Repulsion-based library seeding and subsequent amplification

Abstract

The present invention discloses library inoculation and subsequent amplification based on repulsive force, the method comprises the steps of library vector construction, library vector occupying position on the surface of sequencing chip, amplification and elution, the method can solve the problems of label hopping and reduced stability, and realize simultaneous adaptation of non-array array and array chip.

Description

Technical Field

[0001] The present invention relates to molecular biology; specifically to library construction and sequencing of nucleic acid sequences, and further to library inoculation and subsequent amplification based on repulsive force. Background Art

[0002] In the fields of life and health, species origin, forensic identification, etc., the detection, deciphering and analysis of genetic information have played a significant role. At the same time, in more specific and subdivided fields, genetic information has introduced new research methods and new research results, such as epigenome, metagenome, etc. In addition, in some fields, genetic information provides new research approaches and efficient screening flux in the form of carriers or encoding, such as aptamer research, new drug research, antigen detection, etc. In short, there is a growing demand for genetic information detection, that is, the detection of nucleic acid sequences.

[0003] Among the many nucleic acid sequence detection methods, NGS sequencing, also known as the second-generation sequencing, has a higher market share. In the mainstream second-generation sequencing scheme, the preparation of the genetic material to be tested is an important link. Generally speaking, the genetic material needs to be fragmented to become a nucleic acid library with an appropriate number of bases, and then through different enrichment methods, dispersed sites are formed on the solid surface. When the signal intensity provided by a single nucleic acid library molecule is insufficient, different amplification methods are introduced to convert each nucleic acid library molecule at the dispersed site into a plurality of nucleic acid library clone clusters with the same sequence. Sometimes, amplification is performed directly on the solid surface; sometimes, amplification occurs on a support other than the solid surface, and then the amplified support with a single clone fragment is fixed on the solid surface. In this way, a sequencing chip that can be sequenced is formed, and the sequence information of the nucleic acid library is obtained.

[0004] Generally speaking, sequencing chips can be divided into two categories: non-array or array. Generally speaking, the clone clusters of nucleic acid libraries on non-array chips are arranged in the form of irregular sites, while array chips are arranged in the form of regular sites on the array. When generating nucleic acid library clone clusters on non-array chips, the concentration of the input library is limited by the size of the clone cluster, sequence uniqueness and other requirements, and is usually low, and the arrangement density of the generated sites is also low. On array chips, pattern arrays can be made at a higher density. However, in order to ensure that there is only one nucleic acid library sequence in each site of the pattern array after amplification, without adding additional steps, it is limited by the Poisson distribution, and under the most suitable library concentration, only about 40% of the sites can produce a single sequence of nucleic acid library clone clusters. In response to this situation, different methods have been developed to increase the generation rate of effective sites on array chips, such as kinetic exclusive amplification, which has reached more than about 70% of effective sites. However, this method also brings about phenomena such as index hopping, and cannot be used for non-array chips, which has certain limitations. In another method, the prefabricated nucleic acid library is in the form of nanospheres, which can provide an extremely high site utilization rate of more than about 90%. However, since the complex microstructure of the nucleic acid nanospheres will change with the sequencing process, it will lead to a problem of reduced sequencing stability. Summary of the invention

[0005] Therefore, considering the low cost and ease of manufacture of non-array chips, they complement array chips and have suitable application scenarios. At the same time, considering the defects of the existing array chip method for generating single sequence nucleic acid library clone clusters, the present invention discloses a new nucleic acid library preparation and amplification method, which can solve the problems of label hopping and reduced stability, and achieve simultaneous adaptation of non-array and array chips to meet existing needs.

[0006] The details are as follows:

[0007] A method for preparing single-stranded DNA clusters on a solid support surface, the method comprising the following steps:

[0008] 1) providing a solid support and a library vector comprising a target nucleic acid fragment or a homologous sequence thereof, wherein the solid support and the library vector respectively comprise a first capture sequence and a first gripper sequence complementary thereto;

[0009] 2) The library vector occupies a certain spatial position on the surface of the solid support;

[0010] 3) The first capture sequence and the first gripper sequence are complementary paired, and the target nucleic acid fragment or its complementary sequence in the library vector is used as a template to form a DNA single-stranded cluster on the surface of the solid support by polymerase extension or reverse transcription extension;

[0011] 4) Removing the library vector to obtain a solid support coupled with a DNA single-stranded cluster.

[0012] In a specific embodiment of the present invention, the solid support is selected from a non-array solid support or an array solid support; preferably, the solid support is a part of a fluid pool.

[0013] In a specific embodiment of the present invention, the library carrier has a spatial structure of nanometer or micrometer size; the nanometer size means that the diameter or fluid dynamic diameter of the library carrier is less than 1 micrometer, such as 10nm-900nm; the micrometer size means that the diameter or fluid dynamic diameter is greater than 1 micrometer, such as 10-500 micrometers, preferably 10-90 micrometers, such as 30, 50 or 70 micrometers.

[0014] In a specific embodiment of the present invention, the library vector is a target nucleic acid fragment or its homologous sequence that is amplified to form a nanometer or micrometer-sized amplification product with a certain spatial structure, similar to a sphere rather than a line.

[0015] In another specific embodiment of the present invention, the library carrier is formed into a nanometer or micrometer-sized carrier by biological molecules, organic compounds, inorganic substances or a combination of the above substances, and the target nucleic acid fragment or its complementary sequence is directly or indirectly introduced on the surface of the carrier; preferably, the target nucleic acid fragment or its complementary sequence is introduced directly by chemical coupling; preferably, the target nucleic acid fragment or its complementary sequence is introduced indirectly, that is, primers are modified on the surface of the carrier, and the library to be sequenced is transferred to the surface of the carrier by polymerase extension or reverse transcription extension, thereby forming a library carrier.

[0016] In a specific embodiment of the present invention, when the library carrier is combined with the array or non-array chip surface, the repulsive force generated by the physical properties of the library carrier itself is used to achieve monodispersity of the library carrier and / or different spacing distances between the library carriers (to achieve different seeding densities), and the physical properties are selected from the physical size, steric hindrance, fluid dynamics exclusion volume, charge properties (positive charge or negative charge), charge density, hydrophilicity or magnetism of the library carrier.

[0017] In a specific embodiment of the present invention, the binding of the library carrier to the chip surface is achieved by complementary pairing of the first capture sequence and the first gripper sequence and / or charge interaction and / or hydrophobic interaction;

[0018] Preferably, the library carrier comprises a target nucleic acid fragment or a homologous sequence thereof connected to a first capture sequence, and a target nucleic acid complementary sequence connected to a second capture sequence, and the solid support surface simultaneously comprises a first and a second gripper sequence that are reverse complementary to the first and second capture sequences.

[0019] Preferably, the pH and salt concentration of the buffer are adjusted to achieve the desired seeding density.

[0020] Preferably, multiple inoculations are performed to obtain the desired inoculation density, for example, first using the first library vector for inoculation, and then using the second library vector for inoculation, the size or charge of the second library vector being the same as or different from that of the first library vector; preferably, a library vector with a greater exclusion capacity can be first used for full inoculation, and then a library vector with a smaller exclusion capacity can be used for inoculation.

[0021] In a specific embodiment of the present invention, step 2) and step 3) are performed simultaneously; in another specific embodiment of the present invention, step 2) and step 3) are performed successively; preferably, after repeating step 2) to reach the expected inoculation density, step 3) is performed.

[0022] In a specific embodiment of the present invention, the removal refers to the release of hybridization between the library vector and the first gripper sequence and the extension product, thereby allowing the entire library vector to detach from the surface of the solid support, and the method of releasing hybridization is selected from changing salinity, pH, temperature, solvent compatibility or flow rate.

[0023] In a specific embodiment of the present invention, before step 1), the method further includes: S1) constructing a library to be tested and / or S2) constructing a library vector.

[0024] In a specific embodiment of the present invention, in step S2), a library vector is generated by nucleic acid amplification.

[0025] Preferably, the library vector is formed by RCA rolling circle amplification;

[0026] Preferably, the library vector is formed by RCA-MDA amplification;

[0027] Optionally, after the RCA reaction is completed, a primer complementary to the RCA product is added to initiate an MDA reaction to obtain a library vector consisting of RCA-MDA products;

[0028] Optionally, before the RCA reaction begins, primers that can hybridize with the RCA product are simultaneously added to the system to obtain a library vector consisting of RCA-MDA products;

[0029] Optionally, 2n hybridization sites are designed on the circular library, and the hybridization sites from the 3' end to the 5' end of the circular library interface are recorded as the 1st, 2nd, 3rd ... 2n hybridization sites in sequence. In the RCA-MDA reaction, one or more primers that hybridize with the 1st, 3rd ... 2n-1 hybridization sites are added; at the same time, one or more primers with the same sequence as the 2nd, 4th ... 2n sites are added to carry out the RCA reaction. In this reaction, the primers that hybridize with the 1st, 3rd ... 2n-1 sites are used to generate RCA products; primers with the same sequence as the 2nd, 4th ... 2n sites are used to hybridize with the RCA products and initiate the MDA reaction to produce RCA-MDA products; at the same time, the primers that hybridize with the 1st, 3rd ... 2n-1 sites can further hybridize with the RCA-MDA products to continue the MDA reaction;

[0030] Among them, the RCA product contains the reverse complementary sequence of the target nucleic acid fragment connected to the first capture sequence, the MDA product contains the target nucleic acid fragment connected to the second capture sequence, and the solid support surface contains both the first gripper sequence and the second gripper sequence. During the transfer of the target library sequence to be tested, the target nucleic acid fragment and the reverse complementary fragment will be transferred to the chip surface at the same time.

[0031] In a specific embodiment of the present invention, in step S2), a circular library or a non-circular library carrying a target nucleic acid fragment is subjected to direct rolling circle amplification to obtain a linear single strand having a certain spatial structure, similar to a sphere but not stretched;

[0032] Preferably, in step S1), specific known sequences are added to both ends of the target nucleic acid sequence fragment, and an artificially synthesized nucleic acid oligomer that can simultaneously match the specific known sequences at both ends of the test library is used as a circular primer. Under the action of a ligase, the original linear target test library is converted into a closed circular library.

[0033] Preferably, in step S1), a linker with a nick is used to establish a library to be tested, and in step S2), the displacement enzyme can perform strand displacement and polymerization reactions from the nick of the linker to form a library vector.

[0034] In a specific embodiment of the present invention, in step S2), a library carrier is constructed on the surface of a nano- or micro-carrier.

[0035] In a specific embodiment of the present invention, the nano- or micro-carrier is a biomolecule, an organic compound, an inorganic substance or a combination of the above substances; further preferably, the nano- or micro-carrier is a protein, an antibody, a polymer, a magnetic bead, a gel bead, or a metal nano-micro particle.

[0036] Preferably, the surface of the nano- or micro-carrier and the library to be tested respectively comprise a second capture sequence and a second gripper sequence reversely complementary thereto. After the second capture sequence and the second gripper sequence are paired, a reverse complementary sequence of the target library sequence to be tested is formed on the surface of the nano- or micro-carrier by polymerase extension or reverse transcription extension to obtain a library carrier;

[0037] Preferably, the surface of the nano- or micro-carrier contains only a single second capture or second gripper sequence, and after extension, the surface of the carrier contains only a single target nucleic acid fragment or complementary sequence, and the library carrier thus obtained is a single library carrier;

[0038] Preferably, the surface of the nano- or micro-carrier comprises a single first functional group, and a single second capture sequence is introduced through the first functional group;

[0039] Preferably, the surface of the nano- or micro-carrier further comprises a plurality of second functional groups, and an amplification reaction is performed after a plurality of primers are introduced through the second functional groups to form an amplification cluster on the surface of the nano- or micro-carrier;

[0040] Preferably, in step S1), a reverse complementary sequence of the first capture sequence is introduced at one end of the target nucleic acid fragment or its homologous sequence, so that the library vector obtained after extension contains the first capture sequence.

[0041] In a specific embodiment of the present invention, the nano- or micro-carrier is a protein complex;

[0042] Preferably, the protein complex is AB n A multi-protein complex formed by a protein and an optional linker, wherein A represents a -SH group, B represents a -NH2 group, and n is an integer of 5-50, preferably 10-20;

[0043] Preferably, the AB n The protein is myoglobin;

[0044] Preferably, the second capture sequence or the second gripper sequence is connected through -SH, for example, the third reactive group is introduced through a bifunctional linker containing maleimide and a third reactive group, and the second capture sequence or the second gripper sequence is coupled through the third reactive group to form a first protein monomer; the first protein monomer reacts with an excess (10 to 30:1) of the second protein monomer of the unmodified capture sequence to form a multi-protein complex.

[0045] Preferably, a linker such as a PEG linker (the number of ethoxy units is: 4-2000, preferably 4-800, 4-400, 4-200, 4-100, 4-50 or 4-20, etc.) is introduced during the coupling process of protein monomers to increase the distance between the monomeric proteins of the multi-protein complex and achieve a larger exclusion volume.

[0046] Preferably, the surface of the protein carrier is modified secondary, primers are introduced for amplification, and amplification clusters are formed on the surface of the multi-protein complex.

[0047] In a specific embodiment of the present invention, the nano- or micro-carrier is an artificial microbead;

[0048] Preferably, the artificial microbeads are magnetic beads, gel beads, or metal nano-micro particles;

[0049] Preferably, the artificial microbeads are micrometer-sized.

[0050] In a specific embodiment of the present invention, the nano- or micro-carrier is a positively charged polymer; preferably, the positively charged polymer is PEI or PLL; preferably, the polymer contains a single first functional group for introducing a capture sequence, and / or multiple second or third functional groups for modification or cross-linking to increase repulsion or for introducing primers to form an amplification cluster.

[0051] The second aspect of the present invention provides a method for preparing DNA clone clusters on the surface of a solid support, the method comprising first using the above-mentioned method to form DNA single-stranded clusters on the surface of a solid support, and step 5): amplifying the DNA single-stranded clusters on the surface of the solid support into larger DNA clone clusters by bridge amplification means or isothermal amplification means such as RPA, HDA, LAMP, etc.

[0052] The third aspect of the present invention provides a kit or reagent combination for preparing a DNA single-strand cluster or a clone cluster on a solid support surface, comprising:

[0053] 1) a library vector and 2) a solid support; the library vector and the solid support respectively comprise a first capture sequence and a first gripper sequence that is reverse complementary thereto;

[0054] Preferably, the kit or reagent combination further comprises bridge amplification or isothermal amplification reagents.

[0055] The library vector and solid support are as described in the first aspect of the present invention.

[0056] In a specific embodiment of the present invention, the kit or reagent combination further comprises a reagent for constructing a library vector.

[0057] Preferably, the reagents for constructing the library vector comprise RCA rolling circle amplification primers; preferably, the reagents for constructing the library vector comprise RCA-MDA amplification primers.

[0058] Preferably, the reagent for constructing the library vector comprises a nano- or micro-carrier.

[0059] In a specific embodiment of the present invention, the reagents for constructing the library vector further include reagents for constructing the test library, which, when constructing the test library, add the reverse complementary sequence of the first capture sequence to one end of the target nucleic acid fragment, so that the library vector obtained after extension contains the first capture sequence.

[0060] The fourth aspect of the present invention provides a sequencing method, which comprises preparing a DNA clone cluster by the method of the second aspect of the present invention, and then sequencing the clone cluster;

[0061] Preferably, the sequencing is fluorescence sequencing.

[0062] The fifth aspect of the present invention provides a sequencing kit or reagent combination, wherein the kit comprises the kit or reagent combination of the third aspect of the present invention, and a sequencing reagent.

[0063] Beneficial technical effects

[0064] Embodiments of the present invention relate to methods suitable for preparing single-stranded clusters or clone clusters of nucleic acid libraries in the form of sites on different types of chip surfaces. Based on the difference in chips, such as arrayed or non-arrayed chips, the clone clusters of the nucleic acid library generated can be in the form of arrays with regular patterns or in the form of arrays with irregular patterns.

[0065] When used on an array chip, the present invention can generate a single cloned nucleic acid library cluster in a pattern array, by obtaining enough library copies to generate a sufficient signal intensity for sequencing. After amplification, there is only one nucleic acid library sequence in each site in the array, or only one library sequence is dominant, or only one library sequence accounts for more than 60%, 70%, 80%, or 90%. The proportion of sites containing cloned nucleic acid library clusters generated by this method accounts for more than 40%, 50%, 60%, 70%, 80%, or 90% of all sites, exceeding the statistical probability expectation of Poisson distribution.

[0066] On non-array chips, the present invention can adjust the distribution density of monodispersed nucleic acid libraries occupying random sites on the chip surface, and then generate nucleic acid library clone cluster sites with suitable density and size through bridge amplification, isothermal amplification, etc., to provide sufficient signal intensity and signal density for sequencing. Each nucleic acid library clone cluster contains only one nucleic acid library sequence, or only one library sequence is dominant, or only one library sequence accounts for more than 60%, 70%, 80%, or 90%. The spacing of the center points of the nucleic acid library cluster sites generated by this method can be 10μm, or 1μm, or 500nm, or lower. The density of nucleic acid library clone cluster sites is higher than the site density of common non-array chips.

[0067] In summary, based on the method disclosed in the present invention, nucleic acid clusters with copies of a single nucleic acid sequence with different cluster intervals can be produced on the surface of a non-array chip; or nucleic acid clusters with copies of a single nucleic acid sequence that conform to the array interval can be produced on an array chip. The chip produced in the above manner can be used to determine the sequence of the inserted fragment of the nucleic acid sample to be tested, or to be used in scenarios that require the inclusion of prefabricated random sequence fragments, such as spatial omics, single-cell sequencing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 It is a multi-protein complex with a double-layer spatial structure;

[0069] Figure 2 A polymer library vector for construction;

[0070] Figure 3 This is the electrophoresis diagram of the protein single library vector;

[0071] Figure 4 is a graph showing the relationship between RCA library vector size and site occupancy density;

[0072] Figure 5 The effect of pH on the occupancy of RCA library vectors;

[0073] Figure 6 is the relationship between the vector size and the occupancy density of the RCA-MDA library;

[0074] Figure 7 The occupancy of the RCA-MDA library carrier at pH = 8 on the non-array chip surface;

[0075] Figure 8 The occupancy of the RCA-MDA library vector at pH = 8 on the array chip surface;

[0076] Fig. 9 is the relationship between inoculation density and extension time;

[0077] Fig.10 This is a picture after the extension products of the library vector are completely removed;

[0078] Fig.11 It is a DNA clone cluster generated by isothermal amplification;

[0079] Fig.12 DNA clone clusters generated by HDA;

[0080] Fig.13 It is a DNA clone cluster generated by polymerase chain reaction;

[0081] Fig.14 It is a DNA clone cluster generated by bridge amplification;

[0082] Fig.15 This is a diagram of 8 types of gene tag jumping;

[0083] Fig.16 Compare the fluorescence images of the chip after 600 rounds of fluid flushing;

[0084] Fig.17 This is a fluorescence image of the chip of the present invention after 600 rounds of fluid flushing;

[0085] Fig.18 Comparison of sequencing stability between the chip of the present invention and the comparative chip;

[0086] Fig.19 Comparison of sequencing error rates between the chip of the present invention and the comparison chip;

[0087] Fig. 20 The schematic diagram of the present invention is ( Fig. 20 A shows the library to be tested of the present invention, where capture sequences or their complementary sequences are introduced into two segments of the target nucleic acid fragment. This figure is only a schematic diagram, and those skilled in the art know that functional sequences such as tag sequences and UMI sequences can be introduced into the adapter of the second-generation sequencing; Fig. 20 B illustrates the construction process of a single library vector. A nano- or micro-vector carrying a single gripper sequence captures the target nucleic acid fragment, and then the corresponding homologous sequence is introduced on the surface of the vector through amplification. The R sequence pre-introduced in the target nucleic acid fragment introduces its reverse complementary sequence R-RC into the homologous sequence on the surface of the vector library, so that it can hybridize with the chip surface modified with the R sequence; Fig. 20 C illustrates the construction of the cluster library vector sequence, and amplification is performed after further modifying the primers on the vector surface to form amplification clusters on the vector surface; Fig. 20 D shows the steps of occupying, extending and eluting the vector library to form single-stranded clusters. Fig. 20 D shows a single library vector. In actual use, a cluster library vector is usually used to improve hybridization efficiency. Fig. 20 E illustrates the process of amplifying a single-stranded cluster into a clonal cluster.) DETAILED DESCRIPTION

[0088] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many details are described to enable the present invention to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other elements, materials, methods.

[0089] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0090] The specific implementation steps of the present invention include: construction of a sequencing library, construction and inoculation of a library vector, synthesis of a DNA cluster and sequencing, etc., which are specifically described below in combination with technical terms:

[0091] 1. Construction of Sequencing Library

[0092] As used in the present invention, the nucleic acid being sequenced is referred to as a target nucleic acid (or target). Target nucleic acids include, but are not limited to, DNA and RNA, wherein the DNA includes, but is not limited to, genomic DNA, mitochondrial DNA or cDNA, and the RNA includes, but is not limited to, mRNA or miRNA. The source of the target nucleic acid includes a naturally occurring source or a synthetic source. The nucleic acid can be a PCR product, a cosmid, a plasmid, a naturally occurring or synthetic library member or species, etc. The present invention is not intended to be limited to this aspect. The nucleic acid can be from an animal or pathogen source, including, but not limited to, mammals, such as humans, and microorganisms, such as bacteria, viruses, fungi, parasites and mycobacteria. In some embodiments, the nucleic acid is not a viral nucleic acid. The target nucleic acid can be derived from any body fluid or tissue, including, but not limited to, blood, saliva, cerebrospinal fluid ("CSF"), skin, hair, urine, stool and mucus. The target nucleic acid can also be derived from, but not limited to, environmental samples (such as water samples), food samples, forest samples, which can be fresh samples (e.g., biopsy materials directly subjected to nucleic acid extraction), or samples that have been processed and can be stored, for example, formalin-fixed and / or paraffin-embedded samples (FFPE samples).

[0093] The target nucleic acid is prepared using any method known in the art. For example, genomic DNA can be harvested from a sample according to techniques known in the art. After harvesting, DNA can be broken to obtain nucleic acids of smaller lengths. The length of the resulting fragment can be about hundreds, thousands, or tens of thousands of nucleotides. In some embodiments, the fragment is 50-1000 nucleotides in length, 100-1000 nucleotides in length, 200-1000 base pairs in length, or 300-800 base pairs in length, although they are not limited thereto. Nucleic acid fragmentation can be made by any means, including, but not limited to, mechanical, enzymatic, or chemical methods. Examples include shearing, ultrasound, atomization, and endonuclease (e.g., DNase I) digestion, or any other technique known in the art to produce nucleic acid fragments (preferably, with a desired length). After fragmentation, it can be a size selection technique for enriching or separating fragments of a specific length. These techniques are also known in the art and include, but are not limited to, gel electrophoresis or SPRI.

[0094] Generally, the target nucleic acid is connected to a joint sequence at one or both of the 5' and 3' ends, and the joint sequence is a linear oligonucleotide that can be fused to a nucleic acid molecule by, for example, connection or labeling. In some examples, the joint is substantially not complementary to the 3' end or 5' end of any target sequence present in the sample. In some examples, a suitable joint length is within the range of about 10-100 nucleotides, about 12-60 nucleotides, or about 15-50 nucleotides. Typically, the joint can include any combination of nucleotides and / or nucleic acids. In some aspects, the joint can include one or more shearable groups at one or more positions. On the other hand, the joint can include a sequence complementary to at least a portion of a primer, and the primer is, for example, a primer comprising a universal nucleotide sequence, such as a P5 or P7 sequence. In some examples, the joint can include a barcode (also referred to as a label or index in this article) to assist downstream error correction, identification, or sequencing.

[0095] In a specific embodiment of the present invention, a capture sequence or a gripper sequence is introduced at one or both ends of the target nucleic acid. In the present invention, the capture sequence and the gripper sequence refer to two reverse complementary sequences, which are defined only for the convenience of description, and the terms can be used interchangeably; similarly, in certain specific embodiments of the present invention, the reverse complementary sequence of the capture (gripper) sequence and the gripper (capture sequence) sequence have the same meaning and can be used interchangeably. Preferably, the first capture sequence and the reverse complementary sequence of the second capture sequence are connected to the two ends of the target nucleic acid, respectively, and the first capture sequence is used for the capture of the micro- or nano-carrier; the reverse complementary sequence of the second capture sequence is used to introduce the second capture sequence into the library carrier for pairing with the second gripper sequence on the chip surface. Similarly, the above-mentioned "first" and "second" are also defined for the convenience of description, and in certain schemes of the present invention, they can be used interchangeably.

[0096] In certain specific embodiments of the present invention, the first capture sequence or the first gripper sequence (or the second capture sequence or the second gripper sequence) may refer to a P5 or P7 sequence, or may be an L or R sequence. Optionally, the L sequence is as shown in SEQ ID NO.1, and the R sequence is as shown in SEQ ID NO.6. The conversion may also be performed as needed between the two sets of sequences by template conversion, so that the method of the present invention can be used on existing commercial sequencing platforms.

[0097] In certain embodiments of the present invention, the capture sequence is also used as a primer for a nucleic acid extension reaction.

[0098] In the present invention, the library to be sequenced, the sequencing library or the target library have the same meaning, and all refer to the nucleic acid library obtained through the above steps, and the target nucleic acid fragment therein is also referred to as the target nucleic acid fragment in the present invention.

[0099] 2. Construction and inoculation of library vector

[0100] The library vector in the present invention refers to a vector that contains a target nucleic acid fragment or a homologous sequence thereof and has a nanometer or micrometer-sized spatial structure.

[0101] The spatial structure can be a microsphere or a microbead. The "microsphere" or "microbead" used in the present invention has the usual meaning in the art, that is, a nanometer or micrometer-sized structure formed by biological molecules, organic compounds, inorganic substances or a combination of the above substances, and its shape can be close to or similar to a sphere; this term is only used to express that the biological molecules or organic or inorganic substances that form the library carrier are in a spatial aggregation state, rather than a one-dimensional linear or two-dimensional planar state, such as irregular microspheres that are significantly different from a sphere, which are also applicable in the present invention.

[0102] The library carrier described in the present invention can be amplified by the library to be sequenced to form a nanometer or micrometer-sized amplification product with a certain spatial structure, similar to a sphere rather than a line; or a nanometer or micrometer-sized structure can be formed by biological molecules, organic compounds, inorganic substances or a combination of the above substances, and the structure is used as a carrier to modify the primer on the surface, and the library to be sequenced is transferred to the carrier surface by polymerase extension or reverse transcription extension, thereby forming a library carrier.

[0103] In a specific embodiment of the present invention, the nano- or micron-sized structure formed by biological molecules, organic compounds, inorganic substances or a combination of the above substances is, for example, a polymer or supramolecule produced by coupling, cross-linking, polymerization and other reactions of artificially modified monomers; it can also be generated by intermolecular forces through non-covalent bonds, such as antigen-antibody or biotin-streptavidin; it can also be combined by physical means, such as mutual attraction between opposite charges; it can also be the product of a combination of the above methods.

[0104] The nanometer size described in the present invention is that the diameter or hydrodynamic diameter of the carrier is less than 1 micron, such as 10nm-900nm; the micrometer size is that the diameter (hydrodynamic diameter) is greater than 1 micron, such as 10-500 microns, preferably 10-90 microns, such as 30, 50 or 70 microns. When applied to a preset array chip, its size can be comparable to the size of the array site; when applied to a non-preset array chip, the library carrier size can be selected based on the seeding density, etc.

[0105] The "inoculation" mentioned in the present invention refers to the process of completing the inoculation of the library carrier on the surface of the chip, including three steps: occupying the site, extending and removing.

[0106] The said occupancy refers to that the library carrier occupies a certain spatial position on the chip surface, including but not limited to being fixed on the chip surface by charge attraction, or carrying a reverse complementary sequence modified by the chip surface through hybridization.

[0107] In the invention, when the library carrier is combined with the array or non-array chip surface, the physical properties of the library carrier itself (or the nucleic acid molecule itself) are used to achieve monodispersity of the library carrier and / or different spacing distances between the library carriers, thereby obtaining the expected seeding density. Such physical properties include but are not limited to the physical size, steric hindrance, hydrodynamic exclusion volume, charge properties (positive or negative charge), charge density, hydrophilicity, magnetism, etc. of the library carrier.

[0108] The extension refers to amplification of the fixed library vector.

[0109] The removal refers to the release of hybridization between the library carrier and the capture sequence and the extension product, thereby allowing the entire library carrier to be detached from the chip.

[0110] The library vector of the present invention is described below by taking specific embodiments as examples, but the following embodiments do not constitute any limitation on the structure or preparation method of the library vector.

[0111] (I) Library vectors constructed based on steric hindrance

[0112] 1.RCA library vector

[0113] (1) Construction of RCA library vector

[0114] Rolling circle amplification (RCA) is a common isothermal amplification technology, which is widely used in the fields of second-generation sequencing, in situ sequencing, in situ hybridization and other technical fields. Its principle is that after hybridizing the amplification primer on the circular library, the polymerase with chain displacement function triggers a continuous amplification reaction along the initial circular library. Its product is a repeated single strand carrying the reverse complementary sequence of the starting circular library sequence, and the copy number can be increased by extending the reaction time. In the process of library construction, a designed palindromic sequence of a certain length can be added to the target library to be tested, so as to make the long single-stranded product spontaneously self-hybridize within the chain through the palindromic sequence, and produce a product with a certain spatial structure, similar to a sphere rather than a line.

[0115] Using this principle, the target library to be tested for the second generation sequencing can be added with specific known sequences L sequence and R-RC sequence at both ends by means of second generation sequencing library construction, wherein the L sequence is shown in SEQ ID NO.1, and the R-RC sequence is shown in SEQ ID NO.2. By using artificially synthesized nucleic acid oligomers that can simultaneously match specific known sequences at both ends of the library to be tested as circular primers, the original linear target library to be tested is converted into a closed circular library under the action of DNA ligase. After the circularization is completed, the uncircularized library and the excess circularized primers can be digested by a nuclease specific to the 5' end or the 3' end, leaving only the circular library.

[0116] After direct rolling circle amplification of the circular library carrying the L sequence, the amplified product is a single strand carrying multiple copies, which contains the L-RC sequence, and optionally, the L-RC sequence is shown in SEQ ID NO.3.

[0117] (2) Inoculation of RCA library vector

[0118] ① Placeholder

[0119] By amplifying the multiple L-RC sequences on the single strand, the amplified single strand can be hybridized with a chip with a surface modified with an L' sequence and fixed. Optionally, the L' sequence is as shown in SEQ ID NO.4.

[0120] As mentioned above, the single-stranded product obtained by RCA amplification has a certain spatial structure, a spherical rather than stretched linear product (i.e., DNB well known in the art). The spatial volume occupied by this structure is related to the length of the palindromic sequence, the number of bases in the product, and the GC content of the library. The first two can be artificially controlled during the library construction process and the amplification process. In short, a long palindromic sequence can make the product more compact, the structure is better maintained, and the spatial volume is smaller; a longer reaction time leads to an increased number of bases, thereby increasing the total length and volume of the product. Based on this, the size of the steric hindrance brought about by the spatial volume of the amplified product is controllable to a certain extent, and can meet different requirements for the density of the cluster. When a denser cluster is required, the palindromic sequence in the fixed sequence added to the target library to be tested during the library construction process can be artificially increased; or the amplification time can be shortened, and the total number of bases of the product can be reduced to shorten the total length of the product and reduce the spatial volume occupied by the product. In some examples, the adjustment of pH and salt concentration can also affect the spatial volume of the product, and can also be used to adjust the density of the generated nucleic acid library cluster.

[0121] In the present invention, the method of synthesizing DNB is not subject to any restrictions. For example, the method reported in WO2023050968A1 can also be used. This method uses a linker with a nick, and the displacement enzyme can perform chain displacement and polymerization reactions from the nick of the linker to achieve direct extension of DNA and obtain DNB products, thereby eliminating the PCR and cyclization steps.

[0122] ② Extension

[0123] When using amplification products of appropriate spatial volume to generate clusters, the amplification products must first be hybridized with the randomly distributed L' sequences modified on the chip surface through multiple L-RC sequences. Usually, multiple L' sequences with a lower copy number will fully and sufficiently hybridize with the amplification products. At a suitable temperature (close to the Tm value of the L' sequence corrected by the salt ion concentration), this hybridization is dynamic and constantly changing. Due to differences in copy number, spatial conformation, local microenvironment, and microscopic fluid mechanics, amplification products that cannot complete sufficient hybridization will be repelled and replaced by the spatial volume of other amplification products because they cannot stably occupy the position on the chip surface. Therefore, the final amplification products will be fixed on the chip surface at a certain density through the hybridized L-RC sequences. As the spatial volume increases, the copy number increases, the number of L-RC sequences available for hybridization increases, and the combined force of hybridization increases. However, on the other hand, as the spatial volume increases, the fluid disturbance to the amplification products is stronger, and the diffusion rate decreases, which is not conducive to the fixation of the amplification products. Therefore, the amplification products finally fixed are subject to the feedback regulation of two opposite factors, so that the spatial volume distribution of the amplification products is concentrated in a narrow range.

[0124] After hybridization is completed, the amplified product uses the L' sequence as the starting primer and the amplified product itself as the template. Through polymerase extension, the sequence information of the target fragment to be tested on the library is transferred to the downstream of the L' sequence modified on the chip surface to form a sequencing template. At the same time, this transfer process also includes the transfer of functional preset fixed sequences, such as the R-RC sequence.

[0125] ③Remove

[0126] The hybridization between the amplification product and the L' sequence and the extension product is released, so that the entire amplification product is separated from the chip.

[0127] After the extension is completed, the double-stranded part of the amplification product fixed on the chip surface by hybridization can be denatured by means including but not limited to nucleic acid denaturants, or strong bases, or organic solvents, so that the hybridization between the amplification product and the L' sequence and the extension product is released, so that the amplification product as a whole is separated from the chip. When the amplification product is completely separated from the chip, only DNA single-strand clusters close to the spatial volume of the amplification product are separated on the chip surface, each cluster is composed of multiple single strands, and each single-strand sequence in the cluster is homologous. And each single strand in the cluster can carry a single copy or multiple copies of the target library to be tested. Each copy contains an L'sequence, wherein the starting end L'sequence comes from the L'sequence modified on the chip surface, and in addition to the starting end, the L'sequence extending toward the terminal direction comes from the single strand synthesized by the polymerase using the amplification product as a template. At the same time, each copy contains an R-RC sequence, wherein the starting end R-RC and the R-RC sequence extending toward the terminal direction are both from the single strand synthesized by the polymerase using the amplification product as a template. The L' sequence, the homologous sequence of the target library to be tested, and the R-RC are arranged in order from the starting end to the end in each copy. The copy at the end can be a complete copy containing the L' sequence, the homologous sequence of the target library to be tested, and the R-RC sequence. It can also be a partial copy containing only one or two sequences near the starting end among the L' sequence, the homologous sequence of the target library to be tested, and the R-RC sequence. The sequence contained in the copy at the end (L' sequence, homologous sequence of the target library to be tested, or R-RC sequence) can be complete or incomplete.

[0128] 2.RCA-MDA library vector

[0129] (1) Construction of RCA-MDA library vector

[0130] Based on DNA polymerase with strand displacement function, multiple starting primers can be used to generate isothermal amplification copies equal to the number of primers, which is called multi-strand displacement reaction (Multi-strand-DisplacedAmplification). This technology is often used in conjunction with RCA. Thanks to the chain-like repeated copy fragments produced by RCA, a large number of amplification primer hybridization sites are provided. Through sequence design, the upstream and downstream of the target library can carry multiple functional regions of known sequences for hybridization of amplification primers. Based on this principle, on the basis of the RCA product, bases can be further added to the original RCA product through the MDA reaction to make it reach a larger volume, further increase its steric hindrance, and be used for amplification reactions under larger cluster spacing requirements.

[0131] An optional method includes not terminating the reaction after the RCA reaction is completed. Instead, a primer complementary to the RCA product is added, and optionally, the primer is as shown in SEQ ID NO.5 to trigger the MDA reaction. On each copy, there may be one, two, three or more primer hybridization sites, and when adding complementary primers, only one primer may be added, or two, or three or more complementary primers may be added to trigger a more intense MDA reaction. It should be noted that an excess of a sequence that is reverse complementary to the starting rolling circle primer in the initial RCA reaction may be added to neutralize the starting rolling circle primer. Finally, the RCA-MDA product is obtained.

[0132] Another optional method includes adding primers that can hybridize with the RCA product to the system before the RCA reaction begins, which can be one, two, three or more primers. The reaction temperature is controlled and the primer sequence and length are designed so that the primers that hybridize with the RCA product can hybridize with the generated RCA product. The RCA reaction and the MDA reaction are carried out simultaneously to directly obtain the RCA-MDA product in one step.

[0133] Another optional method includes designing 2n hybridization sites on the circular library, and the hybridization sites from the 3' end to the 5' end of the circular library interface are recorded as the 1st, 2nd, 3rd...2n hybridization sites in sequence. In the RCA-MDA reaction, one or more primers of the primers that hybridize with the 1st, 3rd...2n-1 hybridization sites are added; at the same time, one or more primers of the primers with the same sequence as the 2nd, 4th...2n sites are added to perform RCA reaction. In this reaction, the primers hybridized with the 1st, 3rd...2n-1 sites are used to generate RCA products; the primers with the same sequence as the 2nd, 4th...2n sites are used to hybridize with the RCA products and initiate the MDA reaction to produce RCA-MDA products. At the same time, the primers that hybridize with the 1st, 3rd...2n-1 sites can further hybridize with the RCA-MDA products to continue the MDA reaction.

[0134] (2) Inoculation of RCA-MDA library vector

[0135] The RCA-MDA products obtained by the above methods include a circular initial library, one or more single-stranded RCA single-stranded products, and multiple MDA single-stranded products. The RCA single-stranded product includes an L sequence and an R-RC sequence. The MDA single-stranded product includes an L-RC sequence and an R sequence, and optionally, the R sequence is shown in SEQ ID NO.6.

[0136] Placeholder: The RCA-MDA product can hybridize with the L sequence and R sequence modified on the chip surface at the same time. Therefore, during the transfer of the target library sequence to be tested, the target library sequence to be tested and the sequence that is reverse complementary to the target library sequence to be tested will be transferred to the chip surface at the same time, which can improve the efficiency of subsequent amplification.

[0137] The subsequent extension and removal methods can be the same as those of the above-mentioned RCA library vector.

[0138] 3. Single peptide chain protein

[0139] (1) Construction of single peptide chain protein library vector

[0140] By assembling substances with ABn structure, a structure with a certain hydrodynamic excluded volume is generated. The substances generating the excluded volume can be biological macromolecules, biological micromolecules, polymers, oligomers or a mixture of the above substances.

[0141] Taking myoglobin as an example, its structure contains 154 amino acids, molecular weight 16700, and the sequence is shown in SEQ ID NO: 7.

[0142] It contains one cysteine ​​(C), three asparagines (N), seven glutamines (Q), two arginines (R), and twenty lysines (K). The entire myoglobin has a total of one -SH group and thirty-three -NH2 groups. Due to the influence of steric hindrance and charge distribution, not all thirty-three -NH2 groups are reactive. In actual reactions, about 1 / 2 to 2 / 3 of the -NH2 can react with succinimide esters. Therefore, myoglobin can be represented as AB 20～30 , wherein A represents -SH and B represents -NH2 group. After denaturing myoglobin, its active reactive side chain is fully exposed. On this basis, -SH is modified by maleimide to convert it into other chemical groups available for coupling reaction, such as azide or dibenzocyclooctyne or other alkynes or tetrazine, so as to be subsequently subjected to one-to-one coupling reaction with a nucleic acid oligomer having a specific known sequence (L sequence) modified with the corresponding chemical group capable of coupling reaction. Myoglobin can also be directly connected to a nucleic acid oligomer having a specific known sequence (L sequence) by a maleimide-modified nucleic acid oligomer having a specific known sequence (L sequence). After -SH is modified and fully reacted with a nucleic acid oligomer having a specific known sequence (L sequence), a new coupling reaction active group, such as azide or dibenzocyclooctyne or other alkynes or tetrazine, can be introduced to -NH2 by succinimide ester. At the same time, a new fully denatured myoglobin whose -SH is modified to a corresponding chemical group that can undergo coupling reaction with azide or dibenzocyclooctyne or other alkynes or tetrazine can be added to the reaction system. The newly modified myoglobin can react with the myoglobin whose -SH is fully reacted with a nucleic acid oligomer with a specific known sequence (L sequence) and whose -NH2 is modified to azide or dibenzocyclooctyne or other alkynes or tetrazine in the previous step at a ratio of 10 to 30 to form a multi-protein complex with a double-layer spatial structure, such as Figure 1 shown.

[0143] In the actual use of the multi-protein complex, the reverse complementary sequence (L-RC sequence) of the specific known sequence (L sequence) of the oligomer coupled to the multi-protein complex can be added to the target library to be tested through the library construction process, so that the target library can hybridize with the specific known sequence (L sequence) on the multi-protein complex through the specific known sequence (L-RC sequence), and under the action of DNA polymerase, the reverse complementary sequence of the target library sequence to be tested is transferred to the multi-protein complex. Then, the multi-protein complex carrying the complete reverse complementary sequence of the target library to be tested is contacted with the chip with randomly and evenly distributed L' and R sequences on the surface through another specific known sequence (R sequence) added to the other end of the target library during the library construction process, and hybridized with the R sequence through the R-RC sequence. Once effective and sufficient hybridization occurs, due to the steric hindrance of the multi-protein complex, the second multi-protein complex cannot enter the exclusion volume range, that is, only a single library can exist in an exclusion volume.

[0144] For the library carrier constructed by protein, in addition to the spatial volume of the protein itself, in the modification process of the chemical group with coupling performance, the distance between the monomer proteins of the double-layer multi-protein complex can be artificially increased by coupling reagents containing oligomer PEG, such as PEG4, PEG10, PEG12, PEG18, PEG400, PEG800, PEG2000, etc., to achieve a larger exclusion volume. At the same time, the double-layer multi-protein complex may not be limited to a two-layer structure, and a three-layer, four-layer or even more-layer structure may be achieved by increasing the number of reaction rounds to form a multi-layer multi-protein complex to obtain a larger exclusion volume. At the same time, the reaction ratio of each layer may not be limited to 20 to 30:1, and may be higher or lower. At the same time, by adjusting the degree of modification of -NH2, the reaction ratio between the outer layer and the inner layer can be the same, higher or lower. At the same time, the length of the PEG oligomer in the -SH-modified coupling reagent can be a single 0, 4, 8, 12, 18, 400, 2000, or a mixture of PEGs of several or more lengths. At the same time, the length of the PEG oligomer in the -NH2-modified coupling reagent can be a single length of 0, 4, 8, 12, 18, 400, 2000, or a mixture of several or more lengths. At the same time, the length of the PEG oligomer between each layer can be the same or different.

[0145] It should be pointed out that when the number of layers is too high, the reaction efficiency will be further reduced, and the losses caused by the purification, separation, concentration and other steps will increase sharply. Therefore, in addition to increasing the number of layers, the exclusion volume of the multi-protein complex can also be increased by antibody-antigen specific binding. The double-layer or multi-layer protein complex described above is contacted with a specific myoglobin antibody. After sufficient reaction, a new layer of antibody will be added on the basis of the existing double-layer or multi-layer multi-protein complex to obtain an increase in the exclusion volume. However, it should be noted that the new antibody-multi-protein complex combination lacks direct chemical covalent bonds between the antibody layer and the multi-protein complex, and is only combined through weak interactions such as hydrogen bonds and van der Waals forces. The stability is relatively simple. The environment and conditions of use need to be adjusted accordingly in actual use to avoid the disintegration of the antibody layer in the antibody-multi-protein complex combination.

[0146] In the actual use of the multi-protein complex, the reverse complementary sequence (L-RC sequence) of the specific known sequence (L sequence) of the oligomer coupled to the multi-protein complex can be added to the target library to be tested through the library construction process, so that the target library can hybridize with the specific known sequence (L sequence) on the multi-protein complex through the specific known sequence (L-RC sequence), and under the action of DNA polymerase, the reverse complementary sequence of the target library sequence to be tested is transferred to the multi-protein complex.

[0147] (2) Inoculation of single peptide chain protein library vector

[0148] Occupancy and extension: The multi-protein complex carrying the complete reverse complementary sequence of the target library to be tested contacts the chip with randomly and evenly distributed L' and R sequences on the surface through another specific known sequence (R sequence) added at the other end of the target library during the library construction process, and hybridizes with the R sequence through the R-RC sequence. Once effective and sufficient hybridization occurs, due to the steric hindrance of the multi-protein complex, the second multi-protein complex cannot enter the exclusion volume, that is, only a single library can exist in an exclusion volume.

[0149] Removal: After the transfer of the target library sequence is completed, the multi-protein complex coupled with the library can be released from the chip surface by double-strand denaturation by changing the pH or changing the solvent composition.

[0150] 4. Artificial microbead library vector

[0151] (1) Construction of artificial microbead library vector

[0152] The hydrodynamic volume of carriers produced by DNA chains, proteins, antigens and antibodies is mostly below micrometers, ranging from tens to hundreds of nanometers, as shown in the figure. When a larger spacing, such as micrometers, is required, it is necessary to use structures with larger spatial exclusion volumes, such as micrometer-sized particles, to produce clusters with micrometer-sized spacing. These micrometer-sized particles include but are not limited to magnetic beads, gel beads, metal nano-micrometer particles, etc. Most of them exist in spherical or spherical shapes, and are collectively referred to as microbeads here.

[0153] For example, through emulsion PCR, the microbeads and the diluted library are mixed one-to-one, and the library is amplified by primers modified on the surface of the microbeads, so that different microbeads carry different libraries, and the same microbead carries multiple copies of the same library, thereby achieving the purpose of signal enrichment.

[0154] In addition, in the sequencing of single-cell transcriptomes, encoded microbeads are a common tool. Their characteristic is that each microbead has a single random sequence, and the random sequence carried by each microbead is different, which is also called encoding. This encoding allows each microbead to be identified separately, and at the same time, the captured mRNA also carries the unique encoding on the microbead through transcription. In this way, during the sequencing process, mRNA information with the same encoding can be counted under the same single cell for deeper cellular level analysis.

[0155] In the example here, in terms of nucleic acid sequences, the encoded information on the microbeads used for single-cell transcriptome capture has a similar structure to the microbeads carrying the amplified library in the second-generation sequencing, that is, both contain several known functional sequences (such as P5, P7, PolyA and other sequences and their homologous sequences), as well as random insertion sequences that need to be determined by sequencing (such as barcode sequences, index sequences, umi sequences or library insertion sequences).

[0156] Therefore, this paper uses random barcode simulation as the target library to be tested, which can demonstrate the different spatial exclusion volumes of beads of different sizes. The oligo modified on the beads is covalently linked to the beads at the 5' end. The sequence from the 5' to the 3' end is the R barcode L-RC sequence.

[0157] (2) Inoculation of artificial microbead library vector

[0158] Occupancy and extension: After the microbead library carrier is fully dispersed, it is suspended in a solution suitable for DNA chain hybridization and evenly spread on the surface of the chip coupled with the L' sequence and R sequence. The microbeads that occupy the position can hybridize with the L sequence coupled to the chip surface through the L-RC sequence at the 3' end of the surface-coupled nucleic acid oligomer. The temperature of hybridization is suitable for a temperature slightly lower than, close to or slightly higher than the Tm of the L' sequence. This hybridization process is dynamic for a single DNA strand. However, for microbeads that occupy a dominant position and can fully hybridize, since multiple DNA strands can hybridize at the same time, in general, at any time point during the hybridization process, the surface of the microbeads in the dominant position has a sufficient number of DNA strands in a hybridized state. Therefore, even for a single coupled DNA, the hybridization under hybridization conditions is dynamic, and for microbeads in a dominant position, the overall hybridization of multiple DNA strands is stable and can provide a sufficiently strong fixation force. After the hybridization process is completed, the microbeads that are not hybridized, or are insufficiently hybridized, or have insufficient hybridization force, or have failed hybridization competition in the solution are removed by changing the salinity, pH, temperature, solvent compatibility, flow rate, etc., and only the microbeads that are hybridized and have strong enough hybridization force and have successfully hybridized competition are retained. After a hybridization process is completed, if the density of the microbeads on the chip surface is not high enough, a second, third, or more hybridizations can be performed until the density of the microbeads on the chip surface reaches the expected value.

[0159] After the microbeads on the surface are fully hybridized with the L'sequences on the chip surface through the L-RC sequence, and the microbeads that are not fully hybridized are completely removed, the L' and L-RC sequences on each microbead hybridized with the chip surface can be transformed into a complete hybridization state by changing the salinity, pH, temperature, solvent compatibility, or flow rate. In this state, the L'sequence on the chip surface can transfer the sequence information on the microbead, including the functional sequence containing a specific sequence, the library insert fragment of the random sequence, or the coding information, to the chip surface by DNA polymerase extension, in the form of reverse complementation.

[0160] Removal: After the transfer process is complete, the hybridization is inhibited by further changing the salinity, pH, temperature, solvent compatibility, or flow rate. The double strands in various states on the chip surface are completely denatured, and then the beads lose the ability to be fixed on the chip surface and can be taken out of the chip range with the movement of the fluid. In this way, only single-stranded DNA clusters are retained at the position of the original beads on the chip surface, and there may be a certain degree of hybridized double-stranded regions in the clusters. Each bead corresponds to a single-stranded cluster. The random sequences from the library insert fragment or coding sequence in each single-stranded cluster are the same. This method can be directly applied to array chips with pre-made patterns whose pattern size is similar to the bead size. It can also be used on non-array chips without patterns but uniformly modified with L' sequences and R sequences with sufficient density.

[0161] (II) Library vector based on charge repulsion

[0162] 1. Positively charged polymer library carrier

[0163] (1) Construction of polymer library vector

[0164] In addition to forming a carrier with a certain spatial volume and tiling the library carrier at a certain density by means of fluid mechanics volume exclusion, Coulomb force can also be applied to the mutual exclusion between library carriers. In terms of distance, amino-rich polymers such as PEI and PLL usually have a higher isoelectric point (pI>8) due to their strong ability to bind to protons. Under physiological conditions, at neutral or weakly acidic pH, this type of polymer is positively charged. Generally speaking, in salt buffer, this type of polymer can adsorb a large amount of anions, produce a double layer structure, obtain a charge shielding effect, and thus the overall charge is neutral. However, the formation of the double layer can be destroyed by adjusting the reagent components, or introducing cationic surfactants, or reducing the salt concentration, or changing the reagent temperature, etc., to reduce the charge shielding effect and allow the polymer to produce a stronger mutual repulsion.

[0165] A polymer can carry multiple functional groups. Each functional group can be one or more, but at least one functional group must be one. b C c D d…in the form of. It can be selectively connected to the library directly or indirectly through the A functional group. In practical applications, most of the connections with the library are indirect, so the A functional group can be connected to the precursor of the library, and the precursor includes but is not limited to: a functional group that can directly react with the modified functional group on the library, or a nucleic acid oligomer of a specific sequence that can hybridize with the library, or a functional group modified on a nucleic acid oligomer of a specific sequence that can hybridize with the library. The functional groups mentioned above include but are not limited to common chemical coupling groups, biotin-like small molecules, antigens and antibodies, etc. After the modification of the A functional group is completed, the B, C, and D functional groups can be modified to couple it with the functional groups on the charged polymer, such as PEI, PLL, etc. The molecular weight of the polymer depends on the required electrostatic repulsion. After completing one round of modification, if the electrostatic repulsion of the polymer carrier still needs to be further increased, the residual functional groups can be modified in a similar coupling process, and more rounds of reactions can be carried out with the charged polymer to increase the total charge of the carrier, as follows Figure 2 shown.

[0166] After the vector is synthesized, it can be connected or hybridized with a single-stranded DNA molecule carrying the target library to be tested and a functional known sequence through modification on the A functional group, so that the vector and the library are combined one-to-one, that is, one vector can only contain one single-stranded DNA molecule carrying the target library to be tested and a functional known sequence.

[0167] (2) Inoculation of polymer library vector

[0168] Library seeding based on charge repulsion can be implemented on array chips or non-array chips.

[0169] ① Placeholder

[0170] Taking array chips as an example, the size of the pattern needs to be close to the range of the charge repulsion force of the carrier. The carrier can bind to the active area on the chip with the pattern. The active area can be a region modified with an opposite charge to the carrier, and L' and R sequences are coupled in the region. The functional known sequences in the library contain homologous sequences of L and R sequences, and their directions depend on the connection method between the library and the carrier. In simple terms, the library can hybridize with the L' or R sequence on the active area, and the complete sequence of the library can be transferred to the chip surface by polymerase extension.

[0171] In the case of non-array chips, the density of the library distribution on the chip depends on the charge strength of the carrier. The charge strength is affected by the type and quantity of the polymer used in the carrier, as well as the salt concentration and pH of the carrier solution. In short, higher charge density and more polymers lead to sparser library distribution. High salt will increase the density of the library due to the charge shielding effect. For polymers with high PI values, low pH will enhance the positive charge they carry, thereby increasing charge repulsion and reducing the density of the library, and vice versa. Therefore, on non-array chips, the density of the library can be adjusted as needed based on the above principles. The L'sequence or R sequence modified on the surface of the non-array chip, the functional known sequence in the library contains homologous sequences of the L and R sequences, and its direction depends on the connection method between the library and the carrier. In short, the library can hybridize with the L'or R sequence on the active area, and the complete sequence of the library can be transferred to the chip surface by polymerase extension.

[0172] ② Extension

[0173] When the nucleic acid library comes into contact with the chip, amplification can occur immediately; or the nucleic acid library can be amplified after the inoculation process is completed. In other words, the inoculation of the nucleic acid library on the chip surface and the amplification of the nucleic acid library after inoculation can be two independent processes. Furthermore, this independent process can bring additional advantages, that is, whether on an array chip or on a non-array chip, when the inoculation density of the nucleic acid library does not meet the requirements, the inoculation process can be carried out step by step, multiple times, and repeatedly without interfering with the sites that have been inoculated with the nucleic acid library.

[0174] In particular, on a non-array chip, the nucleic acid library can be inoculated in different orders by changing the size of the nucleic acid library. For example, a carrier with a larger repulsive capacity can be used to fully inoculate first, and then a carrier with a smaller repulsive capacity can be used for inoculation, and on this basis, the number of nucleic acid library inoculations can be increased to achieve a high site density that cannot be achieved by uniform inoculation. In particular, in different batches of inoculations, the carriers of the nucleic acid library can be the same or different.

[0175] ③Remove

[0176] After the transfer of the library sequence is completed, the carrier fixed by hybridization can be removed by a strong base or nucleic acid denaturing reagent such as formamide. If it is on a pattern array chip with an opposite charge to the carrier in the active area, it is necessary to weaken the effect of electrostatic adsorption by adjusting the pH and salt concentration to remove the carrier. After the carrier is completely removed, a nucleic acid cluster that can be sequenced can be formed on the surface of the non-pattern array chip at the position of the original carrier by bridge amplification or isothermal amplification.

[0177] 3. Amplification of DNA Clusters

[0178] After the target library insert sequence information (sequencing template) is transferred to the chip surface with both L' sequence and R sequence, the DNA single-strand cluster can be amplified by conventional bridge amplification means or isothermal amplification means, such as RPA, HDA, LAMP, etc., to form a larger DNA clone cluster with close spacing. The sequences in each cluster are homologous, and there are two completely complementary sequences, positive and negative. There can be a forward single strand in a single-stranded state starting with the L sequence fixed on the chip surface, a reverse single strand in a single-stranded state starting with the R sequence fixed on the chip surface, a double strand in a forward and reverse complementary state starting with the L' sequence and R sequence fixed on the chip surface, and a double strand in a forward and reverse complementary state starting with the L' sequence or R sequence fixed on the chip surface.

[0179] 4. Sequencing

[0180] The present invention has no restrictions on the sequencing method, and the preferred sequencing method is sequencing by synthesis reaction. This means that determining the sequence of the first nucleic acid requires the synthesis of a second nucleic acid using the first nucleic acid as a template. In this way, the sequence of the second nucleic acid is determined based on the order and number of dNTPs incorporated, and the sequence of the first nucleic acid is determined as the complement of the first nucleic acid sequence.

[0181] The preferred sequencing method of the present invention includes fluorescence sequencing.

[0182] Technical terms

[0183] Sequencing chip surface or solid support:

[0184] A solid support refers to a component to which a sequencing library or template is bound or fixed, and can be made of any material, including but not limited to glass or other silica-based materials, plastics or other polymer-based materials, provided that the material is relatively inert to the templates, primers, polymerases, dNTPs and other components used in sequencing reactions and cleaning. A solid support may or may not be rigid. It may be porous. It may or may not be continuous. In some embodiments, a solid support is a glass slide. In some embodiments, the support is a plurality of beads or particles (such as microparticles) that are themselves fixed on a solid support. These beads may be porous. The support may be a screen. In some embodiments, the solid support itself is a detector or sensor, such as but not limited to a contact imager.

[0185] Generally, template must be connected to the observable (or detectable) part on its free end. This part is intended to represent the free end of template, and therefore its position and the movement in the direction of force indicate the length of template. The observable part can be any number of parts, and the present invention is not limited to its property. The property of the observable part will determine the type of sensor or detector suitable for observing (or detecting or monitoring) template length changes. In some important embodiments, the observable part is a bead, such as a microbead, and more specifically, such as a magnetic bead.

[0186] The solid support is part of or adjacent to a circulation cell. As used herein, a circulation cell is a chamber having at least an inlet and an outlet through which liquid moves. Depending on the position of the detection system for viewing the template, the solid support to which the template is tethered can be below, above or beside the circulation cell. The solid support can be a wall of a circulation cell, including a lower wall, a side wall or an upper wall.

[0187] Library transfer:

[0188] In the present invention, two library transfers occur, firstly, the library to be tested is transferred to the library carrier, and then the library carrier is transferred to the sequencing chip.

[0189] In the present invention, preferably, the target nucleic acid fragment or its homologous sequence in the target library is used as a template to transfer the nucleic acid fragment information to the library vector in the form of polymerase amplification or extension.

[0190] In the present invention, preferably, the rolling circle amplification product is used as a template, or the target nucleic acid fragment or homologous sequence on the surface of a micro- or nano-carrier is used as a template, and the nucleic acid fragment information is transferred to the sequencing chip in the form of polymerase amplification to form a sequencing template.

[0191] Single library vectors, cluster library vectors, and amplified clusters

[0192] When constructing a library carrier on the surface of a nano- or micro-carrier, in order to make each library carrier contain only a single target nucleic acid fragment or a homologous fragment thereof, a single capture sequence is introduced on the surface of the nano- or micro-carrier through a single first reaction group. After capturing the single target nucleic acid fragment, the single target nucleic acid fragment is transferred to the carrier surface through amplification or extension to form a single library carrier. In order to improve the inoculation efficiency, amplification primers can be introduced using multiple second reaction groups on the carrier surface to amplify the single library carrier, thereby obtaining a cluster library carrier, and the amplification product formed on its surface is referred to as an amplification cluster in the present invention.

[0193] DNA single-strand cluster: The DNA single-strand cluster of the present invention refers to the state of a DNA single strand or a few DNA single strands on the chip surface after the library vector is inoculated on the chip surface.

[0194] DNA clone cluster: The DNA clone cluster of the present invention refers to the state formed after amplification of a single-stranded DNA cluster. The sequences in each cluster are homologous, and there are two completely complementary sequences, positive and negative.

[0195] Linker: In certain embodiments of the present invention, a linker is used to introduce a capture sequence or a primer sequence to the surface of a carrier, or a linker is used for carrier construction. The concept of a linker is well known in the art, such as a double-ended homofunctional linker or a heterofunctional linker. The present invention preferably uses a heterofunctional linker. Preferably, the linker of the present invention comprises a polyethylene glycol linking group; preferably, the linker of the present invention comprises a reactive group for a thiol such as maleimide, a reactive group for an amine such as an active ester, a group for a click chemistry reaction or a bioorthogonal reaction such as an azide and an alkynyl group, or a tetrazine and a large strained cyclic olefin, etc.

[0196] Example 1 Chip preparation

[0197] The present invention can select non-array chips or array chips, and the chips can be purchased from the market (IlluminaNovaSeq6000; MGI Seq 2000; 10X Genomics Visium), or can be homemade. This embodiment provides references for the preparation of two non-array chips and one array chip, and those skilled in the art can make corresponding optimizations, and the present invention is not limited to this.

[0198] 1.1 Fabrication of non-array chips with positively charged surfaces

[0199] Use 0.1wt% polylysine solution (Sigma, P4832) to soak a standard glass slide. The soaking process is carried out at room temperature for 3 hours. After the soaking time is over, take out the slide, wash the surface with deionized water, and blow dry. Then dry it in an oven at 80°C overnight (>12 hours). The dried slide is further soaked in a PBS solution dissolved with 1mg / mL NHS-N3 (Thermo, 88902) and allowed to stand for 3 hours. After the time is over, take out the slide, wash the surface with deionized water, and dry it. Then soak the slide in a PBS solution dissolved with 1μmol / L DBCO-modified L'sequence (SEQ ID NO.4) nucleic acid oligomer and 1μmol / L DBCO-modified R sequence (SEQ ID NO.6) nucleic acid oligomer, heat to 37°C, and react for 12 hours. After the time is up, the slide is taken out and the surface is washed with deionized water to complete the non-array chip with positive surface charge, which is stored in PBS solution at 4° C. The chip surface modification efficiency can be checked by fluorescently labeled L-RC (SEQ ID NO.3) sequence and R-RC (SEQ ID NO.2) sequence.

[0200] 1.2 Fabrication of non-array chips with negatively charged surfaces

[0201] First, the front of the standard glass slide was infiltrated with 1M KOH solution, and the surface solution was removed after about 30 minutes, and the slide was blown dry with a gas cylinder. Then, acetylene triethoxysilane (Qiyue Bio, Q0201302) was used for chemical precipitation to perform alkyne alkylation on the surface of the slide. The DBCO-modified L' (SEQ ID NO.4) and R (SEQ ID NO.6) sequence primers were fixed on the surface of the chip by Cu (I) catalysis on the surface of the slide, thus completing the preparation of the non-array chip with negative surface charge.

[0202] 1.3 Fabrication of array chips

[0203] The prefabricated pattern array chip in the present embodiment is made based on a customized glass-based slide. It is worth noting that in the specific implementation scheme, the type and method of obtaining the solid support by the researchers are not limited. There are rectangularly distributed patterns on the customized slide. The center of each pattern is a hydrophilic area and the edge is a hydrophobic area. The hydrophilic area is modified with a certain density of L and R sequences by chemical bonding. The hydrophobic area is further modified to reduce the surface energy to inhibit the nonspecific adsorption of the slide to the reagent during the experiment. After that, glue is applied to the slide in a specific manner and bonded to the glass-based cover plate. After curing, the glue can maintain certain mechanical properties and form a flow channel of a certain height between the slide and the cover plate for the flow of reagents.

[0204] This embodiment selects commercially available chips for modification to provide a reference for the production of array chips. Specifically, the spatial omics product Visium chip of 10XGenomics has a nucleic acid cluster pattern array with a diameter of 55 μm and a spacing of 100 μm, which can be used to simulate a chip with a large-interval prefabricated pattern array. First, the Visium chip was modified. According to its public information, the surface modification is P5→Barcode→PolyT sequence, from 5'→3', for compatibility with Illumina sequencers. After sufficient hybridization through the P5 reverse complementary sequence, Thermolabile Exonuclease I (NEB, M0568S) was used to treat the chip, remove the unnecessary Barocde-PolyT sequence, and retain only the P5 sequence. After that, the chip was washed with 0.2M NaOH to restore the P5 single strand. After the L-RC→P5-RC sequence and R-RC→P5-RC sequence mixed in 1:1 were fully hybridized with the chip, T4 DNA Polymerase was used to transfer the L and R sequences to the chip surface, and the ratio was 1:1. The chip was then treated again with 0.2 M NaOH to remove non-fixed nucleic acids, thus completing the transformation of the chip with a prefabricated pattern array with a spacing of 100 μm.

[0205] Example 2 Construction of library vector

[0206] 2.1 Construction of RCA library vector

[0207] The preparation method of the simulated library used for demonstration is as follows: an artificially synthesized simulated library (SEQ ID NO.8) with a sequence order of L-25N-R (RC) from 5' to 3', in which the 5' end is phosphorylated. The synthesized library is dissolved in 50mM Tris-HCl at a concentration of 1nM, and 1μM of loop primer is added at the same time. The mixed solution is set in the PCR instrument at 98℃ (5min) → 60℃ (5min) → 30℃ (5min) → 4℃ (5min), and heated by the temperature control of the PCR instrument. After the heating and cooling process is completed, the treated library is placed on ice for standby use. T4 DNA Ligase (NEB, M0202M), 10mM MgCl2, 1mM ATP, and 10mM DTT are added to the reaction system at an amount of 1μL / 100μL, and the reaction is carried out at 16℃ for 3 hours. After the reaction is completed, it is heated to 65℃ for 20min. Add 3×AMpure XP Beads to the inactivated product for purification and dissolve back to the original volume. Add Exonuclease I (NEB, M0293M) at 1μL / 100μL, and add 67mM Glycine-KOH, 6.7mMMgCl2, 10mMβ-ME, and react at 37°C for 3 hours. After the reaction, heat to 80°C for 20min. The inactivated product is purified using 3×Ampure XP Beads and dissolved back in molecular water, and the concentration is calibrated using the Qubit ssDNA (Thermo, Q10212) kit.

[0208] The purified circular library was converted according to the library length and average base mass. 0.1 pmol of the circular library was dissolved in 100 μL of 1×phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), and a final concentration of 1 μM of the circular primer (SEQ ID NO.9) was added, and it was allowed to stand at room temperature for 30 minutes to allow it to fully hybridize. Then 2 μL of Phi29 polymerase was added to the system, mixed evenly, and heated to 30°C on a PCR instrument. The concentration of the rolling circle product was tracked and observed using the Qubit ssDNA (Thermo, Q10212) kit. When the concentration reached the expected value, the temperature was quickly raised to 80°C, and the Phi29 polymerase was inactivated by high temperature for 15 minutes. After the inactivation was completed, the reaction solution was cooled and stored at 4°C, which was a library vector with a certain electronegativity and spatial volume prepared by rolling circle amplification, named RCA library vector.

[0209] 2.2 Construction of RCA-MDA library vector

[0210] Based on Example 2.1, at the end of the rolling circle reaction, the temperature was not changed, and no heating at 80°C was performed for inactivation. Instead, the activity of Phi29 was temporarily inhibited by adding 1 mM EDTA solution. In addition, 1 μM MDA primer (SEQ ID NO.5) was added simultaneously with the 1 mM EDTA solution. The MDA primer and the rolling circle library vector (SEQ ID NO.10) were incubated at 30°C for 30 minutes and fully hybridized with the rolling circle product. 2 mM Mg was then added. 2+ , the polymerization activity of Phi29 is restored, and the multiple strand displacement reaction is carried out at this time. As time goes on, the concentration of the MDA product can be observed through the Qubit ssDNA reagent. When the ssDNA in the reaction system reaches the expected concentration, the temperature of the reaction system is quickly raised to 80°C and inactivated for 15 minutes. The library vector produced by the rolling circle-multiple strand displacement reaction is obtained and named RCA-MDA library vector.

[0211] 2.3 Construction of protein library vector

[0212] 2.3.1 Construction of protein single library vector

[0213] In actual operation, those skilled in the art can select possible proteins to construct protein library vectors with different spatial volumes, and the present invention does not limit this. In this embodiment, myoglobin (Sigma, M5696) is used as an example to construct a protein single library vector.

[0214] The details are as follows:

[0215] First, myoglobin was dissolved in PBS buffer to form a 1 mg / mL solution, and then 1 mg / mL MAL-PEG3-N3 (Ruixibio, 1858264-36-4) was added and mixed for reaction to modify the -SH on myoglobin to the -N3 group. Then, a DBCO-modified R sequence (SEQ ID NO.6) nucleic acid oligomer was added at a concentration of 1 μM, and a linker that could hybridize with the library was introduced on the -N3 group through a coupling reaction. Then, 1 mg / mL of Sulfo-SMCC (Thermo, 22322) was added to the reaction solution to label the -NH2 group on myoglobin with maleimide. All the above reaction reagents were in excess. The reaction was controlled under low temperature and stirring conditions. After sufficient reaction, the modified myoglobin was purified and concentrated using a 20K MWCO dialysis box (Thermo, 87735). Then, add 30 times the mass ratio of unmodified myoglobin to the concentrated and modified myoglobin solution. Stir thoroughly and react. After sufficient reaction. The product was sampled and analyzed by SDS-PAGE gel. It can be seen that most of the products have a mass of 10 to 25 times that of myoglobin, indicating that the main complex is composed of 10 to 25 myoglobin peptide chains. The product was purified by a 20K MWCO dialysis box to remove unreacted single myoglobin. Use the phosphorylated-modified mock library to hybridize with the protein complex carrying the R sequence, and extend it to transfer the library nucleic acid sequence to the protein complex. The product was then purified by Lambda Exo (NEB, M0262S), the mock library was removed, and after inactivation at 75°C, the protein complex carried the completed mock library complementary chain and became a protein library vector, named protein single library vector. The results are as follows. Figure 3 As shown, the left lane is a marker, and the right lane is the constructed protein single library vector.

[0216] 2.3.2 Construction of protein library cluster vector

[0217] In practical applications, the inoculation efficiency of protein single library vectors is limited. If the inoculation efficiency is high in practical applications, the protein monomer library can be modified to construct a protein library cluster vector to further improve the efficiency of inoculation. Specifically, based on the original protein single library vector, NHS-PEG3500-N3 (Sigma, JKA5086) was used to re-modify the protein library vector, and the -NH2 on the outermost myoglobin of the protein vector was modified to an active coupling group for coupling primers. Then, at 55°C, the DBCO-modified protein vector secondary modified primer (SEQ ID NO.11) was coupled in PBS buffer, and about 300 to 500 partial fragments containing the R sequence (SEQ ID NO.6) were introduced into the protein library vector. Purification was performed by 20KMWCO dialysis cassette to remove unreacted secondary modified primers. After that, the protein library vector was added to the RPA reaction system and reacted at 39°C for 30 minutes to complete the clustering of the nucleic acid library on the surface of the protein library vector, which was named the protein library cluster vector.

[0218] 2.4 Construction of microbead library vector

[0219] The library carrier constructed by using materials such as proteins and nucleic acids is suitable for array chips or non-array chips with nanometer, submicron or near-micron intervals, but in the range of tens to hundreds of microns, the library carrier has poor stability due to its huge volume and complex structure. Therefore, materials with similar size ranges can be considered as library carriers, such as microbeads. As mentioned above, the selection of microbeads is varied (such as magnetic beads, gel beads, metal nano / micro particles, etc.), and the present invention does not limit the type of microbeads. The library carrier constructed by using microbeads is mainly the placeholder of different spatial volumes realized by the different sizes of microbeads.

[0220] This example uses the barcoded beads provided by ChemGene as an example for subsequent experiments to provide a reference for library vectors constructed using artificial microspheres, whose sizes are 30, 50, and 70 μm. Through sequence customization, simulated library sequences are synthesized on the surface of microbeads, in which random sequence fragments can be used as barcodes for applications such as single cell analysis and spatial omics analysis.

[0221] 2.5 Construction of polymer library vector

[0222] 2.5.1 Construction of polymer single library vector

[0223] The positively charged polymer library carrier was constructed by PLL-MAL. First, PLL-PEG-MAL (Qiyuebio, Q0010416) with a total molecular weight of 6000 was reacted with a thiol-modified L sequence (SEQ ID NO.1). It was then purified by a semipermeable membrane of 10K MWCO. After purification, the product was fully reacted with SMCC-LC, purified by dialysis with 10K MWCO, and then grafted with HS-PLL-PEG-NH2 (Qiyuebio, Q0251202) with a total molecular weight of 2000, 4000, and 8000, respectively, and branched on the original PLL chain. After purification using a semipermeable membrane of 20K MWCO, the thiol-modified R sequence containing a flexible chain (SEQ ID NO.6) was coupled to the product by MAL-PEG-NHS (Ponsure, PS2-MH-5K), and the polymer carrier was completed after purification using a semipermeable membrane of 20KMWCO. An excess of custom-synthesized reverse complementary sequences of the simulated library were mixed with the vector. Heat to 37°C and incubate for 30 minutes to allow the reverse complementary sequences of the simulated library to hybridize with the L sequence on the vector. Then, Klenow (exo-) was added to continue the reaction at 37°C, and the simulated library sequence was transferred to the polymer carrier in the form of polymerase extension to obtain a polymer library carrier. At this time, each vector only contained a sequence of the simulated library.

[0224] 2.5.2 Construction of polymer library cluster vector

[0225] In practical applications, the inoculation efficiency of polymer single library vectors is limited. If the inoculation efficiency is high in practical applications, the polymer single library vector can be amplified to construct a polymer library cluster vector to further improve the efficiency of inoculation. Dilute the polymer single library vector in 2.5.1 to an appropriate concentration and add it to the TwistDx RPA Kit for isothermal amplification for about 1 hour. After being fully inactivated at 85°C, a polymer vector carrying a library cluster amplified from a single simulated library sequence can be obtained. The polymer vector of the library cluster is positively charged and is named a polymer library cluster vector.

[0226] Example 3 Inoculation of library vectors on chip surface

[0227] 3.1 Library vector position on the chip surface

[0228] As mentioned above, inoculation includes the steps of occupation, extension and removal. Therefore, after the library vector occupies the site, it is extended to transfer the library sequence to the surface of the stationary phase (i.e., the chip), and then denatured to make the entire library vector fall off. Only single-stranded DNA or single-stranded clusters with intervals close to the spatial volume of the library vector are retained on the chip surface. Therefore, the inoculation status of single-stranded DNA or single-stranded clusters can be inferred by characterizing the occupancy status, and the inoculation status of single-stranded DNA clusters directly affects the subsequent amplification and sequencing of DNA clusters.

[0229] All library vectors of the present invention are applicable to array chips and non-array chips. Due to the limitations of the embodiments, 1 to 2 chips are selected for each library vector for display.

[0230] 3.1.1 RCA library vector space on non-array chip surface

[0231] This example used the non-array chip prepared in Example 1.1 to allow the RCA library vector to react with the chip to explore the factors affecting the occupancy of the RCA library vector.

[0232] A. Size optimization of RCA library vector

[0233] As described in 2.1, different concentrations of RCA library vector solutions can be obtained by different reaction times. RCA library vector solutions of different concentrations are mixed with 10×SSC solution at a volume ratio of 1:1, and then evenly added to the chip and heated to 37°C. After 15 minutes, the RCA library vector solution is removed. Cy3-modified vector monitoring primers are added at 42°C, incubated for 5 minutes, and then the vector monitoring primer solution is removed. The chip is washed with 5×SSC solution, and then SlowFade Diamond mounting medium (Thermo, S36972) is added to take pictures under a fluorescence microscope to observe the occupancy of the library vector.

[0234] like Figure 4 As shown in the figure, when the rolling circle time increases, the occupancy density of the library vector under the same conditions shows a downward trend. However, when the time increases to 2 hours and the ssDNA concentration reaches 140ng / μL, the occupancy density of the library vector no longer decreases. It can be seen that those skilled in the art can control the desired occupancy density by adjusting the concentration, thereby controlling the inoculation density.

[0235] B. Optimization of electronegativity of RCA library vector

[0236] After rolling circle, the surface of the library carrier carries a complex of nucleic acid and protein, the isoelectric point of the nucleic acid is less than 7, and the isoelectric point of the protein in the complex is greater than 7. By changing the occupancy of the library carrier and / or the buffer, and adjusting the pH of the environment, the charge properties of the surface of the RCA library carrier can be changed, thereby affecting the density of occupancy on the non-array chip surface.

[0237] After the rolling circle was performed, the library carrier solution with a concentration of 50 ng / μL was mixed with 10XSSC solution at a volume ratio of 1:1, and then titrated with 2M HCl or 2M KOH standard solution. The solution was then inoculated on the non-array chip surface at room temperature at pH 6.0, 7.0, 8.0, 9.0, and 10.0, incubated for 30 minutes, and the RCA library carrier solution was removed. Cy3-modified carrier monitoring primers were added at 42°C, incubated for 5 minutes, and the carrier monitoring primer solution was removed. The chip was washed with 5×SSC solution, and then SlowFade Diamond mounting medium (Thermo, S36972) was added to take pictures under a fluorescence microscope to observe the occupancy of the library carrier, such as Figure 5 As shown, from left to right, it is pH 6.0-pH 10.0. It can be observed that the occupancy density is the highest at pH 8.0. Under the condition of pH 10, the non-array chip surface and the library carrier are both negatively charged and the occupancy cannot be completed. Therefore, it can be proved that there is an influence of charge between the library carrier and the library carrier, and between the library carrier and the non-array chip surface. The combined influence of the two charges finally determines the density of inoculation. Those skilled in the art can select the appropriate pH concentration according to their own inoculation needs.

[0238] From A and B above, it can be seen that the position of the RCA library vector on the chip surface is affected by the size and electronegativity of the RCA library vector itself. RCA library vectors of different volumes can be obtained by synthesizing solutions of RCA library vectors of different concentrations, and the electronegativity of the RCA library can be adjusted by adjusting the pH of the solution to artificially control the position of the RCA library vector on the chip surface.

[0239] 3.1.2 Positioning of RCA-MDA library vectors on the chip surface

[0240] A. Position occupancy of RCA-MDA library vector on non-array chip surface

[0241] From the results of Example 3.1.1A, it can be seen that the space occupied by the RCA library vector cannot be increased indefinitely by increasing the concentration of the RCA product. It can be considered that the space volume of the RCA library vector has a certain upper limit. When a larger nucleic acid library cluster spacing is required, it is necessary to further increase the space exclusion volume of the library vector by performing multiple strand displacement reactions on the library vector after the rolling circle to obtain a larger inoculation interval. Taking MDA library vectors of different concentrations and inoculating them according to the same process as Example 3.1.1A, it can be found that after inoculation, as the size of the library vector increases, the density of vector inoculation decreases, such as Figure 6 As shown, it is proved that the RCA-MDA library vector has a larger exclusion volume than the RCA library vector and can occupy a larger space. Those skilled in the art can select the appropriate size of the RCA-MDA library vector as needed.

[0242] In addition, this example verifies the occupancy of the RCA-MDA library vector at a concentration of 50 ng / μL and pH=8 on the non-array chip surface. Figure 7 shown.

[0243] It can be seen that even if the pH concentration and the concentration of the library vector are the same, RCA-MDA will show different space-occupying effects due to its larger size compared to RCA. Therefore, when obtaining a chip of DNA single-stranded clusters with a desired inoculation density, those skilled in the art can choose different library vectors according to their own inoculation requirements, and can also optimize and adjust the concentration or electronegativity of the library vector.

[0244] B. The position of RCA-MDA library vector on the array chip surface

[0245] The array chip prepared in Example 1.3 used in this example is closely related to the structure of the array chip itself in addition to the electronegativity and concentration. Therefore, those skilled in the art need to adjust the concentration or electronegativity, combined with the chip itself, to achieve artificial control of the chip surface inoculation. In this example, the RCA-MDA library vector was selected to react with the array chip under the buffer condition of pH=8.0, and multiple FOVs were examined by fluorescence microscopy. The results are as follows: Figure 8 As shown, the efficiency of statistical occupancy can reach about 95%.

[0246] It can be seen from 3.1.2A and B that the method of the present invention can realize artificial control of the inoculation amount and inoculation density on both array chips and non-array chips, and ultimately control to achieve the optimal detection results.

[0247] 3.1.3 Occupancy of protein library cluster carriers on the chip surface

[0248] The protein single library vector or protein library cluster vector prepared in Example 2.3.1 or Example 2.3.2 is dissolved in 1×Phi29 Buffer and incubated at 30°C on the chip prepared in Examples 1.1-1.3 to complete the occupancy on the chip surface. Those skilled in the art can adjust the size and incubation time of the protein single library vector or protein library cluster vector to control the occupancy density on the chip surface.

[0249] 3.1.5 Positioning of microbead library carriers on the chip surface

[0250] The microbead library is the chip prepared in Example 1.3. The microbead library carrier solutions of different concentrations constructed in Example 2.4 are mixed with 10×SSC solution at a volume ratio of 1:1, then evenly added to the chip and heated to 37° C. for 15 minutes.

[0251] 3.1.6 Positioning of polymer library cluster carriers on the chip surface

[0252] This example is preferably carried out on a chip with a negatively charged surface as described in Example 1.2.

[0253] After adjusting the pH of the polymer cluster library carrier solution to neutral, evenly spread the polymer library cluster carrier on the non-array chip with negative surface charge and incubate at 37°C for 1 hour. Then rinse the chip surface with pH 7.4 PBS solution to remove the unadsorbed polymer library cluster carrier. Replace the buffer with 5×SSC, soak and heat to 37°C, and incubate for 30 minutes.

[0254] 3.2 Extension of library vector on chip surface

[0255] Extension based on positional occupancy usually uses the library carried on the library carrier as a template, and uses an oligonucleotide fixed on the chip surface that can hybridize with the library sequence on the library carrier as an initiator primer, and uses DNA polymerase to transfer the library sequence on the library carrier to the chip surface in the form of reverse complement. Therefore, although the type of library carrier is different and the positional occupancy method is different, the extension process is basically the same.

[0256] The type of polymerase in the extension reaction of the present invention is not limited, and any common DNA polymerase can be selected. The selection can also be based on the occupancy conditions of the library vector, such as temperature, salt ion concentration, pH, etc.

[0257] In this embodiment, the DNA polymerase is preferably Phi29 DNA Polymerase, Bst 3.0 DNA Polymerase or Klenow (exo-).

[0258] This example lists several possible extension methods, all of which are applicable to the library vector described herein. Those skilled in the art can adjust and optimize the extension conditions as needed.

[0259] 3.2.1 Extension method I

[0260] Here are the steps:

[0261] (1) Add Klenow (exo-) to the chip where the library vector has been placed for full hybridization;

[0262] (2) Remove the surface solution and clean the chip using pH 8.0 Tris-HCl buffer;

[0263] (3) The chip cleaned in step (2) was immersed in Klenow (exo-) (NEB, M0212M) reaction solution and reacted at 37° C. for 3 hours.

[0264] 3.2.2 Extension method II

[0265] The extension of the library vector can be carried out simultaneously with the site occupation. Phi29 DNA Polymerase with a volume ratio of 2% can be added while the site is occupied, and the extension can be completed by incubating at 30°C for different time periods.

[0266] After the extension is completed, the protein carrier solution can be removed and the seeding density of the surface protein library carrier can be characterized by using a myoglobin antibody coupled to Cy3.

[0267] In this example, a protein single library vector was selected for display, and the results were as follows: Fig. 9 As shown, it can be seen that the inoculation efficiency of the protein single library vector is low. When the occupancy and extension time are prolonged, the inoculation density can be increased. It is speculated that this is due to the low number of libraries carried by the protein single library vector.

[0268] In contrast, the efficiency of protein library cluster vectors carrying multiple libraries was significantly improved during vaccination.

[0269] 3.2.3 Extension method III

[0270] The chip with the library vector occupied was immersed in the Bst 3.0 DNA polymerase (NEB, M0374M) reaction solution, and the temperature was slowly raised from room temperature to 65° C. and maintained for 15 minutes.

[0271] 3.3 Removal of library carrier from chip surface

[0272] The library vector is fixed on the chip surface by charge attraction and DNA hybridization. Therefore, the extended library vector can be removed from the chip surface by weakening the charge attraction or interfering with DNA hybridization. Common formamide, strong alkali or other denaturants can achieve this purpose. Researchers can choose a suitable reagent or a combination of reagents to effectively remove the library vector. Now, taking formamide and NaOH as examples, this example shows the removal process of RCA-MDA library vector and protein library cluster vector.

[0273] 3.3.1 Removal of RCA-MDA library vector using formamide

[0274] After extending the chip in 3.1.2 using the method of Example 3.2.1, this example selects chip B, soaks chip B in formamide solution, heats to 80°C, takes out the chip while it is still hot, and repeats once. The RCA-MDA library vector on the chip surface can be seen to be completely removed by the vector detection primer Fig.10 .

[0275] 3.3.2 Removal of protein library cluster carrier using NaOH

[0276] After the reaction in Example 3.2.2 is completed, the surface reaction solution is removed. After the chip is rinsed with 1×PBS Buffer, the chip is immersed in 0.2M NaOH solution and placed at room temperature for 5 minutes. Thereafter, the excess reaction solution is removed and the chip is washed with 1×PBS Buffer.

[0277] Example 4 Amplification of single-stranded DNA clusters

[0278] After the library vector is constructed and inoculated into the chip, a single-stranded DNA cluster close to the spatial volume of the amplified product will be formed. The single-stranded DNA cluster will be amplified to form a larger DNA clone cluster, which is convenient for the subsequent sequencing process.

[0279] It can be seen from the above embodiments that the seeding density of single-stranded DNA clusters on the chip surface can be artificially controlled, and the chip can be either an array chip or a non-array chip. Those skilled in the art can select different library vectors according to the requirements of seeding density. The present invention provides a variety of possible methods for preparing single-stranded DNA cluster chips, which provides a variety of possibilities for sequencing technology.

[0280] Because the inoculation condition can be controlled, the library vectors in Example 2 can be selected to complete the subsequent DNA cluster amplification and sequencing. In order to illustrate the amplification condition after inoculation, this example randomly selects a library vector: RCA-MDA to prepare a DNA single-stranded cluster chip.

[0281] The amplification methods that can be selected include: RPA, HDA, PCR, bridge amplification, LAMP, etc.

[0282] 4.1 Isothermal amplification (RPA) to generate DNA clone clusters

[0283] The chip from which the RCA-MDA library vector was removed in Example 3.3.1 was washed with pH 8.0 Tris-HCl buffer, and then immersed in a TwistAmp reaction solution (TwistDx, TALQBAS01) supplemented with 1 μM L sequence primer (SEQ ID NO.1) and 1 μM R sequence primer (SEQ ID NO.6), heated to 39°C, and reacted for 1 hour. After washing with pH 8.0 Tris-HCl buffer, the chip was immersed in a User enzyme (NEB, M5505S) reaction solution and heated to 37°C for 3 hours. After the reaction, the chip was washed with 80°C formamide reagent. After hybridizing the nucleic acid library cluster monitoring primer (SEQ ID NO.12), the density and size of the clone cluster can be observed, and the results are as follows. Fig.11 As shown, it can be seen that after the RPA reaction, observable nucleic acid library clone clusters with uniform size and clear boundaries are generated.

[0284] 4.2 HDA to generate DNA clone clusters

[0285] The chip from which the RCA-MDA library vector was removed in Example 3.3.1 can also be used to amplify the chip surface library by HDA to obtain monoclonal oligonucleotide clusters. IIUniversal tHDA Kit, H0110S). After removing the rolling circle library carrier, the chip was washed with pH 8.0 Tris-HCl buffer and placed in a reaction container. Prepare the HDA reaction solution, and add 0.1 μM L sequence primers and R sequence primers to ensure that the chip is completely immersed in the reaction solution. After sealing, place it in an oven and heat it at 65°C for 45 minutes. After the reaction is completed, wash it with pH8.0 Tris-HCl buffer, and then immerse the chip in the User enzyme (NEB, M5505S) reaction solution and heat it to 37°C for 3 hours. After the reaction is completed, use 80°C formamide reagent to wash the chip. After hybridizing the nucleic acid library cluster monitoring primers, the density and size of the clone cluster can be observed, and the results are as follows: Fig.12 As shown, it can be seen that after the HDA reaction, observable nucleic acid library clone clusters with uniform size and clear boundaries are generated.

[0286] 4.3 Polymerase chain reaction (PCR) to generate DNA clone clusters

[0287] After the RCA-MDA library vector on the chip surface is inoculated in Example 3.3.1, the chip surface library can also be amplified by PCR to obtain monoclonal oligonucleotide clusters. TM ThermoFisher ProFlex by Sample Blocks(4484074) TM The 2X96-well thermal cycler is used as an example for demonstration. After the chip is fixed on the Sample Block through the sealing ring, the PCR reaction solution ( Hot Start HiFi PCR MasterMix, M0543L) to ensure that the reaction solution can cover the entire surface of the chip. Set the reaction conditions as follows:

[0288] 98℃(30s)→[98℃(10s)→65℃(75s)]X15→65℃(5min)→4℃(∞)

[0289] After the reaction, the chip was washed with pH 8.0 Tris-HCl buffer and then immersed in User enzyme (NEB, M5505S) reaction solution and heated to 37°C for 3 hours. After the reaction, the chip was washed with 80°C formamide reagent. After hybridization of the nucleic acid library cluster monitoring primer, the density and size of the clone cluster can be observed. The results are as follows: Fig.13 As shown, after the PCR reaction, observable, uniform-sized, and clearly-bounded nucleic acid library clone clusters were generated.

[0290] 4.4 Bridge amplification to generate DNA clone clusters

[0291] After the RCA-MDA library vector on the chip surface is inoculated in Example 3.3.1, bridge amplification can also be performed on the chip surface library through bridge amplification to obtain monoclonal oligonucleotide clusters. Bridge amplification is now demonstrated in the form of an immersion reaction. First, fix the chip with a clamp. Then prepare three reaction pools in a constant temperature oil bath: an amplification pool, a denaturation pool, and a washing liquid pool. The three reaction pools are respectively filled with amplification reagents (Bst 2.0 DNA Polymerase, M0537M + Isothermal Amplification Buffer, B0537S + dNTP Mix, N0447S), denaturants (formamide) and washing liquid (5XSSC). Heat the constant temperature oil bath to 65°C, and use the clamp to operate the chip to immerse it in each reaction pool in the following order:

[0292] Washing pool (2min) → Amplification pool (5min) → Washing pool (30s) → Denaturation pool (5min)

[0293] Repeat the process for a total of 15 rounds to complete bridge amplification. After the reaction is completed, wash with pH 8.0 Tris-HCl buffer, and then soak the chip in User enzyme (NEB, M5505S) reaction solution and heat to 37°C for 3 hours. After the reaction is completed, wash the chip with 80°C formamide reagent. After hybridizing the nucleic acid library cluster monitoring primer, the density and size of the clone cluster can be observed. The results are as follows: Fig.14 As shown, it can be seen that after the PCR reaction, observable nucleic acid library clone clusters with uniform size and clear boundaries are generated.

[0294] It can be seen from Examples 4.1-4.4 that different amplification methods can achieve good amplification effects on the chip of single-stranded DNA clusters prepared by the present invention, so those skilled in the art can randomly select a suitable amplification method.

[0295] Example 5 Index-Hoping Detection

[0296] In this embodiment, the library design, acquisition and construction are performed with reference to the method mentioned in the existing literature [Li, Q., Zhao, X., Zhang, W. et al. Reliable multiplex sequencing with rare index mis-assignment on DNB-based NGS platform. BMC Genomics 20, 215 (2019)], and the DNA single-stranded clusters obtained on the chip are obtained according to the method described in the previous text of this patent. Through a commercial fluid control system, a temperature control system and a camera system based on a fluorescence microscope, corresponding biochemical reagents (which may include but are not limited to: dNTPs that carry labels and reversibly modified dNTPs that can hinder, delay, or interfere with the base incorporation reaction in which the polymerase participates; corresponding chemical or biological reagents that can reversely modify the above modifications; corresponding chemical or biological reagents that can silence, quench, remove or invalidate the labels), image recognition software and signal processing algorithms, the obtained chip is subjected to SBS sequencing and the label jumps generated in the sequencing results are analyzed with reference to the literature listed above. In particular, in this embodiment, the sequencing data analysis principles, methods and processes refer to the publicly disclosed sequencing data analysis principles, methods and processes. Therefore, unless otherwise noted, the meanings of the terms and indicators in the analysis results are similar, consistent or interchangeable to a certain extent with the meanings of the commonly disclosed corresponding terms and indicators.

[0297] In this embodiment, the library construction process is briefly described as follows: using the corresponding PCR primers, 8 gene fragments of about 400 bases are selected for selective amplification, purification, enrichment, and quantification (Table 1). After that, dA addition and labeling adapter connection are completed independently through commercial kits purchased from outside. In particular, 16 different sequence tags are designed in the labeling adapter connection, numbered 1 to 16 respectively. Among them, tags 1 to 8 are connected to 8 gene fragments during the labeling adapter connection process, and then each gene fragment carries a tag, correspondingly marked as Gene1 to 8. And tags 9 to 16 are added to the fragments of similar size obtained from the commercial Human Genomic DNA through a similar process as a balanced library. After the tag addition is completed, the 8 library sequences and the balanced library are independently PCR amplified, purified, and quantified, and after quantification, they are mixed in proportion according to the concentration. After mixing, a suitable library vector form is selected for library vector preparation.

[0298] In particular, in this example, the RCA-MDA library vector was selected and the chip described in Example 1.3 was used, combined with Examples 3.1.2B, 3.1.3, 3.2.1, 3.3.1, 4.1, etc. as demonstration. The insert fragment was sequenced with a read length of 30 bp, the tag was sequenced with a read length of 10 bp, and the results with a Q30 lower than 70% were filtered. In the result analysis, the tag jumping was observed by matching the genes on the tag and the insert fragment alignment, and the results are shown in Table 2 and Fig.15. It can be seen from this example that for the 8 selected genes, the proportion of label jumping events that occurred varies from gene to gene, ranging from 0.0096% to 0.034%, which is significantly lower than the 0.25% to 7% label jumping proportion reported in similar literature based on Illumina ExAmp technology amplification [Costello, M., Fleharty, M., Abreu, J. et al. Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms. BMC Genomics 19, 332 (2018)] [Effects of Index Misassignment on Multiplexing and Downstream Analysis (white paper)] [Sinha, Rahul et al. "Index switching causes "spreading-of-signal" among multiplexed samples in Illumina HiSeq 4000 DNA sequencing." bioRxiv (2017): n. pag.]

[0299] Table 1 Tags of 8 gene fragments

[0300]

[0301] Table 2 8 gene tag jumping situations

[0302]

[0303] Example 6 Nucleic Acid Cluster Stability Detection

[0304] In the current sequencing market, label hopping is a difficult problem that plagues detection accuracy. Therefore, in order to overcome label hopping, existing technologies have proposed the use of prefabricated pattern array chips with high throughput and high inoculation rate. However, in actual operation, the technicians of the present invention found that although it can overcome certain label hopping problems due to the prefabricated pores, it still has the problem of detection stability. In order to overcome the problems of label hopping and poor stability, the technical solution of the present invention is proposed. The solution described in the present invention not only shows that it can control the efficient inoculation rate and reduce the label hopping rate; but also because of the space occupation of the library carrier of the present invention, it shows stronger stability under the same fluid conditions.

[0305] In this embodiment, the stability of the chip is verified by performing fluid testing and sequencing quality testing on the chip.

[0306] A. Fluid Testing

[0307] The fluid test is to continuously flow 1×PBS solution through the chip while performing variable temperature cycles to simulate the sequencing process. In this test, the flow rate is three times the normal sequencing flow rate to simulate a faster sequencing environment.

[0308] First, a slide adapter is made by 3D printing, through which the array chip with the packaged self-made pattern can be fixed on the MGI SEQ2000 stage and connected to the fluid.

[0309] Comparative chip: There is no oligonucleic acid modification on the array chip of the self-made pattern, and the -NH2 group is retained in the pattern area. Starting with the circular starting library carrying L and R sequences, the MGI SEQ2000 kit is used to perform rolling circle amplification according to the instructions, and the product is passed into the array chip of the self-made pattern without oligonucleic acid modification. On the MGI SEQ2000 machine, after loading the PE100 kit, only the "Post Loading" link is set to perform post-loading processing on the rolling circle amplification product loaded on the self-made pattern array chip without oligonucleic acid modification through the slide adapter.

[0310] The chip of the present invention, the array chip of the self-made pattern is modified with a special oligonucleotide, and the same circular library as the comparison chip is introduced as the RCA-MDA library vector to be initially prepared, and the inoculation of the library vector and the amplification of the DNA single-stranded clusters are completed according to the methods mentioned in Examples 3.1.2B, 3.1.3, 3.2.1, 3.3.1, and 4.1.

[0311] The stability test results are as follows Fig.16 and Fig.17 As shown, it can be seen that the comparison chip ( Fig.16 ) After 600 rounds of fluid flushing, the DNB morphology was greatly damaged, while the chip made using the method described in this article ( Fig.17 ), no major morphological changes occurred, and the DNA clone clusters were stable on the chip surface.

[0312] B Sequencing quality detection

[0313] The two chips prepared in A were used for PE150 sequencing experiments on MGI SEQ2000 sequencer, respectively, wherein the sequencing primers were as shown in SEQ ID NO.13.

[0314] Sequencing results comparison Fig.18 and 19As shown, the black triangle represents the method of the present invention, and the hollow circle represents the comparative method. It can be proved that when the scheme described in the present invention is used, the signal is more stable and the sequencing accuracy is higher in the case of longer read length.

[0315] Sequence information 5'→3':

[0316] L sequence: SEQ ID NO.1

[0317] AATGATACGGCGACCACCGAGATCTACAC

[0318] R-RC sequence: SEQ ID NO.2

[0319] ATCTCGTATGCCGTCTTCTGCTTG

[0320] L-RC sequence: SEQ ID NO.3

[0321] GTGTAGATCTCGGTGGTCGCCGTATCATT

[0322] L'sequence coupled to the chip surface: SEQ ID NO.4

[0323] AAUGAUACGGCGACCACCGAGACTACAC

[0324] MDA primer: SEQ ID NO.5

[0325] GCTCTTCCGATCTATCTCGTATGC

[0326] R sequence: SEQ ID NO.6

[0327] CAAGCAGAAGACGGCATACGAGAT

[0328] SEQ ID NO.7

[0329] MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG

[0330] Simulation library (5' phosphorylation modification): SEQ ID NO.8

[0331] Pho-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNNNNNNNNNNN NNN GCTCTTCCGATCTATCTCGTATGCCGTCTTCTGCTTG

[0332] Loop primer: SEQ ID NO.9

[0333] CGCCGTATCATTCAAGCAGAAGACG

[0334] Rolling circle library vector monitoring primer (5'Cy3 modified): SEQ ID NO.10

[0335] Cy3-CGAGATAGATCGGAAGAGC

[0336] Protein carrier secondary modification primer: SEQ ID NO.11

[0337] CAAGCAGAAGACG

[0338] Nucleic acid library clone cluster monitoring primer (5'Cy3 modified): SEQ ID NO.12

[0339] ACGGCGACCACCGAGACTACAC

[0340] Sequencing primer: SEQ ID NO.13

[0341] ACGGCGACCACCGAGACTACAC

Claims

1. A method for preparing single-stranded DNA clusters on a solid support surface, the method comprising the following steps: 1) providing a solid support and a library vector comprising a target nucleic acid fragment or a homologous sequence thereof, wherein the solid support and the library vector respectively comprise a first capture sequence and a first gripper sequence complementary thereto; 2) The library vector occupies a certain spatial position on the surface of the solid support; 3) The first capture sequence and the first gripper sequence are complementary paired, and the target nucleic acid fragment or its complementary sequence in the library vector is used as a template to form a DNA single-stranded cluster on the surface of the solid support by polymerase extension or reverse transcription extension; 4) Removing the library vector to obtain a solid support coupled with a DNA single-stranded cluster.

2. The method according to claim 1, wherein the library carrier has a nanometer or micrometer-sized spatial structure; the nanometer size refers to a diameter or a hydrodynamic diameter of the library carrier that is less than 1 micrometer, such as 10nm-900nm; the micrometer size refers to a diameter or a hydrodynamic diameter that is greater than 1 micrometer, such as 10-500 micrometers, preferably 10-90 micrometers, such as 30, 50 or 70 micrometers; Preferably, the library vector is a target nucleic acid fragment or its complementary sequence that is amplified to form a nanometer or micrometer size and has a certain spatial structure, similar to a sphere rather than a line; Preferably, the library carrier is formed into a nano- or micron-sized carrier by biological molecules, organic compounds, inorganic substances or a combination of the above substances, and the target nucleic acid fragment or its homologous sequence is directly or indirectly introduced on the carrier surface; preferably, the target nucleic acid fragment or its homologous sequence is introduced directly by chemical coupling or affinity interaction; or the target nucleic acid fragment or its homologous sequence is introduced indirectly; preferably, each carrier surface contains a single target nucleic acid fragment or its homologous sequence.

3. The method according to any one of claims 1 to 2, wherein when the library vector is bound to the surface of a solid support, the repulsive force generated by the physical properties of the library vector itself is used to achieve monodispersity of the library vector and / or different seeding densities of the library vector; the physical properties are selected from the physical size, steric hindrance, fluid dynamics exclusion volume, charge properties, charge density, hydrophilicity or magnetism of the library vector. Preferably, the library carrier is bound to the surface of the solid support by one or more of complementary pairing, charge interaction or hydrophobic interaction between the first capture sequence and the first gripper sequence; Preferably, the library carrier comprises a target nucleic acid fragment connected to a first capture sequence, and a target nucleic acid complementary sequence connected to a second capture sequence, and the surface of the solid support simultaneously comprises a first and a second gripper sequence which are reverse complementary to the first and second capture sequences; Preferably, the expected inoculation density is obtained by adjusting the magnitude of the repulsive force or the number of inoculations; Preferably, the desired inoculation density is obtained by adjusting the pH and salt concentration of the buffer; Preferably, multiple inoculations are performed to obtain the desired inoculation density, for example, first using the first library vector for inoculation, and then using the second library vector for inoculation, the size or charge of the second library vector being the same as or different from that of the first library vector; preferably, a library vector with a greater exclusion capacity can be first used for full inoculation, and then a library vector with a smaller exclusion capacity can be used for inoculation.

4. The method according to any one of claims 1 to 3, wherein the removal refers to the release of hybridization between the library vector and the first gripper sequence and the extension product, thereby allowing the entire library vector to detach from the surface of the solid support, and the method of releasing hybridization is selected from changing salinity, pH, temperature, solvent compatibility or flow rate.

5. The method according to any one of claims 1 to 4, further comprising before step 1): S1) step of constructing a library to be tested and / or S2) step of constructing a library vector; Preferably, in step S2), a library vector is generated by nucleic acid amplification; Preferably, the library vector is formed by RCA rolling circle amplification; Preferably, the library vector is formed by RCA-MDA amplification; Optionally, after the RCA reaction is completed, a primer complementary to the RCA product is added to initiate an MDA reaction to obtain a library vector consisting of RCA-MDA products; Optionally, before the RCA reaction begins, primers that can hybridize with the RCA product are simultaneously added to the system to obtain a library vector consisting of RCA-MDA products; Optionally, 2n hybridization sites are designed on the circular library, and the hybridization sites from the 3' end to the 5' end of the circular library interface are recorded as the 1st, 2nd, 3rd ... 2n hybridization sites. In the RCA-MDA reaction, one or more primers that hybridize with the 1st, 3rd ... 2n-1 hybridization sites are added; at the same time, one or more primers with the same sequence as the 2nd, 4th ... 2n sites are added to perform RCA reaction. In this reaction, the primers hybridized with the 1st, 3rd ... 2n-1 sites are used to generate RCA products; Primers with the same sequence as the 2nd, 4th, ... 2nth sites are used to hybridize with the RCA product and initiate the MDA reaction to produce RCA-MDA products; at the same time, primers hybridizing with the 1st, 3rd, ... 2n-1th sites can further hybridize with the RCA-MDA product to continue the MDA reaction; Preferably, in step S2), a circular library or a non-circular library carrying the target nucleic acid fragment is used as a template for rolling circle amplification to obtain a linear single strand having a certain spatial structure, similar to a sphere but not stretched; Preferably, in step S1), specific known sequences are added to both ends of the target nucleic acid sequence fragment, and an artificially synthesized nucleic acid oligomer that can simultaneously match the specific known sequences at both ends of the test library is used as a circular primer, and under the action of a ligase, the original linear target test library is converted into a closed circular library; Preferably, in step S1), a linker with a nick is used to establish a library to be tested, and in step S2), the displacement enzyme can perform strand displacement and polymerization reactions from the nick of the linker to form a library vector; Preferably, in step S2), a library carrier is constructed on the surface of a nano- or micro-carrier; Preferably, the nano- or micro-carrier is a biomolecule, an organic compound, an inorganic substance or a combination of the above substances; further preferably, the nano- or micro-carrier is a protein, an antibody, a polymer, a magnetic bead, a gel bead, or a metal nano-micro particle. Preferably, the surface of the nano- or micro-carrier and the library to be tested respectively comprise a second capture sequence and a second gripper sequence which is reversely complementary thereto. After the second capture sequence and the second gripper sequence are paired, a complementary sequence of the target library sequence to be tested is formed on the surface of the nano- or micro-carrier by polymerase extension or reverse transcription extension to obtain a library carrier; Preferably, the surface of the nano- or micro-carrier contains only a single second capture sequence or second gripper sequence, and after extension, the surface of the carrier contains only a single target nucleic acid fragment or complementary sequence, and the library carrier thus obtained is a single library carrier; Preferably, the surface of the nano- or micro-carrier comprises a single first functional group, and a single second capture sequence is introduced through the first functional group; Preferably, the surface of the nano- or micro-carrier further comprises a plurality of second functional groups, and an amplification reaction is performed after introducing a plurality of primers on the surface of the single library carrier through the second functional groups, so as to form an amplification cluster on the surface of the nano- or micro-carrier and obtain a cluster library carrier; Preferably, in step S1), a reverse complementary sequence of the first capture sequence is introduced at one end of the target nucleic acid fragment, so that the library vector obtained after extension contains the first capture sequence. Preferably, the nano- or micro-carrier is a protein complex; Preferably, the protein complex is AB n A multi-protein complex formed by a protein and an optional linker, wherein A represents a -SH group, B represents a -NH2 group, and n is an integer of 5-50, preferably 10-20; Preferably, the AB n The protein is myoglobin; Preferably, the second capture sequence or the second gripper sequence is introduced through -SH, for example, the third reactive group is introduced through a bifunctional linker containing maleimide and a third reactive group, and the second capture sequence or the second gripper sequence is coupled through the third reactive group to form a first protein monomer; the first protein monomer reacts with an excess (10 to 30:1) of an unmodified capture sequence second protein monomer to form a multi-protein complex; Preferably, a linker such as a PEG linker (the number of ethoxy units is: 4-2000, preferably 4-800, 4-400, 4-200, 4-100, 4-50 or 4-20, etc.) is introduced during the coupling process of the protein monomers to increase the distance between the monomeric proteins of the multi-protein complex to achieve a larger exclusion volume; Preferably, the surface of the protein carrier is modified for the second time, and amplification is performed after primers are introduced to form an amplification cluster on the surface of the protein complex. Preferably, the nano- or micro-carrier is an artificial microbead; Preferably, the artificial microbeads are magnetic beads, gel beads, or metal nano-micro particles; Preferably, the artificial microbeads are micrometer-sized. Preferably, the nano- or micro-carrier is a positively charged polymer; preferably, the positively charged polymer is PEI or PLL; preferably, the polymer comprises a single first functional group for introducing a capture sequence, and / or multiple second or third functional groups for modification or cross-linking to increase repulsion or for introducing primers to form an amplification cluster.

6. A method for preparing DNA clone clusters on the surface of a solid support, the method comprising first forming a DNA single-stranded cluster on the surface of a solid support using the method described in any one of claims 1 to 5, and step 5): amplifying the DNA single-stranded cluster on the surface of the solid support into a larger DNA clone cluster by bridge amplification means or isothermal amplification such as RPA, HDA, LAMP, etc.

7. A kit or reagent combination for preparing single-stranded DNA clusters or cloned clusters on a solid support surface, comprising: 1) a library vector and 2) a solid support; the library vector and the solid support respectively comprise a first capture sequence and a first gripper sequence that is reverse complementary thereto; and optionally, bridge amplification or isothermal amplification reagents; The library vector is as described in any one of claims 1 to 5; Preferably, the kit or reagent combination further comprises reagents for constructing a library vector; Preferably, the reagents for constructing the library vector comprise RCA rolling circle amplification primers; Preferably, the reagents for constructing the library vector comprise RCA-MDA amplification primers; Preferably, the reagents for constructing the library vector comprise nano- or micro-vectors; Preferably, the reagents for constructing the library vector further include reagents for constructing the test library, which, when constructing the test library, add the reverse complementary sequence of the first capture sequence to one end of the target nucleic acid fragment so that the library vector obtained after extension contains the first capture sequence.

8. A sequencing method, comprising: Preparing a DNA clone cluster on a solid support surface by the method of claim 6, and then sequencing the clone cluster; Preferably, the sequencing is fluorescence sequencing.

9. A sequencing kit or reagent combination, comprising the kit or reagent combination for preparing a DNA clone cluster according to claim 7, and a sequencing reagent.

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