Preparation method of sequencing library and reagent

Abstract

The invention relates to the technical field of sequencing, in particular to a preparation method of a sequencing library and a reagent. The invention provides a preparation method of a sequencing library, which comprises the following steps of: additionally adding an additive or an additive combination capable of reducing a DNA secondary structure and / or reducing single-stranded DNA degradation in single-stranded cyclization reaction and / or rolling circle amplification reaction; an additive or an additive combination capable of reducing a DNA secondary structure, reducing single-stranded DNA degradation and / or reducing double-stranded DNA double-helix stability is additionally added in a double-stranded cyclization library rolling circle amplification reaction, so that the binding capacity of DNA ligase and / or strand displacement DNA polymerase and target nucleic acid is improved, and the situation that the rolling circle amplification efficiency of different circular DNAs is not uniform due to the secondary structure is reduced; the probability that the loose single-stranded DNA is easy to degrade is reduced, and the amplification uniformity of different GC regions and different sequence characteristic regions of the library is improved, so that the sequencing quality and the sequencing data yield are improved.

Description

A method and reagent for preparing a sequencing library Technical Field

[0001] The present disclosure relates to the field of sequencing technology, and in particular, to a method and reagents for preparing a sequencing library. Background Art

[0002] Both library amplification and sequencing without amplification based on circular libraries require circularization of the linear DNA with attached sequencing adapters. There are two types of circularization: 1) circularization of single-stranded linear DNA into single-stranded circular DNA; and 2) circularization of double-stranded linear DNA into double-stranded circular DNA.

[0003] When a circular library is used as a template for library amplification and sequencing, a rolling circle amplification reaction is required; when a circular library is directly sequenced, a rolling circle amplification reaction is not required and single-molecule sequencing is performed directly.

[0004] Generally speaking, the cyclization process is affected by many factors, such as the length of linear DNA, the secondary structure of the base sequence with different combinations and arrangements, the pH environment of the cyclase-catalyzed reaction substrate and enzyme, temperature, the activity of the enzyme itself, and the working concentration of the substrate reaction. There are differences in the cyclization efficiency of converting linear DNA into circular DNA.

[0005] Current circularization methods have low circularization efficiencies. For example, PCR-based library construction has a single-strand circularization efficiency of approximately 10-20%, and a double-strand circularization efficiency of approximately 20-40%. Theoretically, if all single strands are circularized, the theoretical circularization efficiency can reach a maximum of 50%, and if all double strands are circularized, the theoretical circularization efficiency can reach a maximum of 100%.

[0006] When using circular libraries as templates for library amplification and sequencing, the rolling circle replication of the circular DNA is also affected by multiple factors, including the template's secondary structure, the pH environment of the rolling circle amplification enzymatic reaction substrate and enzyme, temperature, enzyme activity, and substrate reaction working concentration. This can lead to variations in the efficiency of template-binding primers and the efficiency and fidelity of the rolling circle amplification reaction. The subsequent sequencing process is also constantly affected by multiple factors, including the template's secondary structure, the pH environment of the sequencing enzyme reaction substrate and enzyme, temperature, enzyme activity, and substrate reaction working concentration, leading to variations in the efficiency and stability of template-binding primers and the efficiency and fidelity of the sequencing enzyme reaction.

[0007] Although circular library sequencing has optimized its recipe to overcome the aforementioned difficulties, largely addressing a range of issues caused by these factors, low circularization efficiency and poor sequencing quality in specific sequence regions (such as stem-loop structures, continuous base sequences, and palindromes) remain challenges that need to be optimized and addressed. In particular, the conversion of most Cs in the genome of a methylated library to Ts (for example, human genomic DNA has a GC content of approximately 42% and a C content of approximately 21%. If 5% of the Cs are methylated, then after the unmethylated Cs are converted to Us, the inserted DNA in the methylated library will have a C content of approximately 1% and a T content of approximately 49%). This results in low sequencing signals in the C channel and poor sequencing quality. Furthermore, single-stranded circular libraries (particularly methylated libraries, where the conversion of most Cs to Ts reduces sequence secondary structure and leaves most regions in a loose state) also face the problem of their loose single-stranded structure being easily degraded during the reaction, thus affecting library quality.

[0008] Therefore, there is an urgent need for a method to improve library circularization efficiency and sequencing quality (especially methylation libraries).

[0009] Summary of the Invention

[0010] The present disclosure aims to solve one of the technical problems in the related art at least to a certain extent.

[0011] To this end, the present disclosure provides a first aspect of a method for preparing DNB, the method comprising:

[0012] (a) performing a cyclization reaction on a stranded DNA library to obtain a circular DNA library, wherein the cyclization reaction includes a single-stranded cyclization reaction or a double-stranded cyclization reaction, and the circular DNA library includes a single-stranded circular DNA library or a double-stranded circular DNA library;

[0013] (b) performing rolling circle amplification on the circular DNA library to obtain DNBs,

[0014] in,

[0015] When the cyclization reaction in step (a) is a single-chain cyclization reaction,

[0016] Step (a) further comprises: adding at least one of the following additives to the single-stranded circularization reaction to obtain a single-stranded circular DNA library with higher yield and more uniform coverage:

[0017] 1) Additives that can reduce DNA secondary structure;

[0018] 2) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0019] And / or step (b) further comprises: performing a rolling circle amplification reaction using the single-stranded circular DNA library produced as a product of step (a), wherein at least one of the following additives is added to the rolling circle amplification reaction:

[0020] 3) Additives that can reduce DNA secondary structure;

[0021] 4) additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0022] When the cyclization reaction in step (a) is a double-chain cyclization reaction,

[0023] Step (b) further comprises: performing a rolling circle amplification reaction using the double-stranded circular DNA library produced in step (a), wherein at least one of the following additives is added to the rolling circle amplification reaction:

[0024] 5) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0025] 6) Additives that can reduce the double helix stability of circular double-stranded DNA.

[0026] The present disclosure provides a method for improving sequencing quality, which increases the binding ability of DNA ligase and / or strand displacement DNA polymerase to target nucleic acid by adding additives that reduce DNA secondary structure during the cyclization reaction process and / or the rolling circle amplification reaction process, thereby increasing the binding ability of DNA ligase and / or strand displacement DNA polymerase to DNA sequence fragments with high GC content, continuous AT or GC repeat regions, palindromic sequences, etc. that are difficult to untie hydrogen chains and / or easily form secondary structures. At the same time, the method stabilizes the stability of DNA sequence fragments with low GC content and loose structure and reduces the possibility of degradation by adding additives that stabilize single-stranded structures and prevent them from being degraded; the method improves the melting efficiency and amplification uniformity of the double-stranded circular library in the rolling circle isothermal replication process by adding additives that reduce the stability of the double-stranded DNA double helix. The method effectively improves the cyclization and enrichment uniformity of different GC regions and different sequence feature regions of the library, thereby improving sequencing quality, sequencing data output and sequencing data coverage uniformity. In particular, the methylation sequencing library adopts the above-mentioned inventive method, combined with the optimized base recognition algorithm, which can significantly improve sequencing data output, sequencing data quality and sequencing data coverage uniformity.

[0027] A second aspect of the present disclosure provides a method for rolling circle amplification, comprising performing a rolling circle amplification reaction on a DNA library to obtain single-stranded linear DNA,

[0028] Wherein, when the DNA library is a single-stranded circular DNA library, at least one of the following additives is added to the rolling circle amplification reaction:

[0029] 7) Additives that can reduce DNA secondary structure,

[0030] 8) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0031] When the DNA library is a double-stranded circular DNA library, at least one of the following additives is added to the rolling circle amplification reaction:

[0032] 9) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA,

[0033] 10) Additives that can reduce the double helix stability of circular double-stranded DNA.

[0034] According to the embodiments of the present disclosure, the chain DNA library includes, in addition to the whole genome methylation library and the target region methylation library, any one of the whole genome WGS library (PCR and / or PCR-free library construction method), target region capture library (exon and / or panel library), amplicon type library, RNA library, and stLFR library.

[0035] According to an embodiment of the present disclosure, the additive capable of reducing the secondary structure of DNA includes at least one of a non-ionic detergent, betaine, and DMSO (dimethyl sulfoxide).

[0036] According to an embodiment of the present disclosure, the non-ionic detergent includes at least one selected from Triton-X100, Tween-20, and NP40.

[0037] According to an embodiment of the present disclosure, the additive capable of reducing DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one of SSB (Single-Stranded DNA Binding Protein) and HSP (Heat Shock Protein).

[0038] According to an embodiment of the present disclosure, the additive for reducing the double helix stability of circular double-stranded DNA includes at least one of formamide, ammonium sulfate, urea, and TMAH (tetramethylammonium hydroxide).

[0039] According to an embodiment of the present disclosure, the additive in the single-chain cyclization reaction includes at least one selected from SSB, non-ionic detergent, betaine, and DMSO.

[0040] According to an embodiment of the present disclosure, the additive in the single-chain cyclization reaction is a mixture of a non-ionic detergent and betaine.

[0041] According to an embodiment of the present disclosure, the additive in the single-chain cyclization reaction is a mixture of a non-ionic detergent and DMSO.

[0042] According to an embodiment of the present disclosure, the additive in the single-chain cyclization reaction is a mixture of SSB and a non-ionic detergent and / or betaine.

[0043] According to an embodiment of the present disclosure, the additive in the rolling circle amplification reaction of the single-stranded circular DNA library includes at least one selected from SSB, a mixture of SSB and a non-ionic detergent and / or betaine.

[0044] According to an embodiment of the present disclosure, the additives in the rolling circle amplification reaction of the double-stranded circular DNA library include at least one selected from SSB and formamide.

[0045] According to an embodiment of the present disclosure, the working concentration of SSB added to the single-stranded circularization reaction is 5 ng / μL-50 ng / μL.

[0046] According to an embodiment of the present disclosure, the working concentration of the non-ionic detergent added to the single-chain cyclization reaction is 0.05%-2.00% (V / V).

[0047] According to an embodiment of the present disclosure, the working concentration of DMSO added to the single-chain cyclization reaction is 1.5%-4.0% (V / V).

[0048] According to an embodiment of the present disclosure, the working concentration of betaine added to the single-chain cyclization reaction is 0.1M-1M.

[0049] According to an embodiment of the present disclosure, the working concentration of SSB added to the rolling circle amplification reaction of the single-stranded circular DNA library is 5 ng / μL-130 ng / μL.

[0050] According to an embodiment of the present disclosure, the working concentration of betaine added to the rolling circle amplification reaction of the single-stranded circular DNA library is 0.1M-1M.

[0051] According to an embodiment of the present disclosure, the working concentration of the non-ionic detergent added to the rolling circle amplification reaction of the single-stranded circular DNA library is 0.05%-2.00% (V / V).

[0052] According to an embodiment of the present disclosure, the working concentration of SSB added to the rolling circle amplification reaction of the double-stranded circular DNA library is 5 ng / μL-130 ng / μL.

[0053] According to an embodiment of the present disclosure, the working concentration of formamide added to the rolling circle amplification reaction of the double-stranded circular DNA library is 1.5-4% (V / V).

[0054] The third aspect of the present disclosure provides a sequencing library prepared by the above method. By using the sequencing library provided by the present disclosure for sequencing, sequencing data with higher sequencing data output and better sequencing quality can be obtained.

[0055] The fourth aspect of the present disclosure provides a sequencing method for performing nucleic acid sequence sequencing using the sequencing library provided in the third aspect.

[0056] The fifth aspect of the present disclosure provides a single-stranded DNA cyclization reaction system, which includes an oligonucleic acid, a DNA ligase, a ligation reaction buffer, and an additive that can reduce the secondary structure of DNA and / or can reduce the secondary structure of DNA while maintaining the stability of single-stranded DNA.

[0057] According to an embodiment of the present disclosure, the DNA ligase includes any one of T4 DNA ligase and Taq ligase.

[0058] According to an embodiment of the present disclosure, the single-stranded circularization sequencing reaction system further includes a sequencing chip, and the oligonucleic acid is fixed on the sequencing chip.

[0059] According to an embodiment of the present disclosure, the single-strand circularization sequencing reaction system further includes magnetic beads, and the oligonucleic acid is fixed on the magnetic beads and / or is free in the magnetic bead suspension.

[0060] According to an embodiment of the present disclosure, the additive capable of reducing the secondary structure of DNA includes at least one of a non-ionic detergent, betaine, and DMSO.

[0061] According to an embodiment of the present disclosure, the non-ionic detergent includes at least one selected from Triton-X100, Tween-20, and NP40.

[0062] According to an embodiment of the present disclosure, the additive capable of reducing DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one of SSB and HSP.

[0063] According to an embodiment of the present disclosure, the additive in the single-chain cyclization reaction system includes at least one selected from SSB, non-ionic detergent, betaine, and DMSO.

[0064] According to an embodiment of the present disclosure, the additive is a mixture of a non-ionic detergent and betaine.

[0065] According to an embodiment of the present disclosure, the additive is a mixture of a non-ionic detergent and DMSO.

[0066] According to an embodiment of the present disclosure, the additive is a mixture of SSB with a non-ionic detergent and / or betaine.

[0067] In a sixth aspect, the present disclosure provides a rolling circle amplification reaction system for a single-stranded circular DNA library, wherein the rolling circle amplification reaction system includes a polymerase, a rolling circle amplification reaction buffer, and an additive capable of reducing DNA secondary structure, and / or an additive capable of reducing DNA secondary structure while maintaining the stability of single-stranded DNA.

[0068] According to an embodiment of the present disclosure, the polymerase includes any one of Phi29 DNA polymerase and Bst DNA polymerase, and the polymerase can be used for rolling circle amplification.

[0069] According to a specific embodiment of the present disclosure, the rolling circle amplification reaction system further includes a sequencing chip, and the rolling circle amplification reaction is performed on the chip surface.

[0070] According to a specific embodiment of the present disclosure, the rolling circle amplification reaction system further comprises magnetic beads, and the rolling circle amplification reaction is performed on the magnetic beads and / or in a magnetic bead suspension.

[0071] According to a specific embodiment of the present disclosure, the additive capable of reducing DNA secondary structure includes at least one of a non-ionic detergent, betaine, and DMSO.

[0072] According to a specific embodiment of the present disclosure, the non-ionic detergent includes at least one selected from Triton-X100, Tween-20, and NP40.

[0073] According to a specific embodiment of the present disclosure, the additive capable of reducing DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one of SSB and HSP.

[0074] According to a specific embodiment of the present disclosure, the additive in the rolling circle amplification reaction system includes at least one selected from SSB, a mixture of SSB and a non-ionic detergent and / or betaine.

[0075] In a seventh aspect, the present disclosure provides a rolling circle amplification reaction system for a double-stranded circular DNA library, wherein the rolling circle amplification reaction system includes a polymerase, a rolling circle amplification reaction buffer, and an additive that can reduce the secondary structure of DNA while maintaining the stability of single-stranded DNA, and / or an additive that can reduce the double helix stability of circular double-stranded DNA.

[0076] According to a specific embodiment of the present disclosure, the additive capable of reducing DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one selected from SSB, a mixture of SSB and a non-ionic detergent and / or betaine.

[0077] According to a specific embodiment of the present disclosure, the additive capable of reducing the double helix stability of circular double-stranded DNA includes at least one of formamide, ammonium sulfate, urea, and tetramethylammonium hydroxide.

[0078] According to an embodiment of the present disclosure, the polymerase includes any one of Phi29 DNA polymerase and Bst DNA polymerase, and the polymerase can be used for rolling circle amplification.

[0079] In an eighth aspect, the present disclosure provides use of any one of the methods described in the first and second aspects, the DNB described in the third aspect, the single-chain cyclization reaction system described in the fifth aspect, the rolling circle amplification reaction system described in the sixth aspect, and the rolling circle amplification reaction system described in the seventh aspect in sequencing.

[0080] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0082] FIG1 shows the GC-Bias results of optimized sequencing of the methylated single-stranded circle library in Example 1 of the present disclosure;

[0083] FIG2 shows the GC-Bias results of optimized sequencing of the methylated single-stranded circle library in Example 2 of the present disclosure;

[0084] FIG3 shows the GC-Bias results of optimized sequencing of the methylated single-stranded circle library in Example 11 of the present disclosure;

[0085] FIG4 shows the GC-Bias results of optimized sequencing of the methylated single-stranded circle library in Example 12 of the present disclosure;

[0086] FIG5 shows the GC-Bias results of optimized sequencing of the methylated double-stranded circle library in Example 13 of the present disclosure.

[0087] Detailed Description of the Invention

[0088] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0089] 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 being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0090] In this disclosure, the term "SSB" refers to a single-stranded DNA binding protein (SSB), which has a high affinity for single-stranded DNA (ssDNA) and can wrap around ssDNA, thereby preventing DNA degradation. SSB can also maintain the single strands of circular DNA and prevent the formation of secondary structures in ssDNA.

[0091] The present disclosure provides a method for preparing DNB, the method comprising:

[0092] (a) performing a cyclization reaction on a stranded DNA library to obtain a circular DNA library, wherein the cyclization reaction includes a single-stranded cyclization reaction or a double-stranded cyclization reaction, and the circular DNA library includes a single-stranded circular DNA library or a double-stranded circular DNA library;

[0093] (b) performing rolling circle amplification on the circular DNA library to obtain DNBs,

[0094] in,

[0095] When the cyclization reaction in step (a) is a single-chain cyclization reaction,

[0096] Step (a) further comprises: adding at least one of the following additives to the single-stranded circularization reaction to obtain the single-stranded circular DNA library:

[0097] 1) Additives that can reduce DNA secondary structure,

[0098] 2) additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0099] And / or step (b) further comprises: performing a rolling circle amplification reaction using the single-stranded circular DNA library produced as a product of step (a), wherein at least one of the following additives is added to the rolling circle amplification reaction:

[0100] 3) Additives that can reduce DNA secondary structure,

[0101] 4) additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0102] When the cyclization reaction in step (a) is a double-chain cyclization reaction,

[0103] Step (b) further comprises: performing a rolling circle amplification reaction using the double-stranded circular DNA library produced in step (a), wherein at least one of the following additives is added to the rolling circle amplification reaction:

[0104] 5) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA,

[0105] 6) Additives that can reduce the double helix stability of circular double-stranded DNA.

[0106] In another aspect, the present disclosure provides a method for rolling circle amplification, wherein a DNA library is subjected to rolling circle amplification reaction to obtain single-stranded linear DNA.

[0107] Wherein, when the DNA library is a single-stranded circular DNA library, at least one of the following additives is added to the rolling circle amplification reaction:

[0108] 7) Additives that can reduce DNA secondary structure,

[0109] 8) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA;

[0110] When the DNA library is a double-stranded circular DNA library, at least one of the following additives is added to the rolling circle amplification reaction:

[0111] 9) Additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA,

[0112] 10) Additives that can reduce the double helix stability of circular double-stranded DNA.

[0113] To address the low circularization efficiency of circular libraries, this paper significantly improves the yield of circularized libraries and the uniformity of coverage across different GC regions by using additives that facilitate secondary structure unwinding in the circularization system. In particular, coverage of high GC regions in methylated single-stranded circular libraries is significantly enhanced, which helps improve the accuracy of methylation rate detection.

[0114] In particular, to address the issues of small amount and low quality of sequencing data from circular methylation libraries, the present invention, based on the optimization of algorithms and sequencing scripts, introduces a method of adding additives to the rolling circle amplification process of circular libraries, which further significantly improves the amount and quality of sequencing data from single-stranded and double-stranded circular methylation libraries.

[0115] In the process of single-stranded cyclization and circular library rolling circle amplification, the double-stranded structure of DNA is opened, and single-stranded DNA easily forms a secondary structure of intramolecular folding, which blocks the reaction binding site of ligase and / or polymerase, resulting in a decrease in ligation efficiency and / or polymerization reaction efficiency. The present invention utilizes additives and additive combinations that can reduce DNA secondary structure, and adds them to the cyclization reaction and / or the circular library rolling circle amplification reaction, so that the cyclization efficiency and / or sequencing quality of the circular library are significantly improved. At the same time, the additive that protects the stability of single-stranded DNA protects some single-stranded DNA that is too loose (such as a methylated library that converts non-methylated C into U) from being easily degraded during the reaction, so that the cyclization efficiency and / or sequencing quality of the circular library are significantly improved.

[0116] In particular, during rolling circle amplification of a double-stranded circularized library, although the DNA polymerase capable of rolling circle amplification has a melt function, some difficult-to-melt regions (such as high GC regions) may not melt effectively. The present disclosure utilizes additives that can reduce the stability of the master DNA double helix to improve the melting efficiency of isothermal rolling circle amplification, effectively initiating replication in the difficult-to-melt regions and improving the sequencing quality of the library.

[0117] In some specific embodiments, the present disclosure provides a method for improving the efficiency of single-chain circularization, which is as follows:

[0118] 1) Homogenize the double-stranded DNA with TE buffer or sterile water;

[0119] 2) Perform a high-temperature thermal denaturation reaction on a temperature-controlled instrument (e.g., PCR instrument, thermostat, water bath, etc.) to melt the double-stranded DNA into single strands;

[0120] 3) Rapidly place the denatured reaction mixture on ice or cool it to 4°C, and add the cyclization reaction mixture. The cyclization reaction mixture contains a splint oligo complementary to the ends of the single-stranded DNA adapter, DNA ligase (e.g., T4 DNA ligase), a cyclization buffer compatible with the ligase, and an additive that reduces DNA secondary structure, an additive that reduces DNA secondary structure while maintaining the stability of single-stranded DNA, or a combination of these two additives.

[0121] 4) Perform a single-chain cyclase reaction on a temperature controller (e.g., PCR instrument, thermostat, water bath, etc.);

[0122] 5) After the reaction is completed, place on ice or quickly cool to 4°C;

[0123] 6) Depending on the sequencing application's requirements for library construction time and library purity, you can choose to perform linear digestion followed by purification of the circular DNA library, or you can skip linear digestion and directly take a portion of the circularization reaction mixture for the next step of library amplification.

[0124] 7) If you choose to perform linear digestion followed by purification of the circular DNA library, you will need to use a fluorescence quantification instrument and supporting quantitative reagents to quantify the concentration of the single-stranded circular library and calculate the circularization efficiency;

[0125] Circularization efficiency = total amount of purified single-stranded circularized library / total amount of circularized double-stranded DNA input,

[0126] Among them, additives that reduce DNA secondary structure include Triton-X100, Tween-20, betaine, DMSO, etc., and additives that reduce DNA secondary structure and protect single-strand stability include SSB or additives or additive combinations with equivalent effects, and additive combinations include but are not limited to SSB+Triton-X100, SSB+Tween-20, SSB+Triton-X100+Tween-20, etc.

[0127] In addition to the methods described in the aforementioned examples, methods for improving single-strand circularization efficiency also include other similar modified methods, such as adding additives that can reduce DNA secondary structure and / or additives that can reduce DNA secondary structure while maintaining the stability of single-stranded DNA in step 2), not adding additives in step 3), or adding the aforementioned additives or combinations of additives in both steps 2) and 3). Modified methods, such as replacing the T4 DNA ligase used in step 3) with Taq DNA ligase, should be noted that this operation requires cooling the denatured reaction solution to the optimal reaction temperature of Taq DNA ligase (e.g., 45°C).

[0128] In some specific embodiments, the present disclosure provides a technical solution for improving the sequencing quality of circular methylation libraries as follows:

[0129] ① Homogenize the circular methylation library (circularization methods include single-strand circularization, the above-mentioned single-strand circularization optimization scheme, and double-strand circularization) with Low TE buffer or sterile water;

[0130] ② If it is a single-stranded circular library, it is necessary to add rolling circle amplification primers complementary to the circularized adapter and perform high-temperature thermal denaturation reaction and primer hybridization reaction on a temperature-controlled instrument (such as a PCR instrument, constant temperature reactor, water bath, etc.). If it is a double-stranded circular library with one chain closed and one chain gapped, proceed directly to the next step;

[0131] ③ Add the rolling circle replication reaction mixture, mix thoroughly, centrifuge, and then perform the rolling circle amplification reaction on a temperature-controlled instrument (e.g., a PCR instrument, a thermostat, a water bath, etc.). The rolling circle replication reaction mixture for the single-stranded circularization library contains a polymerase for rolling circle amplification, an amplification reaction buffer compatible with the polymerase, and an additive that reduces DNA secondary structure while maintaining the stability of single-stranded DNA. The rolling circle replication reaction mixture for the double-stranded circularization library contains a polymerase for rolling circle amplification, an amplification reaction buffer compatible with the polymerase, and an additive that reduces DNA secondary structure while maintaining the stability of single-stranded DNA, and / or an additive that reduces the double helix stability of circular double-stranded DNA.

[0132] ④ Add the stop reaction solution and mix the amplified product by slowly pipetting with a wide-mouth pipette tip;

[0133] ⑤ Use a fluorescence quantitative instrument and supporting quantitative reagents to quantify the single-strand concentration, and calculate the sampling volume of the amplified library loaded onto the sequencing chip according to the needs of the sequencing platform;

[0134] ⑥ Sequencing of circular methylation libraries using a sequencer optimized for base imbalance algorithm and sequencing script.

[0135] Among them, additives that reduce the secondary structure of DNA while maintaining the stability of single-stranded DNA include SSB or additives or additive combinations with equivalent effects, additives that reduce the secondary structure of DNA include betaine or additives or additive combinations with equivalent effects, and additives that reduce the double helix stability of circular double-stranded DNA include formamide or additives or additive combinations with equivalent effects.

[0136] In some specific embodiments, the above-mentioned technical solutions and modified methods for improving the circularization efficiency and sequencing quality of circular methylation libraries are also applicable to whole-genome WGS libraries.

[0137] According to the embodiments of the present disclosure, when the working concentration of SSB is between 5ng / μL-130ng / μL, the sequencing quality and data output of the library can be greatly improved. Preferably, the working concentration of SSB is 15ng / μL-50ng / μL for the best effect.

[0138] According to the embodiments of the present disclosure, when the working concentration of betaine is between 0.1M and 1M, the high GC region coverage of the library can be greatly improved. Preferably, the working concentration of betaine is 0.4M-0.7M for the best effect.

[0139] According to the embodiments of the present disclosure, when the working concentration of formamide is between 0.5-4% (V / V), the sequencing quality and data output of the library can be greatly improved. Preferably, the working concentration of formamide is 2-3% (V / V) for the best effect.

[0140] During the single-stranded DNA circularization process, the addition of additives that reduce DNA secondary structure, or additives that reduce DNA secondary structure and maintain single-stranded DNA stability, or a combination of additives, can significantly improve the yield and circularization efficiency of single-stranded circle libraries. This allows a single single-stranded circle library to be sequenced multiple times, eliminating the need for repeated library construction, saving library construction costs and time, and further reducing the starting amount of library construction.

[0141] During the rolling circle replication process of the circular methylation library, the additional addition of additives that can reduce the secondary structure of DNA can reduce the impact of secondary structures (such as stem-loop structures, continuous base sequences, palindromic sequences, etc.) on the efficiency of rolling circle amplification, improve the uniformity of the rolling circle amplification efficiency of different circular DNA molecules, and reduce the signal differences of different circular DNA molecules amplified into DNBs, thereby improving the quality of sequencing data and reducing the coverage bias of sequencing data. In particular, after most of the C in the single-stranded methylation library is converted to T, the sequencing signal of the C channel is low, and there is also the problem of poor sequencing quality, although this problem can be solved by optimizing the sequencing algorithm. However, since most of the C in this type of library is converted to T, although the probability of forming a secondary structure is lower than that of a conventional library, this loose configuration has the problem of being easily degraded during the reaction process, resulting in the inability to produce normal DNB products and signal loss during sequencing. The present invention further improves the sequencing data quality, yield, and uniformity of sequencing data coverage of single-stranded circularized libraries (particularly methylated libraries, including whole-genome WGS libraries, etc.) by adding additives or additive combinations that can both open up DNA secondary structures and protect single-stranded loose DNA from degradation. The present invention further improves the sequencing data quality, yield, and uniformity of sequencing data coverage of double-stranded circularized libraries (particularly methylated libraries, including whole-genome WGS libraries, etc.) by adding additives or additive combinations that can both open up DNA secondary structures and protect single-stranded loose DNA from degradation and / or additives that reduce the double helix stability of circular double-stranded DNA.

[0142] This allows a methylation library to generate more data using fewer sequencing reagents, reducing the sequencing cost of the methylation library. Furthermore, the improved uniformity of sequencing data further increases the detection rate of difficult regions in methylation libraries and whole-genome WGS libraries, and improves sequencing accuracy.

[0143] The additives used in the present disclosure are not limited to SSB, Triton-X100, Tween-20, betaine, DMSO, NP40, and formamide single-dose formulations of different concentrations and cross-combined mixed formulations of different concentrations. It can also be other additives and combination formulations with equivalent effects that can reduce the secondary structure of single-stranded library DNA or can both reduce the secondary structure of single-stranded library DNA and protect the single strand from degradation. In particular, during the rolling circle amplification of the double-stranded circularized library, it can also be other additives or combinations of additives with equivalent effects that can reduce the double helix stability of the circular double-stranded DNA. In addition, in the steps of circularization (including those that do not consume linear DNA and those that consume linear DNA), library replication and amplification steps, circularization + library replication one-tube reaction steps, sequencing hybridization reaction steps, etc., where it is necessary to reduce the interference of secondary structure to improve the reaction efficiency and reaction uniformity, the conversion efficiency and sequencing quality of the circularized library can also be improved by fine-tuning the reaction temperature or fine-tuning the reaction temperature with different formulations of additives.

[0144] The library types used in the present disclosure are not limited to whole genome WGS PCR-free, PCR library, whole genome methylation library, third-party whole genome methylation library, but can also be target region capture library, target region methylation library, amplicon type library (such as multiplex PCR, long fragment PCR, olink amplicon, immune library amplicon, 16s amplicon, HPV amplicon, etc.), RNA library, stLFR library and other application type libraries.

[0145] The optimization effect disclosed herein is applicable to sequencing platforms not limited to the MGISEQ-2000 platform (also known as DNBSEQ-G400). Other platforms based on the same DNB sequencing technology, such as DNBSEQ-E5, DNBSEQ-E25, DNBSEQ-G50, NBSEQ-G99, NBSEQ-G200, DNBSEQ-T7, DNBSEQ-T10, DNBSEQ-T20, etc., which are on sale, under development, or in the future, as well as sequencing platforms based on similar circular library sequencing principles, may also be achievable.

[0146] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0147] Example 1 Optimization experiment-1 to improve the single-strand circularization efficiency and high GC region coverage uniformity of the whole genome methylation library

[0148] The reagents used in this experiment are shown in Table 1

[0149] Table 1 Reagents required for single-strand circularization experiment of whole genome methylation library

[0150] The specific experimental methods are as follows:

[0151] 1) Transfer 163.8 ng of PCR product to a new 0.2 mL PCR tube and add TE Buffer to a total volume of 48 μL.

[0152] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 5 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0153] 3) Prepare the single-stranded cell cyclization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step. For the control group, do not add SSB. For the experimental group, add 1 μL / tube or 1.5 μL / tube of SSB (500 ng / uL). Mix and centrifuge. Place the PCR tube in a thermal cycler and perform the single-stranded cell cyclization reaction at 37°C for 30 min; hold at 4°C.

[0154] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0155] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0156] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0157] 7) Purify the DNA using 170 μL of MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 22 μL of TE Buffer, and transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0158] 8) Use The ssDNA Assay Kit was used for single-stranded circle quantification. The quantitative results are shown in Table 2. Compared with the control group without SSB addition, the circularization efficiency of the methylated single-stranded circle library with SSB addition increased by at least 5 percentage points.

[0159] Table 2 Single-strand circularization results of whole genome methylation library before and after optimization

[0160] The constructed whole-genome methylated single-stranded circular DNA library was prepared using DNA nanoballs and sequenced on the MGISEQ-2000PE100. Sequencing was performed according to the MGISEQ-2000PE100 standard protocol. GC bias analysis of the off-line data, shown in Figure 1, demonstrates significant improvement in GC coverage uniformity in the optimized groups supplemented with 1 μl or 1.5 μl of SSB (500 ng / μL) compared to the control group without SSB.

[0161] Example 1 shows that the additional addition of Tth SSB at a working concentration of 8.18-12.18 ng / uL in the single-strand circularization reaction can improve the single-strand circularization efficiency of the whole genome methylation library and improve the coverage uniformity of the high GC regions of the library.

[0162] Example 2: Optimization experiment 2 to improve single-strand circularization efficiency and high GC region coverage uniformity of whole genome methylation library

[0163] The reagents used in this experiment are shown in Table 3

[0164] Table 3 Reagents required for genome-wide methylation single-strand circularization experiments

[0165] The specific experimental methods are as follows:

[0166] 1) Transfer 300 ng of PCR product to a new 0.2 mL PCR tube and perform the experiment as shown in Table 4. For the control group, no additives were added and the total volume was made up to 48 μL with TE Buffer. For experimental group 1, 6 μL of 5 M betaine was added and the total volume was made up to 42 μL with TE Buffer. For experimental groups 2 and 3, 2.5 μL of 10% Tween-20 and 6 μL of 5 M betaine were added and the total volume was made up to 39.5 μL with TE Buffer.

[0167] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 5 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0168] 3) Prepare the single-stranded circularization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step. Then, add 2 μL / tube of SSB (500 ng / uL) to the product from Experimental Protocol 3. Mix well and centrifuge. Place the PCR tube in a PCR machine and perform the single-stranded circularization reaction: 37°C for 30 min; hold at 4°C.

[0169] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0170] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0171] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0172] 7) Purify the DNA using 170 μL of MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 22 μL of TE Buffer, and transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0173] 8) Use Single-stranded circularization was quantified using the ssDNA Assay Kit, and the results are shown in Table 4. Compared with the control group without the addition of other reagents, the single-stranded circularization efficiency of the whole-genome methylation library in the experimental groups with the addition of betaine, betaine + Tween-20, and betaine + Tween-20 + SSB was significantly improved.

[0174] Table 4 Single-strand circularization results of whole genome methylation library before and after optimization

[0175] The constructed whole-genome methylated single-stranded circular DNA library was prepared using DNA nanoballs and sequenced on the MGISEQ-2000PE100. Sequencing was performed according to the MGISEQ-2000PE100 standard operating procedure. GC bias analysis of the off-line data, shown in Figure 2, demonstrates improved GC coverage uniformity in the optimized groups supplemented with betaine, Tween-20 + betaine, and Tween-20 + betaine + SSB, compared to the control group without additional reagents.

[0176] Example 2 demonstrates that the additional combined formulation, when the working concentrations of the single-strand circularization reaction are 0.48-0.50 M betaine, 0.40%-0.42% Tween-20, and 16.10 ng / uL Tth SSB, can improve the efficiency of single-strand circularization of the whole-genome methylation library and enhance the coverage uniformity of high GC regions in the library. Tth SSB contributes most significantly to improving high GC coverage.

[0177] Example 3: Optimization experiment to improve single-strand circularization efficiency of whole genome methylation library-3

[0178] The reagents used in this experiment are shown in Table 5.

[0179] Table 5 Reagents required for single-strand circularization experiment of whole genome methylation library

[0180] The specific experimental methods are as follows:

[0181] 1) Transfer 200 ng of PCR product to a new 0.2 mL PCR tube and add various additives according to Table 6. For PCR products of test numbers 1 to 4, make up to 48 μL with TE Buffer.

[0182] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 5 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0183] 3) Prepare the single-stranded circularization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step. Mix thoroughly, centrifuge, and place the PCR tube in a thermal cycler for single-stranded circularization: 37°C for 30 min; hold at 4°C.

[0184] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0185] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0186] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0187] 7) Purify the DNA using 170 μL MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 27 μL TE Buffer, and transfer 25 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0188] 8) Single-stranded circle quantification was performed using the Qubit ssDNA Assay Kit. The quantitative results are shown in Table 6. Compared with the control group, the concentration and circularization efficiency of the methylated single-stranded circle library in the experimental groups with different additive test schemes were improved to a certain extent, with experimental group 3 showing a relatively significant improvement.

[0189] Table 6 Single-strand circularization results of whole genome methylation library before and after optimization

[0190] Example 3 shows that the additional additives and / or combination of additives can improve the efficiency of single-strand circularization of the whole genome methylation library when the working concentrations of the single-strand circularization reaction are 2.50% DMSO, 0.83% Triton X-100, and 0.42% Tween-20, respectively.

[0191] Example 4: Optimization experiment for improving single-strand circularization efficiency of whole-genome PCR-free library-1

[0192] The reagents used in this experiment are shown in Table 7

[0193] Table 7 Reagents required for whole genome PCR-free library single-stranded circularization experiment

[0194] The specific experimental methods are as follows:

[0195] 1) Transfer 150 ng of whole-genome PCR-free ligation product (after shearing and double selection) to a new 0.2 mL PCR tube and add TE Buffer to a total volume of 48 μL.

[0196] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0197] 3) Prepare the single-stranded cell cyclization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step, as shown in Table 8. For the experimental group, add an additional 1 μL / tube, 1.5 μL / tube, or 2 μL / tube of SSB (500 ng / uL). Mix and centrifuge. Place the PCR tube in a thermal cycler and perform the single-stranded cell cyclization reaction at 37°C for 30 min; hold at 4°C.

[0198] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0199] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0200] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0201] 7) Purify the DNA using 170 μL MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 22 μL TE Buffer, and transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0202] 8) Use Single-stranded circularization was quantified using the ssDNA Assay Kit. The results are shown in Table 8. Compared to the control group without SSB, the concentration and circularization efficiency of the whole-genome PCR-free single-stranded circularization library with varying volumes of SSB increased significantly by approximately 10%. The addition of 1 μL of SSB (500 ng / μL) achieved relatively ideal results.

[0203] Table 8 Single-stranded circularization results of whole genome PCR-free libraries before and after optimization

[0204] Example 4 shows that the additional addition of Tth SSB at a working concentration of 8.18-16.10 ng / uL in the single-strand circularization reaction can significantly improve the single-strand circularization efficiency of the whole genome PCR-free library, and saturation is reached at a working concentration of Tth SSB of 8.18 ng / uL.

[0205] Example 5: Optimization experiment 2 to improve the efficiency of single-stranded circularization of whole genome PCR-free library

[0206] The reagents used in this experiment are shown in Table 9

[0207] Table 9 Reagents required for single-stranded circularization experiment of whole genome PCR-free library

[0208] The specific experimental methods are as follows:

[0209] 1) Transfer 96.6 ng of whole-genome PCR-free ligation product (single selection after interruption) to a new 0.2 mL PCR tube, as shown in Table 10. For the control group, no additives were added and the tube was filled with TE Buffer to a total volume of 48 μL. For experimental groups 1 and 2, 2.5 μL and 5 μL of 10% Tween-20 were added, respectively, and the tube was filled with TE Buffer to a total volume of 45.5 μL and 43 μL, respectively. For experimental groups 3 and 4, 2.5 μL and 5 μL of 20% Triton-X100 were added, respectively, and the tube was filled with TE Buffer to a total volume of 45.5 μL and 43 μL, respectively. For experimental groups 5 and 6, 2.5 μL and 5 μL of 10% NP40 were added, respectively, and the tube was filled with TE Buffer to a total volume of 45.5 μL and 43 μL, respectively.

[0210] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 5 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0211] 3) Prepare the single-stranded circularization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform the single-stranded circularization reaction at 37°C for 30 minutes; hold at 4°C.

[0212] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0213] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0214] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0215] 7) Purify the DNA using 170 μL of MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 22 μL of TE Buffer, and transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0216] 8) Use Single-stranded circularization was quantified using the ssDNA Assay Kit, and the results are shown in Table 10. Compared with the control group without the addition of other reagents, the experimental group with the addition of Tween-20, Triton-X100, and NP40 showed a certain degree of improvement in the single-stranded circularization concentration and circularization efficiency of the whole-genome methylation library.

[0217] Table 10 Single-stranded circularization results of whole genome PCR-free libraries before and after optimization

[0218] Example 5 shows that the additional addition of Tween-20, Triton-X100, and NP40 at a working concentration of 0.42-0.84% ​​in the single-strand circularization reaction can significantly improve the single-strand circularization efficiency of the whole genome PCR-free library.

[0219] Example 6: Optimization experiment 2 to improve the efficiency of single-stranded circularization of whole genome DNA library (PCR library construction method)

[0220] The reagents used in this experiment are shown in Table 11

[0221] Table 11 Reagents required for single-stranded circularization experiment of whole genome DNA library (PCR library construction method)

[0222] The specific experimental methods are as follows:

[0223] 1) Transfer 155.5 ng of WGS PCR product (double barcoded) to a new 0.2 mL PCR tube as shown in Table 12. For the control group, no additives were added and the total volume was made up to 48 μL with TE Buffer. For experimental groups 1, 2, and 3, 2.5 μL of 10% Tween-20, 2.5 μL of 20% Triton-X100, and 2.5 μL of 10% NP40 were added, respectively, and the total volume was made up to 45.5 μL with TE Buffer.

[0224] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 5 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0225] 3) Prepare the single-stranded cell circularization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step. Mix well and centrifuge. Place the PCR tube in a PCR instrument and perform the single-stranded cell circularization reaction at 37°C for 30 minutes; hold at 4°C.

[0226] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0227] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0228] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0229] 7) Purify the DNA using 170 μL MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 22 μL TE Buffer, and transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0230] 8) Use Single-stranded circularization was quantified using the ssDNA Assay Kit, and the results are shown in Table 12. Compared with the control group without the addition of other reagents, the experimental group with the addition of Tween-20, Triton-X100, and NP40 showed a certain improvement in the single-stranded circularization concentration and circularization efficiency of the whole-genome methylation library.

[0231] Table 12 Single-stranded circularization results of whole genome DNA library (PCR library construction method) before and after optimization

[0232] Example 6 shows that the additional addition of Tween-20, Triton-X100, and NP40 at a working concentration of 0.42% in the single-stranded circularization reaction can slightly improve the single-stranded circularization efficiency of the whole genomic DNA library (double barcode).

[0233] Example 7: Optimization experiment for improving the sequencing quality of whole-genome methylation double-stranded circularized library

[0234] The reagents used in this experiment are shown in Table 13

[0235] Table 13 Reagents required for DNB preparation and sequencing of whole genome methylation double-stranded circle library

[0236] The specific experimental methods are as follows:

[0237] 1) Add 26.4 ng of the whole-genome methylated double-stranded circularized library to a new 0.2 mL PCR tube. Bring the volume to 10 μL with Low TE Buffer. Add the following reagents sequentially on ice: 10 μL WGBS DNB Buffer, 20 μL DNB polymerase mix I, and 2 μL DNB polymerase mix II (LC), as shown in Table 14. For the experimental group, add X μL SSB (2 ug / uL) (X can be 0.5 μL, 1 μL, 1.5 μL, or 2 μL).

[0238] 2) Mix well and centrifuge, then perform DNB preparation reaction on a PCR instrument at 30°C for 15 min, and hold at 4°C;

[0239] 3) When the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not vortex or pipette vigorously. Store at 4°C until needed.

[0240] 4) Use ssDNA Assay Kit fluorescent quantification kit for single-stranded circle quantification;

[0241] 5) Then, 450 ng of DNB was loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE150 (with optimized sequencing algorithm and sequencing script);

[0242] 6) The sequencing results are shown in Table 14. With the increase in the volume of additional Tth SSB (2ug / uL), the total reads yield and total Q30% of the sequencing were improved and approached the sequencing data volume and Q30% of the PCR Free control library.

[0243] Table 14 DNB preparation optimization sequencing results of the whole genome methylation double-stranded circle library MGISEQ-2000PE150 optimized version (optimized base imbalance sequencing algorithm and corresponding sequencing script)

[0244] Example 7 shows that when the working concentration of additional Tth SSB in the DNB preparation reaction is 23.53-90.91 ng / uL, sequencing using the base imbalance sequencing algorithm and the corresponding optimized MGISEQ-2000 sequencing script can significantly improve the sequencing quality and sequencing data output of the whole genome methylation double-stranded circularized library.

[0245] Example 8: Optimization experiment for improving the sequencing quality of whole-genome methylation single-stranded circularized library

[0246] The reagents used in this experiment are shown in Table 15

[0247] Table 15 Reagents required for DNB preparation and sequencing of whole genome methylation single-stranded circle library

[0248] The specific experimental methods are as follows:

[0249] 1) Take a new 0.2 mL PCR tube, add 12 ng of the whole genome methylation single-stranded circularized library, add Low TE Buffer to the volume to 10 μL, and add 10 μL of DNB preparation buffer;

[0250] 2) Mix well and centrifuge. Perform denaturation and annealing reactions on a PCR instrument: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C.

[0251] 3) After the reaction is complete, centrifuge briefly and add the following reagents to the previous PCR tubes on ice in order: 20 μL DNB polymerase mix I, 2 μL DNB polymerase mix II (LC), as shown in Table 16. For the experimental group, add an additional 1 μL SSB (2 μg / uL).

[0252] 4) Mix well and centrifuge, then perform DNB preparation reaction on a PCR instrument at 30°C for 15 min, and hold at 4°C;

[0253] 5) When the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not shake or pipette vigorously. Store at 4°C until needed.

[0254] 6) Use ssDNA Assay Kit fluorescent quantification kit for single-stranded circle quantification;

[0255] 7) Then, 450 ng / lane DNB was loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE150 (with optimized sequencing algorithm and sequencing script);

[0256] 8) Sequencing Results As shown in Table 16, the sequencing quality of the methylation library prepared with 1 μL of SSB (2 μg / μL) added to the DNB preparation reaction was significantly improved in terms of total read yield and total Q30% compared to the control group without SSB addition, and was close to the sequencing data yield and Q30% of the WGS PCR Free control library.

[0257] Table 16 DNB preparation optimization sequencing results of the optimized version of the whole genome methylation single-chain circle library MGISEQ-2000PE150 (optimized sequencing algorithm and sequencing script)

[0258] Example 8 shows that when the working concentration of additional Tth SSB in the DNB preparation reaction is 46.51 ng / uL, sequencing using the base imbalance sequencing algorithm and the corresponding optimized MGISEQ-2000 sequencing script can significantly improve the sequencing quality and sequencing data output of the whole genome methylation single-stranded circularized library.

[0259] Example 9: Experimental study on improving circularization efficiency and sequencing quality of whole genome methylation single-strand circularization library

[0260] The reagents used in this experiment are shown in Table 17

[0261] Table 17 Reagents required for DNB preparation and sequencing of whole genome methylation single-stranded circle library

[0262] The specific experimental methods are as follows:

[0263] 1) Take a new 0.2 mL PCR tube and add 13 ng of the whole-genome methylation single-stranded circularized library. As shown in Table 18, the single-stranded circularized libraries of the control group and Optimization Protocol 2 were prepared in the same tube, and no additional additives were added during single-stranded circularization. Optimization Protocols 1 and 3 were prepared in the same tube, but 1.5 μL of SSB (500 ng / μL) was added during single-stranded circularization. The volume was brought to 10 μL with Low TE Buffer, and 10 μL of DNB preparation buffer was added.

[0264] 2) Mix well and centrifuge. Perform denaturation and annealing reactions on a PCR instrument: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C.

[0265] 3) After the reaction is complete, centrifuge briefly and add the following reagents to the previous PCR tubes on ice: 20 μL DNB polymerase mix I, 2 μL DNB polymerase mix II (LC), and, if needed, 1 μL SSB (500 ng / uL).

[0266] 4) Mix well and centrifuge, then perform DNB preparation reaction on a PCR instrument at 30°C for 15 min, and hold at 4°C;

[0267] 5) When the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not shake or pipette vigorously. Store at 4°C until needed.

[0268] 6) Use ssDNA Assay Kit fluorescent quantification kit for single-stranded circle quantification;

[0269] 7) Then, 450 ng / lane DNB was loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE100 (with optimized sequencing algorithm and sequencing script);

[0270] 8) Sequencing results, as shown in Table 18, show that the sequencing quality, total read yield, and total Q30% of the methylation library with the addition of 1 μL SSB (500 ng / uL) during make DNB (Optimization Protocol 2) were significantly improved compared to the control group without SSB. Furthermore, compared to the control group, the addition of 1.5 μL SSB (500 ng / uL) during circularization and 1 μL SSB (500 ng / uL) during make DNB (Optimization Protocol 3) significantly improved both circularization efficiency and sequencing quality.

[0271] Table 18 DNB preparation optimization sequencing results of the whole genome methylation single-chain circle library MGISEQ-2000PE100 optimized version (optimized sequencing algorithm and sequencing script)

[0272] Example 9 demonstrates that the addition of Tth SSB at a working concentration of 12.18 ng / uL in the single-strand circularization reaction significantly improves the circularization efficiency of a whole-genome methylation single-strand circular library. Furthermore, the addition of Tth SSB at a working concentration of 11.63 ng / uL in the DNB preparation reaction improves the sequencing quality and data output of a whole-genome methylation single-strand circular library using an MGISEQ-2000 sequencing system optimized for base imbalance sequencing and the corresponding sequencing script. Adding Tth SSB to both the single-strand circularization reaction and the DNB preparation reaction improves both the circularization efficiency and the sequencing quality and data output of a whole-genome methylation single-strand circular library.

[0273] Example 10: Optimization experiment on the APP-A conversion circularization efficiency and sequencing quality improvement of a third-party whole-genome methylation library (based on Illumina adapter construction)

[0274] The reagents used in this experiment are shown in Table 19

[0275] Table 19 Reagents required for third-party whole genome methylation library App-A conversion, single-stranded circle library preparation and sequencing

[0276] The specific experimental methods are as follows:

[0277] 1) Take 231 ng of third-party whole-genome methylation App-A conversion PCR product based on Illumina adapter library construction (two PCR protocols, the first protocol is PCR 6 cycles, the second protocol is PCR 8 cycles) into a new 0.2 mL PCR tube and add TE Buffer to a total volume of 48 μL;

[0278] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 5 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0279] 3) Prepare the single-stranded cyclization reaction solution: Add 11.6 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step, as shown in Table 20. No additives were added to the control group, while an additional 1.5 μL of SSB was added to the optimized group. Mix and centrifuge. Place the PCR tube in a thermal cycler and perform the single-stranded cyclization reaction at 37°C for 30 min; hold at 4°C.

[0280] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0281] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 30 minutes; hold at 4°C.

[0282] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0283] 7) Purify the DNA using 170 μL of MGIEasy DNA Purification Magnetic Beads, dissolve the DNA in 22 μL of TE Buffer, and transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0284] 8) Use Single-stranded circularization was quantified using the ssDNA Assay Kit, and the quantitative results are shown in Table 20. Compared with the circularization control group without SSB addition, the circularization efficiency of the single-stranded circularization library in the circularization optimization group with SSB addition was significantly improved.

[0285] Table 20 Single-stranded circularization results of PCR products converted from third-party whole genome methylation libraries before and after optimization

[0286] 9) DNB preparation and sequencing: 12 ng of the constructed single-stranded circular DNA library was used for DNB preparation. 450 ng / lane DNB was loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE150 (with optimized sequencing algorithm and sequencing script).

[0287] 10) Sequencing results As shown in Table 21, the total reads yield and total Q30% of the optimized group third-party methylation library with the addition of 1.5 μL SSB (500 ng / uL) in the single-strand circularization step were significantly improved compared to the control group without addition.

[0288] Table 21 Sequencing results of third-party whole genome methylation single-stranded circularization libraries before and after optimization on the MGISEQ-2000PE150 optimized version (optimized sequencing algorithm and sequencing script)

[0289] Example 10 shows that when the working concentration of the additional Tth SSB in the single-strand circularization reaction is 12.18 ng / uL, the circularization efficiency of the whole genome methylation single-stranded circular library converted based on the Illumina library construction method can be significantly improved, and the sequencing quality and sequencing data output can be significantly improved.

[0290] Example 11: Optimization experiment for improving the sequencing quality of whole-genome methylation single-stranded circularized library

[0291] The reagents used in this experiment are shown in Table 22

[0292] Table 22 Reagents required for DNB preparation and sequencing of whole genome methylation single-stranded circle library

[0293] The specific experimental methods are as follows:

[0294] 1) Add 8 ng of the whole-genome methylated single-stranded circularized library to a new 0.2 mL PCR tube. For Experimental Groups 1, 2, and 3, add 2.5 μL of 5% DMSO, 1 μL of 100% formamide, and 5 μL of 5 M betaine, as listed in Table 23. Bring the volume to 10 μL with Low TE Buffer and add 10 μL of DNB Preparation Buffer.

[0295] 2) Mix well and centrifuge. Perform denaturation and annealing reactions on a PCR instrument: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C.

[0296] 3) After the reaction is complete, centrifuge briefly and add the following reagents to the previous PCR tubes on ice: 20 μL DNB polymerase mix I and 2 μL DNB polymerase mix II (LC);

[0297] 4) Mix well and centrifuge, then perform DNB preparation reaction on a PCR instrument at 30°C for 15 min, and hold at 4°C;

[0298] 5) When the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not shake or pipette vigorously. Store at 4°C until needed.

[0299] 6) Use DNB quantification was performed using the ssDNA Assay Kit fluorescent quantification kit;

[0300] 7) All DNBs were loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE100 (with optimized sequencing algorithm and sequencing script);

[0301] 8) The sequencing results are shown in Table 23. Compared with the control group without additives during DNB preparation, the experimental group with the addition of three additives (DMSO, formamide, and betaine) did not significantly improve the sequencing quality and total read yield of the methylation library.

[0302] 9) At the same time, as shown in Figure 3, the sequencing quality (GC-Bias) was significantly improved when 5 μl of 5M betaine was added during DNB preparation (experimental group solution 3) compared to the control group.

[0303] Table 23 DNB preparation optimization sequencing results of the whole genome methylation single-chain circle library MGISEQ-2000PE100 optimized version (optimized sequencing algorithm and sequencing script)

[0304] Example 11 shows that the additional addition of DMSO, formamide, and betaine to the DNB preparation reaction does not significantly improve the sequencing quality of the whole-genome methylated single-stranded circle library. However, when the working concentration of betaine is 0.60M, the coverage uniformity of the high GC region of the whole-genome methylated single-stranded circle library can be significantly improved.

[0305] Example 12: Whole-genome methylation single-stranded circularized library sequencing quality improvement optimization experiment

[0306] The reagents used in this experiment are shown in Table 24

[0307] Table 24 Reagents required for DNB preparation and sequencing of whole genome methylation single-stranded circle library

[0308] The specific experimental methods are as follows:

[0309] 1) Add 12 ng of the whole-genome methylated single-stranded circularized library (as shown in Table 25) to a new 0.2 mL PCR tube. For Experimental Group 2, add 5 μL of 5 M betaine. Bring each reaction to 10 μL with Low TE Buffer and add 10 μL of DNB preparation buffer.

[0310] 2) Mix well and centrifuge. Perform denaturation and annealing reactions on a PCR instrument: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C.

[0311] 3) After the reaction is complete, centrifuge briefly and add the following reagents to the control PCR tube from the previous step on ice: 20 μL DNB polymerase mix I and 2 μL DNB polymerase mix II (LC); add the following reagents to the PCR tubes from the two experimental groups from the previous step: 1 μL Tth SSB (2 μg / uL), 19.5 μL DNB polymerase mix I, and 2 μL DNB polymerase mix II (LC);

[0312] 4) Mix well and centrifuge, then perform DNB preparation reaction on a PCR instrument at 30°C for 15 min, and hold at 4°C;

[0313] 5) When the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not shake or pipette vigorously. Store at 4°C until needed.

[0314] 6) Use DNB quantification was performed using the ssDNA Assay Kit fluorescent quantification kit;

[0315] 7) Then, half the volume of DNB was loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE150 (with optimized sequencing algorithm and sequencing script);

[0316] 8) As shown in Table 25 and Figure 4, compared with the control group, the addition of SSB (2 μg / μL) to DNB preparation (Optimization 1) significantly increased Total reads and Q30, but reduced the uniformity of coverage in high GC regions. However, when SSB and betaine were added in combination (Optimization 2), both Total reads and Q30 were significantly increased, and coverage in high GC regions did not decrease compared with the control group.

[0317] Table 25 DNB preparation optimization sequencing results of the optimized version of the whole genome methylation single-chain circle library MGISEQ-2000PE150 (optimized sequencing algorithm and sequencing script)

[0318] Example 12 demonstrates that the addition of Tth SSB (working concentration 46.51 ng / uL) and betaine (working concentration 0.58 M) to the DNB preparation reaction significantly improves the sequencing quality and sequencing data output of the whole-genome methylated single-stranded circle library, as well as the uniformity of high GC region coverage.

[0319] Example 13: Whole-genome methylation double-stranded circularized library sequencing quality improvement optimization experiment

[0320] The reagents used in this experiment are shown in Table 26

[0321] Table 26 Reagents required for DNB preparation and sequencing of whole genome methylation double-stranded circle library

[0322] The specific experimental methods are as follows:

[0323] 1) Add 23.76 ng of the whole-genome methylation double-stranded circularized library (see Table 27) to a new 0.2 mL PCR tube. For the experimental group, add 1 μL of 100% formamide. Bring each reaction to 10 μL with Low TE Buffer and add 10 μL of WGBS DNB Buffer.

[0324] 2) Mix thoroughly and centrifuge. On ice, add the following reagents to the previous PCR tube in order: 20 μL DNB polymerase mix I, 2 μL DNB polymerase mix II (LC);

[0325] 3) Mix well and centrifuge, then perform DNB preparation reaction on a PCR instrument at 30°C for 15 min, and hold at 4°C;

[0326] 4) When the PCR instrument temperature reaches 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not shake or pipette vigorously. Store at 4°C until needed.

[0327] 5) Use DNB quantification was performed using the ssDNA Assay Kit fluorescent quantification kit;

[0328] 6) Then, 25 μL of DNB was loaded onto the chip and sequenced using the optimized version of MGISEQ-2000PE150 (with optimized sequencing algorithm and sequencing script);

[0329] 7) Sequencing Results As shown in Table 27 and Figure 5, the sequencing quality and total read yield of the methylation library in the experimental group that added 1 μL of 100% formamide during DNB preparation were significantly improved compared to the control group without addition, and high GC coverage was slightly improved.

[0330] Table 27 DNB preparation optimization sequencing results of the whole genome methylation double-stranded circle library MGISEQ-2000PE150 optimized version (optimized sequencing algorithm and sequencing script)

[0331] Example 13 shows that the additional addition of formamide (working concentration 2.38%) to the DNB preparation reaction can significantly improve the sequencing quality and sequencing data output of the whole-genome methylated double-stranded circle library, and can also slightly improve the coverage uniformity of the high GC regions of the whole-genome methylated double-stranded circle library.

[0332] Example 14: Optimization experiment for improving single-strand circularization efficiency and sequencing quality of whole-genome PCR-free libraries

[0333] The reagents used in this experiment are shown in Table 28

[0334] Table 28 Reagents required for whole genome PCR-free library single-stranded circularization experiment

[0335] The specific experimental methods are as follows:

[0336] 1) Transfer 167 ng of whole-genome PCR-free ligation product (after shearing and single selection) to a new 0.2 mL PCR tube and add TE Buffer to a total volume of 48 μL;

[0337] 2) After mixing and centrifugation, place the PCR tube in a PCR instrument and react at 95°C for 3 minutes and then at 4°C for 10 minutes. After the reaction is complete, immediately transfer the PCR tube to ice and centrifuge briefly.

[0338] 3) Prepare the single-stranded cell cyclization reaction solution: Add 11.5 μL / tube of Splint Buffer and 0.5 μL / tube of DNA Rapid Ligase to the reaction solution from the previous step, as shown in Table 29. For the experimental group, add an additional 1 μL / tube of Tth SSB (500 ng / μL). Mix and centrifuge. Place the PCR tube in a thermal cycler and perform the single-stranded cell cyclization reaction at 37°C for 10 min and hold at 4°C.

[0339] 4) After the reaction is complete, centrifuge briefly and transfer the PCR tube to ice for the next enzymatic digestion step.

[0340] 5) Add 1.4 μL / tube of Digestion Buffer and 2.6 μL / tube of Digestion Enzyme to the cyclized product, mix well, and centrifuge. Place the PCR tube in a PCR instrument and perform enzyme digestion reaction: 37°C for 10 minutes; hold at 4°C.

[0341] 6) After the enzyme digestion reaction is completed, centrifuge briefly, transfer the PCR tube to ice, and add 7.5 μL of Digestion Stop Buffer to the PCR tube. Vortex three times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube;

[0342] 7) Purify the DNA using 130 μL MGIEasy DNA Purification Magnetic Beads and dissolve it in 25 μL TE Buffer.

[0343] 8) Use Single-stranded circularization was quantified using the ssDNA Assay Kit, and the results are shown in Table 29. Compared with the control group without SSB, the experimental group with the additional addition of 1 μL Tth SSB (500 ng / uL) showed a significant improvement in the circularization efficiency of the whole-genome PCR-free single-stranded circularization library.

[0344] Table 29 Whole genome PCR-free library single-stranded circularization results before and after optimization

[0345] 9) Prepare DNBs using the DNB preparation reagent in the MGISEQ-2000RS High-Throughput Sequencing Kit (PE150). Take 5 ng of the single-stranded circle library, bring the volume to 10 μL with TE buffer, and add 10 μL / tube of DNB preparation buffer.

[0346] 12) Mix well and centrifuge. Perform denaturation and annealing reactions on a PCR instrument: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C.

[0347] 13) After the reaction is complete, centrifuge briefly and add the following reagents to the previous PCR tubes on ice: 20 μL DNB polymerase mix I, 2 μL DNB polymerase mix II (LC). As shown in Table 30, the control group does not need to add Tth SSB. The experimental group should add 1 μL / tube of Tth SSB (2 μg / uL).

[0348] 14) Mix well and centrifuge. Perform DNB preparation reaction on a PCR instrument at 30°C for 25 min and hold at 4°C.

[0349] 15) When the PCR instrument temperature drops to 4°C, immediately add 10 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly 5-8 times with a wide-bore pipette. Do not shake or pipette vigorously. Store at 4°C until needed.

[0350] 16) Use DNB quantification was performed using the ssDNA Assay Kit fluorescent quantification kit;

[0351] 17) Load 400 ng of DNB onto the chip and sequence using the optimized version of MGISEQ-2000PE150 (with optimized sequencing algorithm and sequencing script);

[0352] 18) DNB quantification and sequencing results are shown in Table 31. For the single-strand circularization step and DNB preparation step, the total read yield and total Q30% of the PCR-free library added with 1 μL SSB (2 μg / μL) were significantly improved compared to the control group without SSB.

[0353] 19) Sequencing data was captured at 95 GHz and analyzed using MGI WGAA2.1 WGS analysis software (reference genome version hg19). The results are shown in Table 31. The experimental group (1 μL added for both circularization and DNB preparation) showed improved coverage uniformity and variant detection performance, as evidenced by improved 20x coverage at 30x depth and improved SNP and InDel F-measures.

[0354] Table 30 Sequencing results of whole genome PCR-free library before and after optimization on MGISEQ-2000PE150 (optimized sequencing algorithm and sequencing script)

[0355] Table 31 WGS data analysis results of whole genome PCR-free library on MGISEQ-2000PE150 (optimized sequencing algorithm and sequencing script) before and after optimization

[0356] Example 14 demonstrates that the additional addition of Tth SSB (working concentrations of 8.20 ng / uL and 46.51 ng / uL, respectively) to the circularization reaction and DNB preparation reaction significantly improves the circularization efficiency of the whole-genome PCR-free library, slightly improves sequencing quality and sequencing data output, and also improves the coverage and variant detection of the whole-genome PCR-free library.

[0357] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0358] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A method for preparing DNB, the method comprises: (a) performing a cyclization reaction on a linear DNA library to obtain a circular DNA library, wherein the cyclization reaction includes a single-strand cyclization reaction or a double-strand cyclization reaction, and the circular DNA library includes a single-stranded circular DNA library or a double-stranded circular DNA library; (b) performing a rolling circle amplification reaction on the circular DNA library to obtain DNB, wherein, when the cyclization reaction in step (a) is a single-strand cyclization reaction, step (a) further comprises: adding at least one of the following additives to the single-strand cyclization reaction to obtain the single-stranded circular DNA library: 1) an additive capable of reducing the DNA secondary structure, 2) an additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA; and / or step (b) further comprises: performing a rolling circle amplification reaction using the product single-stranded circular DNA library of step (a), wherein at least one of the following additives is added in the rolling circle amplification reaction: 3) an additive capable of reducing the DNA secondary structure, 4) an additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA; when the cyclization reaction in step (a) is a double-strand cyclization reaction, step (b) further comprises: performing a rolling circle amplification reaction using the product double-stranded circular DNA library of step (a), wherein at least one of the following additives is added in the rolling circle amplification reaction: 5) an additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA, 6) an additive capable of reducing the double helix stability of circular double-stranded DNA.

2. A method for rolling circle amplification, characterized in that, performing a rolling circle amplification reaction on a DNA library to obtain single-stranded linear DNA, wherein, when the DNA library is a single-stranded circular DNA library, at least one of the following additives is added in the rolling circle amplification reaction: 7) an additive capable of reducing the DNA secondary structure, 8) an additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA; when the DNA library is a double-stranded circular DNA library, at least one of the following additives is added in the rolling circle amplification reaction: 9) an additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA, 10) an additive capable of reducing the double helix stability of circular double-stranded DNA.

3. The method according to claim 1 or 2, characterized in that, the additive capable of reducing the DNA secondary structure includes at least one of non-ionic detergents, betaine, and DMSO; optionally, the non-ionic detergent includes at least one selected from Triton-X100, Tween-20, and NP40; optionally, the additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one of SSB and HSP; optionally, the additive capable of reducing the double helix stability of circular double-stranded DNA includes at least one of formamide, ammonium sulfate, urea, and tetramethylammonium hydroxide.

4. The method according to claim 3, characterized in that, The additives in the single-stranded circularization reaction include at least one selected from SSB, non-ionic detergents, betaine, and DMSO; Optionally, the additives in the single-stranded circularization reaction are a mixture of non-ionic detergents and betaine; Optionally, the additives in the single-stranded circularization reaction are a mixture of non-ionic detergents and DMSO; Optionally, the additives in the single-stranded circularization reaction are a mixture of SSB and non-ionic detergents and / or betaine.

5. The method according to claim 1 or 2, wherein, the additives in the rolling circle amplification reaction of the single-stranded circular DNA library include at least one selected from SSB, and a mixture of SSB and non-ionic detergents and / or betaine.

6. The method according to claim 3, wherein, the additives in the rolling circle amplification reaction of the double-stranded circular DNA library include at least one selected from SSB and formamide.

7. The method according to claim 4, wherein, the working concentration of SSB added to the single-stranded circularization reaction is 5 ng / μL - 50 ng / μL; Optionally, the working concentration of the non-ionic detergent added to the single-stranded circularization reaction is 0.05% - 2.00% (V / V); Optionally, the working concentration of DMSO added to the single-stranded circularization reaction is 1.5% - 4.0% (V / V); Optionally, the working concentration of betaine added to the single-stranded circularization reaction is 0.1 M - 1 M.

8. The method according to claim 5, wherein, the working concentration of SSB added to the rolling circle amplification reaction of the single-stranded circular DNA library is 5 ng / μL - 130 ng / μL.

9. The method according to claim 8, wherein, the working concentration of betaine added to the rolling circle amplification reaction of the single-stranded circular DNA library is 0.1 M - 1 M; Optionally, the working concentration of the non-ionic detergent added to the rolling circle amplification reaction of the single-stranded circular DNA library is 0.05% - 2.00% (V / V).

10. The method according to claim 6, wherein, the working concentration of SSB added to the rolling circle amplification reaction of the double-stranded circular DNA library is 20 ng / μL - 100 ng / μL; Optionally, the working concentration of formamide added to the rolling circle amplification reaction of the double-stranded circular DNA library is 1.5 - 4% (V / V).

11. A sequencing library prepared by the method according to any one of claims 1 - 10.

12. A sequencing method, wherein, nucleic acid sequence determination is performed on the sequencing library according to claim 11.

13. A single-stranded circularization reaction system, wherein, the single-stranded circularization reaction system includes oligonucleic acids, DNA ligase, ligation reaction buffer, and additives that can reduce the DNA secondary structure, and / or additives that can reduce the DNA secondary structure while maintaining the stability of single-stranded DNA.

14. The single-stranded circularization reaction system according to claim 13, wherein, The DNA ligase includes any one of T4 DNA ligase and Taq ligase.

15. The single-stranded circularization reaction system according to claim 13, characterized in that, it further includes a sequencing chip, and the oligonucleic acid is fixed on the sequencing chip.

16. The single-stranded circularization reaction system according to claim 13, characterized in that, it further includes magnetic beads, and the oligonucleic acid is fixed on the magnetic beads and / or free in the magnetic bead suspension.

17. The single-stranded circularization reaction system according to claim 13, characterized in that, the additive capable of reducing the DNA secondary structure includes at least one of non-ionic detergents, betaine, and DMSO; Optionally, the non-ionic detergent includes at least one selected from Triton-X100, Tween-20, and NP40; Optionally, the additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one of SSB and HSP.

18. The single-stranded circularization reaction system according to claim 13, characterized in that, the additive in the single-stranded circularization reaction system includes at least one selected from SSB, non-ionic detergents, betaine, and DMSO; Optionally, the additive is a mixture of non-ionic detergent and betaine; Optionally, the additive is a mixture of non-ionic detergent and DMSO; Optionally, the additive is a mixture of SSB and non-ionic detergent and / or betaine.

19. A rolling circle amplification reaction system for a single-stranded circular DNA library, characterized in that, the rolling circle amplification reaction system includes a polymerase, a rolling circle amplification reaction buffer, and an additive capable of reducing the DNA secondary structure, and / or an additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA.

20. The rolling circle amplification reaction system according to claim 19, characterized in that, the polymerase includes any one of Phi29 DNA polymerase and Bst DNA polymerase.

21. The rolling circle amplification reaction system according to claim 19, characterized in that, it further includes a sequencing chip, and the rolling circle amplification reaction is carried out on the chip surface.

22. The rolling circle amplification reaction system according to claim 19, characterized in that, it further includes magnetic beads, and the rolling circle amplification reaction is carried out on the magnetic beads and / or in the magnetic bead suspension.

23. The rolling circle amplification reaction system according to claim 19, characterized in that, the additive capable of reducing the DNA secondary structure includes at least one of non-ionic detergents, betaine, and DMSO; Optionally, the non-ionic detergent includes at least one selected from Triton-X100, Tween-20, and NP40; Optionally, the additive capable of reducing the DNA secondary structure while maintaining the stability of single-stranded DNA includes at least one of SSB and HSP.

24. The rolling circle amplification reaction system according to claim 19, characterized in that, the additive in the rolling circle amplification reaction system includes at least one selected from SSB and a mixture of SSB and non-ionic detergent and / or betaine.

25. A rolling circle amplification reaction system for a double-stranded circular DNA library, characterized in that, the rolling circle amplification reaction system comprises a polymerase, a rolling circle amplification reaction buffer, and an additive that can reduce the DNA secondary structure while maintaining the stability of single-stranded DNA and / or an additive that can reduce the double-helix stability of circular double-stranded DNA as described in any one of the methods of claims 1-10.

26. The rolling circle amplification reaction system according to claim 25, characterized in that, the additive that can reduce the DNA secondary structure while maintaining the stability of single-stranded DNA comprises at least one selected from SSB, a mixture of SSB and a non-ionic detergent and / or betaine.

27. The rolling circle amplification reaction system according to claim 25, characterized in that, the additive that can reduce the double-helix stability of circular double-stranded DNA comprises at least one of formamide, ammonium sulfate, urea, and tetramethylammonium hydroxide.

28. The rolling circle amplification reaction system according to claim 25, characterized in that, the polymerase comprises any one of the rolling circle amplification polymerases Phi29 DNA polymerase and Bst DNA polymerase.

29. Use of any one of the methods of any one of claims 1-10, the sequencing library of claim 11, the single-strand circularization reaction system of any one of claims 13-18, the rolling circle amplification reaction system of claims 19-24, and the rolling circle amplification reaction system of any one of claims 25-28 in sequencing.

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