Cross-linked complex, preparation method therefor and use thereof in sequencing

Abstract

The present application provides a cross-linked complex, a preparation method therefor, and a use thereof in sequencing. The cross-linked complex comprises: a protein and a semiconductor chip, the protein being connected to the semiconductor chip by means of a group A, the group A having a structure as shown in formula (A), or a stereoisomer, a tautomer or a salt of the structure shown in formula (A), the protein being connected to the group A by means of an amide bond, and the semiconductor chip being connected to the group A by means of an amide bond, W being -(CH2)n1- or -(CH2CH2O)n2CH2CH2-, and n1 and n2 each being a positive integer.

Description

Cross-linked complexes, their preparation methods, and their applications in sequencing Technical Field

[0001] This application relates to the field of biotechnology. Specifically, this application relates to a cross-linked complex and a method for preparing the same. More specifically, this application relates to a cross-linked complex, a method for preparing the same, a sequencing method, a reagent kit, and their applications. Background Technology

[0002] DNB (DNA nanosphere) sequencing, a high-throughput sequencing technology, amplifies single-stranded circular DNA into DNBs through rolling circle replication. This generates multiple single-stranded copies of each DNA fragment, thereby enhancing signal strength and effectively avoiding error accumulation during amplification. One of the key steps in DNB sequencing is loading the DNBs onto a patterned array sequencing slide.

[0003] Sequencing slides are silicon wafers made of photoresist material with amino-modified coatings. Existing methods for loading DNB rely on the Coulombic adsorption of DNB by the modified groups on the surface of the sequencing slide. This method is relatively unstable and easily detaches from the sequencing slide due to the biochemical environment during sequencing, thus affecting the overall sequencing performance.

[0004] Therefore, there is a need to develop methods that can increase the binding tightness between DNB and sequencing slides, increase the binding stability between DNB and sequencing slides, and improve the DNB loading efficiency.

[0005] Summary of the Invention

[0006] This application was made by the inventor based on the following discoveries:

[0007] Existing DNA nanosphere loading methods have drawbacks such as unstable binding of DNB to sequencing slides, strict requirements for biochemical conditions, and susceptibility to detachment from the slides due to biochemical environmental influences, thereby limiting sequencing efficiency and affecting sequencing quality.

[0008] Therefore, the purpose of this application is to provide a means to effectively increase the stability and tightness of protein binding to semiconductor chips. More specifically, the purpose of this application is to provide a means to effectively increase the binding stability of DNA nanospheres (DNB) to sequencing slides during sequencing; a means to effectively increase the binding tightness of DNB to sequencing slides; and a means to effectively improve the loading efficiency of DNB on sequencing slides.

[0009] In a first aspect of this application, a cross-linking complex is proposed. According to an embodiment of this application, the cross-linking complex comprises: a protein and a semiconductor chip, wherein the protein and the semiconductor chip are linked by a group A, the group A having a structure shown in formula (A) or a stereoisomer, tautomer, or salt thereof of the structure shown in formula (A), the protein and the group A are linked by an amide bond, and the semiconductor chip is linked by the group A by an amide bond;

[0010] Where W stands for -(CH2). n1 -or-(CH2CH2O) n2 CH2CH2-, n1 and n2 are positive integers.

[0011] According to embodiments of this application, the presence of group A makes the binding of the protein to the semiconductor chip tighter and more stable. According to some specific embodiments of this application, when this cross-linked complex is applied in a sequencing scenario, the presence of group A causes the amino groups on the silicon wafer surface to covalently cross-link with the polymerase in the DNA nanospheres, forming stable amide bonds. This enhances the tightness of the binding between DNA nanospheres (DNB) and the sequencing substrate during sequencing, improves the stability of DNB binding on the sequencing substrate, significantly increases the loading efficiency of DNB on the sequencing substrate, and improves the base recognition rate.

[0012] According to embodiments of this application, the crosslinked complex may further include at least one of the following additional technical features:

[0013] According to embodiments of this application, n1 and n2 can each be independently selected from any integer between 1 and 10. According to some specific embodiments of this application, n1 and n2 can each be independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some preferred embodiments of this application, n1 and n2 are each independently selected from 5 or 6. When n1 and n2 are each independently selected from 5 or 6, the crosslinked composite exhibits higher stability.

[0014] According to embodiments of this application, the protein is a strand displacement polymerase. According to some specific embodiments of this application, the strand displacement polymerase is a strand displacement RNA polymerase or a strand displacement DNA polymerase.

[0015] According to embodiments of this application, the strand substitution DNA polymerase is Phi29 polymerase or BST enzyme. According to a specific embodiment of this application, the strand substitution DNA polymerase is Phi29 polymerase.

[0016] According to an embodiment of this application, the semiconductor chip is a sequencing carrier.

[0017] According to embodiments of this application, the protein is incorporated into the DNA nanospheres as a component in the preparation of DNA nanospheres (DNB).

[0018] According to specific embodiments of this application, in some sequencing scenarios, existing methods for loading DNB onto sequencing slides rely on Coulombic adsorption to connect the modified groups on the surface of the sequencing slide to DNB, resulting in poor stability of the formed complex and low loading efficiency. In contrast, this invention covalently cross-links the polymerase bound to DNB with the modified groups on the surface of the sequencing slide through chemical bonds, significantly increasing the binding tightness and stability of DNB to the sequencing vector, and thus significantly improving the DNB loading efficiency.

[0019] In a second aspect of this application, a method for preparing the crosslinked complex described in the first aspect of this application is provided. According to an embodiment of this application, the method includes: loading a protein to be crosslinked onto a semiconductor chip to be crosslinked, wherein the protein to be crosslinked has free amino groups, and the surface of the semiconductor chip to be crosslinked has free amino groups; and contacting a crosslinking compound with the semiconductor chip to be crosslinked on which the protein to be crosslinked is loaded to obtain the crosslinked complex; wherein the crosslinking compound has the structure shown in Formula (B) or a stereoisomer, tautomer, or salt thereof of the structure shown in Formula (B).

[0020] Where X and Y are independent R1 is selected from hydrogen, -OH, -CN, -(C1-C6)alkyl, or... m is 1 or 2; W is -(CH2) n1 -or-(CH2CH2O) n2 CH2CH2-, n1 and n2 are positive integers.

[0021] According to embodiments of this application, n1 and n2 can each be independently selected from any integer between 1 and 10. According to some specific embodiments of this application, n1 and n2 can each be independently selected from any integer among 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0022] According to some specific embodiments of this application, the X group and Y group are independently N-hydroxysulfosuccinimide (NHS).

[0023] According to embodiments of this application, the above method further includes at least one of the following technical features:

[0024] According to embodiments of this application, X and Y are each independently...

[0025] According to embodiments of this application, X and Y are each independently... W stands for -(CH2) n1 -

[0026] According to embodiments of this application, X and Y are each independently... W stands for -(CH2) n1 -

[0027] According to embodiments of this application, X and Y are each independently... W stands for -(CH2CH2O) n2 CH2CH2-.

[0028] According to embodiments of this application, the crosslinking compound is selected from compounds shown in formulas (I)-(IV).

[0029] According to the embodiments of this application, the method for preparing the cross-linked complex is simple to operate and has a short reaction time. This cross-linked complex can be applied in sequencing scenarios and is suitable for various sequencing platforms, including but not limited to those of BGI Group and Illumina.

[0030] According to some specific embodiments of this application, the cross-linking compounds shown in formulas (I) to (IV) can be used in sequencing to avoid changes in protein (e.g., polymerase) activity. Meanwhile, the terminal N-hydroxysulfosuccinimide or succinimide can specifically react with the primary amine in the protein, act as a leaving group in the nucleophilic acyl substitution reaction, and form a stable amide bond with the primary amine, thereby tightly and stably connecting the protein to the semiconductor chip.

[0031] According to an embodiment of this application, the method further includes: before loading the protein to be cross-linked onto the semiconductor chip to be cross-linked, binding the protein to be cross-linked with single-stranded circularized DNA, performing rolling circle amplification to form a sequencing template (DNA nanospheres), so that the protein to be cross-linked is bound to the sequencing template.

[0032] According to an embodiment of this application, the loading is performed in a DNB loading buffer.

[0033] According to embodiments of this application, the DNB loading buffer is DNB loading buffer II or DNB loading buffer IV. According to some specific embodiments of this application, different DNB loading buffers are selected for different sequencing protocols.

[0034] According to an embodiment of this application, the contact treatment is performed in a crosslinking buffer.

[0035] According to embodiments of this application, the crosslinking buffer is at least one of phi29 buffer and PBS buffer.

[0036] According to embodiments of this application, the cross-linking buffer is a PHI29 buffer. Sequencing experiments have verified that, compared to PBS buffer, choosing PHI29 buffer yields more sequencing reads and a higher Q30 value.

[0037] According to an embodiment of this application, the contact treatment is performed at 20–30°C for 25–35 minutes. Sequencing experiments have verified that the number of sequencing reads and the Q30 value are optimal at a temperature of 25°C.

[0038] According to an embodiment of this application, the contact treatment is performed at 25°C for 30 minutes. The inventors experimented with different temperatures to obtain the number of sequencing reads and Q30 values, ultimately determining that the optimal contact treatment conditions were achieved by performing the treatment at 25°C for 30 minutes, which yielded the highest number of sequencing reads and the highest Q30 value.

[0039] In a third aspect of this application, a sequencing method is proposed. According to an embodiment of this application, the method includes: sequencing the cross-linked complex described in the first aspect of this application. The cross-linked complex has high stability and is not easily affected by the biochemical environment, resulting in higher DNA nanosphere loading efficiency. This is reflected in the sequencing results as a higher signal-to-noise ratio and higher sequencing accuracy.

[0040] According to embodiments of this application, the sequencing method further includes at least one of the following technical features:

[0041] According to an embodiment of this application, the cross-linked complex is obtained by: loading a sequencing template system, which includes a strand displacement polymerase, preferably a DNA polymerase, onto a sequencing slide; and contacting the sequencing slide with the cross-linked compound to obtain the cross-linked complex. The DNA polymerase reacts with the free amino groups on the surface of the sequencing slide, forming a cross-linked complex through amide bonds.

[0042] According to embodiments of this application, after the contact treatment and before the sequencing treatment, the process further includes a second round of amplification of the template to be sequenced. By performing a second amplification on the sequencing template, the intensity of the sequencing signal is increased, thereby enabling more accurate detection and identification of the target sequence.

[0043] According to embodiments of this application, the sequencing template is obtained by using single-stranded circular DNA as a starting template and then performing rolling circle amplification. This step generates more DNA copies through rolling circle amplification to facilitate subsequent sequencing.

[0044] According to an embodiment of this application, the sequencing template is a DNA nanosphere (DNB).

[0045] According to some preferred embodiments of this application, before loading the sequencing template system onto the sequencing slide, the strand displacement polymerase is combined with single-stranded circularized DNA and rolled circle amplification is performed to form the sequencing template (DNA nanospheres), so that the strand displacement polymerase is bound to the sequencing template.

[0046] According to embodiments of this application, the sequencing template includes a one-strand sequencing template and a two-strand sequencing template. The one-strand and two-strand sequencing templates represent the two complementary strands of DNA. By simultaneously sequencing these two complementary strands, the sequencing results can be cross-validated, thereby improving sequencing accuracy.

[0047] According to embodiments of this application, the crosslinking compound has the structure shown in Formula (B) or a stereoisomer, tautomer, or salt thereof of the structure shown in Formula (B).

[0048] Where X and Y are independent R1 is selected from hydrogen, -OH, -CN, -(C1-C6)alkyl, or... m is 1 or 2; W is -(CH2) n1 -or-(CH2CH2O) n2 CH2CH2-, where n1 and n2 are positive integers, preferably, n1 and n2 are independently selected from any integer between 1 and 10. Specifically, n1 and n2 are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0049] According to embodiments of this application, the contact treatment is performed in a cross-linking buffer. The cross-linking buffer can maintain the acid-base balance of the reaction system and preserve enzyme activity.

[0050] According to embodiments of this application, the crosslinking buffer is at least one of phi29 buffer and PBS buffer.

[0051] According to an embodiment of this application, the cross-linking buffer is a PHI29 buffer. Experimental verification has shown that using a PHI29 buffer as the cross-linking buffer yields more sequencing data and significantly reduces the base error rate (increases the Q30 value).

[0052] According to an embodiment of this application, the contact treatment is performed at 20–30°C for 25–35 minutes.

[0053] According to an embodiment of this application, the contact treatment is performed at 25°C for 30 minutes. Experimental verification has shown that reacting at 25°C for 30 minutes yields more sequencing data and a lower base error rate.

[0054] In a fourth aspect, this application provides a kit. According to embodiments of this application, the kit comprises: a compound having the structure shown in Formula (B) or a stereoisomer, tautomer, or salt thereof of the structure shown in Formula (B).

[0055] Where X and Y are independent R1 is selected from hydrogen, -OH, -CN, -(C1-C6)alkyl, or... m is 1 or 2; W is -(CH2) n1 -or-(CH2CH2O) n2 CH2CH2-, where n1 and n2 are positive integers. According to embodiments of this application, n1 and n2 can be independently selected from any integer between 1 and 10. According to some specific embodiments of this application, n1 and n2 can be independently selected from any integer among 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0056] According to embodiments of this application, the kit can be used in sequencing and has advantages such as portability and low cost.

[0057] According to embodiments of this application, the kit further includes at least one of DNA polymerase, cross-linking buffer, DNB loading buffer, multiple displacement amplification primers, and sequencing primers.

[0058] According to embodiments of this application, the DNA polymerase is a strand displacement DNA polymerase.

[0059] According to embodiments of this application, the strand substitution DNA polymerase is Phi29 polymerase or BST enzyme. In some preferred embodiments of this application, the strand substitution DNA polymerase is Phi29 polymerase.

[0060] According to embodiments of this application, the cross-linking buffer is selected from at least one of PHI29 buffer and PBS buffer. According to some specific embodiments of this application, the cross-linking buffer is PHI29 buffer.

[0061] According to embodiments of this application, the DNB loading buffer is DNB loading buffer II or DNB loading buffer IV.

[0062] In a fifth aspect of this application, an application of a reagent kit in sequencing is proposed, said reagent kit being the same as described in the fourth aspect. According to embodiments of this application, the reagent kit can be used for sequencing and has the advantages of being easy to operate and portable.

[0063] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0064] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0065] Figure 1 is a schematic diagram of cross-linked assisted loading of DNB according to an embodiment of the present invention;

[0066] Figure 2 is a schematic diagram showing the comparison results of Q30 between the experimental group and the control group according to an embodiment of the present invention;

[0067] Figure 3 is a schematic diagram showing the comparison results of SNR between the experimental group and the control group according to an embodiment of the present invention;

[0068] Figure 4 is a schematic diagram showing the comparison results of FIT between the experimental group and the control group according to an embodiment of the present invention;

[0069] Figure 5 is a schematic diagram of the synchronous sequencing analysis results of the experimental group and the control group compared according to an embodiment of the present invention;

[0070] Figure 6 is a schematic diagram of the first sequencing round reaction (C001R006 P006 A) after BS3 loading according to an embodiment of the present invention;

[0071] Figure 7 is a schematic diagram of the crosslinking reaction optimization results according to an embodiment of the present invention. Detailed Implementation

[0072] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0073] In this document, unless otherwise stated, the singular forms “a,” “an,” etc., include plural referents (more than one); “a group” or “a plurality” refers to two or more.

[0074] In this document, unless otherwise stated, the terms “first,” “second,” “third,” “fourth,” etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated; features defined with “first,” “second,” etc., may explicitly or implicitly include one or more of the stated features.

[0075] In this application, unless otherwise stated, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis / trans) isomers, and hindered isomers.

[0076] In this application, unless otherwise stated, the terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerization is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers, also known as prototropic tautomers, include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0077] In this application, unless otherwise stated, the term "salt" refers to both organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.

[0078] In this document, unless otherwise stated, the “DNB loading buffer” serves to protect DNA nanospheres and their loads from degradation and damage, maintain proper ion balance to prevent uncontrolled aggregation, regulate pH to maintain the correct structure of DNA, help to uniformly disperse the nanospheres to prevent aggregation, and modulate the surface charge properties of the nanospheres to influence their interactions with other molecules or cells.

[0079] In this article, unless otherwise stated, the term “sequencing read” is used interchangeably with the term “read” or “segment” and refers to a single nucleic acid sequence obtained during sequencing, which is referred to as a “sequencing read” or “read” in this article.

[0080] Unless otherwise stated, the sequencing platforms that can be used for sequencing methods in this article include, but are not limited to, Illumina's HiSeq / Miseq / Nextseq / Novaseq sequencing platforms, Thermo Fisher / Life Technologies' Ion Torrent platform, BGISEQ and MGISEQ / DNBSEQ platforms from BGI Group, and single-molecule sequencing platforms.

[0081] In this document, unless otherwise stated, "loading," "introducing," or "introducing" have the same meaning and refer to introducing or placing a sample or sequencing material into a specific device, system, or environment for further processing, analysis, testing, or manipulation. In one example of this application, the loading operation includes placing DNA nanospheres or a DNA sample onto a sequencing chip for DNA sequencing in microregions on the chip. These microregions include microreactors, micropores, or microchannels.

[0082] In this article, unless otherwise stated, a "semiconductor chip or sequencing slide," also known as a flow cell, sequencing chip, or sequencing channel, is one of the key components used for high-throughput DNA sequencing. It is a miniaturized device, typically made of glass or other materials, used to support the fixation and handling of DNA samples for sequencing reactions and signal acquisition. The design and structure of sequencing slides can vary depending on the requirements of different sequencing platforms and technologies, but their primary function is to provide a controlled environment within the sequencer for efficient DNA sequencing.

[0083] In this document, unless otherwise stated, "crosslinking reaction" refers to the combination of two or more molecules or biomacromolecules through a chemical covalent bond. This reaction is widely used for the crosslinking, immobilization, modification, and labeling of proteins and peptides. The principle is that a biocrosslinking agent with reactive terminals reacts chemically with specific active groups (such as thiol groups, primary amino groups, etc.) on proteins or other molecules to form covalent bonds. It is widely used for surface immobilization on solid supports, biotinylation, protein structure studies, and metabolic labeling. The crosslinked complex described in this application is obtained by a crosslinking reaction between proteins, semiconductor chips, and a crosslinking agent.

[0084] sequencing methods

[0085] In one aspect of this application, a sequencing method is proposed. According to an embodiment of this application, the sequencing method includes: performing sequencing processing on the cross-linked complex described in this application.

[0086] According to an embodiment of this application, as shown in Figure 1, in this cross-linking complex, stable amide bonds are formed between the sequencing slide, the protein, and the cross-linking agent. Applying this cross-linking complex to sequencing can improve the binding tightness of DNA nanoballs (DNB) to the sequencing slide during sequencing, enhance the loading efficiency of DNB on the sequencing slide, and strengthen the stability of DNB bound to the sequencing slide during sequencing, thereby improving sequencing quality.

[0087] For ease of understanding, the technical solution of this application will be described in detail below.

[0088] Sequencing Protocol 1: Application of Cross-linked Complexes in Paired-End Sequencing (PE)

[0089] Those skilled in the art will recognize that paired-end (PE) sequencing has become an important tool for studying genome structure and variation. However, the post-processing in traditional PE sequencing workflows is quite cumbersome, involving multiple steps such as sample preparation, slide loading, and subsequent data processing, which limits the improvement of sequencing efficiency and throughput. To optimize this workflow, this application proposes an improved scheme based on cross-linking complexes. The specific scheme is as follows:

[0090] DNB is generated by rolling circle amplification (RCA) using single-stranded circular DNA as a template. The DNB is then mixed with DNB loading buffer II at a volume ratio of 3:1 and loaded onto a sequencing slide. An appropriate cross-linking agent (including but not limited to bis(sulfosuccinimide) octanoate (BS3)) is then pumped in to ensure a complete reaction between the DNB and the cross-linking agent on the slide. The sequencing process then proceeds directly to primer hybridization and sequencing (without a postloading step).

[0091] According to the embodiments of this application, the above sequencing scheme increases the stability of DNB binding to the sequencing slide and eliminates the original postloading process, greatly improving sequencing efficiency and throughput. Importantly, the cross-linking reaction does not negatively affect the multiple displacement amplification (MDA) process of DNA, ensuring the integrity and accuracy of the sequencing sample.

[0092] It should be noted that in high-throughput sequencing technology, "postloading" refers to the key step of DNA fragments binding with primers, amplifying, and sequencing.

[0093] Sequencing Protocol 2: Application of Cross-linked Complexes in Secondary Rolling Circle Amplification (MLG)

[0094] According to a specific embodiment of this application, a rolling circle amplification (RCA) process is performed using single-stranded circular DNA as a template to generate DNA nanospheres (DNB). The DNB is mixed with a weakly acidic buffer (such as DNB loading buffer IV, pH 4.6) and then loaded onto a sequencing slide. By introducing a specific cross-linking agent (including but not limited to bis(sulfosuccinimide) octanoate (BS3)), the DNB on the slide reacts fully with the cross-linking agent, ensuring the stability of the DNB. Then, a weakly alkaline buffer (DNB loading buffer IV, pH 7.75) is added to allow the DNB to undergo secondary rolling circle amplification (MLG) to increase the required amount of DNA. Finally, primer hybridization and sequencing are performed. The unique feature of this scheme is that the cross-linking reaction does not affect the subsequent secondary rolling circle amplification and improves sequencing quality.

[0095] Sequencing Scheme 3: Application of Cross-linked Complexes in Paired-End Simultaneous Sequencing

[0096] According to a specific embodiment of this application, single-stranded circular DNA is used as a template. Rolling circle amplification (RCA) is performed to form DNA nanospheres (DNB). The DNB is mixed with an acidic buffer (DNB loading buffer IV, pH 4.6) and loaded onto a sequencing slide. Then, multiple displacement amplification primers are hybridized. First, a multiple displacement amplification process generates a second-strand template. Then, an alkaline buffer (DNB loading buffer IV, pH 7.75) is pumped in, allowing the DNB to continue rolling circle amplification on the sequencing slide, forming a first-strand template. At this point, both the first-strand and second-strand sequencing templates are generated. Next, a specific cross-linking reagent (including but not limited to bis(sulfosuccinimide) octanoate, BS3) is pumped in. Simultaneous hybridization of the first and second-strand primers enables concurrent sequencing.

[0097] It should be noted that, in addition to BS3 (bis(sulfosuccinimide) octanoate), the cross-linking agent used in the above sequencing scheme can also be its derivatives, including DSS (bis(sulfosuccinimide) octanoate), BS(PEG)5 (polyethylene glycol-modified bis(sulfosuccinimide) octanoate), BS(PEG)9 (polyethylene glycol-modified bis(sulfosuccinimide) octanoate), etc.

[0098] The following examples illustrate this application, but should not be construed as limiting the scope of the subject matter of this application to the following examples. All technologies implemented based on the above content of this application fall within the scope of this application. The compounds or reagents used in the following examples are commercially available or prepared using conventional methods known to those skilled in the art; the experimental instruments used are commercially available.

[0099] Example 1: Application of cross-linked complexes in paired-end (PE) sequencing

[0100] This embodiment is based on sequencing scheme 1.

[0101] 1. Instruments and reagents

[0102] Instruments: MGISEQ-2000 sequencer, MGISEQ-2000 sequencing reagent slides (715nm), mini DNB loading device, PCR instrument, PCR eight-tube set, pipette set, high-speed centrifuge, mini centrifuge, vortex mixer.

[0103] Reagents and sequencing samples: as shown in Table 1.

[0104] Table 1. Reagents and Sequencing Samples

[0105] The BS3 buffer formulation used in this embodiment is shown in Table 2.

[0106] Table 2 BS3 buffer components and concentrations

[0107] In this embodiment, the formulation of DNB loading buffer IV is shown in Table 3.

[0108] Table 3 DNB Loading Buffer IV Formulation

[0109] One-stranded sequencing primer IP1-x1 with linker:

[0110] One-stranded sequencing primers IP1-x2 with linker:

[0111] 2. Sequencing Analysis Procedure

[0112] 2.1 Preparation of DNB

[0113] DNA nanospheres were prepared from the E. coli library according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual".

[0114] 2.2 Loading DNB

[0115] Prepare one MGISEQ-2000 sequencing reagent slide (715nm). Mix the DNB and DNB loading buffer II (included in the MGISEQ-2000RS high-throughput rapid sequencing kit (FCS PE300)) at a volume ratio of 3:1. Then, load the DNB onto the MGISEQ-2000 sequencing reagent slide (715nm) using a mini DNB loading device.

[0116] 2.3 Sequencing Steps

[0117] Experimental group:

[0118] Prepare a sequencing kit according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual"; replace the reagent in well 4 with BS3 loading buffer, and remove the reagents in wells 11, 13, and 14. Place the sequencing kit and slide on the MGI2000-RS sequencer according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual," select the corresponding script, set PE10, and begin sequencing. The sequencing flow is as follows: cross-linking agent incubation (25℃ 30min) → first-strand primer hybridization → first-strand sequencing → MDA process → second-strand primer hybridization → second-strand sequencing.

[0119] Control group:

[0120] Prepare a sequencing kit according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual"; remove reagents from wells 11, 13, and 14. Following the instructions, place the sequencing kit and slide onto the MGI2000-RS sequencer, select the corresponding script, set PE10, and begin sequencing. The sequencing flow is as follows: first-strand primer hybridization → first-strand sequencing → MDA process → second-strand primer hybridization → second-strand sequencing.

[0121] The results are shown in Figures 2, 3, and 4. The introduction of cross-linking reagents successfully simplified the post-loading process before sequencing. Notably, the introduction of cross-linking reagents did not affect the subsequent multiple strand displacement amplification (MDA) process. After treatment with the cross-linking reagents, the ATCG of the cross-linked experimental group was higher than that of the control group (approximately 1) in terms of the signal-to-noise ratio (SNR) of the four bases, indicating that the cross-linking agent did not cause background enhancement or interference, but rather improved the resolution. Furthermore, a significant increase in the FIT value was observed, indicating that more effective DNB was loaded, and a larger proportion of DNB was available for base recognition, reflecting higher DNB loading efficiency. With the increase in sequencing cycle number, the overall Q30 performance remained stable, indicating that the DNB was tightly bound to the sequencing slide during sequencing and exhibited good stability.

[0122] Example 2: Application of cross-linked complex in secondary rolling circle amplification (MLG)

[0123] This embodiment uses sequencing scheme 2. The experimental instruments and reagents are the same as in embodiment 1.

[0124] 1. Sequencing analysis operation steps

[0125] 1.1 Preparation of DNB

[0126] DNA nanospheres were prepared from the E. coli library according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual".

[0127] 1.2 Loading DNB

[0128] Prepare one MGISEQ-2000 sequencing reagent slide (715nm). Mix the DNB and DNB loading buffer IV at a volume ratio of 2:1 and load them onto the MGISEQ-2000 sequencing reagent slide (715nm) using a mini DNB loading device.

[0129] 1.3 Sequencing Steps

[0130] Experimental group:

[0131] Prepare a sequencing kit according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual"; replace the reagent in well 4 with BS3 loading buffer, replace well 6 with DNB loading buffer IV (pH 7.75), and remove the reagents from wells 11, 13, and 14. Place the sequencing kit and slides on the MGI2000-RS sequencer according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual," select the corresponding script, set PE10, and begin sequencing. The sequencing flow is as follows: cross-linking agent incubation (25℃, 30 min) → MLG (25℃, 60 min) → first-strand primer hybridization → first-strand sequencing → MDA process → second-strand primer hybridization → second-strand sequencing.

[0132] Control group:

[0133] Prepare a sequencing kit according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual"; replace well 6 with DNB loading buffer IV (pH 7.75), and remove reagents from wells 11, 13, and 14. Place the sequencing kit and slides on the MGI2000-RS sequencer according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual," select the corresponding script, set PE10, and begin sequencing. The sequencing workflow is as follows: MLG (25℃, 60 min) → first-strand primer hybridization → first-strand sequencing → MDA process → second-strand primer hybridization → second-strand sequencing.

[0134] The results are shown in Table 4. The cross-linking complex had no negative impact on the secondary rolling circle amplification reaction, and the number of loaded DNBs was 136.77w, which was 0.77w more than that of the control group. Signal intensity was comparable between the experimental and control groups for the four bases ATCG. The SNR (signal-to-noise ratio) of the four bases was higher in the cross-linked experimental groups (ATCG) than in the control group (0.2-0.5), indicating that the cross-linking agent did not cause background enhancement or interference. Furthermore, the BIC and FIT values ​​were higher in the cross-linked experimental groups than in the control group, indicating a greater number of effective DNBs loaded and a higher proportion of DNBs available for base recognition, reflecting higher DNB loading efficiency. The overall Q30 and SNR were improved compared to the control group, indicating that the sequencing quality was improved after adding the cross-linking agent.

[0135] Table 4. Effect of BS3 as a crosslinking agent on secondary rolling circle amplification (MLG).

[0136] Example 3: Application of cross-linked complexes in paired-end synchronous sequencing

[0137] This embodiment uses sequencing protocol 5. The experimental instruments and reagents are the same as in embodiment 1.

[0138] 1. Supplementary reagents are shown in Tables 5, 6, and 7.

[0139] Table 5: Configuration of IP1-xlinker

[0140] Table 6. Preparation of Insert Primer Mix for One-Strand and Two-Strand Sequencing Primers

[0141] Table 7.1 Xphi29 buffer configuration

[0142] 2. Sequencing Analysis Procedure

[0143] 2.1 Preparation of DNB

[0144] DNA nanospheres were prepared from the E. coli library according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual".

[0145] 2.2 Loading DNB

[0146] Prepare one MGISEQ-2000 sequencing reagent slide (715nm). Mix the DNB with DNB loading buffer IV (pH 4.6) at a volume ratio of 2:1 and load the mixture onto the MGISEQ-2000 sequencing reagent slide (715nm) using a mini DNB loading device.

[0147] 2.3 Sequencing Steps

[0148] Experimental group:

[0149] Prepare a sequencing kit according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual"; replace the reagent in well 4 with BS3 loading buffer. Replace the reagent in well 13 with the above-mentioned 1μM single-strand sequencing primer working solution IP1-xlinker, replace the reagent in well 6 with DNB loading buffer IV, replace the reagent in well 7 with 1X phi29 buffer, replace the reagent in well 11 with REB reagent, and replace the reagent in well 3 with the single-strand and double-strand sequencing primer mixture Insert Primer mix.

[0150] Following the instructions in the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual," the sequencing kit and slides were placed on the MGI2000-RS sequencer. The corresponding script was selected, SE50 was set, and sequencing was performed. To demonstrate the feasibility of this protocol, the first-strand and second-strand sequencing primers (Insert Primer Mix) were hybridized for simultaneous sequencing (50 bp). The sequencing workflow was as follows: IP1-xlinker primer hybridization → MDA → MLG (25℃, 60 min) → cross-linking agent incubation (25℃, 30 min) → first-strand and second-strand template generation → first-strand and second-strand sequencing primer hybridization → simultaneous sequencing.

[0151] Control group:

[0152] Prepare a sequencing kit according to the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual"; replace the reagent in well 13 with the above-mentioned 1μM single-strand sequencing primer working solution IP1-xlinker, replace the reagent in well 6 with DNB loading buffer IV, replace the reagent in well 7 with 1X phi29 buffer, replace the reagent in well 11 with REB reagent, and replace well 3 with single-strand and double-strand sequencing primer mixture Insert Primer mix.

[0153] Following the instructions in the "MGISEQ-2000RS High-Throughput Sequencing Reagent Kit Instruction Manual," the sequencing kit and slides were placed on the MGI2000-RS sequencer. The corresponding script was selected, SE50 was set, and sequencing was performed. To demonstrate the feasibility of this protocol, the first-strand and second-strand sequencing primers (Insert Primer Mix) were hybridized for simultaneous sequencing (50 bp). The sequencing workflow was as follows: IP1-xlinker primer hybridization → MDA → MLG (25℃, 60 min) → first-strand and second-strand template generation → first-strand and second-strand sequencing primer hybridization → simultaneous sequencing.

[0154] The results are shown in Figure 5. The cross-linking complex is also applicable to the synchronous sequencing method. The experimental group with added BS3 showed a 6% higher overall alignment rate and a 0.2% lower overall error rate compared to the control group. The original images of the experimental group with added BS3 show that the DNB images are clear and uniform, with obvious track lines, no bubbles or impurities, indicating good loading effect.

[0155] Example 4: Optimization of crosslinking reaction conditions

[0156] This embodiment optimizes the cross-linking reaction conditions to further improve sequencing quality. The experimental instruments and reagents are the same as in Example 1. PE10 sequencing was used, and the experimental analysis procedures were also the same as in Example 1, with the only differences being the BS3 preparation buffer system and incubation temperature, as detailed in Table 8.

[0157] Table 8. Correspondence between experimental groups and BS3 buffer components and incubation temperatures

[0158] The results are shown in Figure 7. Compared with PBS buffer, BS3 exhibited more sequencing data (TOTAL reads) and a higher Q30 value in the 1Xphi29 buffer system. When the reaction temperature was increased to 30℃, both TOTAL reads and Q30 decreased significantly. Therefore, 1Xphi29 buffer and 25℃ for 30 min were selected as the crosslinking reaction conditions.

[0159] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0160] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0161] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cross-linked complex, characterized in that, comprising: A protein and a semiconductor chip, wherein the protein is connected to the semiconductor chip through group A, and group A has the structure shown in formula (A) or a stereoisomer, tautomer or salt thereof of the structure shown in formula (A); the protein is connected to group A through an amide bond, and the semiconductor chip is connected to group A through an amide bond; Among them, W is -(CH 2 ) n1 - or -(CH 2 CH 2 O) n2 CH 2 CH 2 -, where n1 and n2 are positive integers respectively.

2. The complex according to claim 1, characterized in that, the n1 and n2 are each independently selected from any integer between 1 and 10, preferably, the n1 and n2 are each independently selected from 5 or 6.

3. The complex according to claim 1, characterized in that, the protein is a strand displacement polymerase.

4. The complex according to claim 3, characterized in that, the strand displacement polymerase is a strand displacement RNA polymerase or a strand displacement DNA polymerase.

5. The complex according to claim 4, characterized in that, the strand displacement DNA polymerase is Phi29 polymerase or BST enzyme.

6. The complex according to claim 1, characterized in that, the semiconductor chip is a sequencing slide.

7. The complex according to claim 1, characterized in that, the protein is used as one of the components for preparing DNA nanospheres and is incorporated into the DNA nanospheres.

8. A method for preparing the cross-linked complex according to any one of claims 1 to 7, characterized in that, comprising: loading the protein to be cross-linked onto the semiconductor chip to be cross-linked, the protein to be cross-linked having free amino groups, and the surface of the semiconductor chip to be cross-linked having free amino groups; contacting the cross-linking compound with the semiconductor chip to be cross-linked loaded with the protein to be cross-linked to obtain the cross-linked complex; Among them, the crosslinked compound has a structure represented by formula (B), a stereoisomer, a tautomer or a salt thereof of the structure represented by formula (B). wherein, X and Y are each independently R 1 selected from hydrogen, -OH, -CN, -(C1-C6) alkyl or m is 1 or 2; W is -(CH 2 ) n1 - or -(CH 2 CH 2 O) n2 CH 2 CH 2 -, where n1 and n2 are positive integers respectively.

9. The method according to claim 8, characterized in that, the n1 and n2 are each independently selected from any integer between 1 and 10.

10. The method according to claim 8, characterized in that, X and Y are each independently 11. The method according to claim 8, characterized in that, X and Y are each independently W is -(CH 2 ) n1 -.

12. The method according to claim 8, characterized in that, X and Y are each independently W is -(CH 2 ) n1 -.

13. The method according to claim 8, characterized in that, X and Y are each independently W is -(CH 2 CH 2 O) n2 CH 2 CH 2 -.

14. The method according to claim 8, characterized in that, The crosslinked compound is selected from compounds represented by formula (I) - formula (IV), 15. The method according to claim 8, characterized in that, the method further comprises: before loading the protein to be cross-linked onto the semiconductor chip to be cross-linked, binding the protein to be cross-linked with single-stranded circular DNA and performing rolling circle amplification to form a template to be sequenced, so that the protein to be cross-linked is incorporated into the template to be sequenced.

16. The method according to claim 8, characterized in that, the loading is carried out in DNB loading buffer.

17. The method according to claim 16, characterized in that, the DNB loading buffer is DNB loading buffer II or DNB loading buffer IV.

18. The method according to claim 8, characterized in that, the contact treatment is carried out in cross-linking buffer.

19. The method according to claim 18, characterized in that, the cross-linking buffer is at least one of phi29 buffer and PBS buffer.

20. The method according to claim 19, characterized in that, the cross-linking buffer is phi29 buffer.

21. The method according to claim 8, characterized in that, the contact treatment is carried out at 20-30 °C for 25-35 minutes.

22. The method according to claim 21, wherein, the contact treatment is carried out at 25 °C for 30 minutes.

23. A sequencing method, wherein, comprising: performing a sequencing treatment on the cross-linked complex according to any one of claims 3 to 7.

24. The sequencing method according to claim 23, wherein, the cross-linked complex is obtained by the following method: loading the template system to be sequenced onto a sequencing slide, the template system to be sequenced comprising a strand displacement polymerase, preferably the strand displacement polymerase is a DNA polymerase, and the surface of the sequencing slide has free amino groups; performing a contact treatment on the sequencing slide loaded with the template system to be sequenced with a cross-linking compound so as to obtain the cross-linked complex.

25. The sequencing method according to claim 24, wherein, after the contact treatment and before the sequencing treatment, it further comprises performing a second round of amplification on the template to be sequenced.

26. The sequencing method according to claim 24, wherein, the template to be sequenced starts with a single-stranded circular DNA and is obtained after a rolling circle amplification treatment.

27. The sequencing method according to claim 26, wherein, the template to be sequenced is a DNA nanosphere; preferably, before loading the template system to be sequenced onto the sequencing slide, the strand displacement polymerase is bound to the single-stranded circularized DNA, and a rolling circle amplification is performed to form the template to be sequenced, such that the strand displacement polymerase is bound in the template to be sequenced.

28. The sequencing method according to claim 26, wherein, the template to be sequenced comprises a first-strand sequencing template and a second-strand sequencing template.

29. The sequencing method according to claim 24, wherein, The cross-linked complex has a structure represented by formula (B), or a stereoisomer, tautomer or salt thereof of the structure represented by formula (B). wherein, X and Y are each independently R 1 selected from hydrogen, -OH, -CN, -(C1-C6)alkyl or m is 1 or 2; W is -(CH 2 ) n1 - or -(CH 2 CH 2 O) n2 CH 2 CH 2 -, where n1 and n2 are positive integers respectively.

30. The sequencing method according to claim 29, wherein, n1 and n2 are each independently selected from any integer between 1 and 10.

31. The sequencing method according to claim 24, wherein, the contact treatment is carried out in a cross-linking buffer.

32. The sequencing method according to claim 30, wherein, the cross-linking buffer is at least one of phi29 buffer and PBS buffer.

33. The sequencing method according to claim 32, wherein, the cross-linking buffer is phi29 buffer.

34. The sequencing method according to claim 24, wherein, the contact treatment is carried out at 20 - 30 °C for 25 - 35 minutes.

35. The sequencing method according to claim 34, wherein, the contact treatment is carried out at 25 °C for 30 minutes.

36. A kit, wherein, comprising: A compound, said compound having the structure shown by formula (B) or a stereoisomer, tautomer or salt thereof of the structure shown by formula (B), wherein, X and Y are each independently R 1 selected from hydrogen, -OH, -CN, -(C1-C6) alkyl or m is 1 or 2; W is -(CH 2 ) n1 - or -(CH 2 CH 2 O) n2 CH 2 CH 2 -, where n1 and n2 are positive integers respectively.

37. The kit according to claim 36, wherein, n1 and n2 are each independently selected from any integer between 1 and 10.

38. The kit according to claim 36, wherein, the kit further comprises at least one of a DNA polymerase, a cross-linking buffer, a DNB loading buffer, a multiple displacement amplification primer, and a sequencing primer.

39. The kit according to claim 38, wherein, the DNA polymerase is a strand displacement DNA polymerase.

40. The kit according to claim 39, wherein, the strand displacement DNA polymerase is Phi29 polymerase.

41. The kit according to claim 38, wherein, the cross-linking buffer is selected from at least one of phi29 buffer and PBS buffer.

42. The kit according to claim 38, wherein, the DNB loading buffer is DNB loading buffer II or DNB loading buffer IV.

43. Use of the kit according to any one of claims 36 to 42 in sequencing.

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