Base calibration reagent and kit for next-generation sequencing and application of base calibration reagent and kit

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a base calibration reagent for next-generation sequencing, a kit and application of the base calibration reagent. The base calibration reagent comprises streptavidin modified by fluorophores with different wavelengths and anti-digoxin, and the fluorophores are connected with the streptavidin and the anti-digoxin, so that the number and the variety of the fluorophores can be adjusted to achieve a stronger fluorescence signal and a higher signal-to-noise ratio, and the data quality of DNA sequencing is further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, in particular to gene sequencing, and specifically relates to a base calibration reagent and a kit for second-generation sequencing and applications thereof. Background Art

[0002] The first generation of DNA sequencing technology, also known as Sanger sequencing or dideoxy chain termination method, is characterized by long sequencing read length and high sequencing accuracy, but the sequencing of each DNA chain needs to be prepared separately, so there is no way to achieve large-scale high-throughput sequencing.

[0003] The second generation DNA sequencing technology uses the "sequencing by synthesis" strategy. During the sequencing process, the DNA sample to be tested (such as chromosomal DNA) is first cut into fragments of several hundred to one thousand bases in length using physical or molecular biological methods, and the two ends of these fragments are then bonded to universal DNA connectors to form a DNA library to be sequenced. The DNA library is then input onto the surface of the sequencing chip, which has surface DNA primers pre-connected by covalent bonds. These primers hybridize with the connectors of the molecules in the library to bind the DNA molecules to the surface of the chip. The DNA molecules to be tested on the surface then undergo DNA replication and amplification reactions to form molecular clusters composed of several identical DNA molecules in close physical positions. These DNA molecular clusters are then hybridized with sequencing primers, which are then connected to a fluorescently modified nucleotide under the catalysis of DNA polymerase. By taking fluorescent photos, the type of bases connected to each molecular cluster on the sequencing chip can be known. These sequencing nucleotides also have a protective group on the 3'-OH group, so only one nucleotide can be connected at a time. After the photo is taken, the fluorescent groups on these protective groups and bases will be removed by chemical methods at the same time, and then the next cycle of DNA extension and base discrimination can be carried out. After several cycles, the sequence of each DNA cluster on the sequencing chip can be detected. This method of synthesizing and detecting sequences uses a large range of images for signal acquisition. It can sequence hundreds of millions to tens of billions of DNA clusters at the same time, has a very high throughput, and is currently the most widely used DNA sequencing technology in the fields of biotechnology and health.

[0004] In the current second-generation DNA sequencing methods, the fluorescent groups that label the bases are directly connected to the bases. Most fluorescent groups have a certain charge and a large conjugated electron system (π bond), which will have a certain impact on the efficiency of nucleotides connecting to primers under the catalysis of DNA polymerase.

[0005] Therefore, there is an urgent need for a base calibration method to improve the quality of DNA sequencing data. Summary of the invention

[0006] The present invention provides a base calibration method for sequencing, wherein the base is not directly connected to a fluorescent group, but is combined through a biotin-streptavidin interaction and a digoxin-anti-digoxin interaction. The interaction between biotin and streptavidin is the strongest non-covalent bond interaction known in nature, and has a strong binding force and high specificity. Digoxin is a glycoside molecule, and anti-digoxin is an antibody to digoxin. Therefore, the connection of dNTP catalyzed by DNA polymerase is less affected, and the sequencing reaction efficiency is higher.

[0007] One aspect of the present invention provides a base calibration reagent for second-generation sequencing, characterized in that the reagent includes streptavidin modified with fluorescent group 1 and anti-digoxigenin modified with fluorescent group 2.

[0008] In some embodiments, the excitation wavelengths of the fluorescent group 1 and the fluorescent group 2 are different.

[0009] In some embodiments, the types and quantities of the fluorescent group 1 and the fluorescent group 2 can be adjusted.

[0010] Another object of the present invention is to provide a base calibration kit for second-generation sequencing, characterized in that the kit includes the reagents described above.

[0011] In some embodiments, the kit further comprises a base substrate, wherein the first base in the base substrate is modified with biotin, the second base is modified with digoxigenin, the third base is modified with both biotin and digoxigenin, and the fourth base is not modified with biotin or digoxigenin groups; optionally, the third base is a mixture of bases modified with biotin and bases modified with digoxigenin.

[0012] Another object of the present invention is to provide a sequencing method, using the bases obtained by the base calibration method for sequencing or the kit for sequencing, characterized in that the method comprises the following steps: Step 1: The DNA sample to be tested is fixed on the surface of the sequencing chip, and the 3' end of the sequence to be tested is hybridized with the sequencing primer; Step 2: adding a solution of a base substrate and a DNA polymerase to the surface of a sequencing chip, wherein the first base in the base substrate is modified with biotin, the second base is modified with digoxigenin, the third base is modified with both biotin and digoxigenin, and the fourth base is not modified with a biotin or digoxigenin group; optionally, the third base is a mixture of a base modified with biotin and a base modified with digoxigenin; Step 3: After the base is added to the 3' end of the sequencing primer, streptavidin and anti-digoxigenin modified with fluorescent groups of different wavelengths are added to the surface of the sequencing chip, and the fluorescent groups are made to emit fluorescence by the excitation light; Step 4: Take a fluorescent photo and measure the base type at each position; Step 5: Perform the next round of extension and repeat this process to complete the DNA sequencing.

[0013] In some embodiments, the 3'-OH of the base substrate is modified with a protecting group.

[0014] In some embodiments, in step 4, the base type of each position on the sequencing chip in this round can be inferred according to the fluorescence wavelength of the newly added base position, the base distribution of streptavidin-biotin, the base distribution of anti-digoxigenin-digoxigenin, and the pairing principle of AT and GC.

[0015] In some embodiments, in step 5, before the next round of extension, the protecting groups at the 3' end of the nucleic acid and the biotin and digoxigenin groups are removed.

[0016] Another object of the present invention is to provide the reagent as described above, or the use of the kit as described above in DNA sequencing.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are: 1. In the present invention, dNTP is not directly connected to the fluorescent group, but connected to biotin and digoxigenin. Biotin and digoxigenin groups are not charged and have no electron conjugation system, so they have little effect on the connection of dNTP catalyzed by DNA polymerase, and the sequencing reaction efficiency is higher.

[0018] 2. In the present invention, based on the Illumina second-generation sequencing platform, the method of base calibration by biotin and digoxigenin group modification is creatively adopted for the first time to perform excitation light sequencing. The fluorescent groups are connected to streptavidin and anti-digoxigenin, so the number and type of fluorescent groups can be adjusted to achieve stronger fluorescent signals and higher signal-to-noise ratio, thereby improving the data quality of DNA sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0020] Figure 1 Base calibration methods and sequencing steps for sequencing; Figure 2 Data comparing the sequencing method of the present invention with the conventional sequencing method using fluorescent groups to directly link bases. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments: To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0022] Example 1: A base calibration method for sequencing of the present invention, wherein the four nucleotides (A, T, C, G) used in sequencing are modified with different combinations of biotin and digoxigenin: the first base is modified with only biotin, the second base is modified with only digoxigenin, the third base is modified with both biotin and digoxigenin groups, and the fourth base has no biotin or digoxigenin groups. Figure 1 Taking one of the multiple possible combinations as an example, dATP is modified with biotin, dTTP is modified with digoxigenin, dGTP is not modified with biotin or digoxigenin groups, and dCTP is modified with both biotin and digoxigenin groups (it can be that one dCTP molecule is modified with both biotin and digoxigenin, or it can be a mixture of dCTP molecules modified with biotin and dCTP molecules modified with digoxigenin).

[0023] Example 2: Sequencing method: During the DNA sequencing process, after the 3' end of the sequence to be tested hybridizes with the sequencing primer, a solution of DNA polymerase and dNTP is added to the surface of the sequencing chip. The dNTP used for sequencing is modified with a protecting group on the -OH at the 3' end, so under the catalysis of DNA polymerase, the dNTP will increase the length of the 3' end of the sequencing primer by one base. The four nucleotides (A, T, C, G) used in sequencing are modified with different combinations of biotin and digoxigenin: the first base is only modified with biotin, the second base is only modified with digoxigenin, the third base is modified with both biotin and digoxigenin groups, and the fourth base has no biotin or digoxigenin groups. Figure 1 Taking one of the multiple possible combinations as an example, dATP is modified with biotin, dTTP is modified with digoxigenin, dGTP is not modified with biotin or digoxigenin groups, and dCTP is modified with both biotin and digoxigenin groups (it can be that one dCTP molecule is modified with both biotin and digoxigenin, or it can be a mixture of dCTP molecules modified with biotin and dCTP molecules modified with digoxigenin).

[0024] After the sequencing primer is extended by one base length by the modified dNTP, the excess DNA polymerase and dNTP are eluted from the surface of the sequencing chip, and then fluorescent group-modified streptavidin and anti-digoxigenin are added to the chip surface. The fluorescent wavelengths of the fluorescent groups on streptavidin and anti-digoxigenin are different. For the convenience of description, the fluorescent wavelength on streptavidin is wavelength 1, and the fluorescent wavelength on anti-digoxigenin is wavelength 2. The base modified with biotin (A in the figure as an example) will bind to streptavidin; the base modified with digoxigenin (T in the figure as an example) will bind to anti-digoxigenin; the base modified with both biotin and digoxigenin (C in the figure as an example) will bind to both streptavidin and anti-digoxigenin; the base not modified with biotin or digoxigenin (G in the figure as an example) will not bind to any molecule with a fluorescent group.

[0025] After the excess streptavidin and anti-digoxigenin are washed off the chip, a fluorescent photo will be taken on the surface of the sequencing chip. If there is only fluorescence at wavelength 1 (streptavidin) for a DNA molecular cluster at a certain position, it means that the added base is only modified with biotin (A); if there is only fluorescence at wavelength 2 (anti-digoxigenin), it means that the added base is only modified with digoxigenin (T); if there is fluorescence at both wavelength 1 and wavelength 2, it means that the added base is modified with both biotin and digoxigenin (C); if there is no fluorescence at wavelength 1 and no fluorescence at wavelength 2, it means that the base is not modified with biotin or digoxigenin (G). In this way, the base types of all DNA molecular clusters in the photo in the previous steps can be determined.

[0026] After the photography and decoding steps, the biotin and digoxigenin groups on the DNA, and the protecting group on the 3'-OH can be cut off at the same time using chemical methods, so that the 3' of the primer that has been extended by one base length can be extended again, and then enter the next cycle, using the same method to detect the next base in the DNA template to be tested.

[0027] Example 3: The base calibration method of the present invention was applied to an actual DNA sequencing experiment and compared with the traditional method of directly connecting fluorescent groups to bases (control experiment). In the two experiments, except for the different solutions involved in base calibration, all other reagents were the same. The sequencing library was the phiX standard library, and the concentration and total amount used in the two experiments were the same, and the density of the DNA clusters generated on the surface of the sequencing chip was consistent ( Figure 2 c). The two experiments were performed three times each, and the average of the three experiments was used for comparison.

[0028] It can be seen from the experimental results that in the experiment using the base calibration method of the present invention, the fluorescence intensity (intensity) emitted by the DNA molecule cluster is stronger than that of the traditional method in which the fluorescent group is directly attached to the base ( Figure 2a, 2b). The percentage of data with Q_Score greater than 30 (Q30) in the results of the present invention is also better than that of the control group ( Figure 2 d). A Q_Score greater than 30 means the confidence level of the result is greater than 99.9%.

[0029] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0030] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A base calibration reagent for second-generation sequencing, characterized in that: The reagent includes streptavidin modified with fluorescent group 1 and anti-digoxigenin modified with fluorescent group 2.

2. The reagent according to claim 1, characterized in that The fluorescent group 1 and the fluorescent group 2 have different excitation wavelengths.

3. The reagent according to claim 1, characterized in that The types and quantities of the fluorescent group 1 and the fluorescent group 2 can be adjusted.

4. A base calibration kit for second-generation sequencing, characterized in that: The kit comprises the reagent according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes a base substrate, in which the first base is modified with biotin, the second base is modified with digoxigenin, the third base is modified with both biotin and digoxigenin, and the fourth base is not modified with biotin or digoxigenin groups; optionally, the third base is a mixture of bases modified with biotin and bases modified with digoxigenin.

6. A sequencing method, characterized in that: Sequencing is performed using the reagent according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5, the method comprising the following steps: Step 1: The DNA sample to be tested is fixed on the surface of the sequencing chip, and the 3' end of the sequence to be tested is hybridized with the sequencing primer; Step 2: adding a solution of a base substrate and a DNA polymerase to the surface of a sequencing chip, wherein the first base in the base substrate is modified with biotin, the second base is modified with digoxigenin, the third base is modified with both biotin and digoxigenin, and the fourth base is not modified with a biotin or digoxigenin group; optionally, the third base is a mixture of a base modified with biotin and a base modified with digoxigenin; Step 3: After the base is added to the 3' end of the sequencing primer, streptavidin and anti-digoxigenin modified with fluorescent groups of different wavelengths are added to the surface of the sequencing chip, and the fluorescent groups are made to emit fluorescence by the excitation light; Step 4: Take a fluorescent photo and measure the base type at each position; Step 5: Perform the next round of extension and repeat this process to complete the DNA sequencing.

7. The sequencing method according to claim 6, characterized in that: The 3'-OH terminal of the base substrate is modified with a protecting group.

8. The sequencing method according to claim 6, characterized in that In step 4, the base type of each position on the sequencing chip in this round can be inferred according to the fluorescence wavelength of the newly added base position, the base distribution of streptavidin-biotin, the base distribution of anti-digoxigenin-digoxigenin, and the pairing principle of AT and GC.

9. The sequencing method according to claim 6, characterized in that: In step 5, before the next round of extension, the protecting groups at the 3' end of the nucleic acid and the biotin and digoxigenin groups are removed.

10. Use of the reagent according to any one of claims 1 to 3, or the kit according to any one of claims 4 to 5 in DNA sequencing.

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