CN115141880A

CN115141880A - Method, reaction system and kit for improving nucleic acid polymerization sequencing quality

Info

Publication number: CN115141880A
Application number: CN202210628687.8A
Authority: CN
Inventors: 李计广; 马可心; 邱敏; 陈奥; 徐崇钧; 章文蔚
Original assignee: MGI Tech Co Ltd
Current assignee: MGI Tech Co Ltd
Priority date: 2017-01-10
Filing date: 2017-01-10
Publication date: 2022-10-04
Also published as: WO2018129644A1; CN109937259B; CN109937259A; CN114958998A

Abstract

The application discloses a method, a reaction system and a kit for improving nucleic acid polymerization sequencing quality. The method for improving the quality of nucleic acid polymerization sequencing comprises the step of adding a second type of nucleotide to a reaction solution of the polymerization sequencing, wherein the second type of nucleotide has a blocking modification on a 3' sugar hydroxyl group but does not have a fluorescent modification. According to the method for improving the nucleic acid polymerization sequencing quality, dNTPs only with blocking modification are added into a reaction solution, and the dNTPs are not subjected to fluorescent modification, so that DNA polymerase identification and synthesis are facilitated, incomplete reaction parts can be supplemented, the synthesis reaction efficiency is improved, and the risk of insufficient reaction is reduced; meanwhile, due to the fact that no fluorescence modification exists, signal interference caused by unclean fluorescence excision or unclean fluorescence elution of excision is reduced, excision efficiency is improved, and sequencing error rate is reduced.

Description

Method, reaction system and kit for improving nucleic acid polymerization sequencing quality

The application is a divisional application of the invention application with the application number of 201780068192.1, the application date of 2017, 01 month and 10 days, and the invention name of the method, the reaction system and the kit for improving the nucleic acid polymerization sequencing quality.

Technical Field

The application relates to the field of nucleic acid sequencing, in particular to a method, a reaction system and a kit for improving the quality of nucleic acid polymerization sequencing.

Background

Nucleic acid polymerase sequencing, sequencing by synthesis (abbreviated SBS), refers to the addition of fluorescently labeled nucleotides during SBS (Metzker et al., genome Res 15 (12): 1767-1776 (2005)) to aid in the recognition of the template DNA bases (Prober et al., science 238 336-341 (1987)) to give information on the DNA sequence. In recent years, polymerase sequencing has gained favor with higher throughput and lower cost than other sequencing methods. In this sequencing method, bases that terminate reversibly and carry a fluorescent signal are synthesized by a DNA polymerase; specifically, 4 different bases were distinguished by fluorescent signals using nucleotides with modifications at the 3' sugar hydroxyl to block the addition of other nucleotides. After removal of the blocking and fluorescent groups, the natural free 3' hydroxyl group is restored for addition of the next nucleotide and the fluorescent signal is removed to facilitate synthesis and detection of the next base. In SBS, the synthesis efficiency is very high, and once incomplete synthesis occurs in multiple copies, signal confusion can result, which affects the read length and accuracy of sequencing. The nucleotide with reversible blocking and fluorescent group is required to be added in the process of synthesizing and sequencing, the molecule of the nucleotide is larger due to the increase of modification, the molecular structure is more special, and compared with the nucleotide without modification, the efficiency of recognizing and synthesizing the dNTPs with modification by DNA polymerase is lower, so that the reading length and the accuracy of polymerization sequencing are limited.

Disclosure of Invention

The purpose of the present application is to provide an improved and novel method for improving the quality of nucleic acid polymerization sequencing and application thereof.

In order to achieve the purpose, the following technical scheme is adopted in the application:

the application discloses a method for improving the quality of nucleic acid polymerization sequencing, which comprises the step of adding a second type of nucleotide into a reaction solution of the polymerization sequencing, wherein the second type of nucleotide has a blocking modification on a 3' sugar hydroxyl group but does not have a fluorescent modification.

It is important to note that dNTPs having only blocking modification, i.e., the second type of nucleotides, are added to the reaction solution for sequencing by polymerization. The influence of blocking modified dNTPs on the recognition and synthesis of DNA polymerase is relatively small, so that the addition of the second type of nucleotides can supplement the incomplete part of the reaction, improve the efficiency of the synthesis reaction and reduce the risk of insufficient reaction; in addition, because the second type of nucleotides are not subjected to fluorescent modification, the signal interference caused by unclean fluorescent excision or unclean eluted excised fluorescence is reduced, the excision efficiency is improved, and the sequencing error rate is reduced; thereby playing a role in improving the sequencing quality. It is understood that the key to the method of the present application is that only the second type of nucleotides with blocking modifications are added to the reaction solution for polymerization sequencing, and that reference may be made to existing polymerization sequencing methods for other components of the reaction solution, as well as specific reaction conditions.

Preferably, the second type of nucleotide is used in the same amount as the first type of nucleotide in the reaction solution, the first type of nucleotide being a nucleotide having a blocking modification at the 3' sugar hydroxyl group and having a fluorescent modification.

It should be noted that the first type of nucleotide is the nucleotide normally added in the polymerase sequencing, and in the preferred embodiment of the present invention, the second type of nucleotide and the first type of nucleotide are added in equal amounts. It is understood that in some special design schemes, the amount of the second type of nucleotide can be adjusted according to the requirement, but the second type of nucleotide can be added to improve the synthesis reaction efficiency and reduce the sequencing error rate to some extent.

In another aspect of the present application, the use of the methods of the present application in nucleic acid sequencing is disclosed. The methods of the present application are applicable to nucleic acid sequence sequencing of the human genome and other animal, plant and microbial species; the main application comprises sequencing of WES, WGS, RNA, DNA and the like; in one embodiment of the present application, the methods of the present application are used for DNA sequencing of e.

In another aspect, the present application discloses a method for sequencing nucleic acids, comprising adding two types of nucleotides simultaneously to a reaction solution during sequencing by synthesis, wherein the first type of nucleotide is a nucleotide having a blocking modification at the 3 'sugar hydroxyl group and having a fluorescent modification, and the second type of nucleotide is a nucleotide having a blocking modification at the 3' sugar hydroxyl group but not having a fluorescent modification.

It can be understood that the method for improving the quality of nucleic acid polymerization sequencing can be applied to various sequencing platforms based on synthesis-while-sequencing, and only the second type of nucleotide of the application needs to be added into the reaction solution under the condition of not changing the sequencing conditions and parameters.

The application also discloses a reaction system for improving the nucleic acid sequencing quality, which comprises a reaction liquid for sequencing while synthesis, wherein a second type of nucleotide is added into the reaction liquid, and the second type of nucleotide has a blocking modification on a 3' sugar hydroxyl group but has no fluorescent modification.

Preferably, the reaction solution comprises a reaction buffer, a DNA polymerase and a first type of nucleotide, the first type of nucleotide being a nucleotide having a blocking modification on the 3' sugar hydroxyl group and having a fluorescent modification.

In yet another aspect, the present application discloses a nucleic acid sequencing kit comprising the reaction system of the present application.

It can be understood that the key inventive concept of the present application is to add dNTPs with only blocking modification, i.e. second type nucleotides, to the reaction solution while synthesizing and sequencing to improve the synthesis efficiency; based on the thought of the invention, in order to be convenient to use, the application further provides a reaction system for improving the nucleic acid sequencing quality, namely a reaction solution which is added with a second type of nucleotide and is synthesized and sequenced at the same time. By adopting the reaction reagent system, the sequencing quality can be effectively improved. Similarly, the reagent system of the present application may be used as a nucleic acid sequencing kit for convenience of use.

Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:

according to the method for improving the nucleic acid polymerization sequencing quality, dNTPs only with blocking modification are added into the reaction liquid, and the dNTPs are not subjected to fluorescent modification, so that the DNA polymerase can be identified and synthesized more conveniently, the incomplete part of the reaction can be supplemented, the efficiency of the synthesis reaction is improved, and the risk of insufficient reaction is reduced; meanwhile, due to the fact that no fluorescence modification exists, signal interference caused by unclean fluorescence excision or unclean fluorescence elution of excision is reduced, excision efficiency is improved, and sequencing error rate is reduced.

Drawings

FIG. 1 is a schematic diagram of the structure of a second class of nucleotides in the examples of the present application;

FIG. 2 is a schematic diagram of the structure of a first type of nucleotide in the examples of the present application;

FIG. 3 is a graph showing the change in fluorescence signal per cycle in the examples of the present application;

FIG. 4 is a plot of sequencing mass change per cycle in an example of the application;

FIG. 5 is a plot of the sequencing error rate per cycle in the examples of the present application.

Detailed Description

The key point of sequencing while synthesizing is that dNTPs with blocking modification and fluorescence modification are added into a reaction solution, however, through a large amount of experiments and researches, the inventors of the present application find that the dNTPs with the blocking modification and the fluorescence modification influence the recognition and synthesis efficiency of DNA polymerase, so that the reading length and the accuracy of polymerization sequencing are limited. The fluorescent modification is found to be the main reason for influencing the recognition and synthesis efficiency of DNA polymerase through intensive research; therefore, the application proposes that dNTPs only with blocking modification are added on the basis of the existing polymerization sequencing reaction solution; therefore, on one hand, the incomplete part of the reaction can be supplemented by the second type of nucleotide, so that the synthesis efficiency is ensured, and the risk of insufficient reaction is reduced; on the other hand, because the second type of nucleotides are not subjected to fluorescent modification, signal interference caused by unclean fluorescent excision or unclean excised fluorescent elution is reduced, excision efficiency is improved, and sequencing error rate is reduced.

The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

The nucleic acid sequencing method is carried out on a BGISEQ-500 sequencing platform, and sequencing samples are standard libraries of escherichia coli. The specific scheme is as follows:

(1) Using a sequencing platform of BGISEQ-500 and using a sequencing reagent matched with the platform according to the instruction;

(2) Preparing dNTP1 mixed liquor according to a formula shown in a table 1, namely first type nucleotide; the first type of nucleotide in this example has both a fluorescent dye and a blocking group as shown in FIG. 2, wherein A, T, G, and C represent adenine nucleotide, thymine nucleotide, guanine nucleotide, and cytosine nucleotide, respectively.

Table 1 dNTP1 mixed liquor formula table

dNTP1 mixture	Final concentration (nmol/L)
		dCTP_1	100
dGTP_1	100
		dATP_1	100
dTTP_1	100

In the table, dCTP _1 refers to cytosine nucleotides having both blocking modification and fluorescence modification, dGTP _1 refers to guanine nucleotides having both blocking modification and fluorescence modification, dATP _1 refers to adenine nucleotides having both blocking modification and fluorescence modification, and dTTP _1 refers to thymine nucleotides having both blocking modification and fluorescence modification.

(3) Preparing dNTP 2 mixed solution according to a formula shown in a table 2, namely second type nucleotides; in the second class of nucleotides, as shown in FIG. 1, the nucleotides have only blocking groups and no fluorescent dye, wherein A, T, G and C respectively represent adenine nucleotide, thymine nucleotide, guanine nucleotide and cytosine nucleotide.

TABLE 2 dNTP 2 mixed liquor formula table

In the table, dCTP _2 refers to cytosine nucleotides having only blocking modification, dGTP _2 refers to guanine nucleotides having only blocking modification, dATP _2 refers to adenine nucleotides having only blocking modification, and dTTP _2 refers to thymine nucleotides having only blocking modification.

(4) The BGISEQ-500 platform is utilized, dNTP 2 mixed liquor is added for realizing the effect of the complementary reaction, and under the condition that other reagents are not changed, the reagents of the No. 5 and No. 6 hole sites of the kit are taken out and replaced by the reagents needed by an experimental group;

(5) Preparing a synthetic reagent 1 according to the table 3, and uniformly mixing for later use;

table 3 synthetic reagent 1 formula table

Synthesis of reagent 1	Dosage (mL)
		Reaction buffer	48
DNA polymerase	1
		dNTP1 mixture	1

(6) Preparing a synthetic reagent 2 according to the table 4, and uniformly mixing for later use;

table 4 synthetic reagent 2 formula table

Synthesis reagent 2	Dosage (mL)
		Reaction buffer	48
DNA polymerase	1
		dNTP 2 mixture	1

(7) Adding an equal amount of synthetic reagent 1 into a #5 hole site and a #6 hole site of the kit of the control group respectively; synthetic reagent 1 is added into a hole #5 of the kit in the experimental group, synthetic reagent 2 is added into a hole #6 of the kit, and the dosage ratio of the two reagents is 1: 1.

(8) Sequencing of SE50 is carried out by using a BGISEQ-500 platform, a test group added with a synthetic reagent 1 is used as a control group, a test group added with a synthetic reagent 2 is used as an experimental group, and a fluorescence signal change curve of each cycle, a sequencing quality change curve of each cycle and a sequencing error rate change curve of each cycle in two groups of tests are respectively subjected to statistical analysis.

The sequencing results are shown in FIGS. 3 to 5. Wherein the fluorescence signal change curve for each cycle is shown in FIG. 3, wherein the abscissa is the sequencing read length, the ordinate is the sequencing signal, ". Smallcircle" is the curve for the experimental group, ". Tangle-solidup" is the curve for the control group; the results show that as the sequencing cycle increases, the sequencing signal slowly decreases, and the fluorescence signal of the experimental group decreases more slowly than that of the control group, indicating that sequencing synthesis and excision are more complete.

The curve of the sequencing mass change per cycle is shown in FIG. 4, in which the abscissa is the sequencing read length, the ordinate is the sequencing mass value, ". Smallcircle" is the curve of the experimental group, ". Tangle-solidup" is the curve of the control group; the results show that as the sequencing cycle increases, the sequencing quality decreases, and the sequencing quality value of the experimental group decreases more slowly than that of the control group, indicating that the sequencing quality of the experimental group is higher.

The curve of the change in the sequencing error rate at each cycle is shown in FIG. 5, in which the abscissa is the sequencing read length, the ordinate is the sequencing error rate, ". Smallcircle" is the curve for the experimental group, and "a-solidup" is the curve for the control group; the results show that as the sequencing cycle increases, the sequencing error rate begins to increase, and the error rate of the experimental group increases more slowly than the control group, indicating that the error rate of the experimental group is lower.

Therefore, the dNTPs only with blocking modification, namely the second type of nucleotides, are added into the reaction solution of polymerization sequencing, so that the sequencing quality can be effectively improved, and the sequencing error rate is reduced.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions can be made without departing from the concept of the present application, which should be considered as belonging to the protection scope of the present application.

Claims

1. A method for improving the quality of nucleic acid sequencing by polymerization, wherein the method comprises adding a first type of nucleotide and a second type of nucleotide to a reaction solution of sequencing by polymerization, the first type of nucleotide has a blocking modification on a 3 'sugar hydroxyl group and has a fluorescent modification, the second type of nucleotide has a blocking modification on a 3' sugar hydroxyl group but has no fluorescent modification, and the first type of nucleotide and the second type of nucleotide are not added at the same time.

2. The method of claim 1, wherein the second type of nucleotide is used in the same amount as the first type of nucleotide.

3. The method of claim 1, wherein the second type of nucleotide is used in an amount that is different from the amount used for the first type of nucleotide.

4. The method according to any one of claims 1 to 3, wherein the reaction solution for the polymerase sequencing further comprises a reaction buffer and/or a DNA polymerase.

5. The method according to any one of claims 1-4, wherein the first type of nucleotide is a dNTP and/or the second type of nucleotide is a dNTP.

6. The method of any one of claims 1-5, wherein the first type of nucleotide is selected from adenine nucleotides, thymine nucleotides, guanine nucleotides and/or cytosine nucleotides, and/or the second type of nucleotide is selected from adenine nucleotides, thymine nucleotides, guanine nucleotides and/or cytosine nucleotides.

7. The method of any one of claims 1-6, wherein the fluorescent modification of the first type of nucleotide is attached to the base of the nucleotide.

8. The method of claim 7, wherein the bases are selected from A, T, C, G.

9. Use of the method according to any one of claims 1-8 in nucleic acid sequencing.

10. The use of claim 9, wherein the nucleic acid sequencing comprises WES, WGS, RNA or DNA sequencing.

11. Use of the method according to any one of claims 1 to 8 for nucleic acid sequencing of human genomes, animals, plants and/or microorganisms.

12. A method for sequencing a nucleic acid, wherein the method comprises adding two types of nucleotides to a reaction solution during sequencing by synthesis, the first type of nucleotide being a nucleotide having a blocking modification at the 3 'sugar hydroxyl group and having a fluorescent modification, the second type of nucleotide being a nucleotide having a blocking modification at the 3' sugar hydroxyl group and having no fluorescent modification, and the two types of nucleotides are not added at the same time.

13. The method of claim 12, wherein the method has the features defined in any one of claims 2 to 8.