Novel DNA nanosphere preparation reagent for improving sequencing coverage and sequencing quality

Abstract

The invention relates to the technical field of high-throughput sequencing, in particular to an additive-containing DNB preparation system for improving sequencing coverage and sequencing quality and application of the additive-containing DNB preparation system, the system comprises an additive and a DNB preparation buffer solution, and the additive is selected from a group consisting of betaine, betaine derivatives and betaine analogues. The additive-containing DNB preparation system and method provided by the invention can improve downstream CG preference for different libraries and effectively improve the sequencing quality.

Description

Technical Field

[0001] The present application relates to the technical field of high-throughput sequencing, and specifically to a DNB preparation system containing additives for improving sequencing coverage and sequencing quality and its application. Background Art

[0002] BGI-SEQ cPAS technology is a second-generation sequencing technology with the advantages of high throughput, low cost, and automation. It uses sequencing technology based on rolling circle replication (RCR) and combined probe anchoring polymerization (CPAS). Unlike PCR exponential amplification, the amplification errors of rolling circle amplification technology will not accumulate, so it is superior to other sequencing technologies in terms of sequencing accuracy and has been increasingly widely used. In addition, BGI-SEQ cPAS technology uses a regular array chip with a special surface modification. Only one DNA nanoball (DNB) is fixed at each modification site. The spacing between the modification sites of the array chip is uniform, which can ensure that the optical signals of different nanoballs will not interfere with each other, thereby greatly improving the accuracy of signal processing.

[0003] However, in the DNB-based CPAS sequencing technology, due to the structural specificity and complexity of DNB, some high-CG libraries are still prone to shortcomings during sequencing. High GC requires high energy for melting, denaturation is difficult, and stable secondary structures are easily formed in the template to hinder DNA polymerase from synthesizing DNA on the template. At the same time, it is easier to produce GC preference, resulting in poor sequencing quality. This defect affects the downstream analysis of bioinformatics, making it unable to meet scientific research needs and clinical applications.

[0004] Therefore, the research and development of DNB preparation systems and kits with improved production performance have both important industrial and scientific research value. Summary of the invention

[0005] The following is only an overview of some aspects of the present application, but the present application is not limited thereto. All references in this specification are incorporated herein by reference in their entirety. When the disclosure of this specification differs from the citation, the disclosure of this specification shall prevail.

[0006] The first aspect of the present application provides a DNB preparation system, comprising: an additive and a DNB preparation buffer, wherein the additive is selected from the group consisting of betaine, a betaine analogue and a betaine derivative.

[0007] In some embodiments, the final concentration of the additive is 0.1 M to 5 M. In some embodiments, the final concentration of the additive is 2M.

[0008] In some embodiments, the DNB preparation buffer comprises tris(hydroxymethyl)aminomethane or tris(hydroxymethyl)aminomethane hydrochloride. In some embodiments, the mass fraction of the buffer is 1-5%.

[0009] In some embodiments, the DNB preparation buffer further comprises a mediator sequence (Splint oligo), and the DNB preparation system hybridizes the mediator sequence with the library at 53° C.-60° C. In some embodiments, the mediator sequence hybridizes with the library at 57° C.

[0010] In some embodiments, the DNB preparation buffer further comprises an enzyme mix, wherein the enzyme mix comprises a DNA polymerase and a DNA ligase.

[0011] The second aspect of the present application provides a one-step DNB preparation kit for DNBSEQ, wherein the kit comprises the DNB preparation system described in any embodiment of the first aspect of the present application.

[0012] In some embodiments, the kit further comprises a DNB stop buffer, wherein the DNB stop buffer comprises ethylenediaminetetraacetic acid.

[0013] The third aspect of the present application provides a method for preparing DNB, which comprises the following steps:

[0014] S1. Configuring DNB preparation reaction system 1: mixing the library and the DNB preparation system described in any embodiment of the first aspect of the present application to obtain the DNB preparation reaction system 1;

[0015] S2. Sequence hybridization: annealing the DNB preparation reaction system 1 to allow the mediating sequence to hybridize with the library;

[0016] S3. Rolling circle amplification: adding an enzyme mixture to the hybridized DNB preparation reaction system 1 to obtain a DNB preparation reaction system 2, and performing rolling circle amplification to obtain DNBs, wherein the enzyme mixture comprises a DNA polymerase and a DNA ligase.

[0017] In some embodiments, the step S3 further comprises: adding a DNB termination buffer to the DNB preparation reaction system 2 to terminate the reaction. In some embodiments, the DNB termination buffer comprises ethylenediaminetetraacetic acid.

[0018] In some embodiments, in step S1, the DNB preparation system comprises an additive selected from betaine, a betaine analogue and a betaine derivative.

[0019] In some embodiments, the final concentration of the additive is 0.1 M to 5 M. In some embodiments, the final concentration of the additive is 2M.

[0020] In some embodiments, in step S2, the annealing is performed at 53° C.-60° C. In some embodiments, the annealing is performed at 57° C.

[0021] In some embodiments, the library is a double-stranded DNA library, and the step S2 further comprises: before annealing the DNB preparation reaction system 1, denaturing the DNB preparation reaction system 1 to denature the double-stranded DNA library into a single-stranded DNA library for hybridization with the mediating sequence.

[0022] This application achieves the following technical effects:

[0023] The DNB preparation system and DNB preparation method containing additives provided in the embodiments of the present application can significantly improve the downstream CG preference. By introducing additives into the DNB preparation system, the formation of the secondary structure of the template during the preparation process is reduced, and the chance of the primer binding to the template chain at a temperature lower than its melting temperature during the annealing process is increased; at the same time, by changing the annealing temperature, the downstream GC preference is improved, the sequencing quality is effectively improved, and the sequencing read length can be further improved while ensuring high sequencing quality, thereby expanding the application scope of the DNB preparation system.

[0024] The DNB preparation system and DNB preparation method containing additives provided in the embodiments of the present application can improve the downstream CG preference for different libraries and improve the sequencing quality, thereby achieving the purpose of obtaining high-quality sequencing data.

[0025] The foregoing merely summarizes certain aspects disclosed herein and is not intended to be limiting in nature. These and other aspects of the present application as well as additional embodiments, features and advantages will become apparent through the following detailed description and through practice of the application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a schematic diagram of the process for preparing DNB in ​​the embodiment of the present application;

[0028] Figure 2 This is a sequencing result analysis diagram in the comparative example of this application;

[0029] Figure 3 This is a diagram showing the analysis of sequencing results in Example 1 of the present application;

[0030] Figure 4 This is a diagram for analyzing the sequencing results in Example 2 of the present application. DETAILED DESCRIPTION

[0031] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field and do not constitute a limitation of the present application in any way.

[0032] This application is made by the inventor based on the following understanding:

[0033] In related technologies, due to the structural specificity and complexity of DNBs, sequencing of some high-CG application libraries will easily produce GC preference and poor sequencing quality, which is not conducive to downstream bioinformatics analysis, scientific research needs and clinical applications. At the same time, due to the wide variety of application libraries on the market, the DNB preparation methods based on related technologies perform differently for different libraries, making it difficult to obtain stable and good downstream results.

[0034] In this regard, the inventors conducted a large number of related experiments and tests, and surprisingly found that the introduction of additives, especially betaine and its derivatives or analogs, into the DNB preparation system can significantly improve the downstream CG preference, reduce the formation of secondary structures of templates and primers, and increase the chance of primers binding to the template chain at a temperature lower than their melting temperature, effectively improving the sequencing quality, thereby further improving the sequencing read length while ensuring high sequencing quality, and having a relatively stable and good performance for different libraries, thereby expanding the application range of DNB preparation and sequencing. In addition, in the experiment, the inventors also found that in the DNB preparation process, by changing the annealing temperature between the circularized primer and the template (library), the downstream CG preference for different libraries can be effectively improved, thereby obtaining high-quality downstream sequencing data.

[0035] Based on this, the embodiment of the first aspect of the present application provides a DNB preparation system, comprising: an additive and a DNB preparation buffer, wherein the additive can be selected from the group consisting of betaine, a betaine derivative and a betaine analog. It is understandable that by adding additives, DNA polymerase can be helped to pass through certain complex structures of library DNA (such as supercoiling) smoothly, preventing DNA polymerase from dissociating from the template library DNA during extension; at the same time, the addition of additives can eliminate the dependence of denaturation temperature on bases (i.e., it can effectively denature AT and GC base pairs at the same denaturation temperature), which is beneficial to PCR amplification of GC-rich templates (increasing the hydration of guanine and cytosine in the guanine and cytosine-rich regions in the minor groove of DNA, affecting the DNA molecular structure, changing the flexibility of DNA, and helping DNA polymerase to extend along the DNA template), reducing the Tm value of library DNA, thereby improving the specificity of binding between primers and library templates, thereby effectively improving the quality of downstream sequencing data.

[0036] In the present application, "Betaine" is an alkaloid, the chemical name of which is N,N,N-trimethylglycine. Its chemical structure is similar to that of amino acids and it belongs to the quaternary ammonium base class. Its molecular formula is C5H 11 NO2. Betaine is commonly found in animals and plants. It is an intermediate product of animal metabolism and plays a very important role in the metabolism of nutrients. Betaine is a byproduct of metabolism and an osmotic regulating substance. It is very important for plants to enhance stress resistance, such as salt and alkali resistance and drought resistance. Betaine is also used in cleaning skin care products. A new use of betaine is proposed in the examples of the present application. It can be used as an additive in the DNB preparation system and can significantly improve the GC preference of the DNB preparation process, effectively improving the quality of sequencing data based on the prepared DNB.

[0037] In the embodiments of the present application, "betaine derivatives" may be alkyl betaine, alkyl amide betaine, sulfopropyl betaine, hydroxysulfopropyl betaine and / or phosphate betaine, etc., derivatives with other modifications based on betaine. In some embodiments, alkyl betaine is obtained by quaternization of aliphatic tertiary amines, that is, by reacting N-alkyl-N,N-dimethylamine with sodium chloroacetate in an aqueous solution, and its general formula is RC4H8NO2, wherein R is an alkyl group with a carbon number of 12 to 18, such as dodecyl dimethyl betaine, tetradecyl dimethyl betaine, octadecyl dimethyl betaine, dodecyl dihydroxyethyl betaine, octadecyl dihydroxyethyl betaine, etc. In some embodiments, the general formula of alkyl amide betaine is RCONH(CH2) nC4H8NO2, wherein R is a carbon number of 12 to 18, n = 2 or 3, such as lauroylamidopropyl betaine, cocamidopropyl betaine, octadecylamidopropyl betaine, etc. In some embodiments, sulfobetaine has a quaternary ammonium salt cation and a sulfonic acid anion, and the general formula is RC5H 12 NSO3, wherein R is an alkyl group with 12 to 18 carbon atoms, such as dodecyl sulfopropyl betaine, tetradecyl sulfopropyl betaine, hexadecyl sulfopropyl betaine, octadecyl sulfopropyl betaine, etc. In some embodiments, hydroxysulfopropyl betaine has both anionic and cationic groups with hydroxyl groups in its structure, and the general formula is RC5H 12 NSO4, wherein R is an alkyl group with 12 to 18 carbon atoms. In some embodiments, phosphobetaine is composed of two parts, one part is a nitrogen-containing cationic part, which is composed of a long-chain derivative of an amine or a quaternary ammonium; the other part is an anionic part, which is composed of a phosphate salt, and the general formula is RC5H 13 NPO5, in which R is an alkyl group with 12 to 18 carbon atoms, such as dodecyl hydroxypropyl betaine phosphate, tetradecyl hydroxypropyl betaine phosphate, etc.

[0038] In the embodiments of the present application, the "betaine analog" may be sulfobetaine, sulfobetaine bromide, and the like.

[0039] It can be understood that the betaine derivatives or analogs proposed in the examples of the present application are based on betaine and thus have the basic physical and chemical properties of betaine. Therefore, they can also be used as additives in the DNB preparation system and can significantly improve the GC preference of the DNB preparation process, thereby effectively improving the quality of sequencing data based on the prepared DNB.

[0040] In some embodiments, the final concentration of the additive can be 0.1-5 M. In some embodiments, the final concentration of the additive is 2 M. In some embodiments, the addition of the additive at the above final concentration effectively improves GC preference and improves the quality of sequencing data.

[0041] In some embodiments, the DNB preparation buffer may include tris(hydroxymethyl)aminomethane or tris(hydroxymethyl)aminomethane hydrochloride. In some embodiments, the mass fraction of the buffer is 1-5%. It is understood that by adding additives to the DNB preparation buffer, the GC preference is effectively improved and the quality of sequencing data is improved.

[0042] In some embodiments, the DNB preparation buffer may further include a mediating sequence (Splint oligo). In the present application embodiment, the mediating sequence may be a cyclization primer, that is, by sequentially denaturing and annealing the DNA library and the mediating sequence in the same system (if the library itself is single-stranded, denaturation is not required), the mediating sequence and the denatured single-stranded DNA are complementary paired at both ends; a ligase and a polymerase are simultaneously introduced into the system, the two ends of the single-stranded DNA are connected under the action of the ligase, and a rolling circle amplification reaction is performed under the action of the polymerase with the mediating sequence as a primer and the single-stranded DNA as a template to obtain a DNA nanoball.

[0043] In the examples of the present application, the DNB preparation system performs hybridization between the mediating sequence and the library at 53°C-60°C. In some embodiments, the DNB preparation system performs hybridization between the mediating sequence and the library at 57°C. In the examples of the present application, by changing the hybridization temperature (also called annealing temperature) between the mediating sequence (cyclization primer) and the library, the CG preference for the downstream of different libraries is effectively improved, so that the preparation method and system of DNB proposed in the examples of the present application are universal, that is, applicable to a variety of library types, thereby expanding the scope of application of DNB sequencing. At the same time, the change in annealing temperature combined with the addition of additives can help DNA polymerase pass through certain complex secondary structures of DNA smoothly and prevent DNA polymerase from dissociating from template DNA; at the same time, it can eliminate the dependence of denaturation temperature on bases (that is, it can effectively denature both AT and GC base pairs at the same denaturation temperature), which is beneficial to PCR amplification of GC-rich templates (increasing the hydration of guanine and cytosine in the guanine- and cytosine-rich regions in the minor groove of DNA, affecting the DNA molecular structure, changing the flexibility of DNA, and helping DNA polymerase extend along the DNA template), reducing the Tm value of library DNA, thereby improving the specificity of binding between primers and library templates, and effectively improving the sequencing quality during the sequencing process, thereby obtaining high-quality sequencing data.

[0044] In some embodiments, the DNB preparation buffer may further comprise an enzyme mixture, wherein the enzyme mixture comprises DNA polymerase and / or DNA ligase. It is to be understood that the DNA polymerase and / or DNA ligase used in the embodiments of the present application can achieve the connection between a single base and a nucleic acid fragment and the connection between nucleic acid fragments respectively, and the present application is not intended to limit its specific type.

[0045] The DNB preparation system containing additives proposed in the examples of the present application can significantly improve the downstream CG preference: by introducing additives into the DNB preparation system, the formation of secondary structures of templates and primers during the preparation process is reduced, and the chance of primers annealing and binding with template chains at temperatures below their melting temperatures during the annealing process is increased; at the same time, by changing the annealing temperature, the downstream GC preference is improved, and the sequencing quality is effectively improved, which can further improve the sequencing read length while ensuring high sequencing quality, and expand the application scope of the DNB preparation system. The DNB preparation system containing additives provided in the examples of the present application can improve the downstream CG preference for different libraries and improve the sequencing quality, thereby achieving the purpose of obtaining high-quality sequencing data.

[0046] The embodiment of the second aspect of the present application provides a one-step DNB preparation kit for DNBSEQ, the kit comprising the DNB preparation system described in any embodiment of the first aspect. In some embodiments, the kit allows for simultaneous cyclization and rolling circle replication in one reaction system to quickly and conveniently obtain DNBs.

[0047] In some embodiments, the kit further comprises a DNB termination buffer, wherein the DNB termination buffer comprises ethylenediaminetetraacetic acid. It is understood that the rolling circle amplification is terminated by adding the termination buffer, and the obtained DNB can be loaded on a sequencing platform for subsequent sequencing.

[0048] The third aspect of the present application provides a method for preparing DNB. Figure 1 is a schematic diagram of a method for preparing DNB according to an embodiment of the present application, such as Figure 1 As shown, the method includes the following steps S1-S3.

[0049] S1. Preparing DNB preparation reaction system 1: mixing the library and the DNB preparation system described in any one of the embodiments of the first aspect to obtain DNB preparation reaction system 1.

[0050] In the embodiments of the present application, the DNB preparation system includes an additive, wherein the additive is selected from the group consisting of betaine, betaine derivatives and betaine analogs. In some embodiments, the final concentration of the additive can be 0.1-5 M. In some embodiments, the final concentration of the additive can be 2 M.

[0051] In the embodiments of the present application, the library refers to a single-stranded or double-stranded DNA containing a sequence to be tested, a linker sequence, etc. In some embodiments, the library may also contain a forward sequencing primer binding sequence, a reverse sequencing primer binding sequence, a barcode sequence (for distinguishing sample information, etc.), etc. The present application does not limit the type of library, the fragments contained, and the applicable platform.

[0052] In the embodiment of the present application, when the library is a double-stranded DNA library, the DNB preparation reaction system 1 can be denatured to denature the double-stranded DNA library into a single-stranded DNA library for subsequent primer hybridization.

[0053] S2. Sequence hybridization: Annealing the DNB preparation reaction system 1 to allow the mediating sequence to hybridize with the library.

[0054] In some embodiments, annealing is performed at 53°C-60°C. In some embodiments, annealing is performed at 57°C. It is understandable that by changing the annealing temperature when an additive is added, the formation of secondary structures in GC-rich regions can be effectively reduced, thereby improving DNA amplification, eliminating the dependence of DNA dissolution on base pair composition, thereby enhancing the specificity of the polymerase chain reaction, and improving the GC preference downstream of sequencing, while effectively improving sequencing quality. S3. Rolling circle amplification: Add an enzyme mixture to the hybridized DNB preparation reaction system 1 to obtain a DNB preparation reaction system 2, and perform rolling circle amplification to obtain DNBs, wherein the enzyme mixture comprises a DNA polymerase and a DNA ligase.

[0055] In some embodiments, step S3 further comprises: adding a DNB termination buffer to the DNB preparation reaction system 2 to terminate the reaction. In some embodiments, the DNB termination buffer comprises ethylenediaminetetraacetic acid.

[0056] It should be noted that the explanation of the DNB preparation system proposed in any embodiment of the first aspect of this application above is also applicable to the one-step DNB preparation kit for DNBSEQ proposed in any embodiment of the second aspect and any embodiment of the third aspect of this application provides a method for preparing DNB, and the technical advantages achieved are the same, which will not be repeated in this application.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0058] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0059] Comparative Example

[0060] 1.1 Experimental Materials

[0061] 1) Library DNA: NA12878 library (PCR product);

[0062] 2) DNBSEQ One-Step DNB Preparation Kit (OS-SB, Catalog No.: 1000020563), which includes DNB Preparation Buffer, TE Buffer, DNB Polymerase Mix I, DNB Polymerase Mix II and DNB Termination Buffer;

[0063] 3) DNBSEQ-E5RS sequencing kit (SE100, catalog number: 1000026249).

[0064] 1.2 Experimental equipment

[0065] 1)DNBSEQ-E5RS(ALPHA 0.2FPGA);

[0066] 2) Qubit instrument (Life Technologies);

[0067] 3) PCR amplifier (ABI).

[0068] 1.3 Experimental steps for preparing DNB

[0069] 1) Prepare DNB reaction system 1:

[0070] Take the DNB preparation buffer in the DNBSEQ one-step DNB preparation kit and prepare the DNB preparation reaction system 1 in a PCR tube according to Table 1 below:

[0071] Table 1 DNB preparation reaction system 1

[0072]

[0073] 2) Sequence hybridization: The DNA preparation reaction system 1 configured in step 1) was mixed by vortex oscillator, centrifuged in a mini centrifuge for 5 seconds, and placed in a PCR instrument for sequence hybridization (denaturation and annealing in sequence). The sequence hybridization conditions are shown in Table 2 below, where the reaction temperature I (annealing temperature / primer hybridization temperature) is 40°C.

[0074] Table 2 DNB preparation reaction sequence hybridization conditions

[0075]

[0076] 3) Add enzyme mixture to the DNB preparation reaction system 1 after sequence hybridization:

[0077] First, take DNB polymerase mixed solution II (LC), centrifuge briefly for 5 seconds, and place on ice for later use.

[0078] After the sequence hybridization in step 2) is completed (PCR instrument reaches 4°C), take out the PCR tube containing DNB preparation reaction system 1, centrifuge for 5 seconds in a mini centrifuge, and then add 40 μL of DNB polymerase mixture I and 4 μL of DNB polymerase mixture II to the PCR tube on ice to obtain DNB preparation reaction system 2.

[0079] 4) Rolling circle amplification: The DNB preparation reaction system 2 prepared in step 3) was mixed by vortex oscillator, centrifuged for 5 seconds in a mini centrifuge, and immediately placed in a PCR instrument for rolling circle amplification. The reaction conditions are shown in Table 3 below:

[0080] Table 3 DNB preparation rolling circle amplification conditions

[0081]

[0082] When the temperature of the PCR instrument reaches 4°C, 20 μL of DNB termination buffer is immediately added to the DNB preparation reaction system 2 after rolling circle amplification, and the mixture is slowly pipetted and mixed 5-8 times with a wide-mouth pipette tip to prepare DNB, and its concentration is 7.3 ng / μl detected by Qubit.

[0083] 1.4 Sequencing

[0084] The DNB prepared in this example was used for sequencing with reference to the "DNBSEQ-E5RS Sequencing Reagent Set (SE100) V1.0 User Manual".

[0085] 1.5 Analysis of sequencing results

[0086] The sequencing results were analyzed using the basecall (http: / / 10.49.56.64 / EcoliDoc / TEMP / BaseCall) provided with the instrument. The specific results are shown in Tables 7 and Figure 2 shown.

[0087] Example 1

[0088] 1.1 Experimental Materials

[0089] 1) Library DNA: NA12878 library (PCR product);

[0090] 2) DNBSEQ One-Step DNB Preparation Kit (OS-SB, Catalog No.: 1000020563), which includes DNB Preparation Buffer, TE Buffer, DNB Polymerase Mix I, DNB Polymerase Mix II and DNB Termination Buffer;

[0091] 3) DNBSEQ-E5RS sequencing kit (SE100, catalog number: 1000026249).

[0092] 1.2 Experimental equipment

[0093] 1)DNBSEQ-E5RS(ALPHA 0.2FPGA);

[0094] 2) Qubit instrument;

[0095] 3) PCR amplifier.

[0096] 1.3 Experimental steps for preparing DNB

[0097] 1) Prepare DNB reaction system 1:

[0098] Prepare reaction system 1 by preparing DNB in ​​a PCR tube according to Table 4 below.

[0099] Table 4 DNB preparation reaction system 1

[0100]

[0101] 2) Sequence hybridization: The DNA prepared in step 1) was mixed by vortex oscillator, centrifuged in a mini centrifuge for 5 seconds, and placed in a PCR instrument for sequence hybridization (denaturation and annealing in sequence). The sequence hybridization conditions are shown in Table 5 below, where the reaction temperature I (annealing temperature / primer hybridization temperature) was 57°C.

[0102] Table 5 DNB preparation reaction sequence hybridization conditions

[0103]

[0104] 3) Add enzyme mixture to the DNB preparation reaction system 1 after sequence hybridization:

[0105] First, take DNB polymerase mixed solution II (LC), centrifuge briefly for 5 seconds, and place on ice for later use.

[0106] After the sequence hybridization in step 2) is completed (PCR instrument reaches 4°C), take out the PCR tube containing DNB preparation reaction system 1, centrifuge for 5 seconds in a mini centrifuge, and then add 40 μL of DNB polymerase mixture I and 4 μL of DNB polymerase mixture II to the PCR tube on ice to obtain DNB preparation reaction system 2.

[0107] 4) Rolling circle amplification: The DNB preparation reaction system 2 prepared in step 3) was mixed by vortex oscillator, centrifuged for 5 seconds in a mini centrifuge, and immediately placed in a PCR instrument for rolling circle amplification. The reaction conditions were the same as those in Table 3 in the comparative example.

[0108] When the temperature of the PCR instrument reaches 4°C, 20 μL of DNB termination buffer is immediately added to the DNB preparation reaction system 2 after rolling circle amplification, and the mixture is slowly pipetted and mixed 5-8 times with a wide-mouth pipette tip to obtain DNB, and its concentration is 6.9 ng / μl detected by Qubit.

[0109] 1.4 Sequencing

[0110] The DNB prepared in this comparative example was sequenced on a machine according to the "DNBSEQ-E5RS Sequencing Reagent Set (SE100) V1.0 User Manual".

[0111] 1.5 Analysis of sequencing results

[0112] The sequencing results were analyzed using the basecall (http: / / 10.49.56.64 / EcoliDoc / TEMP / BaseCall) provided with the instrument. The specific results are shown in Tables 7 and Figure 3 shown.

[0113] Example 2

[0114] 1.1 Experimental Materials

[0115] 1) Library DNA: NA12878 library (PCR product);

[0116] 2) DNBSEQ One-Step DNB Preparation Kit (OS-SB), which includes DNB Preparation Buffer, TE Buffer, DNB Polymerase Mix I, DNB Polymerase Mix II and DNB Termination Buffer;

[0117] 3) DNBSEQ-E5RS sequencing kit (SE100);

[0118] 4) Betaine.

[0119] 1.2 Experimental equipment

[0120] 1)DNBSEQ-E5RS(ALPHA 0.2FPGA);

[0121] 2) Qubit instrument;

[0122] 3) PCR amplifier.

[0123] 1.3 Experimental steps for preparing DNB

[0124] 1) Prepare DNB reaction system 1:

[0125] Take the DNB preparation buffer in the DNBSEQ one-step DNB preparation kit, add betaine thereto to prepare the DNB preparation buffer containing the additive, and configure the DNB preparation reaction system 1 in the PCR tube according to Table 6 below. The final concentration of betaine in the DNB preparation buffer containing the additive in this embodiment is 2M.

[0126] Table 6 DNB preparation reaction system 1

[0127]

[0128] 2) Sequence hybridization: The DNA preparation reaction system 1 configured in step 1) was vortexed and mixed, centrifuged in a mini centrifuge for 5 seconds, and placed in a PCR instrument for sequence hybridization. The sequence hybridization conditions were the same as those in Table 5 in Example 1, that is, the reaction temperature I (annealing temperature / primer hybridization temperature) in this embodiment was 57°C.

[0129] 3) Add enzyme mixture to the DNB preparation reaction system 1 after sequence hybridization:

[0130] First, take DNB polymerase mixed solution II (LC), centrifuge briefly for 5 seconds, and place on ice for later use.

[0131] After the sequence hybridization in step 2) is completed (PCR instrument reaches 4°C), take out the PCR tube containing DNB preparation reaction system 1, centrifuge for 5 seconds in a mini centrifuge, and then add 40 μL of DNB polymerase mixture I and 4 μL of DNB polymerase mixture II to the PCR tube on ice to obtain DNB preparation reaction system 2.

[0132] 4) Rolling circle amplification: The DNB preparation reaction system 2 prepared in step 3) was mixed by vortex oscillator, centrifuged for 5 seconds in a mini centrifuge, and immediately placed in a PCR instrument for rolling circle amplification. The reaction conditions were the same as those in Table 3 in the comparative example.

[0133] When the temperature of the PCR instrument reaches 4°C, 20 μL of DNB termination buffer is immediately added to the DNB preparation reaction system 2 after rolling circle amplification, and the mixture is slowly pipetted and mixed 5-8 times with a wide-mouth pipette tip to obtain DNB, and its concentration is 5.73 ng / μl detected by Qubit.

[0134] 1.4 Sequencing

[0135] The DNB prepared in this comparative example was sequenced on a machine according to the "DNBSEQ-E5RS Sequencing Reagent Set (SE100) V1.0 User Manual".

[0136] 1.5 Analysis of sequencing results

[0137] The sequencing results were analyzed using the basecall (http: / / 10.49.56.64 / EcoliDoc / TEMP / BaseCall) provided with the instrument. The specific results are shown in Tables 7 and Figure 4 shown.

[0138] Table 7 Test results of different reaction temperatures

[0139] Group Comparative Example Example 1 Example 2 Reference sequence Ecoli Ecoli Ecoli Cycle times 50 50 50 Q30％ 91.31 91.53 92.47 Lag% 0.07 0.07 0.07 Runon% 0.08 0.08 0.06 ESR% 79.14 79.25 83.52 InitialFIT% 85.55 87.29 88.27

[0140] Table 7 shows various indicators of the output data in Comparative Example 1 and Examples 1-2, wherein the Q30 value refers to the percentage of bases with a quality value ≧30 (i.e., an error rate of 0.1% and an accuracy rate of 99.99%), and the higher the Q30 value, the higher the data quality; ESR (Effective Spot Rate) refers to the ratio of the number of reads (Total Reads) obtained after filtering and the number of DNBs (DNB Number) that can be identified by base call (Basecall), i.e., ESR=Total Reads / DNB Number×100%, wherein the higher the ESR value, the more data obtained after filtering and the higher the sequencing quality; InitialFIT is used to reflect the distribution of the signal and noise of each base. The more concentrated the light intensity (Intensity) distribution of each base after correction (the light intensity of one base is very high and the light intensity of the other three bases is very low), the higher its FIT value; the more discrete the light intensity distribution, the lower the FIT value, that is, the higher the InitialFIT value, the more accurate the judgment of the base type of the data is, and the higher the accuracy of the data is.

[0141] As can be seen from the data in Table 7, compared with the comparative example, after changing the primer hybridization temperature, the sequencing data in Example 1 shows a higher Q30 value, the ESR value is also suggested, and the InitialFIT value is greatly improved, indicating that the change (improvement) of the hybridization temperature in the DNB preparation method proposed in the embodiment of the present application is conducive to the hybridization of primers in the DNB preparation process, and the DNB prepared based on this temperature effectively improves the sequencing process and improves the sequencing quality. Further, compared with the comparative example, by adding additives, the output data Q30, ESR value and InitialFIT value of Example 2 show a higher improvement, indicating that the addition of additives in the DNB preparation method proposed in the embodiment of the present application can improve the preparation quality of DNB, and effectively improve the sequencing process and improve the sequencing quality; at the same time, the combination of the change of primer hybridization temperature and the addition of additives is more conducive to obtaining high-quality sequencing results.

[0142] Figure 2-Figure 4The improvement of GC preference of the output data in the comparative example and Examples 1-2 of the present application is shown respectively, wherein the horizontal axis represents the GC content distribution diagram with a window size of 100 bases. The gray horizontal line (central axis) represents the ideal normalized coverage, expressed as 1.0, and the dotted line represents the actual normalized coverage of the sample. The closer the dotted line is to 1.0, the better the uniformity of the genome coverage of the sample. However, for the application samples, some high GC sites cannot be detected by the comparative example, and the coverage of the high GC part is low. Figure 2 and Figure 3 It can be seen that compared with the comparative example, when the primer hybridization temperature is changed, the coverage of the high GC region is better and the primer binding temperature is better; Figure 2 and Figure 4 It can be seen that compared with the comparative example, when the primer hybridization temperature is changed and additives are further added, the coverage of the high GC region is better and can cover the site to be tested.

[0143] Throughout the specification, references to "embodiment", "some embodiments", "one embodiment", "another example", "example", "specific example" or "some examples" refer to specific features, structures, materials or characteristics described in conjunction with the embodiment or example in at least one embodiment or example of the present application. Therefore, the appearance of words such as "in some embodiments", "in one embodiment", "in an embodiment", "in another example", "in an example", "in a specific example" or "in some examples" throughout the specification does not necessarily refer to the same embodiment or example of the present application. In addition, specific features, structures, materials or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0144] Although exemplary embodiments have been shown and described, those skilled in the art will appreciate that the above embodiments should not be construed as limiting the present invention and that changes, substitutions and modifications may be made to the embodiments without departing from the spirit, principle and scope of the present invention.

Claims

1. A DNB preparation system, characterized in that: The DNB preparation system comprises: an additive and a DNB preparation buffer, wherein the additive is selected from the group consisting of betaine, betaine analogs and betaine derivatives.

2. The DNB preparation system according to claim 1, characterized in that: The final concentration of the additive is 0.1M-5M; preferably, the final concentration of the additive is 2M.

3. The DNB preparation system according to claim 1 or 2, characterized in that: The DNB preparation buffer contains tris(hydroxymethyl)aminomethane or tris(hydroxymethyl)aminomethane hydrochloride); preferably, the mass fraction of the buffer is 1-5%.

4. The DNB preparation system according to any one of claims 1 to 3, characterized in that The DNB preparation buffer further comprises a mediating sequence (Splint oligo), and the DNB preparation system performs hybridization between the mediating sequence and the library at 53°C-60°C, preferably at 57°C.

5. The DNB preparation system according to any one of claims 1 to 4, characterized in that: The DNB preparation buffer further comprises an enzyme mixture, wherein the enzyme mixture comprises DNA polymerase and DNA ligase.

6. A one-step DNB preparation kit for DNBSEQ, characterized in that: The kit comprises the DNB preparation system according to any one of claims 1 to 5, Optionally, the kit further comprises a DNB termination buffer, wherein the DNB termination buffer comprises ethylenediaminetetraacetic acid.

7. A method for preparing DNB, characterized in that: The method comprises the following steps: S1. Configuring DNB preparation reaction system 1: mixing the library and the DNB preparation system according to any one of claims 1 to 4 to obtain the DNB preparation reaction system 1; S2. Sequence hybridization: annealing the DNB preparation reaction system 1 to allow the mediating sequence to hybridize with the library; S3. Rolling circle amplification: adding an enzyme mixture to the hybridized DNB preparation reaction system 1 to obtain a DNB preparation reaction system 2, and performing rolling circle amplification to obtain DNBs, wherein the enzyme mixture comprises a DNA polymerase and a DNA ligase, Optionally, the step S3 further comprises: adding a DNB termination buffer to the DNB preparation reaction system 2 to terminate the reaction. Optionally, the DNB termination buffer comprises ethylenediaminetetraacetic acid.

8. The method according to claim 7, characterized in that In step S1, the DNB preparation system comprises an additive selected from betaine, betaine analogs and betaine derivatives. Optionally, the final concentration of the additive is 0.1M-5M; preferably, the final concentration of the additive is 2M.

9. The method according to claim 7 or 8, characterized in that: In step S2, the annealing is performed at 53°C-60°C, preferably, the annealing is performed at 57°C.

10. The method according to any one of claims 7 to 9, characterized in that The library is a double-stranded DNA library, and the step S2 further comprises: before annealing the DNB preparation reaction system 1, denaturing the DNB preparation reaction system 1 to denature the double-stranded DNA library into a single-stranded DNA library for hybridization with the mediating sequence.

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