METHOD FOR LOADING NUCLEIC ACID MOLECULE ON SOLID SUPPORT

Abstract

Provided are a method for loading a nucleic acid molecule, for example nucleic acid nanoball (e.g., DNA nanoball (DNB)), on a solid support, and a kit for the method.

Claims

1-11. (canceled)

12. A method for loading a nucleic acid molecule on a solid support or for constructing a nucleic acid library that is immobilized on a solid support, comprising: (1) providing a composition comprising a nucleic acid molecule, and the solid support; (2) performing a first amplification of the nucleic acid molecule in the composition to obtain an amplification product comprising the nucleic acid molecule; (3) suspending or slowing down the first amplification; (4) loading the amplification product comprising the nucleic acid molecule onto the solid support; and (5) performing a second amplification of the nucleic acid molecule in the amplification product loaded on the solid support.

13. The method according to claim 12, wherein the method has one or more characteristics selected from the following: (1) the solid support has an array of sites for loading the amplification product; optionally, the sites are arranged on the solid support in a predetermined pattern; (2) the nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA; (3) the composition comprises a single-stranded circular nucleic acid molecule; (4) the composition comprises a single-stranded circular DNA molecule; (5) the composition comprises at least 10, at least 20, at least 50, at least 100, at least 1000, at least 10.sup.4, at least 10.sup.5, at least 10.sup.6, at least 10.sup.7 or more nucleic acid molecules; and (6) the nucleic acid molecule comprises a target nucleic acid fragment with a length of at least 100 bp, at least 200 bp, at least 500 bp, at least 800 bp, at least 1000 bp, or at least 2000 bp.

14. The method according to claim 12, wherein in step (2), the obtained amplification product is a linear nucleic acid molecule.

15. The method according to claim 12, wherein, in step (2), the size of the obtained amplification product does not exceed a predetermined size; optionally, the predetermined size is the maximum size of the nucleic acid molecule that can be accommodated by or attached to a single site in the solid support or is the spacing of adjacent sites in the solid support.

16. The method according to claim 15, wherein the method has one or more characteristics selected from the following: (1) the size of the obtained amplification product does not exceed 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the maximum size or the spacing of adjacent sites; and (2) by controlling the time of amplification, the size of the amplification product does not exceed the predetermined size.

17. The method according to claim 12, wherein, in step (3), the first amplification is suspended or slowed down by one or more methods selected from the group consisting of: (a) controlling or reducing the amount of dNTPs in the reaction mixture for the first amplification; and (b) inhibiting the activity of the nucleic acid polymerase used in the first amplification or inactivating the nucleic acid polymerase used in the first amplification.

18. The method according to claim 17, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by one or more methods selected from the group consisting of: (a) adding an activity inhibitor or denaturing agent of the nucleic acid polymerase; (b) removing a cofactor required for the nucleic acid polymerase to function; (c) adjusting the temperature of the reaction mixture for the first amplification to deviate from the working temperature range of the nucleic acid polymerase; and (d) adjusting the pH of the reaction mixture for the first amplification to deviate from the working pH range of the nucleic acid polymerase.

19. The method according to claim 17, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by removing a cofactor required for the nucleic acid polymerase to function.

20. The method according to claim 19, wherein the method has one or more characteristics selected from the following: (1) the cofactor is a cation; (2) the cofactor is a magnesium ion; (3) the cofactor is removed by adding a cation chelating agent, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase; (4) the cofactor is removed by adding a magnesium ion chelating agent; and (5) the cofactor is removed by adding a cation chelating agent which is selected from the group consisting of NTA, EDTA, HEDP, EDTMPS, DTPMPA, EDDHA, STPP, sodium dextrose and sodium metasilicate.

21. The method according to claim 17, wherein the temperature of the reaction mixture for the first amplification is adjusted to deviate from the working temperature range of the nucleic acid polymerase, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase.

22. The method according to claim 21, wherein the method has one or more characteristics selected from the following: (1) the temperature of the reaction mixture is adjusted to be higher than the upper limit of the working temperature of the nucleic acid polymerase by at least 5° C., at least 10° C., at least 15° C. or more, or, the temperature of the reaction mixture is adjusted to be lower than the lower limit of the working temperature of the nucleic acid polymerase by at least 5° C., at least 10° C., at least 15° C. or more; and (2) the temperature of the reaction mixture is lowered to a temperature of ≤20° C., ≤10° C., ≤4° C., or lower; or, the temperature of the reaction mixture is elevated to a temperature of ≥50° C., ≥60° C., ≥ 70° C., ≥80° C., or higher.

23. The method according to claim 17, wherein the pH of the reaction mixture for the first amplification is adjusted by adding an acidic or basic buffer to deviate from the working pH range of the nucleic acid polymerase, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase.

24. The method according to claim 23, wherein the method has one or more characteristics selected from the following: (1) the pH of the reaction mixture is adjusted to be higher than the upper limit of the working pH range of the nucleic acid polymerase by at least 1, at least 2, at least 3 or more pH units; or, the pH of the reaction mixture is adjusted to be lower than the lower limit of the working pH range of the nucleic acid polymerase by at least 1, at least 2, at least 3 or more pH units; and (2) the acidic or basic buffer is selected from the group consisting of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer and Tris hydrochloride buffer.

25. The method according to claim 17, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by adding an activity inhibitor or denaturing agent of the nucleic acid polymerase.

26. The method according to claim 25, wherein the method has one or more characteristics selected from the following: (1) the activity inhibitor or denaturing agent of the nucleic acid polymerase is a surfactant; and (2) the activity inhibitor or denaturing agent of the nucleic acid polymerase is selected from the group consisting of sodium deoxycholate, sodium lauryl sarcosinate, sodium lauryl sulfate, Tween-20, NP-40 and Tritox-100.

27. The method according to claim 12, wherein the method has one or more features selected from the group consisting of: (a) the solid support is selected from the group consisting of latex bead, dextran bead, polystyrene surface, polypropylene surface, polyacrylamide gel, gold surface, glass surface, chip, sensor, electrode and silicon wafer; (b) the solid support is planar, spherical or porous; (c) the maximum size of the nucleic acid nanoball that can be accommodated by or attached to a single site in the solid support is ≤ 5000 nm, ≤ 2000 nm, ≤ 1000 nm, ≤ 700 nm, ≤ 500 nm, ≤ 300 nm, or ≤ 100 nm; (d) in step (2), the first amplification of the nucleic acid molecule in the composition is carried out by rolling circle amplification; (e) in step (5), the second amplification of the nucleic acid molecule in the amplification product is carried out by rolling circle amplification; (f) the first amplification and the second amplification use the same or different nucleic acid polymerase; (g) the solid support is subjected to pretreatment prior to performing step (4); (h) the solid support is pretreated by using poloxamer, enzyme, or any combination thereof; (i) in step (5), the amplification product obtained by the second amplification is a linear nucleic acid molecule; (j) the product obtained in step (5) is used as a nucleic acid sequencing library; (k) the method further comprises, after step (5), sequencing the nucleic acid molecule loaded on the solid support; and (l) the amplification product is a nucleic acid nanoball.

28. The method according to claim 12, wherein the method has one or more features selected from the group consisting of: (a) in step (5), the second amplification of the nucleic acid molecules in the amplification product loaded on the solid support is performed by adding a reagent required for nucleic acid amplification; and (b) in step (5), the second amplification of the nucleic acid molecule in the amplification product loaded on the solid support is performed by eliminating a condition that causes the first amplification to be suspended or slowed down in step (3).

29. The method according to claim 28, wherein the method has one or more features selected from the group consisting of: (a) the reagent required for nucleic acid amplification is selected from the group consisting of nucleic acid polymerase, dNTPs, working buffer for nucleic acid polymerase, and any combination thereof; (b) in step (3), the first amplification is suspended or slowed down by inhibiting the activity of the nucleic acid polymerase used for the first amplification, and, in step (5), the second amplification is performed by restoring the activity of the nucleic acid polymerase; (c) in step (3), the first amplification is suspended or slowed down by inhibiting the activity of the nucleic acid polymerase used for the first amplification, and, in step (5), the second amplification is performed by eliminating a condition that inhibits the activity of the nucleic acid polymerase; (d) in step (3), the first amplification is suspended or slowed down by adding an activity inhibitor of the nucleic acid polymerase, and, in step (5), the activity inhibitor is removed to restore the activity of the nucleic acid polymerase, and the second amplification is performed; (e) in step (3), the first amplification is suspended or slowed down by removing a cofactor required for the nucleic acid polymerase to function, and, in step (5), the activity of the nucleic acid polymerase is restored by adding the cofactor, and the second amplification is performed; (f) in step (3), the first amplification is suspended or slowed down by adjusting the temperature of the reaction mixture to deviate from the working temperature range of the nucleic acid polymerase, and, in step (5), the activity of the nucleic acid polymerase is restored by adjusting the temperature of the reaction mixture to the working temperature range of the nucleic acid polymerase, and the second amplification is performed; (g) in step (3), the first amplification is suspended or slowed down by adjusting the pH of the reaction mixture to deviate from the working pH range of the nucleic acid polymerase, and, in step (5), the activity of the nucleic acid polymerase is restored by adjusting the pH of the reaction mixture to the working pH range of the nucleic acid polymerase, and the second amplification is performed; and (h) in step (3), the first amplification is suspended or slowed down by denaturing the nucleic acid polymerase, and, in step (5), the second amplification is performed by adding the nucleic acid polymerase.

30. A kit, which comprises: a reagent for amplifying a nucleic acid, a reagent for suspending or inhibiting nucleic acid amplification, and a reagent for loading the nucleic acid molecule onto a solid support; preferably, the kit has one or more features selected from the group consisting of: (1) the reagent for amplifying nucleic acid comprises a nucleic acid polymerase, a working buffer for the nucleic acid polymerase, a cofactor required for the nucleic acid polymerase to function, dNTPs, or any combination thereof; (2) the reagent for suspending or inhibiting nucleic acid amplification may comprise one or more selected from the group consisting of: (a) an activity inhibitor or denaturing agent of the nucleic acid polymerase; (b) a reagent capable of removing a cofactor required for the nucleic acid polymerase to function; and (c) an acidic or alkaline buffer capable of adjusting the pH of solution; (3) the reagent for loading the nucleic acid molecule onto the solid support comprises tripotassium citrate, citric acid, phi29 polymerase, or Pluronic F68; and (4) the kit further comprises a reagent for pretreating the solid support.

31. The kit according to claim 30, wherein the kit has one or more features selected from the group consisting of: (1) the nucleic acid polymerase is a DNA polymerase; (2) the cofactor is a cation; (3) the working buffer is concentrated, e.g by at least 2-fold, at least 5-fold or at least 10-fold; (4) the activity inhibitor or denaturing agent of the nucleic acid polymerase is a surfactant; (5) the reagent capable of removing the cofactor required for nucleic acid polymerase to function is a cation chelating agent; and (7) the reagent for pretreating the solid support is selected from the group consisting of poloxamer, enzyme, or any combination thereof; preferably, the kit has one or more features selected from the group consisting of: (1) the DNA polymerase is a DNA polymerase with strand displacement activity; (2) the DNA polymerase is selected from a Bst DNA polymerase, phi29 DNA polymerase or exo-Klenow; (3) the cation is a magnesium ion; (4) the surfactant is selected from the group consisting of sodium deoxycholate, sodium lauryl sarcosinate, sodium lauryl sulfate, Tween-20, NP-40 and Tritox-100; (5) the cation chelating agent is a magnesium ion chelating agent; (6) the cation chelating agent is selected from the group consisting of NTA, EDTA, HEDP, EDTMPS, DTPMPA, EDDHA, STPP, sodium dextrose and sodium metasilicate; (7) the acidic or basic buffer is selected from the group consisting of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer and Tris hydrochloric acid buffer; and (8) the enzyme is selected from a DNA polymerase, T4 ligase, and BSA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1 shows the general procedure for loading DNB on a slide. In an exemplary embodiment, using a single-stranded circularized DNA library, DNB is obtained by performing rolling circle amplification with primer, dNTP and phi29 DNA polymerase or other polymerase with strand displacement activity under suitable conditions; after the rolling circle amplification is complete, the DNB is loaded directly onto a sequencing slide. Since each DNB may contain different number of copies of the nucleic acid molecule (some DNBs contain a higher number of copies and some DNBs contain a lower number of copies), the size of different DNB varies. During the loading process, due to the influence of Brown/thermal motion and shearing, etc., large DNBs may be loaded on two or more adjacent sites on the slide, while multiple small DNBs may be loaded at the same one site on the slide, and a few DNBs may even be lost into the solution, resulting in no-loading. This significantly affects the subsequent sequencing results (sequencing efficiency and sequencing quality).

[0076] FIG. 2 shows the procedure of loading a DNB onto a slide using the method of the present invention. In an exemplary embodiment, using a single-stranded circularized DNA library, a DNB is obtained by performing a first rolling circle amplification with primer, dNTP and phi29 DNA polymerase or other polymerase with strand displacement activity under suitable conditions. The conditions and time of the first rolling circle amplification are controlled to obtain a DNB of suitable size. In a preferred embodiment, the size of the DNB does not exceed the spacing of adjacent sites on the slide, whereby the DNB can be loaded onto the slide efficiently, avoiding simultaneous loading of multiple DNBs to the same site, or loading of one DNB to two or more adjacent sites on the slide. For example, when the site spacing of the chip is 700 nm, a rolling circle amplification time of no more than 20 min can be used; when the site spacing of the chip is 500 nm, a rolling circle amplification time of no more than 10 min can be used; when the site spacing is further reduced, less rolling circle amplification time can be used to obtain DNBs of suitable size.

[0077] After a predetermined period of rolling circle amplification, various methods can be used to suspend or slow down the first rolling circle amplification. For example, methods that can be used include but are not limited to: (1) adding a certain concentration of EDTA to the reaction mixture to chelate magnesium ions, weaken or stop the polymerization ability of phi29 DNA polymerase, and stop or slow down the growth of DNB; (2) adjusting the temperature of the reaction mixture to a low temperature (e.g, below 20° C., such as 4° C.) to weaken or stop the polymerization ability of phi29 DNA polymerase, and stop or slow down the growth of DNB; (3) adjusting the pH of the reaction mixture to acidity (e.g., using a pH=4.7 mixture of tripotassium citrate and citric acid) to weaken or stop the polymerization ability of phi29 DNA polymerase, and stop or slow down the growth of DNB.

[0078] Subsequently, the DNB is loaded onto a sequencing slide, and a second rolling circle amplification is then performed on the slide to increase the number of copies of nucleic acid molecule contained in the DNB. Various methods can be used to perform the second rolling circle amplification. In certain embodiments, the rolling circle amplification can be restarted by eliminating conditions that inhibit phi29 DNA polymerase activity. For example, when EDTA is used in the previous step to chelate magnesium ions and weaken or stop the polymerization ability of phi29 DNA polymerase, after loading the DNB onto the sequencing slide, an appropriate concentration of magnesium ion buffer can be added to restore the polymerization ability of phi29 DNA polymerase, thereby continuing the rolling circle amplification on the slide. For example, when a low temperature (e.g., below 20° C., for example, 4° C.) is used in the previous step to weaken or stop the polymerization ability of phi29 DNA polymerase, after the DNB is loaded onto the sequencing slide, the temperature of the reaction system can be elevated (e.g., 30-37° C.) to restore the polymerization ability of phi29 DNA polymerase, thereby continuing the rolling circle amplification on the slide. For example, when an acidic buffer (e.g., a mixture of tripotassium citrate and citric acid at pH=4.7) is used to weaken or stop the polymerization ability of phi29 DNA polymerase in the previous step, after the DNB is loaded onto the sequencing slide, the pH of the reaction system can be elevated (for example, by using a working buffer of phi29 DNA polymerase or adding an alkaline buffer) to restore the polymerization ability of phi29 DNA polymerase, thereby continuing the rolling circle amplification on the slide.

[0079] By using the above loading scheme of the present invention, the problem of low loading efficiency caused by the inappropriate size of the DNB used in the loading process (e.g., the size is too large) is effectively avoided, and it is effectively guaranteed that each site corresponds to one DNB; moreover, the number of copies of nucleic acid molecule contained in the DNB loaded at each site is higher, resulting in a stronger signal. Thus, the method of the present invention can improve the DNB loading efficiency and loading quality of each site on the chip (high-density array), improve the signal-to-noise ratio of each site, and allow longer read length and shorter imaging exposure time, and better overall data quality.

[0080] FIG. 3 shows the base signals of the first sequencing cycle of the experimental group 1 chip, the experimental group 2 chip and the control group chip.

[0081] FIG. 4 shows the DNB duplication rates (i.e., the probability that one DNB occupies two sites on the slide) at adjacent sites in the control group chip (FIG. 4A), the experimental group 1 chip (FIG. 4B), and the experimental group 2 chip (FIG. 4C).

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0082] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, they are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer’s indication are conventional products that can be obtained from the market.

[0083] The main equipment used in the examples of the present application were as follows: DNBSEQ-T7RS sequencer (purchased from MGI Co., Ltd. (MGI)), MGIDL-T7RS (purchased from MGI), DNBSEQ-T7RS chip (700 nm, purchased from MGI), Qubit® 3.0 fluorescence quantitative instrument (purchased from Thermo), PCR machine (purchased from Trobot), high-speed centrifuge (purchased from Eppendorf).

[0084] The key reagents used in the examples of the present application were shown in Table 1 below:

TABLE-US-00001 Reagents used Reagent Name Brand One strand sequencing primer MGI DNBSEQ-T7RS high-throughput sequencing kit MGI DNBSEQ-T7RS washing buffer kit MGI phi29 DNA polymerase MGI DNB rapid preparation kit MGI DNBSEQ-T7RS DNB rapid loading kit MGI 25 mM dNTP Mix 10 Pluronic F68 Sigma 1X TE buffer MGI Citric acid Sigma Tripotassium citrate Sigma 10X phi29 buffer MGI DNA nanoball loading buffer II MGI E. coli library MGI

Example 1: Amplification of DNB and Loading

[0085] In this example, experimental group 1, experimental group 2 and control group were set.

[0086] The protocol of experimental group 1 was shown in FIG. 2, and the specific experimental process was as follows:

1.1 First Amplification

[0087] According to the manufacturer’s instructions, the MGIEasy™ DNA library preparation kit (MGI Co., Ltd.) was used to prepare the E. coli single-stranded circular DNA library with the main insert fragment at 300bp. The sequences of the two nucleic acid strands of the library adapter used were: 5′-AGT CGG AGG CCA AGC GGT CTT AGG AAG ACA ATC CTT TTG TAC AAC TCC TTG GCT CAC A-3′ (SEQ ID NO: 1); and 5′-TTG TCT TCC TAA GGA ACG ACA TGG CTA CGA TCC GAC TT-3′ (SEQ ID NO: 2). Then, according to the instructions of the DNB rapid preparation kit (MGI Co., Ltd.), 2 fmol/uL of single-stranded circular DNA library was taken, and then added with 20 uL of DNB primer mixture solution (the specific formula was shown in Table 2), wherein the RCA primer sequence was 5′-GCC ATG TCG TTC TGT GAG CCA AGG-3′ (SEQ ID NO: 3). Primer hybridization was carried out on a PCR machine (95° C., 1 min .fwdarw. 65° C., 1 min .fwdarw. 40° C., 1 min .fwdarw. 4° C., 1 min); then 40 uL of DNA polymerase mixture solution (specific formula was shown in Table 3) and 4ul of phi29 DNA polymerase (MGI Co., Ltd.) were added, and rolling circle amplification was carried out on a PCR machine (30° C., 20 min). The prepared DNB was used for the subsequent loading onto DNBSEQ-T7 chip (the spacing of adjacent sites on the chip was 700 nm, and the maximum size of nucleic acid nanoball that could be accommodated by or attached to a single site was 220 nm).

TABLE-US-00002 DNB primer mixture solution Reagent Name Final concentration/Volume 10X phi29 buffer 1X RCA primer 1 uM H.sub.2O Supplemented to volume of 1 ml

TABLE-US-00003 DNB polymerase mixture solution Reagent Name Final concentration/Volume 10X phi29 buffer 1X 25 mM dNTP Mix 400 uM 10%Pluronic F68 1% 0.1U/ul Pyrophosphatase 0.005U H.sub.2O Supplemented to volume of 1 ml

[0088] After the rolling circle amplification was performed for the above time, the amplification reaction mixture was immediately placed on an ice box, added with 8 .Math.L of 0.5 mM EDTA, and slowly mixed with a wide-mouth pipette for 5-8 times, avoiding shaking and vigorous pipetting. Then, 2 .Math.L of DNB was taken, and Qubit® ssDNA Assay Kit (purchased from Thermo Fisher, art. No. Q10212) and Qubit® 3.0 Fluorometer were used to perform concentration detection. The detection result showed that the nucleic acid concentration was 15 ng/ul. Through 20 min of rolling circle amplification, the size of the formed DNB was about 200 nm. According to the loading volume of the DNBSEQ-T7 chip (MGI Co., Ltd.), a total of 5 tubes of DNB were prepared.

1.2 Preparing DNB Loading System:

[0089] According to the instructions of DNBSEQ-T7RS DNB Rapid Loading Kit (MGI Co., Ltd.), 400 ul of the DNB mixture prepared above was taken, added with 100 ul of PBS, and slowly mixed with a wide-mouth pipette for 5-8 times. Centrifugation, shaking and vigorous pipetting should be avoided.

1.3 Pretreatment of Sequencing Slide and Loading

[0090] First, slide pretreatment reagent 1 (the specific formula was shown in Table 4) was pumped into the chip, allowed to stand at room temperature for 5 minutes, and then slide pretreatment reagent 2 (the specific formula was shown in Table 5) was pumped therein, the two reagents were used for the pretreatment of the chip, which helped to assist in the efficient loading of DNB onto the sequencing slide. After the chip pretreatment was completed, the DNB loading reagent was loaded into MGIDL-T7RS equipment, therefore performing DNB loading. The loading was performed at 4° C. for 30 min.

TABLE-US-00004 Slide pretreatment reagent 1 Reagent Name Final concentration/Volume 1 M tripotassium citrate 0.3 M 1 M citric acid 0.3 M 1X TE buffer Supplemented to 1 ml 10U/ul phi29 polymerase 10 ul

TABLE-US-00005 Slide pretreatment reagent 2 Reagent Name Final concentration/Volume 1 M tripotassium citrate 0.03 M 1 M citric acid 0.03 M 10%Pluronic F68 6 ul 1XTE buffer Supplemented to 1 ml

1.4 Second Amplification

[0091] After the loading was completed, 740 ul of a mixture of magnesium ions with a final concentration of 5 mM and DNB polymerase, and 1 ul of a mixture of phi29 DNA polymerase were pumped therein to reactivate the polymerase, the temperature was set to 30° C., the second amplification was performed on the sequencing slide, and the amplification time was 30 min. After the amplification was completed, the sequencing chip of experimental group 1 was obtained.

[0092] The protocol of experimental group 2 was shown in FIG. 2, and the specific experimental process was as follows:

2.1 The First Amplification

[0093] According to the manufacturer’s instructions, the MGIEasy™ DNA library preparation kit (MGI Co., Ltd.) was used to prepare the E. coli single-stranded circular DNA library with the main insert fragment at 300bp. The sequences of the two nucleic acid strands of the library adapter used were: 5′-AGT CGG AGG CCA AGC GGT CTT AGG AAG ACA ATC CTT TTG TAC AAC TCC TTG GCT CAC A-3′ (SEQ ID NO: 1); and 5′-TTG TCT TCC TAA GGA ACG ACA TGG CTA CGA TCC GAC TT-3′ (SEQ ID NO: 2). Then, according to the instructions of the DNB rapid preparation kit (MGI Co., Ltd.), 2 fmol/uL, of single-stranded circular DNA library was taken, and then added with 20 uL of DNB primer mixture solution (the specific formula was shown in Table 2), wherein the RCA primer sequence was 5′-GCC ATG TCG TTC TGT GAG CCA AGG-3′ (SEQ ID NO: 3). Primer hybridization was carried out on a PCR machine (95° C., 1 min .fwdarw. 65° C., 1 min .fwdarw. 40° C., 1 min .fwdarw. 4° C., 1 min); then 40 uL of DNA polymerase mixture solution (specific formula was shown in Table 3) and 4 ul of phi29 DNA polymerase (MGI Co., Ltd.) were added, and rolling circle amplification was carried out on a PCR machine (30° C., 20 min). The prepared DNB was used for the subsequent loading of DNBSEQ-T7 chip (the spacing of adjacent sites on the chip was 700 nm, and the maximum size of nucleic acid nanoball that could be accommodated by or attached to a single site was 220 nm).

TABLE-US-00006 DNB primer mixture solution Reagent Name Final concentration/Volume 10X phi29 buffer 1X RCA primer 1 uM H.sub.2O Supplemented to volume of 1 ml

TABLE-US-00007 DNB polymerase mixture solution Reagent Name Final concentration/Volume 10X phi29 buffer 1X 25 mM dNTP Mix 400 uM 10%Pluronic F68 1% 0.1 U/ul Pyrophosphatase 0.005U H.sub.2O Supplemented to volume of 1 ml

[0094] After the rolling circle amplification was carried out for the above time, the amplification reaction mixture was immediately placed on an ice box, and gently mixed using a wide-mouth pipette for 5-8 times, avoiding shaking and vigorous pipetting. Then, 2 .Math.L of DNB was taken, and Qubit® ssDNA Assay Kit (purchased from Thermo Fisher, art. No. Q10212) and Qubit® 3.0 Fluorometer were used for concentration detection. The detection result showed that the nucleic acid concentration was 15 ng/ul. Through 20 min of rolling circle amplification, the size of the formed DNB was about 200 nm. According to the loading volume of the DNBSEQ-T7 chip (MGI Co., Ltd.), a total of 5 tubes of DNB were prepared.

2.2 Preparation of DNB Loading System:

[0095] According to the instructions of the DNBSEQ-T7RS DNB Rapid Loading Kit (MGI Co., Ltd.), 500 ul of the DNB mixture prepared above was taken and placed in a loading DNB tube, and immediately placed on ice for later use.

2.3 Pretreatment of Sequencing Slide and Loading

[0096] First, slide pretreatment reagent 1 (the specific formula was shown in Table 4) was pumped in the slide, allowed to stand at room temperature for 5 minutes, and then slide pretreatment reagent 2 (the specific formula was shown in Table 5) was pumped therein, and the two reagents were used to perform the pretreatment of the chip, which helped to assist in the efficient loading of DNB onto the sequencing slide. After the chip pretreatment was completed, the MGIDL-T7RS equipment was used to load the DNB loading system, so as to perform DNB loading. The loading was performed at 4° C. for 30 min.

TABLE-US-00008 Slide pretreatment reagent 1 Reagent Name Final concentration/Volume 1 M tripotassium citrate 0.3 M 1 M citric acid 0.3 M 1X TE buffer Supplemented to 1 ml 10 U/ul phi29 polymerase 10 ul

TABLE-US-00009 Slide pretreatment reagent 2 Reagent Name Final concentration/Volume 1 M tripotassium citrate 0.03 M 1 M citric acid 0.03 M 10%Pluronic F68 6 ul 1XTE buffer Supplemented to 1 ml

2.4 Second Amplification

[0097] After the loading was performed at 4° C. for 30 min, the temperature was set to 30° C., so as to reactivate the phi29 enzyme, the second amplification was performed on the sequencing slide with an amplification time of 30 min. After the amplification was completed, the sequencing chip of experimental group 2 was obtained.

[0098] The protocol of the control group was shown in FIG. 1, and the specific experimental process was as follows:

[0099] The sample used in the control group was the same as that in the experimental group, that was, the E. coli single-stranded circular DNA library with the insert fragment mainly at 300 bp. The sample was subjected to rolling circle amplification using the same kit and the same protocol as the experimental group. Due to the requirements of the sequencing protocol on copy number and sequencing signal, the amplification time in the control group was set to 40 min. The nucleic acid concentration of the amplification product was 35 ng/ul, and the average size of the formed DNB was about 300 nm.

[0100] Subsequently, using the same kit and the same protocol as the experimental group, the amplification product (DNB) was loaded onto the sequencing slide, and the second rolling circle amplification was not performed (i.e., step 1.4 was not performed), thereby obtaining the sequencing chip of control group.

Example 2: Sequencing

[0101] According to the instructions of DNBSEQ-T7RS high-throughput sequencing kit (MGI Co., Ltd.) and DNBSEQ-T7RS washing buffer kit (MGI Co., Ltd.), the sequencing chips obtained in the above experimental group 1, experimental group 2 and control group were placed on the DNBSEQ-T7RS sequencer, and PE100 (paired-end 100bp) sequencing was performed respectively. The sequence of the one-strand sequencing primer (MGI Co., Ltd.) used in the sequencing process was 5′-GC TCA CAG AAC GAC ATG GCT ACG ATC CGA CTT-3′ (SEQ ID NO: 4).

[0102] FIG. 3 shows the comparison results of the base signals of the experimental group 1 chip, the experimental group 2 chip and the control group chip in the first sequencing cycle. The results showed that the signals of the experimental group 1 and experimental group 2 were higher than those of the control group. Table 6 shows the PE100 sequencing report in the experimental group 1, the experimental group 2 and the control group of the present invention. The results showed that the total data volume (total reads) and sequencing quality (Q30) of the experimental group 1 and experimental group 2 using the protocol of the present invention were better than those of the control group. FIG. 4 shows the values of the duplication rate of adjacent DNBs in the experimental group 1, experimental group 2 and control group of the present invention. The higher the value, the more serious the phenomenon that one DNB occupied two site positions. It could be seen from FIG. 4, the duplication rate (dup-rate) of the control group (FIG. 4A) was 10.09%, while the duplication rate (dup-rate) of the experimental group 1 (FIG. 4B) was 1.14%, and the duplication rate (dup-rate) of the experimental group 2 (FIG. 4C) was 1.6%. The results showed that the duplication rates of the experimental groups according to the protocol of the present invention were much lower than that of the control group.

TABLE-US-00010 PE100 sequencing data of experimental groups and control group Control group Experimental group 1 Experimental group 2 Sequencing cycles 210 210 210 Total reads (M) 4813 5355 5200 Q30 87.35 90.72 90.5 Resolution ratio (%) 99.01 99.01 99.00

[0103] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of all the teachings that have been disclosed, and that these changes are all within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.