WO2023240494A1 - Sequencing buffer and method for improving stability of dntp modified with reversible blocking group - Google Patents

Abstract

A sequencing buffer and a method for improving the stability of dNTP modified with a reversible blocking group. The sequencing buffer comprises: a competing reagent, which can compete with dNTP modified with a reversible blocking group in reacting with a specific substance. The competing reagent in the sequencing buffer can compete with dNTP modified with a reversible blocking group in interacting with a specific substance, which reduces a change in the reversible blocking group (for example, being reduced and hydrolyzed, etc.), thereby ensuring the stability of the dNTP, allowing same to be better used in sequencing, helping to improve the quality of sequencing, greatly reducing the occurrence of premature reactions, and reducing the error rate.

Description

Sequencing buffer and method for improving stability of dNTPs modified with reversible blocking groups Technical field

The present invention relates to the biological field. Specifically, the present invention relates to sequencing buffers and methods for improving the stability of dNTPs modified with reversible blocking groups.

Background technique

Sequencing by Synthesis (SBS), the most widely used technology in current high-throughput sequencing, requires the 3'-hydroxyl end of dNTPs to be connected to a removable blocking group (also known as a "reversible blocking group"). "), and this removable blocking group needs to meet two conditions: 1) It does not affect the efficient and accurate polymerization of modified dNTPs to the 3'-hydroxyl end of the sequencing strand by DNA polymerase; 2) It is stable enough: in its In the reaction buffer, the cleavable blocking group is stably connected to the 3'-hydroxyl end of dNTPs without breaking; 3) Easy to cleave: After one cycle of measurement, the cleavable blocking group can be efficiently cleaved. Remove and restore the 3'-hydroxyl structure of the sequencing strand.

In sequencing reactions, the reversible blocking groups of dNTPs are often not stable enough due to the structure of the sequencing buffer or modified dNTPs themselves. For example, the disulfide bond of the reversible blocking group is easily reduced to a sulfhydryl group, and the reversible blocking group The ester bond is easily hydrolyzed, causing the blocking group of dNTPs with reversible blocking groups to fall off easily and lose its blocking function, which in turn causes the sequencing reaction to proceed ahead of schedule (the removable blocking group is lost, causing the polymerization reaction to stop. Stop, one cycle aggregates two or more modified or unmodified dNTPs); this is reflected in the sequencing data, showing a decrease in sequencing quality and an increase in error rate. Therefore, improving the stability of dNTPs modified with reversible blocking groups is one of the key factors for successful sequencing and remains to be studied.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention proposes the application of sequencing buffers and sequencing solutions in sequencing, methods for improving the stability of dNTPs modified with reversible blocking groups, and sequencing kits. The sequencing buffer includes competitive reagents that can be combined with The dNTP modified with the reversible blocking group competitively reacts to reduce the changes of the reversible blocking group (such as being reduced, hydrolyzed, etc.), thus ensuring its stability, better application in sequencing, and helping to improve sequencing Quality, greatly reducing the occurrence of Runon (premature reaction) and reducing the error rate.

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

In the sequencing buffer according to the embodiment of the present invention, by adding a competitive reagent, the dNTP modified with a reversible blocking group competes with the specific substance to react, thereby reducing changes in the reversible blocking group (such as being modified by the blocking group). reduction, hydrolysis, etc.) to ensure its stability, which further helps to improve the quality of sequencing, greatly reduces the occurrence of runon (advanced reactions), and reduces the error rate.

It should be noted that the "specific substance" described in the present invention refers to a substance that can react with a reversible blocking group to change the group. For example, it can be an oxygen-containing compound, an OH ^- containing compound or an H ⁺ -containing compound. The present invention does not strictly limit the source of the "specific substance". For example, it can originate 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, it can be oxidation reaction, reduction reaction or hydrolysis reaction, etc.

According to embodiments of the present invention, the sequencing buffer may also have the following additional technical features:

According to an embodiment of the present invention, the reversible blocking group includes an ester bond and/or a disulfide bond. As a result, a reversible reaction can occur and be reversibly ablated. Any dNTP with ester bonds and/or disulfide bonds can be used in the present invention, and the present invention is not strictly limited to this.

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

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

In another aspect of the invention, the invention provides a sequencing solution. According to an embodiment of the present invention, the sequencing solution includes: the aforementioned sequencing buffer and dNTPs modified with reversible blocking groups. Therefore, in the sequencing solution according to the embodiment of the present invention, the dNTP modified with a reversible blocking group has strong stability and is not prone to hydrolysis, reduction, etc., thus improving the sequencing quality and greatly reducing Runon (premature reaction) occurs, reducing the error rate.

According to an embodiment of the present invention, the concentration of the dNTP is 0.1 μM ~ 50mM, preferably 0.1 μM ~ 10mM. 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 inventor obtained the above concentration through extensive experiments, thereby better improving the stability of the reversible blocking group.

In yet 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 includes: contacting a competitive reagent with the dNTP modified with a reversible blocking group; wherein the competitive reagent can compete with the reversible blocking group to interact with a specific Substances react. By adding competitive reagents, the dNTPs modified with reversible blocking groups can react competitively with specific substances, thereby reducing changes in the reversible blocking groups (such as being reduced, hydrolyzed, etc.) and ensuring their stability. It further helps to maintain the accuracy of sequencing, improve sequencing quality, greatly reduce the occurrence of Runon (premature reaction), and reduce the error rate.

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

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

According to an embodiment of the present invention, in the mixed solution obtained by the contact, the concentration of the dNTP is 0.1 μM ~ 50mM, preferably 0.1 μM ~ 10mM. According to another embodiment of the present invention, in the mixed solution obtained by the contact, the concentration of the competitive reagent is 10 μM ~ 100mM, preferably 1 ~ 10mM. The inventor obtained the above concentration through extensive experiments, thereby better improving the stability of the reversible blocking group.

In yet another aspect of the present invention, 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. Therefore, the dNTP with reversible blocking group modification in the sequencing kit according to the embodiment of the present invention has good stability, helps to improve the sequencing quality, greatly reduces the occurrence of Runon (premature reaction), and reduces the error rate.

In yet another aspect of the present invention, the present invention proposes the application of the aforementioned sequencing buffer, sequencing solution or sequencing kit in sequencing. The dNTPs modified with reversible blocking groups in the sequencing solution or sequencing kit described above have good stability, help to improve sequencing quality, reduce error rates, and are suitable for wide application.

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

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

Detailed ways

The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand 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. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1 2-hydroxyethyl disulfide as competitive reagent

1. Experimental materials

Table 1 Reagents and consumables information

2. Instruments

Table 2 Instrument information

instrument	model
Gene cycler	6337
sequencer	MGISEQ-2000RS
Qubit instrument	Qubit4.0
Electronic balance	MS204TS

3. Experimental samples

Sample name: Standard library reagent V3 (26ng/tube, E320, Barcode 97-104, MGI 1000005033).

4. Experimental design

Conduct the following experiments on HotMPS dNTPs Mix and HotMPS dNTPs Mix II respectively:

HotMPS dNTPs Mix and HotMPS dNTPs Mix II were added to the LTE buffer containing 2-hydroxyethyl disulfide (HED) at a final concentration of 2mM as the test group and to the LTE buffer as the control group, respectively. Put the sequencing enzyme on the machine within half an hour after adding it, and put it on the machine after placing it at 4°C for 24 hours to measure SE50. Add the sequencing enzyme before putting it on the machine. Compare the data at two time points of freshly prepared reagents (within half an hour) and 24 hours at 4°C, especially Runon (%), Q30 (%), AvgErrorRate (%), TotalReads (M) and other indicators.

Continue the concentration gradient test: set the final concentration of HED to 2, 5, 10, and 20mM gradients, add HotMPS dNTPs Mix and HotMPS dNTPs Mix II to the LTE buffer containing HED or the LTE buffer without HED, and proceed. SE50 sequencing. Compare the sequencing data, especially Runon (%), Q30 (%), AvgErrorRate (%), TotalReads (M) and other indicators, and select the most appropriate concentration.

5. Evaluation indicators and standards

Table 3 Evaluation Criteria

Evaluation index	Eligibility criteria
Runon(%)	The lower the better
Q30(%)	The higher the better
AvgErrorRate(%)	The lower the better
TotalReads(M)	The higher the better

6. Experimental steps

6.1 DNB preparation

Add reagents and libraries according to the system in the table below

Table 4 DNB preparation reaction system

Component	Adding volume (μL)
Library ssDNA	3
LTE buffer	17
DNB preparation buffer	20
total capacity	40

Vortex and mix the above reaction system with a vortex shaker, centrifuge for 5 seconds, and place it in a PCR machine for primer hybridization. The reaction conditions are shown in the table below:

Table 5 DNB preparation reaction primer hybridization conditions

temperature	time
Hot lid (105℃)	On
95℃	1min

65℃	1min
40℃	1min
4℃	Hold

Take out the DNB polymerase mixture II (LC) and place it on an ice box, centrifuge briefly for 5 seconds, and place it on an ice box for later use. When the PCR machine reaches 4°C, take out the PCR tube, centrifuge for 5 seconds, and add the following components to the PCR tube on ice:

Table 6 DNB preparation reaction components 2

Components	Adding volume (μL)
DNB polymerase mix I	40
DNB polymerase mix II (LC)	4

The reaction system was vortexed and mixed evenly, centrifuged for 5 seconds, and immediately placed in a PCR machine. The reaction conditions were as follows:

Table 7 DNB preparation rolling circle amplification conditions

temperature	time
Hot lid (35℃)	On
30℃	25min
4℃	Hold

When the temperature of the PCR machine reaches 4°C, immediately add 20 μL of DNB stop buffer and pipet and mix slowly with a wide-mouth pipette 5-8 times.

Use Qubit to measure DNB concentration.

6.2 DNB pooling and loading

6.2.1 Take 100μL of DNB, then add 32μL of DLBⅡ, and then mix evenly with an expanded pipette tip.

6.2.2 DNB loading

Use MGIDL-200H to load DNB onto the slide. After the slide is loaded, remove the slide and wait for sequencing.

6.3 On-machine sequencing

According to Tables 8-1 and 8-2, prepare 4 sets of the same batch of reagents, 2 sets of which are used as controls, and the other 2 sets are added with a final concentration of 2mM 2-hydroxyethyl disulfide as the test group. Half an hour after preparation Test one set of the control and test groups on the computer; test the other set after placing it at 4°C for 24 hours; use the same script for SE50 sequencing.

Testing of additives at different concentrations: According to Tables 8-1 and 8-2, prepare 5 sets of reagents from the same batch, of which 1 set is used as a control, and the other 4 sets are added with final concentrations of 2mM, 5mM, 10mM, and 20mM HED. Select The same script performed SE50 sequencing.

Experimental conditions:

Table 8-1 Hot dNTP reagent tank preparation components

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

Table 8-2 Cold dNTP reagent tank preparation components

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

Script conditions:

Table 9 script conditions

step	Tm(℃)	Time(s)
Hot aggregation	60	120
Cold aggregation	60	120
resection	65	120

7. Experimental results and analysis

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

HED sequencing group: The runon (%) of the freshly prepared reagent was reduced to 0.41%, and after being left for 24 hours, the runon (%) increased to 0.58%, which was much lower than the runon (%) of the control group after being left for 24 hours.

This shows that the addition of 2-hydroxyethyl disulfide is helpful to improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer.

Table 10 Comparison of different additives

As shown in Table 11, the Runon (%) of 5-20mM HED is basically the same, both are lower than the Runon (%) of 2mM; the difference in MappingRate (%) may come from the differences between different chips; Q30 (%) with HED All were better than the control group; there was little difference between different concentrations.

Table 11 HED concentration test comparison

8. Experimental conclusion

HED is helpful to improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer; 5mM, 10mM and 20mM HED have basically the same effect on Runon, with 5-10mM being preferred.

Example 2 5-bromouracil as a competitive reagent

1. Experimental materials

Table 12 Reagents and consumables information

2. Instruments

Table 13 Instrument information

instrument	model
Gene cycler	6337
sequencer	MGISEQ-2000RS
Qubit instrument	Qubit4.0
Electronic balance	MS204TS

3. Experimental samples

Same as Example 1.

4. Experimental design

Configure two SE50 sequencing kits, one kit is used as a control, and a final concentration of 5mM 5-bromouracil is added to the sequencing buffer of one kit. After accelerating at room temperature for 6 hours, perform SE50 sequencing on the MGISEQ-2000RS sequencer. Compare Runon(%), Q30(%), AvgErrorRate(%) and TotalReads(M).

5. Evaluation indicators and standards

Table 14 Evaluation Criteria

Evaluation index

Eligibility criteria

Runon(%)	The lower the better
Q30(%)	The higher the better
AvgErrorRate(%)	The lower the better
TotalReads(M)	The higher the better

6. Experimental steps

6.1 DNB preparation

Same as Example 1.

6.2: DNB pooling and loading

Same as Example 1.

6.3 On-machine sequencing

According to Tables 15-1 and 15-2, prepare two sets of reagents from the same batch, one of which is used as a control, and the other set is added with 5mM 5-bromouracil as a test group. After preparation, the control and test are tested on the machine within half an hour. The group performed SE50 sequencing.

Experimental conditions:

Table 15-1 Hot dNTP reagent tank preparation components

Table 15-2 Cold dNTP reagent tank preparation components

Script conditions:

Table 16 Script Conditions

step	Tm(C)	Time(s)
Hot aggregation	60	120
Cold aggregation	60	120
resection	65	120

7. Experimental results and analysis

Table 17 Experimental results

​	5-Br(5mM)	control
Q30(%)	93.32	92.85
ESR(%)	85.11	84.78
MappingRate(%)	99.37	99.33
AvgErrorRate(%)	0.38	0.43
Lag(%)	0.15	0.17
Runon(%)	0.52	0.58

After 6 hours of acceleration at room temperature, the Runon (%) of the experimental group with 5-bromouracil was reduced by 10%, and the AvgErrorRate (%) was reduced by 11.6%. This shows that the addition of 5-bromouracil helps improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II.

Comparative ratio

The experimental materials (except for 2-hydroxyethyl disulfide), instruments, and experimental samples were the same as in Example 1.

Experimental steps:

1. DNB preparation, DNB pooling, and loading are the same as in Example 1.

2. On-machine sequencing

According to Tables 8-1 and 8-2, prepare 6 sets of reagents from the same batch, of which 2 sets are used as controls, and 2 sets are added with a final concentration of 2mM DMTU (N,N'-dimethylthiourea, brand: Merck, product number : D188700) as test group 1, 2 sets were added with a final concentration of 2mM APDC (ammonium pyrrolidine dithiocarbamate, brand: Merck, product number: P8765) as test group 2, and the control and control were tested within half an hour after preparation. One set of test group 1 and test group 2; test the other set after placing it at 4°C for 24 hours; use the same script (same as Example 1) for SE50 sequencing.

The results are shown in Table 18. It can be seen that adding DMTU and APDTC to the sequencing buffer and leaving it at 4°C for 24 hours was worse than the control group. This shows that DMTU and APDTC cannot improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer.

Table 18 Analysis of sequencing results of DMTU and APDTC

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (19)

1. A sequencing buffer, characterized in that it includes:

   Competitive reagents, which can compete with dNTPs modified with reversible blocking groups to react with specific substances.
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, wherein 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, wherein the reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil.
5. A sequencing solution, characterized by comprising: the sequencing buffer according to any one of claims 1 to 4 and a dNTP modified with a reversible blocking group.
6. The sequencing solution according to claim 5, wherein the concentration of the dNTP is 0.1 μM ~ 50 mM.
7. The sequencing solution according to claim 5 or 6, wherein the concentration of the dNTP is 0.1 μM to 10 mM.
8. The sequencing solution according to claim 5, wherein 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 to 10 mM.
10. A method for improving the stability of dNTPs modified with reversible blocking groups, which is characterized by including:

    Contact the competitive reagent with the dNTP modified with a reversible blocking group;

    Wherein, the competitive reagent can compete with the reversible blocking group to react with a specific substance.
11. The method of claim 10, wherein 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, wherein 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 to 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 by comprising: 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. Application 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 by comprising: using 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.