CN114410764B - Preparation method of long DNA sequence silica microbeads - Google Patents
Preparation method of long DNA sequence silica microbeads Download PDFInfo
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- CN114410764B CN114410764B CN202210137482.XA CN202210137482A CN114410764B CN 114410764 B CN114410764 B CN 114410764B CN 202210137482 A CN202210137482 A CN 202210137482A CN 114410764 B CN114410764 B CN 114410764B
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 93
- 239000011325 microbead Substances 0.000 title claims abstract description 61
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 46
- 108091028043 Nucleic acid sequence Proteins 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 21
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 16
- 230000000295 complement effect Effects 0.000 claims abstract description 15
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 108020004414 DNA Proteins 0.000 claims description 23
- 239000011324 bead Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 238000006911 enzymatic reaction Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000011535 reaction buffer Substances 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000006482 condensation reaction Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 6
- 235000012239 silicon dioxide Nutrition 0.000 abstract description 6
- 238000000137 annealing Methods 0.000 abstract description 3
- 125000000524 functional group Chemical group 0.000 abstract description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000001308 synthesis method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000000018 DNA microarray Methods 0.000 description 2
- 238000006473 carboxylation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
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- 230000035772 mutation Effects 0.000 description 1
- KFDKNTQGTAEZGC-UHFFFAOYSA-N phenanthrene-1-carboxylic acid Chemical compound C1=CC2=CC=CC=C2C2=C1C(C(=O)O)=CC=C2 KFDKNTQGTAEZGC-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 238000004393 prognosis Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
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Abstract
The invention discloses a preparation method of silica microbeads with long DNA sequences. The method uses silicon dioxide as a carrier, adopts a chemical reagent to activate a surface carboxyl functional group, and can be covalently connected with an amino modified oligonucleotide sequence. While subsequent sequences (sequence extension) may be introduced by way of complementary sequence annealing in combination with polymerization. By using the method, various long-sequence encoded silica microbeads can be obtained. Compared with the traditional method, the method has the advantages that the modified oligonucleotide sequences on the silica microbeads are more, and the biological information is more comprehensive.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a preparation method of silica microbeads with long DNA sequences.
Background
Nucleic acids, including deoxyribonucleic acid and ribonucleic acid, are one of the most basic substances in life, and play a vital role in various vital activities such as growth, development, mutation, inflammation, cancer, etc. of organisms. Nucleic acid molecules have a close relationship with the occurrence and development of various diseases affecting human health and play an important role. Therefore, the method has very important significance in developing an accurate and effective method to sensitively and accurately detect the nucleic acid, deeply exploring the functional regulation and control of the nucleic acid, screening medicaments, early detection of related diseases, clinical diagnosis and treatment, prognosis evaluation and the like.
Silicon dioxide is a typical inorganic powder material, and has large specific surface area and good chemical stability. The monodisperse silica microbead has simple preparation process and good biocompatibility, so that the silica microbeads with different sizes and different surface modifications can be applied to different fields of information, biology, medicine and the like.
The traditional sequencing method may result in important information omission due to the limitation of an action mechanism, however, the nucleic acid contains more biological information and has large data volume, so that the oligonucleotide chain coded by the long sequence is modified on a matrix, and the method is further used for DNA sequencing and has important significance for acquiring the nucleic acid information.
Disclosure of Invention
The invention aims to provide a preparation method of silica microbeads with long DNA sequences.
In order to achieve the purpose of the invention, the preparation method of the long DNA sequence silica microbeads provided by the invention comprises the following steps:
(1) Four sets of primers were designed, primer 1, primer 2, primer 3 and UMI primer: the primers are all oligonucleotide single chains, the sequences of the primer 1, the primer 2 and the primer 3 are consistent in length, the GC content is 45-55%, and the Tm value of each primer is guaranteed to be similar;
the 5 end of the primer 1 (first round primer) is modified by amino, and the primer comprises a READ1 sequence, a barcode 1 sequence and a linker 1 sequence of an illumine sequencing platform from 5'-3' (the whole sequence is synthesized by other rounds of primers through reverse complementary polymerization); primer 2 (second round primer) includes a linker 1 reverse complement sequence, a barcode 2 sequence, and a linker2 sequence from 5 '-3'; primer 3 (third round primer) includes linker2 reverse complement sequence, barcode 3 sequence and linker3 sequence from 5 '-3'; UMI primers (fourth primer) include linker3 reverse complement sequence, UMI sequence (random sequence) and polyA sequence from 5 '-3'; wherein, the barcode 1, the barcode 2 and the barcode 3 are different barcode sequences, and the length of polyA is 20-35 nt;
when the primer design is carried out, on one hand, the principle of the reverse complementary synthesis is considered, and on the other hand, the quality inspection of various barcode sequences is required to be large in difference and has no same sequence with the genome;
(2) Activation of carboxylated silica microbeads: placing carboxylated silica microbeads into EDC and NHS mixed solution for activation;
(3) Ligation reaction: placing the activated microbeads into a primer 1 solution for condensation reaction to obtain silica microbeads with different sequences;
Wherein the amino-modified oligonucleotide chain comprises barcode 1 and linker 1;
(4) Synthesis of long DNA sequences: mixing the microbeads obtained in the step (3), the primer 2 and a polymerization reaction reagent, performing DNA chain extension reaction, and then removing the reverse complementary sequence with linker 1; mixing the obtained microbeads with a primer 3 and a polymerization reaction reagent, and further carrying out a DNA chain extension reaction; finally, the microbeads are connected with UMI sequences to obtain the silica microbeads with long DNA sequences.
The step (2) comprises: the carboxylated silica microbeads (50 mg/mL) are centrifuged, and the precipitate is mixed with 20-100 mu L of EDC and NHS mixed solution, and the mixture is subjected to oscillation reaction at 1500-2000rpm for 30min-1h at room temperature.
The preparation method of the EDC and NHS mixed solution comprises the following steps: 1.09mg of EDC and 0.65mg of NHS were dissolved in 0.1M MES 100 ul.
The step (3) comprises: mixing the activated microbeads with 2.5 mu L of 50 mu M amino modified oligonucleotide chain solution uniformly, and oscillating at 2000rpm at room temperature for reaction overnight; after the reaction is completed, the microbeads are collected centrifugally and used for subsequent synthesis reaction after being cleaned.
Bead washing includes: the beads were placed in 0.1M PBS containing 0.02% Tween 20, the beads were collected by centrifugation, and then the beads were washed twice with TE buffer at pH 8.0.
The polymerization reagent of step (4) comprises: dNTPs, klenow enzyme and Klenow enzyme reaction buffer.
The reaction system used for DNA chain extension is as follows: mu.M primer 21. Mu.L, 10 XKlenow enzyme reaction buffer 5. Mu.L, 2.5mM dNTPs 4. Mu.L, 5U/. Mu.L Klenow enzyme 1. Mu.L.
The reaction conditions are as follows: the reaction was carried out at 37℃with shaking at 2000rpm for 30min-1h.
The reaction system used for the connection of the microbeads and the UMI sequences in the step (4) is as follows: 50. Mu.M UMI primer, 5. Mu.L of 10 XKlenow enzyme reaction buffer, 4. Mu.L of 2.5mM dNTPs and 1. Mu.L of 5U/. Mu.L Klenow enzyme.
The reaction conditions are as follows: shaking reaction at 37 ℃ and 2000rpm for 0.5-1h
Preferably, the nucleotide sequence of the primer 1 is shown as SEQ ID NO. 1-5, the nucleotide sequence of the primer 2 is shown as SEQ ID NO. 6-10, the nucleotide sequence of the primer 3 is shown as SEQ ID NO. 11-15, and the nucleotide sequence of the UMI primer is shown as SEQ ID NO. 16.
The carboxylated silica microbeads used in the invention are purchased from Shanghai carboxyphenanthrene biomedical science and technology, inc., and can also be prepared according to a conventional method.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
The invention provides a synthesis method of long sequence coding carboxylated silicon dioxide, which takes silicon dioxide as a carrier, adopts chemical reagent to activate surface carboxyl functional groups, and can be covalently connected with amino modified oligonucleotide sequences. While subsequent sequences (sequence extension) may be introduced by way of complementary sequence annealing in combination with polymerization. By using the method, various long-sequence encoded silica microbeads can be obtained. Compared with the traditional method, the method has the advantages that the modified oligonucleotide sequences on the silica microbeads are more, and the biological information is more comprehensive.
And (II) the activation efficiency is high: EDC, NHS are able to fully activate the carboxyl structure of the silica microbead surface modification.
And (III) the connection efficiency is high: the condensation reaction is effective to link the carboxyl microbeads to the amino oligonucleotide chains.
And (IV) high operation success rate: the operation process is simple, the used reagents are less, and the synthesis efficiency is ensured to the greatest extent.
Drawings
FIG. 1 is a schematic diagram of the structure of a long sequence encoded silica microbead synthesized in a preferred embodiment of the present invention.
Detailed Description
The invention provides a synthesis method of a long-sequence encoded silica microbead. The method adopts carboxylation reaction to connect carboxylated silicon dioxide and amino-bearing sequences, then adds the complementary sequence of the subsequent barcode sequences, and utilizes primer annealing and polymerase polymerization reaction to obtain silicon dioxide microbeads with long sequence codes, so that the obtained microbeads are further applied to DNA chip decoding.
The invention adopts the following technical scheme:
1. designing a primer: the coding primer sequence is designed.
2. Activation of carboxylated silica microbeads: and activating the silica microbeads with carboxyl groups by EDC and NHS to obtain the silica microbeads with good dispersibility and exposed surface carboxyl groups.
3. Ligation reaction: carboxylated silica microbeads and oligonucleotide chains with amino groups modified at the tail ends are connected through a carboxylation reaction to obtain silica microbeads with different sequences.
4. Barcode sequence synthesis: and (3) sequentially connecting the latter sequences to the silica microbeads in the step (3) through polymerization reaction, and finally obtaining the silica microbeads with long sequence codes.
The terms referred to in this invention:
EDC:1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
NHS: n-hydroxysuccinimide.
MES: 2-morpholinoethanesulfonic acid.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.
EXAMPLE 1 Synthesis method of Long sequence encoded silica microbeads
The synthesis method of the long-sequence encoded silica microbeads provided by the embodiment comprises the following steps:
1. activated carboxylated silica
Accurately weighing 1.09mg of EDC and 0.65mg of NHS, simultaneously preparing 0.1M MES, and dissolving the weighed EDC and NHS by using 100ul of MES to obtain an EDC and NHS mixed solution; simultaneously taking 100 mu L of carboxylated silica microbeads (50 mg/mL), washing twice with the prepared MES solution, and then adding the EDC and NHS mixed solution into the microbeads, wherein the final reaction volume is 100 mu L; the microbeads were then left to react at room temperature with metal bath shaking (2000 rpm) for 30min.
2. Ligation of barcode 1 amino-modified oligonucleotide chains
After the microbead reaction, the reaction mixture was divided into 5 tubes, and 2.5. Mu.L of amino-modified oligonucleotide (dissolved in 0.1M MES at a final concentration of 50. Mu.M) was added to each tube, followed by stirring and stirring at room temperature, and then the reaction mixture was subjected to metal bath shaking (2000 rpm) overnight. After completion of the reaction, the beads were collected into 1mL of 0.1M PBS containing 0.02% tween 20, centrifuged, the supernatant carefully removed, and then the beads were washed twice in 1mL of TE buffer (pH 8.0).
3. Barcode 2 connection
The microbeads were washed with water and equally divided into 5 tubes, and then 1. Mu.L of BC2 (dissolved in TE buffer at pH 8.0, final concentration 50. Mu.M), 5. Mu.L of 10 XKlenow enzyme reaction buffer, and a final total volume of 45. Mu.L were added. The oligonucleotide sequences in each tube were: complementary sequence to linker 1 in reverse, a unique bar code and linker 2 sequence, the following procedure was run on a vibrating metal bath (2000 rpm):
After completion of the program run, 2.5mM dNTPs 4. Mu.L, 5U/. Mu.L Klenow enzyme 1. Mu.L, 37℃and 2000rpm were added to each tube and reacted for 1 hour.
After the reaction was completed, the beads were collected in 0.1M PBS containing 0.02% Tween 20, centrifuged, the supernatant was carefully removed, and then the beads were washed twice in TE buffer (pH 8.0), then 0.1M NaOH was added to stand for 2min, the reverse complement to linker2 was removed, the supernatant was rapidly removed after centrifugation, and repeated twice.
4. Barcode 3 and UMI connections
The ligation procedure for barcode 3 was identical to that for barcode 2, with the addition of the oligonucleotide sequence: reverse complement to linker 2, a unique bar code and linker 3 sequence; after the washing of the barcode 3 ligation was completed, 2. Mu.L of UMI was added for the same treatment, wherein the sequence of UMI added was: the reverse complement of linker 3, a unique bar code, a UMI sequence and a poly-T tail. The barcode 2 ligation step was repeated. Microbeads were stored in TE-TW solution (10mM Tris pH 8.0;1mM EDTA,0.01% Tween 20) and stored at 4 ℃.
The experimental results are shown in FIG. 1, and various long-sequence encoded silica microbeads can be obtained.
Together, these silica microspheres there are 384×384×384 kinds the number of final synthesized microspheres is over five tens of millions), can be used for preparing subsequent biochips.
The sequences involved in this example are shown in Table 1 (SEQ ID NOS: 1-16).
TABLE 1
Note that: n, B is a degenerate base, N represents A, T, C or G, and B represents G, T or C.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing Baimaike Biotech Co., ltd
<120> Preparation method of silica microbeads with long DNA sequences
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Claims (8)
1. The preparation method of the silica microbeads with long DNA sequences is characterized by comprising the following steps:
(1) Four sets of primers were designed, primer 1, primer 2, primer 3 and UMI primer: the primers are all oligonucleotide single chains, the sequences of the primer 1, the primer 2 and the primer 3 are consistent in length, the GC content is 45-55%, and the Tm value of each primer is guaranteed to be similar;
The primer 15 ' end is modified by amino, and comprises a READ1 sequence, a barcode 1 sequence and a linker 1 sequence of an illumine sequencing platform from 5' -3 '; primer 2 comprises a linker 2 sequence, a barcode 2 sequence and a linker 1 reverse complement sequence from 5 '-3'; primer 3 comprises a linker 3 sequence, a barcode 3 sequence and a linker 2 sequence from 5 '-3'; UMI primers include from 5'-3' a polyA sequence, UMI sequence and linker 3 sequence; wherein, the barcode 1, the barcode 2 and the barcode 3 are different barcode sequences, and the length of polyA is 20-35 nt;
(2) Activation of carboxylated silica microbeads: placing carboxylated silica microbeads into EDC and NHS mixed solution for activation;
(3) Ligation reaction: placing the activated microbeads into a primer 1 solution for condensation reaction to obtain silica microbeads with different sequences;
(4) Synthesis of long DNA sequences: mixing the microbeads obtained in the step (3), the primer 2 and a polymerization reaction reagent, performing DNA chain extension reaction, and then removing the reverse complementary sequence with linker 1; mixing the obtained microbeads with a primer 3 and a polymerization reaction reagent, and further carrying out a DNA chain extension reaction; finally, the microbeads are connected with UMI sequences to obtain the silica microbeads with long DNA sequences.
2. The method of claim 1, wherein step (2) comprises: centrifuging 50 mg/mL carboxylated silica microbeads, mixing the precipitate with 20-100 mu L of EDC and NHS mixed solution, and carrying out oscillation reaction for 30 min-1h at 1500-2000rpm at room temperature;
The preparation method of the EDC and NHS mixed solution comprises the following steps: 1.09mg of EDC and 0.65mg of NHS were dissolved in 100ul of 0.1M MES.
3. The method of claim 2, wherein step (3) comprises: uniformly mixing the activated microbeads with 2.5 mu L of an oligonucleotide chain solution modified by 50 mu M amino groups, and carrying out oscillation reaction at room temperature of 2000rpm for overnight; after the reaction is completed, the microbeads are collected centrifugally and used for subsequent synthesis reaction after being cleaned.
4. The method of claim 3, wherein the bead washing comprises: the beads were placed in 0.1M PBS containing 0.02% tween 20, collected by centrifugation and then washed twice with TE buffer pH 8.0.
5. A method according to claim 3, wherein the polymerization reagent of step (4) comprises: dNTPs, klenow enzyme and Klenow enzyme reaction buffer.
6. The method according to claim 5, wherein the reaction system for performing the DNA chain extension is: 50. mu M primer 21 mu L,10 XKlenow enzyme reaction buffer 5 mu L,2.5mM dNTPs 4 mu L and 5U/mu L Klenow enzyme 1 mu L;
The reaction conditions are as follows: the reaction was carried out at 37℃with shaking at 2000rpm for 0.5-1h.
7. The method of claim 6, wherein the reaction system used for the ligation of the microbeads to the UMI primers in step (4) is: 50. mu M UMI primer, 10 XKlenow enzyme reaction buffer solution 5 mu L, 2.5mM dNTPs 4 mu L and 5U/mu L Klenow enzyme 1 mu L;
The reaction conditions are as follows: the reaction was carried out at 37℃with shaking at 2000rpm for 0.5-1h.
8. The method according to any one of claims 1 to 7, wherein the nucleotide sequence of primer 1 is shown in SEQ ID NO. 1-5, the nucleotide sequence of primer 2 is shown in SEQ ID NO. 6-10, the nucleotide sequence of primer 3 is shown in SEQ ID NO. 11-15, and the nucleotide sequence of UMI primer is shown in SEQ ID NO. 16.
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