A1 (en) * | 2008-02-04 | 2010-02-04 | Andreas Gnirke | Selection of nucleic acids by solution hybridization to oligonucleotide baits |
| US20150211047A1 (en) * | 2014-01-29 | 2015-07-30 | Agilent Technologies, Inc. | Fast hybridization for next generation sequencing target enrichment |
| CN108456713A (en) * | 2017-11-27 | 2018-08-28 | 天津诺禾致源生物信息科技有限公司 | The construction method of tab closure sequence, library construction Kit and sequencing library |
| CN110564831A (en) * | 2019-08-30 | 2019-12-13 | 北京优迅医学检验实验室有限公司 | Blocking reagent for sequencing library and method for improving targeted capture efficiency |
| CN110600082A (en) * | 2019-11-13 | 2019-12-20 | 上海仁东医学检验所有限公司 | Nucleic acid capture probe for HLA typing and design method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480579A (en) * | 2021-12-28 | 2022-05-13 | 上海英基生物科技有限公司 | Hybridization capture kit for sequencing genome target region, capture method and application |
| CN114480579B (en) * | 2021-12-28 | 2024-05-17 | 上海英基生物科技有限公司 | Hybridization capture kit, capture method and application for genome target region sequencing |
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