Method for improving loading efficiency and quality of nucleic acid nanospheres

Abstract

The invention relates to a method for adjusting the volume of a nucleic acid nanosphere, which comprises the following steps of: carrying out coagulation treatment on nucleic acid (for example, adding a nucleic acid coagulant and / or adjusting the pH value to 3-4.5 to gather positively charged counter ions around the nucleic acid, and neutralizing negative charges on the surface of the nucleic acid by cations through the electrostatic binding action of anions and cations to coagulate the form of the nucleic acid), so that the volume of the nucleic acid nanosphere is adjusted. Further, the form of the nucleic acid nanosphere can be changed, the volume of the nucleic acid nanosphere is adjusted, and the nucleic acid nanosphere is compressed; the nucleic acid nanospheres obtained by adopting the method (carrying out condensation treatment on the nucleic acid nanospheres) are tighter, smaller in shape and more uniform in size, and the method can be applied to any occasion (such as a loading process of the nucleic acid nanospheres (such as DNB) and a sequencing process after loading) where the volume of the nucleic acid nanospheres needs to be adjusted (reduced).

Description

A method for improving the loading efficiency and quality of nucleic acid nanospheres Technical Field

[0001] The present invention belongs to the field of nucleic acid sequencing, and in particular relates to a method for improving the loading efficiency and quality of nucleic acid nanospheres. Background Art

[0002] Microarray DNA nanoball sequencing technology (DNBseq technology) is a type of high-throughput sequencing technology. Its main principle is to use rolling circle amplification (RCA) technology to obtain amplification products, namely DNA nanoballs (DNBs), and then load them onto a regular microarray chip. Finally, the optimized combined probe anchor polymerization technology (cPAS) is used to achieve high-precision gene sequencing.

[0003] Take the DNBseq technology used in MGI sequencers as an example: Single-stranded circular DNA (DNBs) are first generated using RCA technology, using single-stranded circular DNA as a template. The DNBs are then loaded onto a regular array chip via the instrument's gas-liquid system and immobilized. Finally, high-throughput sequencing is achieved through enzyme action and cPAS technology (Figure 1).

[0004] DNBseq technology requires the following three requirements to achieve high sequencing quality: 1) a sufficient number of DNB copies; 2) precise and unique arrangement of DNBs on the microarray chip during loading; and 3) a sufficiently strong and easily distinguishable optical signal. However, practical applications still present challenges. For example, when a sufficiently strong signal is required, the RCA time often needs to be extended to increase the library copy number. Within a certain timeframe, as the RCA time increases, the DNB copy number also increases. During gene sequencing, a high DNB copy number enhances the detection signal, facilitating base call interpretation and improving sequencing quality. However, this increased DNB copy number also increases DNB size, which increases the interaction between DNBs when loaded onto a regular microarray chip. This can lead to problems such as an increased number of empty sites due to incomplete reaction and uneven size of DNBs loaded onto the chip, resulting in poor sequencing quality. Therefore, a method is needed to increase DNB copy number without increasing size, enabling better loading onto the chip, in order to optimize microarray DNBseq technology and improve sequencing quality.

[0005] Summary of the Invention

[0006] The first aspect of the present invention aims to provide a method for adjusting the volume of nucleic acid nanoballs (eg, DNBs).

[0007] The second aspect of the present invention is to provide a reagent for adjusting the volume of nucleic acid nanoballs (eg, DNBs).

[0008] The third aspect of the present invention aims to provide a method for improving the loading efficiency and quality of nucleic acid nanospheres (eg, DNBs).

[0009] The fourth aspect of the present invention aims to provide a sequencing method.

[0010] The fifth aspect of the present invention aims to provide a kit.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] In a first aspect, the present invention provides a method for adjusting the volume of nucleic acid nanospheres (e.g., DNBs), wherein the nucleic acid nanospheres are subjected to a coagulation treatment to obtain nucleic acid nanospheres with a compressed volume;

[0013] The method for agglomerating the nucleic acid nanospheres comprises at least one of a1) to a2):

[0014] a1) adding a nucleic acid condensing agent to the system containing nucleic acid nanospheres;

[0015] a2) adjusting the pH of the system containing the nucleic acid nanospheres to 3-4.5;

[0016] The nucleic acid condensing agent comprises at least one of a multivalent salt, a basic peptide, a basic protein, a polar solvent, a positively charged high molecular polymer, a polyamine, a polyammonium, and a lipid.

[0017] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a1).

[0018] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a2).

[0019] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a1) and a2).

[0020] Preferably, the basic protein comprises at least one of histone and protamine.

[0021] Preferably, the polar solvent comprises an alcohol.

[0022] Preferably, the positively charged high molecular polymer comprises at least one of polyhistidine, polylysine, polyarginine and polyornithine.

[0023] Preferably, the polyamine comprises at least one of spermine, spermidine and putrescine; and further comprises at least one of spermine and putrescine.

[0024] Preferably, the polyammonium comprises hexadimethrinebromide (polybrene).

[0025] Preferably, the lipid comprises at least one of DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE; and further comprises at least one of DOTAP and DC-Chol / DOPE.

[0026] Preferably, the nucleic acid condensing agent in a1) comprises at least one of a polyvalent salt, a polyamine, and a lipid; further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent salt; even further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent metal salt.

[0027] Preferably, the polyvalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; further preferably, the polyvalent metal salt comprises: at least one of a zinc salt, a calcium salt, a magnesium salt, a copper salt, and a cerium salt (for example, it can be ZnCl2, ZnSO4, CaCl2, MgCl2, CuCl2·2H2O, CeCl3·7H2O); even more preferably, the polyvalent metal salt comprises: at least one of a copper salt, a cerium salt, a calcium salt, and a zinc salt.

[0028] Preferably, the final concentration of the nucleic acid condensing agent in a1) in the system is 0.001 to 1000 mM; further 1 to 1000 mM; further 10 to 100 mM; further 12 to 80 mM.

[0029] Preferably, the pH of the system in a2) is 4 to 4.5; further 4.2 to 4.4.

[0030] In a second aspect of the present invention, a reagent for adjusting the volume of nucleic acid nanoballs (e.g., DNBs) is provided.

[0031] The reagent for adjusting the volume of the nucleic acid nanospheres comprises at least one of b1) to b2):

[0032] b1) the reagent for adjusting the volume of nucleic acid nanospheres comprises a nucleic acid condensing agent;

[0033] b2) the pH of the reagent for adjusting the volume of nucleic acid nanospheres is 3 to 4.5;

[0034] The nucleic acid condensing agent comprises at least one of a multivalent salt, a basic peptide, a basic protein, a polar solvent, a positively charged high molecular polymer, a polyamine, a polyammonium, and a lipid.

[0035] The reagent for adjusting the volume of the nucleic acid nanospheres further comprises a nucleic acid nanosphere loading buffer or a sequencing reagent.

[0036] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres comprises b1).

[0037] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres comprises b2).

[0038] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres comprises b1) and b2).

[0039] Preferably, the basic protein comprises at least one of histone and protamine.

[0040] Preferably, the polar solvent comprises an alcohol.

[0041] Preferably, the positively charged high molecular polymer comprises at least one of polyhistidine, polylysine, polyarginine and polyornithine.

[0042] Preferably, the polyamine comprises at least one of spermine, spermidine and putrescine; and further comprises at least one of spermine and putrescine.

[0043] Preferably, the polyammonium comprises hexadimethrinebromide (polybrene).

[0044] Preferably, the lipid comprises at least one of DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE; and further comprises at least one of DOTAP and DC-Chol / DOPE.

[0045] Preferably, the nucleic acid condensing agent in b1) comprises at least one of a polyvalent salt, a polyamine, and a lipid; further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent salt; and even further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent metal salt.

[0046] Preferably, the polyvalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; further preferably, the polyvalent metal salt comprises: at least one of a zinc salt, a calcium salt, a magnesium salt, a copper salt, and a cerium salt (for example, it can be ZnCl2, ZnSO4, CaCl2, MgCl2, CuCl2·2H2O, CeCl3·7H2O); even more preferably, the polyvalent metal salt comprises: at least one of a copper salt, a cerium salt, a calcium salt, and a zinc salt.

[0047] Preferably, the pH of the reagent in b2) is 4 to 4.5; further 4.2 to 4.4.

[0048] Preferably, the method for using the reagent for adjusting the volume of nucleic acid nanospheres is: mixing the reagent for adjusting the volume of nucleic acid nanospheres with nucleic acid nanospheres to obtain a system for adjusting the volume of nucleic acid nanospheres;

[0049] And the system satisfies at least one of c1) to c2):

[0050] c1) the final concentration of the nucleic acid condensing agent in the system for adjusting the volume of nucleic acid nanospheres is 0.001 to 1000 mM (when the reagent for adjusting the volume of nucleic acid nanospheres comprises b1) or comprises both b1) and b2);

[0051] c2) The pH of the system for adjusting the volume of nucleic acid nanospheres is 3-4.5 (when the reagent for adjusting the volume of nucleic acid nanospheres comprises b2) or comprises both b1) and b2)).

[0052] Preferably, the final concentration of the nucleic acid condensing agent in c1) in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 12 to 80 mM.

[0053] Preferably, the pH of the system for adjusting the volume of nucleic acid nanospheres in c2) is 4 to 4.5; further 4.2 to 4.4.

[0054] Preferably, the nucleic acid nanosphere (eg, DNB) loading buffer comprises at least one of a citrate buffer, a MES buffer solution, and a Tris hydrochloride buffer.

[0055] Preferably, the sequencing reagent comprises: at least one of: a dNTPs mixture, a nucleic acid (such as DNA) polymerase mixture, an MDA reagent, and an MDA polymerase mixture; and further comprises: a dNTPs mixture, a nucleic acid (such as DNA) polymerase mixture, an MDA reagent, and an MDA polymerase mixture.

[0056] The third aspect of the present invention provides a method for improving the loading efficiency and quality of nucleic acid nanoballs (such as DNBs), wherein the method for adjusting the volume of nucleic acid nanoballs (such as DNBs) according to the first aspect of the present invention is used during the loading of nucleic acid nanoballs.

[0057] Preferably, the method for improving the loading efficiency and quality of nucleic acid nanospheres comprises the following steps:

[0058] A nucleic acid nanosphere is subjected to a coagulation treatment to obtain a nucleic acid nanosphere with a compressed volume;

[0059] loading the volume-compressed nucleic acid nanospheres onto a solid support;

[0060] The method for agglomerating nucleic acid nanospheres is the method for agglomerating acid nanospheres in the first aspect of the present invention.

[0061] Preferably, the solid support comprises at least one of latex beads, dextran beads, polystyrene surface, polypropylene surface, polyacrylamide gel, gold surface, glass surface, chip, sensor, electrode, silicon wafer; further comprises a chip; further a high-density matrix chip.

[0062] Preferably, the nucleic acid nanoballs (eg, DNBs) are prepared by rolling circle amplification (RCA).

[0063] Preferably, the system comprising nucleic acid nanospheres in a1), and / or a2) further comprises: a nucleic acid nanosphere (eg, DNB) loading buffer.

[0064] Preferably, the nucleic acid nanosphere (eg, DNB) loading buffer is the nucleic acid nanosphere loading buffer in the second aspect of the present invention.

[0065] Preferably, the rolling circle amplification time of the nucleic acid nanoballs (eg, DNBs) is 20 to 120 minutes.

[0066] Preferably, the rolling circle amplification time of the nucleic acid nanoballs (eg, DNBs) is 30 to 120 minutes.

[0067] Preferably, the rolling circle amplification time of the nucleic acid nanoballs (eg, DNBs) is 40 to 120 minutes.

[0068] A fourth aspect of the present invention provides a sequencing method comprising the following steps:

[0069] 1) adjusting the volume of the nucleic acid nanospheres using the method of the first aspect of the present invention;

[0070] 2) Sequencing to obtain the sequence information carried by the nucleic acid nanospheres.

[0071] Preferably, step 1) can be performed in any one or more sequencing processes of the sequencing method.

[0072] Preferably, the sequencing process includes but is not limited to: a nucleic acid nanoball loading process and a nucleic acid nanoball sequencing process.

[0073] Preferably, during the loading process of the nucleic acid nanospheres in step 1), the system comprising the nucleic acid nanospheres further comprises: a nucleic acid nanosphere loading buffer.

[0074] Preferably, the nucleic acid nanosphere loading buffer comprises at least one of citrate buffer, MES buffer solution, and Tris hydrochloride buffer.

[0075] Preferably, during the sequencing process of the nucleic acid nanoballs in step 1), the system comprising the nucleic acid nanoballs further comprises: a sequencing reagent.

[0076] Preferably, the sequencing reagent comprises at least one of a dNTPs mixture, a nucleic acid polymerase mixture, an MDA reagent, and an MDA polymerase mixture.

[0077] A fifth aspect of the present invention provides a kit comprising the reagent for adjusting the volume of nucleic acid nanoballs (eg, DNBs) according to the second aspect of the present invention.

[0078] Preferably, the kit further comprises: a reagent for preparing nucleic acid nanospheres (eg, DNB).

[0079] Preferably, the nucleic acid nanosphere (e.g., DNB) preparation reagent comprises: at least one of: TE buffer, nucleic acid nanosphere (e.g., DNB) preparation buffer, nucleic acid nanosphere (e.g., DNB) polymerase mixture, and nucleic acid nanosphere (e.g., DNB) termination buffer; and further comprises: TE buffer, nucleic acid nanosphere (e.g., DNB) preparation buffer, nucleic acid nanosphere (e.g., DNB) polymerase mixture, and nucleic acid nanosphere (e.g., DNB) termination buffer.

[0080] The beneficial effects of the present invention are:

[0081] The present invention provides a method for adjusting the volume of nucleic acid nanoballs (e.g., DNBs). The method comprises agglomerating nucleic acids (e.g., DNA) (e.g., adding a nucleic acid agglomerating agent (e.g., a multivalent salt, a basic peptide, a basic protein, a polar solvent (e.g., an alcohol), a positively charged polymer, a polyamine, a polyammonium compound, or a lipid) and / or adjusting the pH to 3-4.5, so that positively charged counterions gather around the nucleic acids (e.g., DNA), and through electrostatic binding between anions and cations, the negative surface charge of the nucleic acids (e.g., DNA) is neutralized by cations, thereby causing the nucleic acids (e.g., DNA) to condense). The method can thereby change the morphology of the nucleic acid nanoballs (e.g., DNBs), adjust the volume of the nucleic acid nanoballs (e.g., DNBs), and compress the nucleic acid nanoballs (e.g., DNBs). The nucleic acid nanoballs (e.g., DNBs) obtained by this method (agglomerating nucleic acid nanoballs) are more compact, smaller in size, and more uniform in size. The method can be used in any application requiring the volume adjustment (reduction) of nucleic acid nanoballs (e.g., DNBs) (e.g., any one or more sequencing processes, such as the loading process of nucleic acid nanoballs (e.g., DNBs) and the sequencing process after loading).

[0082] When this method is used in the loading process of nucleic acid nanoballs (e.g., DNBs), it can improve the loading efficiency and quality of nucleic acid nanoballs (e.g., DNBs), so that when the nucleic acid nanoballs (e.g., DNBs) are loaded onto a solid support (e.g., a chip), their distribution is more uniform, with fewer empty sites and uniform size, more concentrated signals, and less impact on adjacent sites. This can lower the Dup value, reduce chip empty sites, and improve sequencing quality. At the same time, it can also improve the detection signal to meet the requirements of long-read sequencing, as follows:

[0083] For nucleic acid nanospheres (e.g., DNBs) with the same RCA time, the method of the present invention for the loading process of nucleic acid nanospheres (e.g., DNBs) has a lower Dup value than the existing technology (nucleic acid nanospheres are not subjected to aggregation treatment, i.e., the method of the present invention is not used);

[0084] Furthermore, by limiting the rolling circle amplification time of nucleic acid nanoballs (e.g., DNBs) to 30 to 120 minutes (increasing the RCA time), for large nucleic acid nanoballs (e.g., large DNBs) with increased RCA time, the method of the present invention is used for the loading process of nucleic acid nanoballs (e.g., DNBs), compared with the original technology (nucleic acid nanoballs are not subjected to agglomeration treatment). Since the morphology of the large nucleic acid nanoballs is compressed, the loading efficiency and quality of the nucleic acid nanoballs (e.g., DNBs) are improved, the arrangement of the nucleic acid nanoballs on the chip is more uniform and regular, the large nucleic acid nanoball signals at each site are more concentrated, the interference of adjacent sites is reduced, and the Dup value is reduced; at the same time, after the volume of the large DNB is compressed, the interference of sites competing with each other due to volume during the loading process is also reduced, and the number of chip vacancies becomes smaller; the signals of the large nucleic acid nanoballs are stronger and more concentrated, which is conducive to base calling and improves sequencing quality. This solves the contradiction between the existing sequencing technology requiring the base signal to be enhanced by increasing the copy number but the sequencing quality deteriorates, and can improve the problem of reduced sequencing quality, and can meet the requirements of long read length sequencing;

[0085] Based on the solution to the contradictory problem that increasing the copy number enhances the base signal but deteriorates the sequencing quality, the method of the present invention, when used in the loading process of nucleic acid nanoballs (such as DNBs), can increase the RCA time while performing agglomeration treatment on the obtained large nucleic acid nanoballs, so that the structure of the large nucleic acid nanoballs (such as DNBs) is more compact and the size is more uniform, thereby improving the loading efficiency and quality of the nucleic acid nanoballs (such as DNBs). When loaded onto a solid support (such as a chip), the distribution is more uniform, the number of empty sites is reduced, the size is more uniform, the signal is more concentrated, and the impact on adjacent sites is reduced, thereby reducing the Dup value and reducing the number of empty sites on the chip. The combination of these two effects improves the sequencing quality of large nucleic acid nanoballs (such as DNBs) and has better sequencing quality than nucleic acid nanoballs (such as DNBs) obtained with a short RCA time but without agglomeration treatment;

[0086] Furthermore, the use of multivalent metal salts as nucleic acid condensing agents can also improve the adsorption capacity of solid supports (e.g., chips);

[0087] When this method is used in the sequencing process after loading, the volume of the nucleic acid nanoballs (eg, DNBs) can be adjusted (eg, to maintain the shape of the DNBs), further improving the sequencing quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] FIG1 is a schematic diagram of the rolling circle amplification process for preparing DNBs in the prior art and loading the DNBs onto a chip.

[0089] FIG2 is a schematic diagram of a method for loading nucleic acid nanospheres of the present invention to perform agglomeration treatment on DNB.

[0090] FIG3 is a result diagram of the effect of metal ions on Dup value and Q30 in Example 1: wherein the left vertical axis is the Dup value, and the right vertical axis is the Q30.

[0091] FIG4 is a graph showing the effect of DLBII pH on Dup value and Q30 in Example 2, wherein the left vertical axis represents the Dup value, and the right vertical axis represents Q30.

[0092] FIG5 is a result diagram showing the effect of metal ion concentration on Dup value and Q30 in Example 3: wherein the left vertical axis is the Dup value, and the right vertical axis is the Q30.

[0093] Figure 6 is a graph of the double-end PE100 sequencing results of the experimental group and the control group in Example 4: wherein A is the Dup value of the double-end PE100 sequencing results of the experimental group and the control group in Example 4 (the horizontal axis is different FOVs, and the vertical axis is the Dup value); B is the A base initial signal (Signal-A) of the double-end PE100 sequencing results of the experimental group and the control group in Example 4 (the horizontal axis is the sequencing cycle number, and the vertical axis is Signal-A); C is the Q30 of the double-end PE100 sequencing results of the experimental group and the control group in Example 4 (the horizontal axis is the sequencing cycle number, and the vertical axis is Q30).

[0094] Figure 7 is an atomic force microscope (AFM) characterization result diagram of the experimental group and the control group in Example 4: wherein, A is an intuitive diagram of the atomic force microscope (AFM) characterization of the control group (the scale is 1 μm); B is an intuitive diagram of the atomic force microscope (AFM) characterization of the experimental group (the scale is 1 μm); C is a result diagram of the height of the DNB of the control group (the horizontal axis is the scanning range of 5 μm, and the vertical axis is the DNB height); D is a result diagram of the height of the DNB of the experimental group (the horizontal axis is the scanning range of 5 μm, and the vertical axis is the DNB height).

[0095] FIG8 is a result diagram showing the effect of metal ion concentration on Dup value and Q30 in Example 5, wherein the left vertical axis is the Dup value and the right vertical axis is the Q30.

[0096] Figure 9 is a graph of the double-end PE50 sequencing results of the experimental group and the control group in Example 6: wherein A is the Dup value of the double-end PE50 sequencing results of the experimental group and the control group in Example 6 (the horizontal axis is different FOVs, and the vertical axis is the Dup value); B is the A base initial signal (Signal-A) of the double-end PE50 sequencing results of the experimental group and the control group in Example 6 (the horizontal axis is the sequencing cycle number, and the vertical axis is Signal-A); C is the Q30 of the double-end PE50 sequencing results of the experimental group and the control group in Example 6 (the horizontal axis is the sequencing cycle number, and the vertical axis is Q30). DETAILED DESCRIPTION

[0097] Definition of terms

[0098] As used herein, the term "nucleic acid" can be any type of nucleic acid. For example, the nucleic acid can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA made from, for example, nucleotide analogs.

[0099] As used herein, the term "nucleic acid nanoball" generally refers to a complex or concatemer comprising multiple copies of a nucleic acid molecule. In certain typical embodiments, these nucleic acid copies can be arranged one by one in a continuous linear chain of nucleotides. This tandem repeat structure, together with the single-stranded nature of nucleic acids (e.g., DNA), causes the nanoball to fold. It is easy to understand that the multiple copies of the nucleic acid molecules in the nucleic acid nanoball can each contain a linker sequence with a known sequence to facilitate amplification or sequencing. The linker sequences of each nucleic acid molecule are generally the same, but may also be different. Nucleic acid nanoballs include, but are not limited to, DNA nanoballs, which are also referred to herein as DNBs.

[0100] Nucleic acid nanospheres can be produced using, for example, rolling circle replication (RCR) / rolling circle amplification (RCA). The RCR process was used to prepare multiple serial copies of the M13 genome (Blanco et al., (1989) J Biol Chem 264:8935-8940). In this method, nucleic acids are replicated in linear concatemers. Those skilled in the art can find guidance on selecting conditions and reagents for RCR reactions in a number of references, including U.S. Patents Nos. 5,426,180, 5,854,033, 6,143,495, and 5,871,921, each of which is incorporated herein by reference in its entirety for all purposes, and in particular for all teachings relating to the preparation of nucleic acid nanospheres using RCR or other methods.

[0101] As used herein, the term "solid support" means any insoluble substrate or matrix to which nucleic acid nanospheres (e.g., DNBs) can be attached, for example, latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers. The surface of the solid support can be of any desired shape, including, for example, planar, spherical, or porous shapes suitable for a particular application; for example, the solid support can be a planar glass surface. The solid support can be fixed, for example, mounted inside a flow cell to allow interaction with a solution containing a reagent. Alternatively, the solid support can also be removable so as to contact with solutions in different reaction vessels.

[0102] Typically, a solid support may comprise an array of sites arranged in a predetermined pattern, wherein each site can be attached to or accommodate a nucleic acid nanoball (e.g., a DNB). When the size of an attached nucleic acid nanoball (e.g., a DNB) exceeds the maximum size that can be accommodated by a single site or exceeds the spacing between adjacent sites in the array, the nucleic acid nanoball (e.g., a DNB) may occupy two or more sites, which may result in reduced utilization of array sites and may affect sequencing efficiency and quality.

[0103] As used herein, the term "loading" when referring to nucleic acid nanoballs (e.g., DNBs) is used, means directly or indirectly attached to a solid support via a covalent bond or a non-covalent bond. In certain embodiments of the present disclosure, the method of the present invention includes fixing nucleic acid nanoballs (e.g., DNBs) on a solid support via covalent attachment. However, generally, it is only necessary that the nucleic acid (e.g., nucleic acid nanoballs, DNBs) remain fixed or attached to the solid support under conditions where a solid support is desired (e.g., in applications requiring nucleic acid sequencing). Preferably, loading can occur by base pairing hybridization or other means, such as the covalent attachment described above. Non-limiting examples of nucleic acid (e.g., nucleic acid nanoballs (DNBs)) and solid support attachment methods include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, etc. Various exemplary methods for immobilizing nucleic acids (e.g., nucleic acid nanospheres (DNBs)) on solid supports can be found in, for example, G. Steinberg-Tatman et al., Bioconjugate Chemistry 2006, 17, 841-848; Xu X. et al., Journal of the American Chemical Society 128 (2006) 9286-9287; U.S. patents or patent applications US 5639603, US 5641658, US2010248991; international patent applications WO 2001062982, WO 2001012862, WO2007111937, WO0006770, all of which are incorporated herein by reference in their entirety for all purposes, in particular for all teachings relating to the preparation of solid supports having nucleic acids immobilized thereon.

[0104] A key metric for evaluating the quality of DNA nanosphere loading onto a chip is the Duplicate value (Dup value). In high-throughput sequencing, the Dup value refers to the proportion of duplicate reads in the total sequence. Duplicate reads are primarily defined by two criteria: 1) the position and base alignment of the sequenced reads to the genome are completely identical; and 2) the orientation of the reads aligned to the reference genome is completely identical. Duplicate reads that meet both of these criteria are considered duplicates. Duplicates are primarily generated by the following: 1) Dup from the sample itself; 2) Dup introduced by amplification during library construction (i.e., PCR Dup); 3) Dup introduced by pre-sequencing signal amplification (the generation process of the fluorescence signal acquisition unit); and 4) optical Dup introduced during chip sequencing. Theoretically, due to the complexity of the sample itself, the probability of Dup from the first two is extremely low, and the latter two, i.e., Dup caused by optical resolution, are more likely to be generated. When DNBs are loaded onto the chip, incomplete reactions may occur due to inconsistent DNB sizes. For example, if a DNB signal is too strong (due to its large size) or the loading position is offset, its signal coverage exceeds the entire DNB region, resulting in the sequences generated by adjacent "DNA nanoballs" being identical, mapped to two wells expressing the same signal. This is an optical Dup, or Neighbor Dup (%). Therefore, it is necessary to balance the relationship between DNB copy number and Dup value to improve sequencing quality.

[0105] In the process of optimizing the microarray DNBseq technology, the inventors discovered that the Dup value is closely related to the DNB copy number. The longer the RCA time, the more DNB copies, the larger the DNB, and the result obtained by sequencing is a larger Dup value. On the contrary, the shorter the RCA time, the fewer DNB copies, the smaller the DNB, and the smaller the Dup value obtained by sequencing. However, there are certain problems in reducing the Dup value by shortening the RCA time. That is, when the RCA time is short, although the Dup value is small, the DNB copy number is small. During sequencing, the DNB signal is weak, which is not conducive to identifying the base sequence and cannot meet the requirements of long read length sequencing. Therefore, it is urgent to solve the problem that the RCA time is increased, the DNB copy number is large but the Dup value does not increase, so as to improve the sequencing quality.

[0106] Another key factor affecting sequencing quality is whether DNBs can be evenly loaded onto a regular microarray chip. While optimizing microarray DNBseq technology, the inventors discovered that as DNB copy number increases, their larger size makes it difficult for a large DNB to be loaded onto one site. This makes it difficult for adjacent sites to load other large DNBs, resulting in more empty sites on the chip and reduced sequencing quality. Therefore, it is necessary to address the issue of increased RCA time, a large number of DNB copies, and an increase in size leading to more empty sites on the chip, thereby improving sequencing quality.

[0107] During their research, the inventors learned that nucleic acids (e.g., DNA) are long-chain polymers composed of repeating deoxynucleotides. In polar solutions, the bases and phosphates in their structures carry a large amount of negative charge. By subjecting nucleic acids (e.g., DNA) to agglomeration treatment (for example, adding a nucleic acid agglomerating agent (e.g., a multivalent salt, basic peptide, basic protein, polar solvent (e.g., alcohol), a positively charged polymer, polyamine, polyammonium, lipid) and / or adjusting the pH to 3-4.5, positively charged counterions gather around the nucleic acids (e.g., DNA), and through the electrostatic binding of anions and cations, the negative charge on the surface of the nucleic acids (e.g., DNA) is neutralized by cations, thereby causing the nucleic acids (e.g., DNA) to agglomerate), the morphology of nucleic acid nanoballs (e.g., DNBs) can be changed, the volume of nucleic acid nanoballs (e.g., DNBs) can be adjusted, and the nucleic acid nanoballs (e.g., DNBs) can be compressed.

[0108] The nucleic acid nanospheres (e.g., DNBs) obtained by this method (agglomeration of nucleic acid nanospheres) are more compact, smaller in size, and more uniform in size. This method can be used in any situation where the volume of DNBs needs to be adjusted (reduced) (e.g., during any one or more sequencing processes, such as the loading of nucleic acid nanospheres (e.g., DNBs) and the subsequent sequencing process).

[0109] When this method is used in the loading process of nucleic acid nanoballs (such as DNBs), the loading efficiency and quality of nucleic acid nanoballs (such as DNBs) can be improved, so that when the nucleic acid nanoballs (such as DNBs) are loaded on a solid support (such as a chip), the distribution is more uniform, the empty sites are reduced, and the size is uniform, the signal is more concentrated, and the impact on adjacent sites is reduced, thereby reducing the Dup value, reducing the empty sites on the chip, and improving the sequencing quality; at the same time, it can also improve the detection signal to meet the requirements of long-read sequencing; when this method is used in the sequencing process after loading, the volume of the nucleic acid nanoballs (such as DNBs) can be adjusted (for example, to keep the DNBs in shape), further improving the sequencing quality.

[0110] Therefore, the inventors applied agglomeration treatment (e.g., adding nucleic acid agglomerating agents (e.g., multivalent salts, basic peptides, basic proteins, polar solvents (e.g., alcohols), positively charged polymers, polyamines, polyammonium, lipids) and adjusting the pH to 3-4.5) to nucleic acid nanoballs (e.g., DNBs), especially multi-copy nucleic acid nanoballs (e.g., nucleic acid nanoballs obtained by increasing the RCA time) to any situation where the volume of DNBs needs to be adjusted (reduced) (e.g., during any one or more sequencing processes, such as the loading process of nucleic acid nanoballs (e.g., DNBs) and the subsequent sequencing process):

[0111] When this method is used in the loading process of nucleic acid nanoballs (such as DNBs), the loading efficiency and quality of nucleic acid nanoballs are improved by improving the morphology of nucleic acid nanoballs (such as DNBs), especially nucleic acid nanoballs with multiple copies (nucleic acid nanoballs obtained by increasing the RCA time, also known as large nucleic acid nanoballs), thereby improving the effect of loading onto the chip (reducing the Dup value and reducing chip empty sites). That is, the RCA time is increased to obtain nucleic acid nanoballs with multiple copies (nucleic acid nanoballs obtained by increasing the RCA time, also known as large nucleic acid nanoballs), and the nucleic acid nanoballs (such as DNBs) are subjected to a cohesion treatment during loading to make the morphology of the nucleic acid nanoballs (such as DNBs) smaller and more uniform, thereby making the nucleic acid nanoballs with multiple copies (large nucleic acid nanoballs) accurately and uniquely arranged on the microarray chip. At the same time, the large nucleic acid nanoballs have stronger and more concentrated signals, which are beneficial to base calling and improve sequencing quality. This solves the contradiction between the existing sequencing technology requiring base signal enhancement by increasing the copy number but the sequencing quality deteriorating, thereby further improving sequencing quality.

[0112] When this method is used in the sequencing process after nucleic acid nanoballs (eg, DNBs) are loaded, the volume of the nucleic acid nanoballs (eg, DNBs) can be adjusted (eg, the DNBs maintain their shape), thereby improving sequencing quality.

[0113] In a first aspect, the present invention provides a method for adjusting the volume of nucleic acid nanospheres (e.g., DNBs), wherein the nucleic acid nanospheres are subjected to a coagulation treatment to obtain nucleic acid nanospheres with a compressed volume;

[0114] The method for agglomerating the nucleic acid nanospheres comprises at least one of a1) to a2):

[0115] a1) adding a nucleic acid condensing agent to the system containing nucleic acid nanospheres;

[0116] a2) adjusting the pH of the system containing the nucleic acid nanospheres to 3-4.5;

[0117] The nucleic acid condensing agent includes at least one of a multivalent salt, a basic peptide, a basic protein (such as histone, protamine), a polar solvent (such as alcohol), a positively charged high molecular polymer (such as polyhistidine, polylysine, polyarginine and polyornithine), a polyamine (such as spermine, spermidine and putrescine), a polyammonium (such as polybrene (hexadimethrinebromide)), and a lipid (such as DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE).

[0118] During their research, the inventors learned that nucleic acids (e.g., DNA) are long-chain polymers composed of repeating arrangements of (deoxy)nucleotides. In polar solutions, the bases and phosphates in their structures carry a large amount of negative charge. By subjecting the nucleic acids (e.g., DNA) to agglomeration treatment (for example, adding a nucleic acid agglomerating agent (e.g., a multivalent salt, basic peptide, basic protein, polar solvent (e.g., alcohol), a positively charged polymer, polyamine, polyammonium, lipid) and / or adjusting the pH to 3-4.5, positively charged counterions gather around the nucleic acids (e.g., DNA), and through the electrostatic binding of anions and cations, the negative charge on the surface of the nucleic acids (e.g., DNA) is neutralized by cations, thereby causing the nucleic acids (e.g., DNA) to agglomerate), the morphology of the nucleic acid nanoballs (e.g., DNBs) can be changed, the volume of the nucleic acid nanoballs (e.g., DNBs) can be adjusted, and the nucleic acid nanoballs (e.g., DNBs) can be compressed, making the nucleic acid nanoballs (e.g., DNBs) more compact, smaller, and more uniform in size.

[0119] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a1).

[0120] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a2).

[0121] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a1) and a2).

[0122] Preferably, the nucleic acid condensing agent in a1) comprises at least one of a polyvalent salt, a polyamine, and a lipid; further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent salt; even further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent metal salt.

[0123] Preferably, the polyvalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; further preferably, the polyvalent metal salt comprises: at least one of a zinc salt, a calcium salt, a magnesium salt, a copper salt, and a cerium salt (for example, it can be ZnCl2, ZnSO4, CaCl2, MgCl2, CuCl2·2H2O, CeCl3·7H2O); even more preferably, the polyvalent metal salt comprises: at least one of a copper salt, a cerium salt, a calcium salt, and a zinc salt.

[0124] Preferably, the polyamine comprises at least one of spermine and putrescine.

[0125] Preferably, the lipid comprises at least one of DOTAP and DC-Chol / DOPE.

[0126] Preferably, the final concentration of the nucleic acid condensing agent in a1) in the system is 0.001 to 1000 mM; further 1 to 1000 mM; further 10 to 100 mM; further 12 to 80 mM.

[0127] Preferably, the final concentration of the zinc salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 80 mM.

[0128] Preferably, the final concentration of the calcium salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 12 to 20 mM.

[0129] Preferably, the final concentration of the copper salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0130] Preferably, the final concentration of the magnesium salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0131] Preferably, the final concentration of the cerium salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0132] Preferably, the final concentration of the polyamine in the system is 0.001 to 10 mM; further 0.01 to 0.04 mM.

[0133] Preferably, the final concentration of the lipid in the system is 0.001 to 10 mM; further 0.003 to 0.007 mM.

[0134] Preferably, the pH of the system in a2) is 4 to 4.5; further 4.2 to 4.4.

[0135] In a second aspect of the present invention, a reagent for adjusting the volume of nucleic acid nanoballs (e.g., DNBs) is provided.

[0136] The reagent for adjusting the volume of the nucleic acid nanospheres comprises at least one of b1) to b2):

[0137] b1) the reagent for adjusting the volume of nucleic acid nanospheres comprises a nucleic acid condensing agent;

[0138] b2) the pH of the reagent for adjusting the volume of nucleic acid nanospheres is 3 to 4.5;

[0139] The nucleic acid condensing agent includes at least one of a multivalent salt, a basic peptide, a basic protein (such as histone, protamine), a polar solvent (such as alcohol), a positively charged high molecular polymer (such as polyhistidine, polylysine, polyarginine and polyornithine), a polyamine (such as spermine, spermidine and putrescine), a polyammonium (such as polybrene (hexadimethrinebromide)), and a lipid (such as DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE).

[0140] By adding a nucleic acid condensing agent and / or adjusting the pH to 3-4.5, positively charged counter ions are gathered around the nucleic acid (e.g., DNA), and the negative charge on the surface of the nucleic acid (e.g., DNA) is neutralized by the cations through the electrostatic binding of anions and cations, thereby causing the nucleic acid (e.g., DNA) to condense, thereby changing the morphology of the nucleic acid nanoballs (e.g., DNBs), adjusting the volume of the nucleic acid nanoballs (e.g., DNBs), and compressing the nucleic acid nanoballs (e.g., DNBs), making the nucleic acid nanoballs (e.g., DNBs) more compact, smaller in shape, and more uniform in size.

[0141] The reagent for adjusting the volume of the nucleic acid nanospheres further comprises a nucleic acid nanosphere loading buffer or a sequencing reagent.

[0142] After being treated with the nucleic acid nanoball volume adjustment reagent, the nucleic acid nanoballs (e.g., DNBs) become more compact, smaller in shape, and more uniform in size. The nucleic acid nanoball volume adjustment reagent can be used in any situation where the DNB volume needs to be adjusted (reduced) (which can be any one or more sequencing processes: for example, the nucleic acid nanoball (e.g., DNB) loading process and the sequencing process after loading).

[0143] When used in the loading process of nucleic acid nanoballs (e.g., DNBs) (i.e., when the reagent for adjusting the volume of nucleic acid nanoballs also includes a nucleic acid nanoball loading buffer), the loading efficiency and quality of nucleic acid nanoballs (e.g., DNBs) can be improved, so that when the nucleic acid nanoballs (e.g., DNBs) are loaded onto a solid support (e.g., a chip), the distribution is more uniform, the empty sites are reduced, the size is uniform, the signal is more concentrated, and the impact on adjacent sites is reduced, thereby reducing the Dup value, reducing the number of empty sites on the chip, and improving the sequencing quality; at the same time, the detection signal can also be improved to meet the requirements of long-read sequencing;

[0144] When it is used in the subsequent sequencing process (ie, when the reagent for adjusting the volume of the nucleic acid nanoballs also includes a sequencing reagent), the nucleic acid nanoballs (eg, DNBs) can be further adjusted (eg, the DNBs maintain their shape), thereby further improving the sequencing quality.

[0145] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres comprises b1).

[0146] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres comprises b2).

[0147] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres comprises b1) and b2).

[0148] Preferably, the nucleic acid condensing agent in b1) comprises: at least one of: a polyvalent salt, a polyamine, and a lipid; further preferably, the nucleic acid condensing agent in b1) comprises: a polyvalent salt; even further preferably, the nucleic acid condensing agent in b1) comprises: a polyvalent metal salt.

[0149] Preferably, the polyvalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; further preferably, the polyvalent metal salt comprises: at least one of a zinc salt, a calcium salt, a magnesium salt, a copper salt, and a cerium salt (for example, it can be ZnCl2, ZnSO4, CaCl2, MgCl2, CuCl2·2H2O, CeCl3·7H2O); even more preferably, the polyvalent metal salt comprises: at least one of a copper salt, a cerium salt, a calcium salt, and a zinc salt.

[0150] Preferably, the polyamine comprises at least one of spermine and putrescine.

[0151] Preferably, the lipid comprises at least one of DOTAP and DC-Chol / DOPE.

[0152] Preferably, the pH of the reagent in b2) is 4 to 4.5; further 4.2 to 4.4.

[0153] Preferably, the reagent for adjusting the volume of nucleic acid nanospheres further comprises instructions, which describe the method for using the reagent for adjusting the volume of nucleic acid nanospheres.

[0154] Preferably, the method for using the reagent for adjusting the volume of nucleic acid nanospheres is: mixing the reagent for adjusting the volume of nucleic acid nanospheres with nucleic acid nanospheres to obtain a system for adjusting the volume of nucleic acid nanospheres;

[0155] And the system satisfies at least one of c1) to c2):

[0156] c1) the final concentration of the nucleic acid condensing agent in the system for adjusting the volume of nucleic acid nanospheres is 0.001 to 1000 mM (when the reagent for adjusting the volume of nucleic acid nanospheres comprises b1) or comprises both b1) and b2);

[0157] c2) The pH of the system for adjusting the volume of nucleic acid nanospheres is 3-4.5 (when the reagent for adjusting the volume of nucleic acid nanospheres comprises b2) or comprises both b1) and b2)).

[0158] Preferably, the final concentration of the nucleic acid condensing agent in c1) in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 12 to 80 mM.

[0159] Preferably, the final concentration of the zinc salt in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 20 to 80 mM.

[0160] Preferably, the final concentration of the calcium salt in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 12 to 20 mM.

[0161] Preferably, the final concentration of the copper salt in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0162] Preferably, the final concentration of the magnesium salt in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0163] Preferably, the final concentration of the cerium salt in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0164] Preferably, the final concentration of the polyamine in the system is 0.001 to 10 mM; further 0.01 to 0.04 mM.

[0165] Preferably, the final concentration of the lipid in the system is 0.001 to 10 mM; further 0.003 to 0.007 mM.

[0166] Preferably, the pH of the system for adjusting the volume of nucleic acid nanospheres in c2) is 4 to 4.5; further 4.2 to 4.4.

[0167] Preferably, the nucleic acid nanosphere (eg, DNB) loading buffer comprises at least one of a citrate buffer, a MES buffer solution, and a Tris hydrochloride buffer.

[0168] Preferably, the pH of the nucleic acid nanosphere (eg, DNB) loading buffer is 4.6-5; further 4.6-4.7.

[0169] Preferably, the sequencing reagent comprises: at least one of: a dNTPs mixture, a nucleic acid (such as DNA) polymerase mixture, an MDA reagent, and an MDA polymerase mixture; and further comprises: a dNTPs mixture, a nucleic acid (such as DNA) polymerase mixture, an MDA reagent, and an MDA polymerase mixture.

[0170] Preferably, the dNTPs mixture comprises: tris(hydroxymethylaminomethane), hydrochloric acid, ethylenediaminetetraacetic acid and chemically modified dNTPs.

[0171] Preferably, the chemical modification includes but is not limited to primary amino modification (such as: 3'NH2C6, 5'NH2C6, 5'NH2C12, IntNH2C6dT, etc.), Int Uni-Link Amino modification and its derivatives, carboxyl modification and its derivatives, aldehyde modification and its derivatives, acrylamide modification and its derivatives, azide modification and its derivatives (such as 5'Azide(N3), 3'Azide(N3), Int Azide-dT, etc.), alkynyl modification and its derivatives (such as 5'CHCH, Int CHCH-dT, 3'CHCH, etc.), diphenylcyclooctyne modification and its derivatives (such as 5'DBCO, 3'DBCO, Int DBCO dT, etc.), maleimide modification (such as 5'Maleimide, 3'Maleimide, etc.), biotin modification (such as 5'Biotin, 3'Biotin, 3'Biotin-TEG, 5-Biotin-TEG, Triple Biotin, etc.), desthiobiotin (such as 5'Desthiobiotin, 3'Desthiobiotin, etc.), SH / HS-SH / sulfhydryl modification (such as 5'SH C6, 3'SH C6, 3'SH C3, 5'HS-SH C6, 3'HS-SH C3, 3'HS-SH C6, Int HS-SHC6, etc.), dithiol modification and its derivatives (5'Dithiol, 3'Dithiol, lntDithiol,Int Dithiol dT, etc.), Ferrocene modification (such as 5'Ferrocene, 3'Ferrocene, Int Ferrocene dT, etc.), C3 / C6 Spacer (5'C3Spacer, 5'C6 Spacer, 3'C3 Spacer, 3'C6 Spacer, Int C6 Spacer, etc.), Spacer 9 / Spacer18 modification (5'Spacer 9, 5'Spacer 18, 3'Spacer 9, 3'Spacer 18, Int Spacer 9, Int Spacer18, etc.), PC Spacer / PC Linker photocleavage spacer, tetrahydrofuran modification, phosphorylation modification (such as 5'P, 3'P, etc.), thiolation modification, phosphorothioate modification, phosphorothioate modification, 2-aminopurine modification, 5-bromodeoxyuridine modification, deoxyuridine modification, inverted dT / dG modification, dideoxycytidine modification, 5-methylcytosine deoxynucleoside modification, 5-hydroxymethyl dC modification, N6 methyladenine nucleotide modification, 5-aza-2-deoxycytidine modification, locked nucleic acid modification, 2-methoxy modified base modification and its derivatives, RNA 2-fluoro RNA modification, pyrrole-deoxycytosine modification, digoxigenin modification, cholesterol modification, azobenzene modification, methylene blue modification, 8-oxo-7-hydrodeoxyguanine modification and its derivatives, pyrene modification, symmetric modification, ruthenium modification, etc.

[0172] Preferably, the nucleic acid (eg DNA) polymerase mixture comprises: nucleic acid (eg DNA) polymerase and glycerol.

[0173] Preferably, the MDA reagent comprises: deoxyribonucleoside triphosphates, dithiothreitol, dimethyl sulfoxide, sucrose and glycerol.

[0174] Preferably, the MDA polymerase mixture comprises: MDA polymerase and glycerol.

[0175] Preferably, the nucleic acid condensing agent exists independently or mixed with the nucleic acid nanoball loading buffer / sequencing reagent, that is, the nucleic acid condensing agent can exist independently of the nucleic acid nanoball loading buffer / sequencing reagent, or can exist in the nucleic acid nanoball loading buffer / sequencing reagent.

[0176] The aforementioned dNTPs mixture, nucleic acid (eg, DNA) polymerase mixture, MDA reagent, and MDA polymerase mixture are conventional reagents in the art and can be purchased commercially or prepared by oneself.

[0177] The third aspect of the present invention provides a method for improving the loading efficiency and quality of nucleic acid nanoballs (DNBs), wherein the method for adjusting the volume of nucleic acid nanoballs (eg, DNBs) according to the first aspect of the present invention is used during the loading of nucleic acid nanoballs.

[0178] Preferably, the method for improving the loading efficiency and quality of nucleic acid nanospheres comprises the following steps:

[0179] A nucleic acid nanosphere is subjected to a coagulation treatment to obtain a nucleic acid nanosphere with a compressed volume;

[0180] loading the volume-compressed nucleic acid nanospheres onto a solid support;

[0181] The method for agglomerating the nucleic acid nanospheres comprises at least one of a1) to a2):

[0182] a1) adding a nucleic acid condensing agent to the system containing nucleic acid nanospheres;

[0183] a2) adjusting the pH of the system containing the nucleic acid nanospheres to 3-4.5;

[0184] The nucleic acid condensing agent includes at least one of a multivalent salt, a basic peptide, a basic protein (such as histone, protamine), a polar solvent (such as alcohol), a positively charged high molecular polymer (such as polyhistidine, polylysine, polyarginine and polyornithine), a polyamine (such as spermine, spermidine and putrescine), a polyammonium (such as polybrene (hexadimethrinebromide)), and a lipid (such as DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE).

[0185] During the research, the inventors learned that nucleic acids (such as DNA) are long-chain polymers composed of repeated arrangements of deoxynucleotides. In polar solutions, the bases and phosphates in their structures carry a large amount of negative charge. By subjecting nucleic acids (such as DNA) to agglomeration treatment (for example, adding nucleic acid agglomerating agents (such as multivalent salts, basic peptides, basic proteins, polar solvents (such as alcohols), positively charged high molecular polymers, polyamines, polyammonium, lipids) and / or adjusting the pH to 3-4.5, positively charged counter ions are gathered around the nucleic acids (such as DNA), and through the electrostatic binding of anions and cations, the negative charge on the surface of the nucleic acids (such as DNA) is neutralized by cations, thereby making the nucleic acids (such as DNA) more stable. (e.g., DNA) morphology condenses), thereby changing the morphology of nucleic acid nanoballs (e.g., DNBs), adjusting the volume of nucleic acid nanoballs (e.g., DNBs), compressing nucleic acid nanoballs (e.g., DNBs), making nucleic acid nanoballs (e.g., DNBs) more compact, smaller in morphology, and more uniform in size, thereby improving the loading efficiency and quality of nucleic acid nanoballs, making the distribution of nucleic acid nanoballs (e.g., DNBs) more uniform when loaded onto a solid support (e.g., a chip), with fewer empty sites, and more concentrated signals after uniform size, with less impact on adjacent sites, thereby reducing the Dup value, reducing chip empty sites, and improving sequencing quality; at the same time, it can also improve the detection signal to meet the requirements of long-read sequencing.

[0186] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a1).

[0187] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a2).

[0188] Preferably, the method of agglomerating nucleic acid nanospheres comprises: a1) and a2).

[0189] Preferably, the nucleic acid condensing agent in a1) comprises at least one of a polyvalent salt, a polyamine, and a lipid; further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent salt; even further preferably, the nucleic acid condensing agent in a1) comprises a polyvalent metal salt.

[0190] Preferably, the polyvalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; further preferably, the polyvalent metal salt comprises: at least one of a zinc salt, a calcium salt, a magnesium salt, a copper salt, and a cerium salt (for example, it can be ZnCl2, ZnSO4, CaCl2, MgCl2, CuCl2·2H2O, CeCl3·7H2O); even more preferably, the polyvalent metal salt comprises: at least one of a copper salt, a cerium salt, a calcium salt, and a zinc salt. The use of multivalent metal salts as nucleic acid condensing agents can not only change the morphology of nucleic acid nanoballs (such as DNBs), compress nucleic acid nanoballs (such as DNBs), make nucleic acid nanoballs (such as DNBs) more compact, smaller in morphology, and more uniform in size, so that when nucleic acid nanoballs (such as DNBs) are loaded onto a solid support (such as a chip), the distribution is more uniform, the number of empty sites is reduced, and the signal is more concentrated after the size is uniform, and the impact on adjacent sites is reduced, thereby reducing the Dup value, reducing chip empty sites, and improving sequencing quality; it can also improve the detection signal to meet the requirements of long-read sequencing; at the same time, it can also improve the adsorption force of the solid support (such as a chip).

[0191] Preferably, the polyamine comprises at least one of spermine and putrescine.

[0192] Preferably, the lipid comprises at least one of DOTAP and DC-Chol / DOPE.

[0193] Preferably, the final concentration of the nucleic acid condensing agent in a1) in the system is 0.001 to 1000 mM; further 1 to 1000 mM; further 10 to 100 mM; further 12 to 80 mM.

[0194] Preferably, the final concentration of the zinc salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 80 mM.

[0195] Preferably, the final concentration of the calcium salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 12 to 20 mM.

[0196] Preferably, the final concentration of the copper salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0197] Preferably, the final concentration of the magnesium salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0198] Preferably, the final concentration of the cerium salt in the system is 1 to 1000 mM; further 10 to 100 mM; further 20 to 30 mM.

[0199] Preferably, the final concentration of the polyamine in the system is 0.001 to 10 mM; further 0.01 to 0.04 mM.

[0200] Preferably, the final concentration of the lipid in the system is 0.001 to 10 mM; further 0.003 to 0.007 mM.

[0201] Preferably, the pH of the system in a2) is 4 to 4.5; further 4.2 to 4.4.

[0202] Preferably, the solid support comprises at least one of latex beads, dextran beads, polystyrene surface, polypropylene surface, polyacrylamide gel, gold surface, glass surface, chip, sensor, electrode, silicon wafer; further comprises a chip; further a high-density matrix chip.

[0203] Preferably, the solid support is planar, spherical or porous.

[0204] Preferably, the material of the solid support includes materials such as glass, polyacrylamide-coated glass, ceramics, silica, silicon, quartz, various plastics, etc.

[0205] Preferably, the solid support is suitable for use with the BGI sequencing platform.

[0206] Preferably, the maximum size of the nucleic acid nanoball (eg, DNB) that can be accommodated or attached at a single site on the solid support is ≤5000 nm, ≤2000 nm, ≤1000 nm, ≤700 nm, ≤500 nm, ≤300 nm, or ≤100 nm.

[0207] Preferably, the distance between adjacent spots in the solid support is ≤5000 nm, ≤2000 nm, ≤1000 nm, ≤700 nm, ≤500 nm, ≤300 nm, or ≤100 nm.

[0208] Preferably, the nucleic acid nanoballs (eg, DNBs) are produced by rolling circle amplification (RCA).

[0209] Preferably, the rolling circle amplification (RCA) is a process of replicating nucleic acid (eg DNA) in large quantities using a single-stranded circular nucleic acid (eg DNA) as a template.

[0210] Preferably, the preparation method of the single-stranded circular nucleic acid (eg, DNA) is a conventional method in the art, that is, circularizing a linear nucleic acid library.

[0211] Preferably, the cyclization and rolling circle amplification can be carried out in the same reaction system (for example, the one-step method for preparing DNBs in patent document CN115852493A), or in different systems (i.e., cyclization is carried out in one reaction system and then rolling circle amplification is carried out in another reaction system; for example, the DNB preparation method commonly used in the art: cyclization, enzyme digestion, magnetic bead purification, denaturation annealing, and rolling circle amplification).

[0212] Preferably, the method for constructing the linear nucleic acid library can be any method for constructing a linear nucleic acid library, including but not limited to the method for constructing a linear nucleic acid library mentioned in the prior art.

[0213] Preferably, the nucleic acid starting material for constructing the linear nucleic acid library can be any suitable DNA, RNA, or a complex of DNA and RNA, particularly DNA, and more particularly genomic DNA. The source of the nucleic acid is not limited and can be any biological source, such as nucleic acids from animals, plants, microorganisms, etc., particularly mammalian sources, especially human nucleic acids, and most preferably human genomic DNA.

[0214] More specifically,

[0215] The nucleic acid can be isolated from a biological sample obtained from an individual (e.g., a test individual). The individual can be any living or non-living organism, including but not limited to humans, non-human animals, plants, bacteria, fungi, protozoa, or pathogens.

[0216] Nucleic acids can be isolated or obtained from any type of suitable biological sample. Nucleic acids can be isolated or obtained from a single cell, a plurality of cells (e.g., cultured cells), a cell culture medium, a conditioned medium, a tissue, an organ, or an organism (e.g., bacteria, yeast, etc.).

[0217] Nucleic acids can be isolated or obtained from existing organisms or animals. In some cases, nucleic acids can be isolated or obtained from extinct (or "ancient") organisms or animals (e.g., extinct mammals, extinct mammals from the genus Homo, paleontological fossils). In some cases, nucleic acids can be obtained as part of a diagnostic assay.

[0218] In some cases, nucleic acids can be isolated or obtained from forensic samples or specimens. Forensic samples or specimens can include any biological material containing nucleic acids. For example, forensic samples or specimens can include blood, semen, hair, skin, sweat, saliva, decomposed tissue, bone, nail scrapings, licked stamps / envelopes, sluff, contact DNA, razor residue, etc. The specimen can be formalin-fixed tissue and / or paraffin-embedded tissue.

[0219] A biological sample can be any sample isolated or obtained from an individual or part thereof (e.g., a human individual, a pregnant female, a cancer patient, a patient suffering from an infection or infectious disease, a transplant recipient, a fetus, a tumor, an infected organ or tissue, a transplanted organ or tissue, a microbiome). In some embodiments, the biological sample is a cervical swab from an individual. The liquid sample or tissue sample from which nucleic acid is extracted can be acellular (e.g., free of cells). In some embodiments, the biological sample can contain cellular components or cell remnants. In some embodiments, the biological sample can include fetal cells or cancer cells.

[0220] Biological sample can be a liquid sample.Liquid sample can include extracellular nucleic acid (for example, circulating cell-free DNA).The example of liquid sample includes but is not limited to blood or blood products (such as serum, plasma, etc.), urine, cerebrospinal fluid, saliva, sputum, biopsy sample (for example, for detecting liquid biopsy of cancer), above-mentioned liquid sample, analog or its combination.In certain embodiments, biological sample is liquid biopsy, it generally refers to the assessment of the presence, absence, progression or alleviation of the liquid sample from individual about disease (for example, cancer).Liquid biopsy can be used in combination with the biopsy (for example, tumor biopsy) sold or used as its substitute.In some cases, extracellular nucleic acid is analyzed in liquid biopsy.

[0221] The biological sample can be a tumor nucleic acid sample (ie, a nucleic acid sample isolated from a tumor).

[0222] Preferably, the system comprising nucleic acid nanospheres in a1), and / or a2) further comprises: a nucleic acid nanosphere (eg, DNB) loading buffer.

[0223] Preferably, the nucleic acid nanosphere (eg, DNB) loading buffer is the nucleic acid nanosphere loading buffer in the second aspect of the present invention.

[0224] Preferably, the single-stranded circular nucleic acid comprises at least one of single-stranded circular DNA libraries such as E.coil, PCR Free, and WGBS.

[0225] Preferably, the nucleic acid nanoballs (eg, DNBs) are prepared using the rolling circle amplification technology of the BGI sequencing platform.

[0226] Preferably, the BGI sequencing platform includes MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-T10, DNBSEQ-T20, DNBSEQ-G99, DNBSEQ-E25, etc.

[0227] Preferably, the rolling circle amplification time of the nucleic acid nanoballs (eg, DNBs) is 20 to 120 minutes, so that nucleic acid nanoballs (eg, DNBs) with at least several hundred copies can be obtained for sequencing, thereby improving the quality of sequencing.

[0228] Under the same RCA time, by performing agglomeration treatment on the nucleic acid nanoballs, the morphology of the nucleic acid nanoballs (e.g., DNBs) can be changed, the volume of the nucleic acid nanoballs (e.g., DNBs) can be adjusted, the nucleic acid nanoballs (e.g., DNBs) can be compressed, and the loading efficiency and quality of the nucleic acid nanoballs (e.g., DNBs) can be improved. As a result, sequencing of the nucleic acid nanoballs treated with agglomeration can obtain a lower Dup value, thereby improving sequencing quality.

[0229] Preferably, the rolling circle amplification time of the nucleic acid nanoballs (eg, DNBs) is 30 to 120 minutes.

[0230] Preferably, the rolling circle amplification time of the nucleic acid nanoballs (eg, DNBs) is 40 to 120 minutes.

[0231] It is well known to those skilled in the art that within a certain time range, as the RCA time increases, the copy number of nucleic acid nanoballs (such as DNBs) also increases accordingly; in the gene sequencing process, when the copy number of nucleic acid nanoballs (such as DNBs) is large, the detection signal becomes stronger, which is conducive to the interpretation of bases and improves the sequencing quality; however, the increase in the copy number of nucleic acid nanoballs (such as DNBs) also makes the volume of nucleic acid nanoballs (such as DNBs) larger, resulting in an increase in the influence between nucleic acid nanoballs (such as DNBs) when they are loaded on a solid support (such as a chip), such as an increase in the number of empty sites due to insufficient reaction, and uneven size of nucleic acid nanoballs (such as DNBs) loaded on a solid support (such as a chip), resulting in poor quality during gene sequencing; and the present invention increases the RCA time (limiting the time of rolling circle amplification to 30 to 120 minutes) to obtain nucleic acid nanoballs (such as DNBs) with increased copy number and increased volume. Agglomeration treatment (Figure 2) changes the morphology of nucleic acid nanoballs (e.g., DNBs), adjusts the volume of nucleic acid nanoballs (e.g., DNBs), and compresses nucleic acid nanoballs (e.g., DNBs), making nucleic acid nanoballs (e.g., DNBs) with increased copy number and larger volume more compact, regular, and uniform in size, thereby improving the loading efficiency and quality of nucleic acid nanoballs (e.g., DNBs). When nucleic acid nanoballs (e.g., DNBs) are loaded onto a solid support (e.g., a chip), their distribution is more uniform, with fewer empty sites and uniform size, resulting in more concentrated signals and less impact on adjacent sites. This can lower the Dup value and reduce chip empty sites, thereby improving the sequencing quality of nucleic acid nanoballs (e.g., DNBs) with increased copy number and larger volume obtained by increasing the RCA time (the rolling circle amplification time is limited to 30 to 120 minutes). This solves the contradictory problem that existing sequencing technologies require enhancing base signals by increasing the copy number, but this deteriorates sequencing quality.

[0232] Based on the solution to the contradictory problem that the base signal is enhanced by increasing the copy number but the sequencing quality deteriorates, the present invention increases the RCA time (limiting the rolling circle amplification time to 30 to 120 minutes, also known as long RCA time), while performing agglomeration treatment on the nucleic acid nanoballs (such as DNBs) with increased copy number and enlarged volume obtained by the long RCA time, changing the morphology of the nucleic acid nanoballs (such as DNBs), adjusting the volume of the nucleic acid nanoballs (such as DNBs), and compressing the nucleic acid nanoballs (such as DNBs), so that the nucleic acid nanoballs (such as DNBs) with increased copy number and enlarged volume obtained by the long RCA time (such as The structure of nucleic acid nanoballs (such as DNBs) is tighter and the size is more uniform, which improves the loading efficiency and quality of nucleic acid nanoballs (such as DNBs). When loaded onto a solid support (such as a chip), the distribution is more uniform, the empty sites are reduced, and the signal is more concentrated after the size is uniform, which reduces the impact on adjacent sites, thereby reducing the Dup value, reducing the empty sites on the chip, and improving the sequencing quality of nucleic acid nanoballs (such as DNBs) obtained with a long RCA time. In addition, the sequencing quality is better than that of nucleic acid nanoballs (such as DNBs) obtained with a short RCA time (15 to 25 minutes) but without agglomeration treatment.

[0233] A fourth aspect of the present invention provides a sequencing method comprising the following steps:

[0234] 1) adjusting the volume of the nucleic acid nanospheres using the method of the first aspect of the present invention;

[0235] 2) Sequencing to obtain the sequence information carried by the nucleic acid nanospheres.

[0236] Preferably, step 1) can be performed in any one or more sequencing processes of the sequencing method.

[0237] Preferably, the sequencing process includes but is not limited to: a nucleic acid nanoball loading process and a nucleic acid nanoball sequencing process.

[0238] Preferably, during the loading process of the nucleic acid nanospheres in step 1), the system comprising the nucleic acid nanospheres further comprises: a nucleic acid nanosphere loading buffer.

[0239] Preferably, the nucleic acid nanosphere loading buffer comprises at least one of citrate buffer, MES buffer solution, and Tris hydrochloride buffer.

[0240] Preferably, during the sequencing process of the nucleic acid nanoballs in step 1), the system comprising the nucleic acid nanoballs further comprises: a sequencing reagent.

[0241] Preferably, the sequencing reagent comprises at least one of a dNTPs mixture, a nucleic acid polymerase mixture, an MDA reagent, and an MDA polymerase mixture.

[0242] Preferably, the methods for agglomerating the nucleic acid nanospheres in step 1) during different sequencing processes may be the same or different.

[0243] By adopting the method for adjusting the volume of nucleic acid nanoballs (e.g., DNBs) according to the first aspect of the present invention (performing agglomeration treatment on nucleic acid nanoballs (e.g., DNBs)) during the loading process of nucleic acid nanoballs, the morphology of nucleic acid nanoballs (e.g., DNBs) can be changed, the volume of nucleic acid nanoballs (e.g., DNBs) can be adjusted, and the nucleic acid nanoballs (e.g., DNBs) can be compressed, making the nucleic acid nanoballs (e.g., DNBs) more compact, smaller in morphology, and more uniform in size, thereby improving the loading efficiency and quality of nucleic acid nanoballs (e.g., DNBs), making the distribution of nucleic acid nanoballs (e.g., DNBs) more uniform when loaded onto a solid support (e.g., a chip), reducing empty sites, and making the signal more concentrated after uniform size, reducing the impact on adjacent sites, thereby reducing the Dup value, reducing chip empty sites, and improving sequencing quality; at the same time, the detection signal can also be improved to meet the requirements of long-read sequencing.

[0244] By adopting the method for adjusting the volume of nucleic acid nanoballs (e.g., DNBs) according to the first aspect of the present invention (agglomerating nucleic acid nanoballs (e.g., DNBs)) during the sequencing process of nucleic acid nanoballs, the volume of nucleic acid nanoballs (e.g., DNBs) can be adjusted (e.g., maintaining the DNBs' shape), thereby improving sequencing quality.

[0245] Preferably, the sequencing is performed using the BGI sequencing platform.

[0246] Preferably, the BGI sequencing platform includes MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-T10, DNBSEQ-T20, DNBSEQ-G99, DNBSEQ-E25, etc.

[0247] A fifth aspect of the present invention provides a kit comprising the reagent for adjusting the volume of nucleic acid nanoballs (eg, DNBs) according to the second aspect of the present invention.

[0248] Preferably, the kit further comprises: a reagent for preparing nucleic acid nanospheres (eg, DNB).

[0249] Preferably, the nucleic acid nanosphere (e.g., DNB) preparation reagent comprises: at least one of: TE buffer, nucleic acid nanosphere (e.g., DNB) preparation buffer, nucleic acid nanosphere (e.g., DNB) polymerase mixture, and nucleic acid nanosphere (e.g., DNB) termination buffer; and further comprises: TE buffer, nucleic acid nanosphere (e.g., DNB) preparation buffer, nucleic acid nanosphere (e.g., DNB) polymerase mixture, and nucleic acid nanosphere (e.g., DNB) termination buffer.

[0250] Preferably, the TE buffer comprises: tris(hydroxymethylaminomethane), hydrochloric acid and ethylenediaminetetraacetic acid.

[0251] Preferably, the nucleic acid nanosphere (eg, DNB) preparation buffer comprises: ammonium sulfate, dithiothreitol, magnesium chloride, tris(hydroxymethyl)aminomethane, and oligonucleotides.

[0252] Preferably, the nucleic acid nanoball (e.g., DNB) polymerase mixture comprises: nucleic acid nanoball (e.g., DNB) polymerase mixture I and nucleic acid nanoball (e.g., DNB) polymerase mixture II; the nucleic acid nanoball (e.g., DNB) polymerase mixture I comprises: dNTP, ammonium sulfate, dithiothreitol, magnesium chloride, tris(hydroxymethylaminomethane), glycerol and single-stranded binding protein; the nucleic acid nanoball (e.g., DNB) polymerase mixture II comprises: tris(hydroxymethylaminomethane), potassium chloride, ethylenediaminetetraacetic acid, nucleic acid nanoball (e.g., DNB) polymerase and glycerol.

[0253] Preferably, the nucleic acid nanosphere (eg, DNB) termination buffer comprises: ethylenediaminetetraacetic acid.

[0254] The sixth aspect of the present invention provides the method for adjusting the volume of nucleic acid nanospheres (e.g., DNBs) according to the first aspect of the present invention, the reagent for adjusting the volume of nucleic acid nanospheres (e.g., DNBs) according to the second aspect of the present invention, and the kit according to the fifth aspect of the present invention, used in any one of items f1) to f3):

[0255] f1) loading nucleic acid nanospheres (DNBs);

[0256] f2) sequencing;

[0257] f3) Adjust the volume of the nucleic acid nanospheres.

[0258] Preferably, in the present invention, the adjustment is reduction.

[0259] The present invention is further described in detail below through specific examples.

[0260] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0261] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0262] In this example, the E. coli and PCR-free libraries used were provided by MGI. Sequencing was performed using the MGISEQ-2000 sequencer, and the instrument and handheld loader were manufactured and provided by MGI. Sequencing primers, sequencing reagents, Make DNB enzyme MIX I and Make DNB enzyme MIX II, stop-run buffer, and DNB loading buffer II (DLBII) were all from the MGISEQ-2000 standard PE100 sequencing reagent set. Sequencing slides were provided by MGI. The ZnCl2 (Sanggong, A501003-0250), CaCl2 (Sanggong, A600506-0100), CuCl2 (Sanggong, A603090-0250), MgCl2 (Sanggong, A100288-0500), and CeCl3 (Sanggong, A610054-0050) used were all purchased from MGI.

[0263] Example 1 Effect of Metal Ions on Dup Value

[0264] 1) Using a PCR-free single-stranded circularized DNA library, the input amount was 6 ng. DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and the instruction manual. The preparation time for RCA was 25 min.

[0265] 2) 1M aqueous solutions of ZnCl2, CuCl2, MgCl2, and CeCl3 were added to the existing DLBII to obtain new DLBII with final concentrations of 80mM ZnCl2 (experimental group 3), CuCl2 (experimental group 1), MgCl2 (experimental group 2), and CeCl3 (experimental group 4), respectively (the control group was left untreated).

[0266] 3) The DNBs prepared in step 1) were mixed with the new DLBII obtained in step 2) so that the final concentration of ZnCl2, CuCl2, MgCl2, and CeCl3 in the mixed solution was 20 mM, and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and single-end SE20 sequencing verification was performed on the MGISEQ-2000 platform).

[0267] The SE20 sequencing results of the new DLBII containing metal ions are shown in Figure 3 and Table 1. Adding various metal ions that can cause DNB morphology to condense into DLBII can change the DNB morphology, adjust the DNB volume, compress the DNB, and make its morphology smaller, thereby obtaining a lower Dup value through sequencing. Moreover, different types of metal ions have different abilities to reduce the Dup value.

[0268] Table 1 SE20 sequencing results of DNBs from PCR-free libraries after loading with the new DLBII containing metal ions

[0269] Example 2 Effect of DLBIIpH on Dup Value

[0270] 1) Using a PCR-free single-stranded circularized DNA library, the input amount was 6 ng. DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and the instruction manual. The preparation time for RCA was 40 min.

[0271] 2) DLBII was prepared with pH values ​​of 4.7 (control group, pH of existing DLBII), 4.4 (experimental group 1), 4.3 (experimental group 2), and 4.2 (experimental group 3).

[0272] 3) The DNB prepared in step 1) was mixed with the DLBII obtained in step 2) and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and double-end PE50 sequencing verification was performed on the MGISEQ-2000 platform).

[0273] The PE50 sequencing results of DLBII at different pH values ​​are shown in Figure 4 and Table 2. Adjusting the pH of DLBII, especially lowering its pH, can change the DNB morphology, adjust the DNB volume, compress the DNB, and make its morphology smaller, thereby obtaining a lower Dup value for sequencing, and the lower the pH value, the smaller the Dup value.

[0274] Table 2 PE50 sequencing results of DNBs from PCR-free libraries after loading with the new DLBII at different pH values

[0275] Example 3 Effect of Metal Ion Concentration on Dup Value

[0276] 1) Using a PCR-free single-stranded circularized DNA library, the input amount was 6 ng. DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and the instruction manual. The preparation time for RCA was 25 min.

[0277] 2) Add 1M CaCl2 aqueous solution to the existing DLBII to obtain new DLBII with final concentrations of 48 (experimental group 1) and 80mM CaCl2 (experimental group 2), respectively (the control group was left untreated).

[0278] 3) The DNB prepared in step 1) was mixed with the new DLBII obtained in step 2) so that the final CaCl2 concentrations in the mixed solution were 12 and 20 mM, respectively, and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and double-end PE50 sequencing verification was performed on the MGISEQ-2000 platform).

[0279] The PE50 sequencing results of DNBs obtained at the same RCA time after loading with new DLBII at different CaCl2 concentrations are shown in Figure 5 and Table 3. Adding various metal ions to DLBII that can cause DNB morphology to condense can adjust (reduce) DNB volume, resulting in lower Dup values ​​during sequencing. This Dup value also decreases with increasing metal ion concentration. To achieve better sequencing quality, the implementation of this technology requires careful consideration of the matching relationship between RCA time and different metal ion concentrations and types, ensuring that the resulting DNB morphology better meets our requirements.

[0280] Table 3 PE50 sequencing results of DNBs from PCR-free libraries after loading with the new DLBII containing different concentrations of metal ions

[0281] Example 4: Using metal ions to improve the sequencing quality of DNBs obtained by RCA 40 min

[0282] 1) Using an E.coil single-stranded circularized library at an input of 40 fmol, DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and its instructions. The RCA preparation time was 40 min.

[0283] 2) Add 1M ZnCl2 aqueous solution to the existing DLBII to obtain a new DLBII with a final concentration of 80mM ZnCl2 (experimental group) (the control group was not treated).

[0284] 3) The DNB prepared in step 1) was mixed with the new DLBII obtained in step 2) so that the final ZnCl2 concentration in the mixed solution was 20 mM, and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and double-end PE100 sequencing verification was performed on the MGISEQ-2000 platform).

[0285] The results of double-end PE100 sequencing of the new DLBII containing metal ions are shown in Figure 6 and Table 4: the Dup value of the experimental group is smaller than that of the control group, indicating that the use of the new DLBII containing metal ions to load DNBs can adjust (reduce) the DNB volume, so that DNBs with long RCA times obtain smaller Dup values; and from the signal point of view, the use of the new DLBII containing metal ions to load DNBs can improve the loading of large DNBs, making their arrangement more neat and regular, reducing mutual interference between DNBs, and more concentrated signals, thereby strengthening the sequencing signal and improving the problem of rapid degradation of sequencing quality; from the sequencing indicators such as Q30 after filtration, the number of sequences separated by [AT], and the average sequencing coverage depth, the use of the new DLBII containing metal ions to load DNBs improves the problem of large DNB loading, so that better sequencing quality can be obtained in high-throughput sequencing.

[0286] Table 4 PE100 sequencing results of DNBs from the E.coil library after loading with the new DLBII containing metal ions

[0287] The sequenced chips were characterized by atomic force microscopy to observe the morphology of DNBs. The results are shown in Figure 7: the DNBs in the control group were uneven in size and diverse in morphology, with large differences in DNB heights. Compared with the control group, the DNBs in the experimental group were more compact in morphology, more uniform in size, more neatly arranged, with fewer empty sites, and smaller differences in DNB heights. This indicates that the new DLBII containing metal ions was used to load DNBs to change the morphology of DNBs, making them more compact, more uniform in size, more neatly arranged, and with fewer empty sites.

[0288] Example 5: Using metal ions to improve the sequencing quality of DNBs obtained by RCA120min

[0289] 1) Using a PCR-free single-stranded circularized DNA library, the input amount was 6 ng. DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and the instruction manual. The preparation time for RCA was 120 min.

[0290] 2) Add 1M ZnCl2 aqueous solution to the existing DLBII to obtain new DLBII with final concentrations of 160mM (experimental group 1) and 320mM (experimental group 2), respectively (the control group was not treated).

[0291] 3) The DNB prepared in step 1) was mixed with the new DLBII obtained in step 2) so that the final ZnCl2 concentrations in the mixed solution were 40 and 80 mM, respectively, and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and double-end PE100 sequencing verification was performed on the MGISEQ-2000 platform).

[0292] The PE100 sequencing results of DNBs obtained by extending the RCA time to 120 minutes and loading them with the new DLBII at different ZnCl2 concentrations are shown in Figure 8 and Table 5. The DNBs obtained by significantly extending the RCA time to 120 minutes are larger than those obtained with a conventional RCA time of 25 minutes. By using the new DLBII of the present invention and a higher ZnCl2 concentration, the DNB size can be adjusted (reduced), resulting in smaller Dup and higher sequencing quality. This indicates that the new DLBII of the present invention can further improve the loading and sequencing quality of DNBs with longer RCA times by adjusting the concentration of the added coagulant.

[0293] Table 5 PE100 sequencing results of DNBs from the PCR-free library after loading with the new DLBII containing different concentrations of ZnCl2

[0294] Example 6 Sequencing quality of DNBs obtained with different RCA times using metal ion treatment

[0295] 1) Using a 6 ng PCR-free single-stranded circularized DNA library, DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and its instructions. RCA times were 25 and 30 minutes, respectively. The concentration of the prepared DNBs was measured using the ssDNA Qubit quantification reagent, denoted as C1. The results are shown in Table 6. As RCA time increased, DNB copy number and concentration increased, resulting in larger DNB morphology.

[0296] Table 6 DNB concentrations obtained from PCR-free libraries after different RCA times

[0297] 2) Add 1M ZnCl2 aqueous solution to the existing DLBII to obtain a new DLBII containing a final concentration of 80mM ZnCl2 (the control group was not treated).

[0298] 3) The DNB prepared in step 1) was mixed with the new DLBII obtained in step 2) (wherein, the control group was: RCA 25 min + DLBII (untreated DLBII, control group); experimental group 1 was: RCA 25 min + new DLBII containing 80 mM ZnCl2 (the final concentration of ZnCl2 in the mixed solution after mixing was 20 mM); experimental group 2 was: RCA 30 min + new DLBII containing 80 mM ZnCl2) (the final concentration of ZnCl2 in the mixed solution after mixing was 20 mM) and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and double-end PE50 sequencing verification was performed on the MGISEQ-2000 platform).

[0299] The results are shown in Figure 9 and Table 7: By comparing experimental group 1 with the control group, it can be seen that: at the same RCA time, using DLBII containing metal ions (ZnCl2) to load DNBs can change the DNB morphology, adjust (reduce) the DNB volume, and make Dup smaller; by comparing experimental group 2 with the control group, it can be seen that: as the RCA time increases, even if the DNB copy number increases and the DNB morphology becomes larger, using DLBII containing metal ions (ZnCl2) to load DNBs can still change the DNB morphology, adjust (reduce) the DNB volume, reduce the Dup value and error rate, and improve Q30, sequencing signal, etc., and the sequencing quality is better than that of the control group. That is, using DLBII containing metal ions (ZnCl2) to load DNBs is suitable for improving the loading of large DNBs obtained with long RCA time.

[0300] Table 7 PE50 sequencing results of DNBs from PCR-free libraries after loading with the new DLBII containing metal ions

[0301] It can be seen that the method of improving the loading efficiency and quality of nucleic acid nanoballs (such as DNBs) involved in the present invention (causing DNBs to condense (for example, adding a nucleic acid condensing agent to the solution and / or adjusting the pH value of the solution)) is more suitable for loading large DNBs with increased RCA time and a large number of copies. It can improve the sequencing signal while reducing the Dup value. The combination of the two improves the sequencing quality of large nucleic acid nanoballs and is conducive to long-read sequencing.

[0302] Example 7 Effect of polyamines on Dup value

[0303] 1) Using a PCR-free single-stranded circularized DNA library, the input amount was 6 ng. DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and the instruction manual. The preparation time for RCA was 25 min.

[0304] 2) Spermine and putrescine were added to the existing DLBII to obtain new DLBII containing spermine and putrescine respectively (the control group was not treated).

[0305] 3) The DNB prepared in step 1) was mixed with the new DLBII obtained in step 2) so that the final concentrations of spermine and putrescine in the mixed solution were 0.003 mg / mL (approximately 0.015 mM and 0.034 mM, respectively, when converted to molar concentrations), and used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and single-end SE20 sequencing verification was performed on the MGISEQ-2000 platform).

[0306] The SE20 sequencing results of the new DLBII containing polyamines in this example are similar to the SE20 sequencing results of the new DLBII containing metal ions in Example 1: adding polyamines (for example, spermine or putrescine) that can cause DNB morphology to condense to DLBII can change the DNB morphology, adjust the DNB volume, compress the DNB, and make its morphology smaller, thereby obtaining a lower Dup value by sequencing.

[0307] Example 8 Effect of lipids on Dup value

[0308] 1) Using a PCR-free single-stranded circularized DNA library, the input amount was 6 ng. DNBs were prepared according to the MGISEQ-2000 DNB Sample Preparation and Loading Kit and the instruction manual. The preparation time for RCA was 25 min.

[0309] 2) DOTAP and DC-Chol / DOPE were added to the existing DLBII (molar ratio of 1:1) to obtain new DLBII containing DOTAP and DC-Chol / DOPE, respectively (the control group was not treated).

[0310] 3) The DNB prepared in step 1) was mixed with the new DLBII obtained in step 2) so that the final concentrations of DOTAP and DC-Chol / DOPE in the mixed solution were 0.005 mg / mL (approximately 0.00646 mM and 0.0039 mM, respectively). The mixed solution was used for further testing (the operating procedures of this experiment were referred to the instruction manual of the gene sequencer (MGISEQ-2000), and single-end SE20 sequencing verification was performed on the MGISEQ-2000 platform).

[0311] The SE20 sequencing results of the new lipid-containing DLBII in this example are similar to the SE20 sequencing results of the new metal ion-containing DLBII in Example 1: adding lipids that can cause DNB morphology to condense (for example, DOTAP or DC-Chol / DOPE) to DLBII can change the DNB morphology, adjust the DNB volume, compress the DNB, and make its morphology smaller, thereby obtaining a lower Dup value during sequencing.

[0312] It can be seen that the method for improving the loading efficiency and quality of nucleic acid nanoballs (such as DNBs) involved in the present invention (causing DNBs to condense (for example: adding a nucleic acid condensing agent to the solution and / or adjusting the pH value of the solution)) can make the distribution of nucleic acid nanoballs (such as DNBs) more uniform when loaded on a solid support (such as a chip), with fewer empty sites, and more concentrated signals after uniform size, with less impact on adjacent sites, thereby reducing the Dup value, reducing chip empty sites, and improving sequencing quality; it is particularly suitable for loading large DNBs with increased RCA time and a large number of copies. It can improve the sequencing signal while reducing the Dup value. The combination of the two improves the sequencing quality of large nucleic acid nanoballs and is conducive to long-read sequencing.

[0313] Furthermore, aggregation treatment can also be performed during the sequencing process (for example, by adding a nucleic acid condensing agent to the sequencing reagent and / or adjusting the pH of the sequencing reagent) to further adjust the volume of the DNB (for example, to maintain the DNB's shape) and further improve the sequencing quality.

[0314] That is, the present invention provides a method for adjusting (reducing) the volume of nucleic acid nanoballs (DNBs), which can be used in any occasion where the volume of DNBs needs to be adjusted (reduced) (it can be any one or more sequencing processes: for example, the loading process of nucleic acid nanoballs (such as DNBs), and the sequencing process after loading): wherein, during the loading process of nucleic acid nanoballs (DNBs), the nucleic acid nanoballs are caused to condense (for example, by adding a nucleic acid condensing agent to the solution and / or adjusting the pH value of the solution), so as to achieve the effect of adjusting (reducing) the volume of nucleic acid nanoballs, making the nucleic acid nanoballs more compact, smaller in shape, and more uniform in size, thereby improving the nucleic acid nanoballs. The efficiency and quality of nanoball loading are improved, so that the distribution of nucleic acid nanoballs loaded on the chip is more uniform, the empty sites are reduced, and the signal is more concentrated after the size is uniform, which has a smaller impact on adjacent sites, thereby reducing the Dup value, reducing chip empty sites, and improving sequencing quality. Moreover, it is more suitable for loading large nucleic acid nanoballs (DNBs) with increased RCA time and large copy number. It can improve the sequencing signal while reducing the Dup value. The combination of the two can meet the requirements of long-read sequencing. In addition, agglomeration treatment can also be performed during the sequencing process to further adjust the volume of nucleic acid nanoballs (DNBs) (for example, to keep the DNBs in shape) to further improve sequencing quality.

[0315] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for adjusting the volume of nucleic acid nanospheres, wherein the nucleic acid nanospheres are subjected to a condensation treatment to obtain nucleic acid nanospheres with compressed volume; The method for agglomerating the nucleic acid nanospheres comprises at least one of a1) to a2): a1) adding a nucleic acid condensing agent to a system containing nucleic acid nanospheres; a2) setting the pH of the system containing the nucleic acid nanospheres to 3-4.5; The nucleic acid condensing agent comprises at least one of a multivalent salt, a basic peptide, a basic protein, a polar solvent, a positively charged high molecular polymer, a polyamine, a polyammonium, and a lipid.

2. The method according to claim 1, characterized in that: The method for agglomerating nucleic acid nanospheres comprises: a1) and a2); Preferably, the basic protein comprises at least one of histone and protamine; and / or The polar solvent comprises alcohol; and / or The positively charged high molecular polymer comprises at least one of polyhistidine, polylysine, polyarginine and polyornithine; and / or The polyamine comprises at least one of spermine, spermidine and putrescine; further comprises at least one of spermine and putrescine; and / or The polyammonium comprises hedimethonium bromide; and / or The lipid comprises at least one of DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE; and further comprises at least one of DOTAP and DC-Chol / DOPE.

3. The method according to claim 1, characterized in that: The nucleic acid condensing agent in a1) comprises at least one of a multivalent salt, a polyamine, and a lipid; Preferably, the nucleic acid condensing agent in a1) comprises: a multivalent metal salt; Preferably, the multivalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; Preferably, the multivalent metal salt comprises at least one of zinc salt, calcium salt, magnesium salt, copper salt and cerium salt.

4. The method according to any one of claims 1 to 3, characterized in that: a1) The final concentration of the nucleic acid condensing agent in the system is 0.001 to 1000 mM; and / or The pH of the system in a2) is 4 to 4.5; Preferably, the final concentration of the nucleic acid condensing agent in a1) in the system is 1 to 1000 mM; Preferably, the final concentration of the nucleic acid condensing agent in a1) in the system is 10 to 100 mM; Preferably, the final concentration of the nucleic acid condensing agent in a1) in the system is 12 to 80 mM; Preferably, the pH of the system in a2) is 4.2-4.

4.

5. A reagent for adjusting the volume of nucleic acid nanospheres, the reagent for adjusting the volume of nucleic acid nanospheres comprising at least one of b1) to b2): b1) the reagent for adjusting the volume of nucleic acid nanospheres comprises a nucleic acid condensing agent; b2) the pH of the reagent for adjusting the volume of nucleic acid nanospheres is 3 to 4.5; The nucleic acid condensing agent comprises at least one of a multivalent salt, a basic peptide, a basic protein, a polar solvent, a positively charged high molecular polymer, a polyamine, a polyammonium, and a lipid.

6. The reagent for adjusting the volume of nucleic acid nanospheres according to claim 5, characterized in that: The reagent for adjusting the volume of the nucleic acid nanospheres further comprises a nucleic acid nanosphere loading buffer or a sequencing reagent; Preferably, the basic protein comprises at least one of histone and protamine; and / or The polar solvent comprises alcohol; and / or The positively charged high molecular polymer comprises at least one of polyhistidine, polylysine, polyarginine and polyornithine; and / or The polyamine comprises at least one of spermine, spermidine and putrescine; further comprises at least one of spermine and putrescine; and / or The polyammonium comprises hedimethicone bromide; and / or The lipid comprises at least one of DOTAP, DC-Chol / DOPE, DOGS / DOPE and DOTMA / DOPE; and further comprises at least one of DOTAP and DC-Chol / DOPE.

7. The reagent for adjusting the volume of nucleic acid nanospheres according to claim 6, characterized in that: The reagents for adjusting the volume of nucleic acid nanospheres include b1) and b2).

8. The reagent for adjusting the volume of nucleic acid nanospheres according to any one of claims 5 to 7, characterized in that: b1) The nucleic acid condensing agent comprises: at least one of a multivalent salt, a polyamine, and a lipid; and / or b2) The pH of the reagent is 4 to 4.5; Preferably, the nucleic acid condensing agent in b1) comprises: a multivalent metal salt; Preferably, the multivalent metal salt comprises: at least one of a divalent metal salt and a trivalent metal salt; Preferably, the multivalent metal salt comprises: at least one of zinc salt, calcium salt, magnesium salt, copper salt and cerium salt; Preferably, the pH of the reagent in b2) is 4.2-4.

4.

9. The reagent for adjusting the volume of nucleic acid nanospheres according to claim 8, characterized in that: The method for using the reagent for adjusting the volume of nucleic acid nanospheres is as follows: mixing the reagent for adjusting the volume of nucleic acid nanospheres with nucleic acid nanospheres to obtain a system for adjusting the volume of nucleic acid nanospheres; And the system satisfies at least one of c1) to c2): c1) the final concentration of the nucleic acid condensing agent in the system for adjusting the volume of nucleic acid nanospheres is 0.001 to 1000 mM; c2) the pH of the system for adjusting the volume of nucleic acid nanospheres is 3 to 4.5; Preferably, the final concentration of the nucleic acid condensing agent in c1) in the system for adjusting the volume of nucleic acid nanospheres is 1 to 1000 mM; further 10 to 100 mM; further 12 to 80 mM; Preferably, the pH of the system for adjusting the volume of nucleic acid nanospheres in c2) is 4 to 4.5; further, 4.2 to 4.4; Preferably, the nucleic acid nanosphere loading buffer comprises at least one of a citrate buffer, a MES buffer solution, and a Tris hydrochloride buffer; Preferably, the sequencing reagent comprises: at least one of a dNTPs mixture, a nucleic acid polymerase mixture, an MDA reagent, and an MDA polymerase mixture.

10. A method for improving the loading efficiency and quality of nucleic acid nanospheres, wherein the method for adjusting the volume of nucleic acid nanospheres according to any one of claims 1 to 4 is used during the loading of nucleic acid nanospheres.

11. The method according to claim 10, characterized in that: The method for improving the loading efficiency and quality of nucleic acid nanospheres comprises the following steps: A nucleic acid nanosphere is subjected to a condensation treatment to obtain a nucleic acid nanosphere with a compressed volume; loading the volume-compressed nucleic acid nanospheres onto a solid support; The method for agglomerating nucleic acid nanospheres is the method for agglomerating nucleic acid nanospheres as described in any one of claims 1 to 4; Preferably, the solid support comprises at least one of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers.

12. The method according to claim 10 or 11, characterized in that: The nucleic acid nanospheres are prepared by rolling circle amplification; Preferably, the rolling circle amplification time of the nucleic acid nanoball is 20 to 120 minutes; Preferably, the rolling circle amplification time of the nucleic acid nanoball is 30 to 120 minutes; Preferably, the rolling circle amplification time of the nucleic acid nanoball is 40 to 120 minutes; Preferably, the system comprising nucleic acid nanospheres in a1), and / or a2) further comprises: a nucleic acid nanosphere loading buffer; Preferably, the nucleic acid nanosphere loading buffer comprises at least one of a citrate buffer, a MES buffer solution, and a Tris hydrochloride buffer.

13. A sequencing method comprising the following steps: 1) adjusting the volume of nucleic acid nanospheres by the method according to any one of claims 1 to 4; 2) Sequencing to obtain the sequence information carried by the nucleic acid nanospheres.

14. The sequencing method according to claim 13, characterized in that: Step 1) can be performed in any one or more sequencing processes of the sequencing method; Preferably, the sequencing process includes but is not limited to: a nucleic acid nanoball loading process and a nucleic acid nanoball sequencing process; Preferably, in step 1), during the loading process of the nucleic acid nanospheres, the system comprising the nucleic acid nanospheres further comprises: a nucleic acid nanosphere loading buffer; Preferably, the nucleic acid nanosphere loading buffer comprises at least one of a citrate buffer, a MES buffer solution, and a Tris hydrochloride buffer; Preferably, in step 1), during the sequencing process of the nucleic acid nanoballs, the system comprising the nucleic acid nanoballs further comprises: a sequencing reagent; Preferably, the sequencing reagent comprises: at least one of a dNTPs mixture, a nucleic acid polymerase mixture, an MDA reagent, and an MDA polymerase mixture.

15. A kit comprising: the reagent for adjusting the volume of nucleic acid nanospheres according to any one of claims 5 to 9; The kit also includes: a nucleic acid nanosphere preparation reagent; Preferably, the nucleic acid nanosphere preparation reagent comprises at least one of: TE buffer, nucleic acid nanosphere preparation buffer, nucleic acid nanosphere polymerase mixture, and nucleic acid nanosphere termination buffer.

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