Sequencing buffer solution and method for improving stability of dNTP modified by reversible blocking group

Abstract

The invention discloses a sequencing buffer solution and a method for improving the stability of dNTP modified by reversible blocking groups, the sequencing buffer solution comprises a competitive reagent, and the competitive reagent and the dNTP modified by the reversible blocking groups can competitively react with a specific substance. The competitive reagent in the sequencing buffer solution can competitively act with a specific substance with the dNTP modified by the reversible blocking group to reduce the change (such as reduction, hydrolysis and the like) of the reversible blocking group, so that the stability of the sequencing buffer solution is ensured, the sequencing buffer solution can be better applied to sequencing, the sequencing quality is favorably improved, advanced reactions are greatly reduced, and the error rate is reduced.

Description

Sequencing buffer and method for improving the stability of dNTP modified with reversible blocking groups Technical Field

[0001] The present invention relates to the biological field, and in particular to a sequencing buffer and a method for improving the stability of dNTPs modified with reversible blocking groups. Background Art

[0002] The most widely used sequencing by synthesis (SBS) technology in current high-throughput sequencing requires that the 3'-hydroxyl end of dNTPs be connected to a removable blocking group (also called a "reversible blocking group"). This removable blocking group must meet two conditions: 1) It must not affect the efficient and accurate polymerization of modified dNTPs by DNA polymerase to the 3'-hydroxyl end of the sequencing chain; 2) It must be sufficiently stable: In its reaction buffer, the removable blocking group must be stably attached to the 3'-hydroxyl end of the dNTPs without breaking; and 3) It must be easily removable: After one cycle of sequencing, the removable blocking group can be efficiently cleaved, restoring the 3'-hydroxyl structure of the sequencing chain.

[0003] During sequencing reactions, the reversible blocking groups of dNTPs are often unstable due to sequencing buffers or the structure of the modified dNTPs themselves. For example, disulfide bonds in the reversible blocking groups are easily reduced to sulfhydryl groups, and ester bonds in the reversible blocking groups are easily hydrolyzed, causing the blocking groups of dNTPs with reversible blocking groups to fall off and lose their blocking function. This can cause the sequencing reaction to proceed prematurely (the removal of the removable blocking group causes the polymerization reaction to not stop when it should, resulting in the polymerization of two or more modified or unmodified dNTPs in a single cycle). This is reflected in the sequencing data as decreased sequencing quality and increased error rates. Therefore, improving the stability of dNTPs modified with reversible blocking groups is a key factor for successful sequencing and remains to be studied.

[0004] Summary of the Invention

[0005] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides a sequencing buffer, the use of a sequencing solution in sequencing, a method for improving the stability of dNTPs modified with reversible blocking groups, and a sequencing kit. The sequencing buffer includes a competitive reagent that can competitively react with the dNTPs modified with reversible blocking groups, reducing changes in the reversible blocking groups (such as reduction or hydrolysis), thereby ensuring their stability, enabling better application in sequencing, helping to improve sequencing quality, significantly reducing the occurrence of runons (premature reactions), and lowering error rates.

[0006] In one aspect, the present invention provides a sequencing buffer. According to an embodiment of the present invention, the sequencing buffer includes a competitive reagent that can react competitively with a dNTP modified with a reversible blocking group and with a specific substance.

[0007] In the sequencing buffer according to an embodiment of the present invention, by adding a competitive reagent, it is made to compete with the dNTP modified with a reversible blocking group to react with a specific substance, thereby reducing changes in the reversible blocking group (such as reduction, hydrolysis, etc.), ensuring its stability, further contributing to improving sequencing quality, greatly reducing the occurrence of runons (premature reactions), and reducing error rates.

[0008] It should be noted that the "specific substance" described in the present invention refers to a substance that can react with the reversible blocking group to change the group, such as an oxygen-containing compound, an OH-containing - Compounds or H + Compounds. The present invention does not strictly limit the source of the "specific substance". For example, it can come from other components in the sequencing buffer (such as oxygen dissolved in the solution) or the environment in which the sequencing buffer is located (such as OH contained in acidic or alkaline environments). - or H + ); The type of “reaction” is not strictly limited and may be an oxidation reaction, a reduction reaction, or a hydrolysis reaction, etc.

[0009] According to an embodiment of the present invention, the sequencing buffer may also have the following additional technical features:

[0010] According to an embodiment of the present invention, the reversible blocking group includes an ester bond and / or a disulfide bond. Thus, a reversible reaction can be performed and the dNTP can be reversibly removed. Any dNTP having an ester bond and / or a disulfide bond can be used in the present invention, and the present invention is not strictly limited to this.

[0011] According to an embodiment of the present invention, the reversible blocking group includes a disulfide bond, and the competitive reagent includes 2-hydroxyethyl disulfide. This competitive reagent can be competitively reduced with the disulfide-modified dNTP, making the disulfide bond less susceptible to reduction, thereby improving the stability of the protected disulfide bond.

[0012] According to an embodiment of the present invention, the reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil. The competitive reagent can be hydrolyzed competitively with the ester-modified dNTP, making the ester bond less susceptible to hydrolysis, thereby improving the stability of the protected ester bond.

[0013] In another aspect, the present invention provides a sequencing solution. According to an embodiment of the present invention, the sequencing solution comprises the aforementioned sequencing buffer and dNTPs modified with reversible blocking groups. Thus, in the sequencing solution according to an embodiment of the present invention, the dNTPs modified with reversible blocking groups are highly stable and less susceptible to hydrolysis and reduction, thereby improving sequencing quality, significantly reducing runons, and lowering error rates.

[0014] According to an embodiment of the present invention, the concentration of the dNTP is 0.1 μM to 50 mM, preferably 0.1 μM to 10 mM. According to another embodiment of the present invention, the concentration of the competitive reagent is 10 μM to 100 mM, preferably 1 to 10 mM. The inventors obtained these concentrations through extensive experiments, thereby better improving the stability of the reversible blocking group.

[0015] In another aspect of the present invention, the present invention proposes a method for improving the stability of dNTPs modified with reversible blocking groups. According to an embodiment of the present invention, the method comprises: contacting a competitive reagent with the dNTP modified with a reversible blocking group; wherein, the competitive reagent can react competitively with the reversible blocking group with a specific substance. By adding a competitive reagent, the dNTP modified with a reversible blocking group reacts competitively with the specific substance, thereby reducing the occurrence of changes in the reversible blocking group (such as being reduced, hydrolyzed, etc.), ensuring its stability, further contributing to maintaining the accuracy of sequencing, improving sequencing quality, greatly reducing the occurrence of Runon (advanced reaction), and reducing the error rate.

[0016] According to an embodiment of the present invention, the reversible blocking group includes a disulfide bond, and the competitive reagent includes 2-hydroxyethyl disulfide. This competitive reagent can be competitively reduced with the disulfide-modified dNTP, making the disulfide bond less susceptible to reduction, thereby improving the stability of the protected disulfide bond.

[0017] According to an embodiment of the present invention, the reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil. The competitive reagent can be hydrolyzed competitively with the ester-modified dNTP, making the ester bond less susceptible to hydrolysis, thereby improving the stability of the protected ester bond.

[0018] According to an embodiment of the present invention, the concentration of the dNTP in the mixed solution obtained by contacting is 0.1 μM to 50 mM, preferably 0.1 μM to 10 mM. According to another embodiment of the present invention, the concentration of the competitive reagent in the mixed solution obtained by contacting is 10 μM to 100 mM, preferably 1 to 10 mM. The inventors obtained these concentrations through extensive experiments, thereby further improving the stability of the reversible blocking group.

[0019] In yet another aspect, the present invention provides a sequencing kit. According to an embodiment of the present invention, the sequencing kit includes the aforementioned sequencing buffer or sequencing solution. Thus, the dNTPs modified with reversible blocking groups in the sequencing kit according to embodiments of the present invention exhibit excellent stability, contribute to improved sequencing quality, significantly reduce the occurrence of runons (premature reactions), and lower error rates.

[0020] In yet another aspect, the present invention provides the use of the aforementioned sequencing buffer, sequencing solution, or sequencing kit in sequencing. The dNTPs modified with reversible blocking groups in the aforementioned sequencing solution or sequencing kit exhibit excellent stability, help improve sequencing quality, reduce error rates, and are suitable for a wide range of applications.

[0021] In yet another aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the method comprises using the aforementioned sequencing buffer, sequencing solution, or sequencing kit. Thus, the sequencing method according to an embodiment of the present invention has high sequencing quality and low error rate, and is suitable for wide application.

[0022] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0023] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0024] Example 1 2-Hydroxyethyl disulfide as a competitive reagent

[0025] 1. Experimental Materials

[0026] Table 1 Reagents and consumables information

[0027]

[0028] 2. Instruments

[0029] Table 2 Instrument information

[0030] Instrument model: Gene Cycler 6337 Sequencer MGISEQ-2000RS Qubit Instrument Qubit 4.0 Electronic Balance MS204TS

[0031] 3. Experimental samples

[0032] Sample name: Standard Library Reagent V3 (26 ng / tube, E320, Barcode 97-104, MGI 1000005033).

[0033] 4. Experimental Design

[0034] The HotMPS dNTPs Mix and HotMPS dNTPs Mix II were used for the following experiments:

[0035] HotMPS dNTPs Mix and HotMPS dNTPs Mix II were added to LTE buffer containing 2-hydroxyethyl disulfide (HED) at a final concentration of 2 mM as the test group and to LTE buffer as the control group. Sequencing enzymes were added before loading the instrument, with the reagents added within half an hour and after 24 hours at 4°C. The data from the freshly prepared reagents (within half an hour) and after 24 hours at 4°C were compared, particularly for metrics such as Runon (%), Q30 (%), AvgErrorRate (%), and TotalReads (M).

[0036] Continue with a concentration gradient test: Set the final HED concentration to 2, 5, 10, and 20 mM. Add HotMPS dNTPs Mix and HotMPS dNTPs Mix II to either HED-containing or HED-free LTE buffer and perform SE50 sequencing. Compare sequencing data, particularly metrics such as Runon (%), Q30 (%), AvgErrorRate (%), and TotalReads (M), to determine the optimal concentration.

[0037] 5. Evaluation indicators and standards

[0038] Table 3 Evaluation criteria

[0039] Evaluation index qualification standard Runon (%) The lower the better Q30 (%) The higher the better AvgErrorRate (%) The lower the better TotalReads (M) The higher the better

[0040] 6. Experimental steps

[0041] 6.1 DNB preparation

[0042] Add reagents and library according to the system in the table below

[0043] Table 4 DNB preparation reaction system

[0044] Component volume (μL) Library ssDNA 3 LTE buffer 17 DNB preparation buffer 20 Total volume 40

[0045] The above reaction system was mixed by vortex oscillator, centrifuged for 5 seconds, and placed in a PCR instrument for primer hybridization. The reaction conditions are shown in the table below:

[0046] Table 5 DNB preparation reaction primer hybridization conditions

[0047] Temperature time hot cover (105℃)On95℃1min

[0048] 65℃1min40℃1min4℃Hold

[0049] Remove the DNB polymerase mix II (LC) and place it on ice. Centrifuge briefly for 5 seconds and place it on ice until ready to use. When the PCR instrument reaches 4°C, remove the PCR tube and centrifuge for 5 seconds. Then, add the following components to the PCR tube on ice:

[0050] Table 6 DNB preparation reaction components 2

[0051] Component addition amount (μL)DNB polymerase mixture I40DNB polymerase mixture II (LC)4

[0052] The reaction system was mixed by vortexing, centrifuged for 5 seconds, and immediately placed in a PCR instrument. The reaction conditions were as follows:

[0053] Table 7 DNB preparation rolling circle amplification conditions

[0054] Temperature Time Hot Cover (35℃) On 30℃ 25min 4℃ Hold

[0055] When the PCR instrument temperature reaches 4°C, immediately add 20 μL of DNB stop buffer and mix thoroughly by slowly pipetting up and down 5-8 times with a wide-mouth pipette tip.

[0056] DNB concentration was measured using Qubit.

[0057] 6.2 DNB pooling and loading

[0058] 6.2.1 Take 100 μL of DNB and add 32 μL of DLBⅡ, then mix thoroughly using a flared pipette tip.

[0059] 6.2.2 DNB Loading

[0060] Use MGIDL-200H to load DNB onto the slide. After the slide loading is completed, remove the slide and prepare it for sequencing.

[0061] 6.3 Sequencing

[0062] Prepare 4 sets of reagents from the same batch according to Tables 8-1 and 8-2. 2 sets were used as controls, and the other 2 sets were added with 2-hydroxyethyl disulfide at a final concentration of 2 mM as test groups. Within half an hour after preparation, test one of the control and test sets on the machine; test the other set after incubation at 4°C for 24 hours; and use the same script for SE50 sequencing.

[0063] Test of additives at different concentrations: Prepare 5 sets of reagents from the same batch according to Tables 8-1 and 8-2. One set is used as a control, and the other 4 sets are added with HED at final concentrations of 2mM, 5mM, 10mM, and 20mM, respectively. Use the same script for SE50 sequencing.

[0064] Experimental conditions:

[0065] Table 8-1 Hot dNTP reagent tank preparation components

[0066] HotMPS dNTPs Mix conc. 0.6μM Enzyme DNA polymerase Enzyme conc. 10μg / mL

[0067] Table 8-2 Cold dNTP reagent tank preparation components

[0068] HotMPS dNTPs Mix II conc. 2μM Enzyme DNA polymerase Enzyme conc. 10μg / mL

[0069] Script conditions:

[0070] Table 9 Script conditions

[0071] Step Tm (℃) Time (s) Hot polymerization 60120 Cold polymerization 60120 Excision 65120

[0072] 7. Experimental results and analysis

[0073] As shown in Table 10, in the control group test: the freshly prepared reagent had a runon (%) of 0.54%; after being placed at 4°C for 24 hours, the runon (%) increased to 0.73%;

[0074] HED sequencing group: The runon (%) of the freshly prepared reagent decreased to 0.41%. After 24 hours, the runon (%) increased to 0.58%, which was much lower than the runon (%) of the control group after 24 hours.

[0075] This indicates that the addition of 2-hydroxyethyl disulfide helps improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer.

[0076] Table 10 Comparison of different additives

[0077]

[0078]

[0079] As shown in Table 11, the Runon (%) of 5-20 mM HED was basically the same, and all were lower than the Runon (%) of 2 mM; the difference in MappingRate (%) may be due to the difference between different chips; the Q30 (%) of HED was better than that of the control group; and there was little difference between different concentrations.

[0080] Table 11 HED concentration test comparison

[0081]

[0082] 8. Experimental Conclusion

[0083] HED helps improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer; 5mM, 10mM, and 20mM HED have similar effects on Runon, with 5-10mM being preferred.

[0084] Example 2 5-Bromouracil as a competitive reagent

[0085] 1. Experimental Materials

[0086] Table 12 Reagents and consumables information

[0087]

[0088]

[0089] 2. Instruments

[0090] Table 13 Instrument information

[0091] Instrument model: Gene Cycler 6337 Sequencer MGISEQ-2000RS Qubit Instrument Qubit 4.0 Electronic Balance MS204TS

[0092] 3. Experimental samples

[0093] Same as Example 1.

[0094] 4. Experimental Design

[0095] Two SE50 sequencing kits were prepared, one kit was used as a control, and a final concentration of 5 mM 5-bromouracil was added to the sequencing buffer of the other kit. After acceleration for 6 hours at room temperature, SE50 sequencing was performed on the MGISEQ-2000RS sequencer to compare Runon (%), Q30 (%), AvgErrorRate (%), and TotalReads (M).

[0096] 5. Evaluation indicators and standards

[0097] Table 14 Evaluation criteria

[0098] Evaluation index qualification standards

[0099] The lower the Runon (%), the better. The higher the Q30 (%), the better. The lower the AvgErrorRate (%), the better. The higher the TotalReads (M), the better.

[0100] 6. Experimental steps

[0101] 6.1 DNB preparation

[0102] Same as Example 1.

[0103] 6.2: DNB pooling and loading

[0104] Same as Example 1.

[0105] 6.3 Sequencing

[0106] Prepare two sets of reagents from the same batch according to Tables 15-1 and 15-2, one set as a control and the other set with 5mM 5-bromouracil added as a test group. Perform SE50 sequencing on the control and test groups within half an hour of preparation.

[0107] Experimental conditions:

[0108] Table 15-1 Hot dNTP Reagent Tank Preparation Components

[0109]

[0110] Table 15-2 Cold dNTP reagent tank preparation components

[0111]

[0112] Script conditions:

[0113] Table 16 Script conditions

[0114] Step Tm(C)Time(s)Hot polymerization 60120Cold polymerization 60120Excision 65120

[0115] 7. Experimental results and analysis

[0116] Table 17 Experimental results

[0117] 5-Br (5 mM) Control Q30 (%) 93.32 92.85 ESR (%) 85.11 84.78 Mapping Rate (%) 99.37 99.33 Avg Error Rate (%) 0.38 0.43 Lag (%) 0.15 0.17 Runon (%) 0.52 0.58

[0118] After 6 hours of accelerated reaction at room temperature, the Runon (%) of the experimental group with 5-bromouracil decreased by 10% and the AvgErrorRate (%) decreased by 11.6%, indicating that the addition of 5-bromouracil helps improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II.

[0119] Comparative Example

[0120] The experimental materials (except 2-hydroxyethyl disulfide), instruments, and experimental samples are the same as those in Example 1.

[0121] Experimental steps:

[0122] 1. DNB preparation, DNB pooling, and loading were the same as in Example 1.

[0123] 2. Sequencing

[0124] According to Tables 8-1 and 8-2, 6 sets of reagents from the same batch were prepared, 2 sets of which were used as controls, 2 sets were added with DMTU (N,N′-dimethylthiourea, brand: Merck, catalog number: D188700) at a final concentration of 2 mM as test group 1, and 2 sets were added with APDC (ammonium pyrrolidine dithiocarbamate, brand: Merck, catalog number: P8765) at a final concentration of 2 mM as test group 2. Within half an hour after preparation, one of the control, test group 1, and test group 2 was tested on the machine; the other set was tested after being placed at 4°C for 24 hours; and the same script (same as in Example 1) was used for SE50 sequencing.

[0125] The results are shown in Table 18. It can be seen that the addition of DMTU and APDTC to the sequencing buffer after 24 hours at 4°C resulted in a worse stability than the control group. This indicates that DMTU and APDTC do not improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in the sequencing buffer.

[0126] Table 18 Sequencing analysis of DMTU and APDTC

[0127]

[0128]

[0129] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 invention. 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0130] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A sequencing buffer, characterized in that include: A competitive reagent can react with a specific substance competitively with a dNTP modified with a reversible blocking group.

2. The sequencing buffer according to claim 1, wherein The reversible blocking group includes an ester bond and / or a disulfide bond.

3. The sequencing buffer according to claim 1 or 2, characterized in that The reversible blocking group includes a disulfide bond, and the competitive reagent includes 2-hydroxyethyl disulfide.

4. The sequencing buffer according to claim 1 or 2, characterized in that The reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil.

5. A sequencing solution, characterized in that include: The sequencing buffer according to any one of claims 1 to 4 and dNTPs modified with reversible blocking groups.

6. The sequencing solution according to claim 5, characterized in that The concentration of the dNTP is 0.1 μM to 50 mM.

7. The sequencing solution according to claim 5 or 6, characterized in that The concentration of the dNTP is 0.1 μM to 10 mM.

8. The sequencing solution according to claim 5, characterized in that The concentration of the competitive reagent is 10 μM to 100 mM.

9. The sequencing solution according to any one of claims 5 to 8, wherein The concentration of the competitive reagent is 1-10 mM.

10. A method for improving the stability of dNTPs modified with reversible blocking groups, characterized in that: include: contacting a competitive reagent with the dNTP modified with a reversible blocking group; The competitive reagent can react with the reversible blocking group competitively with the specific substance.

11. The method according to claim 10, characterized in that The reversible blocking group includes a disulfide bond, and the competitive reagent includes 2-hydroxyethyl disulfide.

12. The method according to claim 10 or 11, characterized in that The reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil.

13. The method according to claim 10, characterized in that In the mixed solution obtained by the contact, the concentration of the competitive reagent is 10 μM to 100 mM.

14. The method according to claim 10 or 13, characterized in that In the mixed solution obtained by the contact, the concentration of the competitive reagent is 1-10 mM.

15. The method according to claim 10, characterized in that In the mixed solution obtained by the contact, the concentration of the dNTP is 0.1 μM to 50 mM.

16. The method according to claim 10 or 15, characterized in that In the mixed solution obtained by the contact, the concentration of the dNTP is 0.1 μM to 10 mM.

17. A sequencing kit, characterized in that: include: The sequencing buffer according to any one of claims 1 to 4 or the sequencing solution according to any one of claims 5 to 9.

18. Use of the sequencing buffer according to any one of claims 1 to 4, the sequencing solution according to any one of claims 5 to 9, or the sequencing kit according to claim 17 in sequencing.

19. A sequencing method, characterized in that: include: Use the sequencing buffer according to any one of claims 1 to 4, the sequencing solution according to any one of claims 5 to 9, or the sequencing kit according to claim 17.