Uncoupling reagent composition, kit, continuous sequencing method and application

Abstract

The invention discloses an uncoupling reagent composition, a kit, a continuous sequencing method and application. Wherein the uncoupling reagent composition is prepared from cyclodextrin and nuclease. The method can solve the problems that in the prior art, the input amount of an uncoupling reagent is large, the uncoupling effect is poor, and cross contamination is caused by sample residues, and is suitable for the field of high-throughput sequencing.

Description

Uncoupling reagent composition, kit, and continuous sequencing method and application Technical Field

[0001] The present invention relates to the field of high-throughput sequencing, and in particular to an uncoupling reagent composition, a kit, and a continuous sequencing method and application. Background Art

[0002] Nanopore detection technology originated in the 1980s and has garnered widespread attention in recent years, with research and applications spanning numerous fields. In terms of sequencing, nanopore sequencing offers advantages such as long length, high speed, high throughput, high integration, and excellent portability. By increasing chip array density or the number of chips arrayed, throughput can be significantly increased, with data output reaching terabytes.

[0003] The principle of nanopore sequencing technology is as follows: a transmembrane protein embedded in an artificial membrane serves as the detection unit. The transmembrane protein is immersed in a buffer solution suitable for sequencing. A pair of electrodes, such as silver chloride electrodes, are located on either side of the membrane. When the DNA or RNA to be tested passes through the transmembrane protein under the action of an electric field, this causes a change in the ion flow through the nanopore protein. The DNA or RNA is read by analyzing this change in ion flow (using current as a characteristic parameter). Because DNA and RNA pass through the nanopore at very high speeds, sequence reading is difficult. Therefore, motor proteins are used to control the speed of DNA and RNA permeation, thereby improving resolution and enabling sequence reading. To further reduce the input of the analyte to be tested, an anchor molecule containing cholesterol is used to connect the sequencing library DNA and the artificial membrane. Buffers suitable for sequencing often contain high concentrations of potassium chloride or sodium chloride (e.g., above 0.5 M) to achieve a sufficiently high signal-to-noise ratio and resolution to enable reading of DNA or RNA sequences. In addition, the nanopore sequencing system is relatively sensitive, and many substances can cause detection failure, such as enzymes that degrade proteins, compounds that interact with or dissolve artificial membranes (phospholipid membranes), etc.

[0004] Regarding the decoupling method of the anchoring molecule connected to the artificial membrane, the existing document CN201580029651B discloses that the cholesterol buried in the artificial membrane can be separated from the membrane by using cyclodextrin, thereby achieving the decoupling effect.

[0005] Summary of the Invention

[0006] The present application has been accomplished based on the following findings of the inventors.

[0007] According to prior art, cyclodextrin can be used to remove cholesterol embedded in artificial membranes. However, after multiple experimental tests, the inventors of this application found that using cyclodextrin as a decoupling reagent composition after sequencing resulted in poor decoupling and a high level of sample residue. Furthermore, the sequencing library may interact with the microfluidic channel surface, causing it to remain on the surface due to adsorption, which can easily lead to sample cross-contamination when a different library is used for a second sequencing run.

[0008] It is generally believed that nucleases are enzymes used to digest DNA or RNA. In addition, nucleases are generally used in reaction environments with low salt concentrations. For example, according to the official websites of NEB, Thermo Fisher, etc., it is recommended to use DNase I when the salt concentration in the buffer does not exceed 0.1M. When the salt concentration increases, the activity of DNase I decreases. The inventors of the present application attempted to use nucleases for the uncoupling of samples after sequencing, but after multiple experimental tests, they found that in an environment with a higher salt concentration such as sequencing, the activity of nucleases was low and the uncoupling effect was poor; if the input amount of nucleases was increased, the artificial membrane (phospholipid) would be unstable, causing the detection unit to fail and unable to be used for subsequent sequencing. In addition, due to the steric hindrance effect of protein in the enzyme action, short oligonucleotide chains anchored on the artificial membrane are easily left on the side close to the membrane, and then when different libraries are used for the second sequencing, cross contamination of samples is easily caused.

[0009] Therefore, the main purpose of the present invention is to provide an uncoupling reagent composition, a kit, and a continuous sequencing method and application to solve the problems in the prior art of large uncoupling reagent input, poor uncoupling effect, and cross contamination due to sample residue.

[0010] In order to achieve the above object, according to a first aspect of the present invention, a decoupling reagent composition is provided, which comprises cyclodextrin and nuclease.

[0011] Furthermore, the cyclodextrin is one or more of methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, mono-(6-amino-6-deoxy)-β-cyclodextrin or 2,6-di-O-methyl-β-cyclodextrin.

[0012] Furthermore, the concentration of cyclodextrin in the uncoupling reagent composition is 0.1 mM to 3 mM.

[0013] Furthermore, the nuclease is a DNA nuclease and / or an RNA nuclease; preferably, the nuclease is a nuclease that acts on double-stranded DNA or double-stranded RNA.

[0014] Furthermore, the concentration of the nuclease in the uncoupling reagent composition is 0 to 100 U / μL.

[0015] Furthermore, the DNA nuclease is DNase I, Benzonase Nuclease and / or S1 nuclease.

[0016] Furthermore, the RNA nuclease is RNase H, RNase A, RNase E and / or RNase One.

[0017] Furthermore, the uncoupling reagent composition further comprises potassium ions, magnesium ions, calcium ions and a buffer solution.

[0018] Furthermore, the uncoupling reagent composition comprises potassium salt, magnesium salt, calcium salt, nonionic amphoteric buffer, cyclodextrin and nuclease.

[0019] Furthermore, the uncoupling reagent composition comprises KCl, CaCl2, MgCl2, HEPES, methyl-β-cyclodextrin and DNase I.

[0020] Furthermore, the uncoupling reagent composition comprises 300 mM KCl, 2 mM CaCl2, 10 mM MgCl2, 15 mM HEPES, 3 mM methyl-β-cyclodextrin, 0.0067 U / μL DNase I, and a pH of 8.0; or the contents of the above components are proportional multiples within 10% of the above values, and the pH is in the range of 7.8 to 8.2.

[0021] To achieve the above-mentioned object, according to a second aspect of the present invention, a continuous sequencing method is provided, which comprises: after determining the sequence of a first library using a sequencing chip, cleaning the sequencing chip using the above-mentioned uncoupling reagent composition, and then using the cleaned sequencing chip to sequence a second library, wherein the first library and the second library are the same or different.

[0022] Furthermore, cleaning the sequencing chip using the uncoupling reagent composition includes: placing a container containing the sequencing chip in the uncoupling reagent composition for incubation.

[0023] Furthermore, the incubation time is 1 min to 60 min, preferably 10 min to 30 min.

[0024] Furthermore, the number of incubations is at least 1 time, preferably 1 time.

[0025] Furthermore, before sequencing the second library, the sequencing chip is flushed with a sequencing buffer to remove the uncoupling reagent composition.

[0026] Furthermore, the sequencing chip is a chip based on nanopore sequencing.

[0027] Furthermore, the container is a chip-flow tank.

[0028] Furthermore, the first library and / or the second library is a DNA library and / or an RNA library.

[0029] Furthermore, the first library and the second library are different, and the first library is derived from a first sample, and the second library is derived from a second sample.

[0030] Furthermore, after sequencing the second library, the method further includes: sequencing the third library up to the Nth library using a sequencing chip, and performing a "sequencing-cleaning" process in sequence according to the order of the libraries until all the libraries to be tested are sequenced, wherein N is a positive integer greater than three, and the libraries are different from each other.

[0031] In order to achieve the above object, according to the third aspect of the present invention, a sequencing kit is provided, which comprises the above uncoupling reagent composition.

[0032] Furthermore, the kit further comprises a sequencing buffer.

[0033] In order to achieve the above-mentioned object, according to a fourth aspect of the present invention, there is provided a use of the above-mentioned uncoupling reagent composition or kit in sequencing.

[0034] Furthermore, the sequencing is nanopore-based high-throughput sequencing.

[0035] The uncoupling reagent composition of the present invention can not only reduce the amount of nuclease input to avoid detection unit failure and reduce costs, but also avoid the poor uncoupling effect caused by the use of cyclodextrin or nuclease alone, which in turn causes the problem of sample residue. Compared with the prior art, the uncoupling reagent composition of the present invention has higher uncoupling efficiency and less sample residue. Using the uncoupling reagent composition of the present invention, continuous sequencing of samples can be achieved, and it is particularly suitable for continuous sequencing of multiple different samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0037] FIG1 shows a workflow diagram of multi-sample sequencing in one embodiment of the present invention.

[0038] Figure 2 shows a current characteristic graph of the sequencing library according to Example 1 of the present invention, wherein A in Figure 2 is a current signal characteristic graph captured when SEQ1 is added for sequencing, and B in Figure 2 is a current signal characteristic graph captured when SEQ2 is added for sequencing.

[0039] Figure 3 shows a current characteristic graph during the sequencing process according to Example 1 of the present invention; wherein, Figure 3 A is one of the current signal characteristic graphs captured when SEQ1 was added for 30 minutes of sequencing; Figure 3 B is a current signal characteristic graph captured when sequencing buffer 1 was pushed into the nanopore chip-flow tank system after the library was cleaned with the uncoupling reagent composition A for 30 minutes of sequencing. Most of the time during the sequencing process is in the non-sequencing stage; Figure 3 C is one of the current signal characteristic graphs captured when the second library SEQ2 was re-added for 30 minutes of sequencing after cleaning with the uncoupling reagent composition A; Figure 3 D is a statistical graph of the number of sequencing signals generated by adding SEQ1 for 30 minutes of sequencing, adding sequencing buffer for 30 minutes after decoupling, and adding SEQ2 for 30 minutes of sequencing. When SEQ1 was added for sequencing, an average of 144 sequencing signals were collected per channel, when sequencing was added with sequencing buffer after decoupling, an average of 0.3 sequencing signals were collected per channel, and when sequencing was performed after SEQ2 was added, an average of 124 sequencing signals were collected per channel.

[0040] Figure 4 shows the current characteristic graph during the sequencing process according to Comparative Example 1 of the present invention; wherein, Figure 4 A is one of the current signal characteristic graphs intercepted when SEQ1 was added for 30 minutes of sequencing; Figure 4 B is one of the current signal characteristic graphs intercepted when 1 ml of sequencing buffer 1 was re-injected for sequencing after 5 ml of sequencing buffer 2 was used to rinse and soak the nanopore chip-flow tank system; Figure 4 C is a statistical graph of sequencing signals after adding SEQ1 for 30 minutes of sequencing, pushing a large amount of sequencing buffer for rinsing and soaking, and then sequencing for 30 minutes, wherein when SEQ1 was added for 30 minutes of sequencing, an average of 143 signals were collected per channel, and when the library was washed with a large amount of buffer 2 for 30 minutes of sequencing, an average of 149 signals were collected per channel.

[0041] Figure 5 shows the current characteristic graph during the sequencing process according to Comparative Example 2 of the present invention; wherein, Figure 5 A is one of the current signal characteristic graphs intercepted when SEQ1 was added for 30 minutes of sequencing; Figure 5 B is a current signal characteristic graph intercepted when the sequencing buffer was re-introduced after the library was cleared using the uncoupling reagent B for 30 minutes of sequencing; Figure 5 C is a statistical graph of the number of sequencing signals after adding SEQ1 for 30 minutes of sequencing and adding the sequencing buffer for 30 minutes after decoupling, wherein when SEQ1 was added for sequencing, an average of 147 signals were collected per channel, and after sequencing was performed using the uncoupling reagent composition B to clear the library, an average of 15 signals were collected per channel.

[0042] Figure 6 shows the current characteristic graph during the sequencing process according to Comparative Example 3 of the present invention; wherein, Figure 6 A is one of the current signal characteristic graphs intercepted when SEQ1 was added for 30 minutes of sequencing; Figure 6 B is a current signal characteristic graph intercepted when the library was cleared using the uncoupling reagent C and the sequencing buffer was re-injected for 30 minutes of sequencing; Figure 6 C is a statistical graph of the number of sequencing signals after adding SEQ1 for sequencing for 30 minutes and then adding the sequencing buffer for sequencing for 30 minutes after cleaning, wherein when SEQ1 was added for sequencing, an average of 152 signals were collected per channel, and after sequencing was performed using the cleaning reagent to clear the library, an average of 13 signals were collected per channel. DETAILED DESCRIPTION

[0043] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0044] As mentioned in the background, while prior art discloses cyclodextrin as an uncoupling reagent for decoupling artificial membranes from anchor molecules in nanopore sequencing, the uncoupling effect is poor and there are issues with cross-contamination due to sample residue. Therefore, in this application, the inventors sought to develop a new uncoupling reagent composition with improved uncoupling effect and efficient sample removal, thus proposing a series of protection schemes in this application.

[0045] In a first typical embodiment of the present application, a decoupling reagent composition is provided, which comprises cyclodextrin and nuclease.

[0046] In the above-mentioned uncoupling reagent composition, cyclodextrin and nuclease can work together to efficiently remove the sequencing samples bound to the sequencing chip, obtain a good removal rate and efficiency, and avoid the residual samples from affecting the sequencing of subsequent samples to be tested.

[0047] In a preferred embodiment, the cyclodextrin is one or more of methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, mono-(6-amino-6-deoxy)-β-cyclodextrin or 2,6-di-O-methyl-β-cyclodextrin.

[0048] In the present application, cyclodextrin refers to a dextrin derivative having a cyclodextrin structure ring, including but not limited to one or more of the above-mentioned cyclodextrins, all of which can play a role in clearing samples bound to the sequencing chip.

[0049] In a preferred embodiment, the concentration of cyclodextrin in the uncoupling reagent composition is 0.1 mM to 3 mM.

[0050] In a preferred embodiment, the nuclease is a DNA nuclease and / or an RNA nuclease; preferably, the nuclease is a nuclease that acts on double-stranded DNA or double-stranded RNA.

[0051] In a preferred embodiment, the concentration of the nuclease in the uncoupling reagent composition is 0-100 U / μL.

[0052] The above nucleases can be flexibly adjusted and combined according to the type of sequencing sample, using DNA nuclease or RNA nuclease to decompose residual samples without affecting the sequencing performance of the sequencing chip.

[0053] In a preferred embodiment, the DNA nuclease is DNase I, Benzonase Nuclease and / or S1 nuclease.

[0054] In a preferred embodiment, the RNA nuclease is RNase H, RNase A, RNase E and / or RNase One.

[0055] Flexible combinations of the aforementioned DNA or RNA nucleases can yield a variety of uncoupling reagent compositions, enabling efficient decomposition of different samples to be uncoupled. In practical applications, different nuclease compositions and concentrations can be flexibly selected based on the type of sample to be uncoupled, such as DNA or RNA. This allows for efficient removal of residual sample and cleaning of sequencing chips while reducing nuclease usage and saving cleaning costs.

[0056] In a preferred embodiment, the uncoupling reagent composition further comprises potassium ions, magnesium ions, calcium ions and a buffer.

[0057] In a preferred embodiment, the uncoupling reagent composition comprises potassium salt, magnesium salt, calcium salt, nonionic amphoteric buffer, cyclodextrin and nuclease.

[0058] In a preferred embodiment, the uncoupling reagent composition comprises KCl, CaCl2, MgCl2, HEPES, methyl-β-cyclodextrin and DNase I.

[0059] In a preferred embodiment, the components of the uncoupling reagent composition include 300mM KCl, 2mM CaCl2, 10mM MgCl2, 15mM HEPES, 3mM methyl-β-cyclodextrin, 0.0067U / μL DNase I, and a pH of 8.0; or the contents of the above components are proportional multiples of the above values ​​within a range of 10% above and below, and the pH is in the range of 7.8 to 8.2.

[0060] In a second typical embodiment of the present application, a continuous sequencing method is provided, which comprises: after determining the sequence of a first library using a sequencing chip, cleaning the sequencing chip using the above-mentioned uncoupling reagent composition, and then using the cleaned sequencing chip to sequence a second library, wherein the first library is the same as or different from the second library.

[0061] The sequencing chips in the prior art can produce a large amount of data. Therefore, using the same sequencing chip to sequence different samples (libraries) one after another can greatly reduce the cost of the sequencing chips used for sequencing. In order to ensure sequencing accuracy, different samples must not affect each other. Therefore, between sequencing different samples, the sequencing chip needs to be cleaned to completely remove the residual samples bound to the sequencing chip to prevent it from affecting the sequencing of subsequent samples. With the continuous improvement of sequencing throughput and sequencing speed in the prior art, the time required for sequencing a single sample has also gradually decreased. Therefore, under the premise of ensuring the quality of the cleaning, reducing the time required for the cleaning step between samples is an important method to improve sequencing efficiency.

[0062] The above-mentioned multi-sample continuous sequencing method, utilizing the above-mentioned uncoupling reagent composition, can efficiently remove residual samples on the sequencing chip in a relatively short period of time without affecting the sequencing of subsequent samples. It can shorten the cleaning time required between library sequencing, increase the data output of sequencing per unit time, and improve sequencing efficiency. If the sequencing throughput of the sequencing chip has not reached the design upper limit, after the sequencing of the second library is completed, the above-mentioned multi-sample continuous sequencing method can be reused, using the uncoupling reagent composition for cleaning and then sequencing, thereby achieving the completion of sequencing of multiple samples using a single sequencing chip until the sequencing chip reaches the sequencing throughput.

[0063] In a preferred embodiment, cleaning the sequencing chip using the uncoupling reagent composition includes: placing a container containing the sequencing chip in the uncoupling reagent composition for incubation, including but not limited to incubating the nanopore chip-flow tank system where the sequencing chip is located with the uncoupling reagent composition.

[0064] In a preferred embodiment, the incubation time is 1 min to 60 min, preferably 10 min to 30 min.

[0065] In a preferred embodiment, the number of incubations is at least 1; preferably 1.

[0066] The uncoupling reagent composition is used to incubate the nanopore chip-flow cell system housing the sequencing chip, eliminating the need for additional experimental conditions. This facilitates the experimental procedure and ensures stable operation of the sequencing chip during subsequent sequencing. Furthermore, the uncoupling reagent composition cleaning and incubation process can be easily automated for existing sequencing devices, further improving sequencing efficiency.

[0067] In a preferred embodiment, before sequencing the second library, the sequencing chip is flushed with a sequencing buffer to remove the uncoupling reagent composition.

[0068] In order to prevent the residual uncoupling reagent composition from affecting the binding and sequencing of the second library (i.e., the subsequent sequencing sample) to the sequencing chip, the sequencing chip is rinsed with sequencing buffer before sequencing, so that the uncoupling reagent composition can be removed without residue, preventing the cyclodextrin and / or nuclease in the uncoupling reagent composition from affecting the stability and sequencing effect of the second library sequencing.

[0069] In a preferred embodiment, the sequencing chip is a nanopore sequencing-based chip.

[0070] In a preferred embodiment, the container is a chip-flow channel.

[0071] In a preferred embodiment, the first library and / or the second library is a DNA library and / or an RNA library.

[0072] In a preferred embodiment, the first library and the second library are different, and the first library is derived from a first sample, and the second library is derived from a second sample.

[0073] In a preferred embodiment, after sequencing the second library, the method further comprises: sequencing the third library up to the Nth library using a sequencing chip, and performing a "sequencing-cleaning" process in sequence according to the order of the libraries until all the libraries to be tested are sequenced, wherein N is a positive integer greater than three, and the libraries are different from each other.

[0074] As shown in the workflow diagram of multi-sample sequencing in Figure 1, when performing multi-library sequencing, first, a library is added to the nanopore chip-flow tank system (i.e., container), and voltage is applied to perform nanopore sequencing. After the sequencing of the library is completed, an uncoupling reagent composition is added to clean the library remaining in the container. Buffer is then added to the container to remove the residual uncoupling reagent composition to prevent it from affecting the subsequent library to be sequenced. After washing with buffer, another library can be added to the container for sequencing, and this cycle is repeated to achieve multi-sample sequencing.

[0075] In a third typical embodiment of the present application, a sequencing kit is provided, which comprises the above-mentioned uncoupling reagent composition.

[0076] In a preferred embodiment, the kit further comprises a sequencing buffer.

[0077] In a fourth typical embodiment of the present application, a use of the above-mentioned uncoupling reagent composition or the above-mentioned kit in sequencing is provided.

[0078] In a preferred embodiment, the sequencing is nanopore-based high-throughput sequencing.

[0079] The beneficial effects of the present application will be further explained in detail below in conjunction with specific examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. Reagents or instruments used that do not indicate the manufacturer are conventional products available on the market. The experimental methods used are conventional methods unless otherwise specified.

[0080] Example 1

[0081] Library 1 used in this example consists of a 2.7 kbp double-stranded DNA (SEQ1, sequence shown in SEQ ID NO: 1). SEQ1 is the sequence of plasmid pUC57. This plasmid was transfected into competent DH5α cells, amplified by single clone selection, and purified using a plasmid extraction kit (Tiangen). After double enzyme digestion and linearization, the target SEQ1 sequence was obtained for subsequent library construction. Library 2 consists of a 2.8 kbp double-stranded DNA (SEQ2, sequence shown in SEQ ID NO: 2).

[0082] SEQ ID NO: 1:

[0083]

[0084]

[0085] SEQ ID NO: 2:

[0086]

[0087]

[0088] The sequencing signals of the two DNA fragments on the nanopore are obviously different (SEQ1 is shown in FIG2A and SEQ2 is shown in FIG2B ), and SEQ1 and SEQ2 can be distinguished based on the original sequencing signals during sequencing.

[0089] The reagent formula in this embodiment 1 is as follows:

[0090] Sequencing buffer 1: 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0;

[0091] Sequencing buffer 2: 470 mM KCl, 25 mM HEPES, 1 mM EDTA, pH 8.0;

[0092] Uncoupling reagent composition A: 300 mM KCl, 2 mM CaCl2, 10 mM MgCl2, 15 mM HEPES, 3 mM methyl-β-cyclodextrin, 0.0067 U / μL DNase I.

[0093] In this embodiment 1, a nanopore sequencing chip constructed based on a phospholipid membrane is used for data collection. 100 ng of DNA analyte SEQ1 and 6 μL of 1 μM anchoring molecules (containing cholesterol, which helps the DNA analyte to be enriched near the nanopore and membrane) are added to 300 μL of sequencing buffer 1 and pushed into the nanopore chip-flow trough system, and 30 min data are collected at 180 mV voltage (the result is shown in A in Figure 3). After the first data collection is completed, the voltage is adjusted to 0 mV, and 300 μL of uncoupling reagent composition A is pushed into the nanopore chip-flow trough system and incubated at 28 degrees Celsius for 30 min. Excess uncoupling reagent composition A is pushed away with 5 mL of sequencing buffer 2. 1 mL of sequencing buffer 1 is pushed in, the voltage is re-adjusted to 180 mV, and the sample is run empty for 30 min for a second data collection (the result is shown in B in Figure 3). 100 ng of DNA analyte SEQ2 and 6 μL of 1 μM anchor molecule were reintroduced into the nanopore chip-flow cell system, and data were collected again for 30 min (the results are shown in FIG3C ).

[0094] The sequencing data of A, B and C in Figure 3 are shown in Figure 3D. Within 30 minutes of normal sequencing, an average of (144±22) sequencing signals can be collected per well. After incubation with the uncoupling reagent composition A, an average of (0.3±1.0) sequencing signals can be collected per well within 30 minutes of sequencing, and the average number of sequencing signals (2B) is 0.21% of the normal sequencing (2A). After re-adding the DNA analyte SEQ2, an average of (124±17) sequencing signals can be collected per well within 30 minutes of sequencing. This shows that the addition of the uncoupling reagent composition A can effectively reduce the number of the first library, and the second library can be re-added for sequencing analysis after clearing the first library, thereby achieving the purpose of continuous analysis of multiple samples.

[0095] Comparative Example 1

[0096] Compared with Example 1, the DNA analyte used in this Comparative Example 1 is SEQ1, and a large amount of sequencing buffer is used instead of the uncoupling reagent composition A for sample cleaning.

[0097] The reagent formula in this comparative example 1 is as follows:

[0098] Sequencing buffer 1: 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0;

[0099] Sequencing buffer 2: 470 mM KCl, 25 mM HEPES, 1 mM EDTA, pH 8.0;

[0100] In this comparative example 1, a nanopore sequencing chip constructed based on a phospholipid membrane is used for data collection. 100 ng of DNA library 1 (SEQ1) and 6 μL of 1 μM anchor molecules (containing cholesterol, which helps the DNA analyte to be enriched near the nanopore and membrane) are added to 300 μL of sequencing buffer 1, mixed evenly and pushed into the nanopore chip-flow tank system, and 30 min data is collected at 180 mV voltage (the result is shown in A in Figure 4). After completing 30 min of data collection, the voltage is adjusted to 0 mV, 5 mL of sequencing buffer 2 is pushed in and soaked at 0 V for 30 min, and then 1 mL of sequencing buffer 1 is pushed in. The voltage is re-applied to 180 mV, and data collection is performed for the second time, and data collection continues for 30 min (the result is shown in B in Figure 4).

[0101] The sequencing data for Figures A and B in Figure 4 are shown in Figure 4C. Within 30 minutes of normal sequencing, an average of (143±12) sequencing signals were collected per well. After rinsing and soaking with 5 mL of buffer, an average of (149±16) sequencing signals were collected per well within 30 minutes of sequencing. The average number of sequencing signals (shown in Figure 4B) did not decrease compared to normal sequencing (shown in Figure 4A), and a large amount of SEQ1 was still captured by the nanopore. This indicates that pushing in a large amount of sequencing buffer cannot effectively reduce the number of sequenced DNA analytes, and therefore cannot achieve the purpose of continuous analysis of multiple samples.

[0102] Comparative Example 2

[0103] Compared with Example 1, the DNA analyte used in Comparative Example 2 is SEQ1, and the uncoupling reagent composition B is used instead of the uncoupling reagent composition A for sample cleaning.

[0104] The reagent formula in this comparative example 2 is as follows:

[0105] Uncoupling reagent composition B: 300 mM KCl, 2 mM CaCl2, 10 mM MgCl2, 15 mM HEPES, 3 mM methyl-β-cyclodextrin.

[0106] In this example, a nanopore sequencing chip constructed based on a phospholipid membrane was used for data collection. 100 ng of DNA analyte SEQ1 and 6 μL of 1 μM anchoring molecule were added to 300 μL of sequencing buffer 1, and pushed into the nanopore chip-flow tank system, and data was collected for 30 minutes at a voltage of 180 mV (the results are shown in A in Figure 5). After the first data collection, the voltage was adjusted to 0 V, and 300 μL of uncoupling reagent composition B was pushed into the nanopore chip-flow tank system and incubated at 28 degrees Celsius for 30 minutes. Excess uncoupling reagent composition B was pushed away with 5 mL of sequencing buffer 2. 1 mL of sequencing buffer 1 was pushed in, the voltage was re-adjusted to 180 mV, and the sample was run empty for 30 minutes for a second data collection (the results are shown in B in Figure 5).

[0107] The sequencing data for A and B in Figure 5 are shown in Figure 5C. Within 30 minutes of normal sequencing, an average of (147±22) sequencing signals were collected per well. After incubation with uncoupling reagent composition B, an average of (15±11) sequencing signals were collected per well within 30 minutes of sequencing. The average number of sequencing signals (as shown in Figure 5B) was 10.2% of that of normal sequencing (as shown in Figure 5A). Compared to the 0.21% empty run data after uncoupling in Example 1, the empty run signal after clearing the library using this method increased by 49 times, indicating that the cleaning effect of uncoupling reagent composition B is not as good as that of uncoupling reagent composition A.

[0108] Comparative Example 3

[0109] Compared with Example 1, the DNA analyte used in Comparative Example 2 is SEQ1, and the uncoupling reagent composition C is used instead of the uncoupling reagent composition A for sample cleaning.

[0110] The reagent formula in this comparative example 3 is as follows:

[0111] Uncoupling reagent composition C: 300 mM KCl, 2 mM CaCl2, 10 mM MgCl2, 15 mM HEPES, 0.0067 U / μL DNase I.

[0112] In this example, a nanopore sequencing chip constructed based on a phospholipid membrane was used for data collection. 100 ng of DNA analyte SEQ1 and 6 μL of 1 μM anchoring molecule were added to 300 μL of sequencing buffer 1, and pushed into the nanopore chip-flow tank system, and 30 mm data was collected at a voltage of 180 mV (the results are shown in A in Figure 6 ). After the first data collection, the voltage was adjusted to 0 V, and 300 μL of uncoupling reagent composition C was pushed into the nanopore chip-flow tank system and incubated at 28 degrees Celsius for 30 minutes. Excess uncoupling reagent composition C was pushed away with 5 mL of sequencing buffer 2. 1 mL of sequencing buffer 1 was pushed in, the voltage was re-adjusted to 180 mV, and the sample was run empty for 30 minutes for a second data collection (the results are shown in B in Figure 6 ).

[0113] The sequencing data for A and B in Figure 6 are shown in Figure 6C. Within 30 minutes of normal sequencing, an average of (152±19) sequencing signals were collected per well. After incubation with coupling reagent composition C, an average of (13±9) sequencing signals were collected per well within 30 minutes, and the average number of sequencing signals (as shown in Figure 6B) was 8.6% of the normal sequencing (as shown in Figure 6A). Compared to the 0.21% empty run data after uncoupling in Example 1, the empty run signal after cleaning the library using this method increased by 41 times, indicating that the cleaning effect of uncoupling reagent composition C is not as good as that of uncoupling reagent composition A.

[0114] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: In this application, the applicant creatively discovered that the uncoupling reagent composition containing cyclodextrin and nuclease has excellent cleaning efficiency when cleaning the artificial membrane used in nanopore sequencing. Compared with the use of cyclodextrin, nuclease or buffer alone, the uncoupling efficiency is higher and the sample residue is less. And the cost of using this uncoupling reagent composition is low. While the cleaning efficiency is improved, the cost is much lower than that of reagents that only use nucleases. The use of this uncoupling reagent composition can solve the problem of sample cross-contamination in continuous sequencing, and is suitable for continuous sequencing such as nanopore high-throughput sequencing.

[0115] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A decoupling reagent composition, characterized in that The uncoupling reagent composition comprises cyclodextrin and nuclease.

2. The uncoupling reagent composition according to claim 1, characterized in that The cyclodextrin is one or more of methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, mono-(6-amino-6-deoxy)-β-cyclodextrin or 2,6-di-O-methyl-β-cyclodextrin.

3. The uncoupling reagent composition according to claim 2, characterized in that The concentration of the cyclodextrin in the decoupling reagent composition is 0.1 mM to 3 mM.

4. The uncoupling reagent composition according to claim 1, characterized in that The nuclease is a DNA nuclease and / or an RNA nuclease; Preferably, the nuclease is a nuclease that acts on double-stranded DNA or double-stranded RNA.

5. The uncoupling reagent composition according to claim 4, characterized in that The concentration of the nuclease in the decoupling reagent composition is 0 to 100 U / μL.

6. The uncoupling reagent composition according to claim 4 or 5, characterized in that The DNA nuclease is DNase I, Benzonase Nuclease and / or S1 nuclease.

7. The uncoupling reagent composition according to claim 4 or 5, characterized in that The RNA nuclease is RNase H, RNase A, RNase E and / or RNase One.

8. The uncoupling reagent composition according to claim 1, characterized in that The uncoupling reagent composition further comprises potassium ions, magnesium ions, calcium ions and a buffer.

9. The uncoupling reagent composition according to claim 8, characterized in that The uncoupling reagent composition comprises potassium salt, magnesium salt, calcium salt, non-ionic amphoteric buffer, cyclodextrin and nuclease.

10. The uncoupling reagent composition according to claim 9, characterized in that The uncoupling reagent composition comprises KCl, CaCl2, MgCl2, HEPES, methyl-β-cyclodextrin and DNase I.

11. The uncoupling reagent composition according to claim 10, characterized in that The uncoupling reagent composition comprises 300 mM KCl, 2 mM CaCl2, 10 mM MgCl2, 15 mM HEPES, 3 mM methyl-β-cyclodextrin, 0.0067 U / μL DNase I, and a pH of 8.0; or the contents of the above components are proportional multiples of the above values ​​within a range of 10% above and below, and the pH is in the range of 7.8 to 8.

2.

12. A continuous sequencing method, characterized in that: The continuous sequencing method comprises: after determining the sequence of a first library using a sequencing chip, cleaning the sequencing chip using a decoupling reagent composition described in any one of claims 1 to 11, and then sequencing a second library using the cleaned sequencing chip, wherein the first library is the same as or different from the second library.

13. The continuous sequencing method according to claim 12, characterized in that: Using the uncoupling reagent composition to clean the sequencing chip includes: placing a container containing the sequencing chip in the uncoupling reagent composition for incubation.

14. The continuous sequencing method according to claim 13, characterized in that: The incubation time is 1 min-60 min, preferably 10 min-30 min.

15. The continuous sequencing method according to claim 13, characterized in that: The incubation is performed at least once, preferably once.

16. The continuous sequencing method according to claim 12, characterized in that: Before sequencing the second library, the sequencing chip is washed with a sequencing buffer to remove the uncoupling reagent composition.

17. The continuous sequencing method according to claim 12, characterized in that: The sequencing chip is a chip based on nanopore sequencing.

18. The continuous sequencing method according to claim 12, characterized in that: The container is a chip-flow trough.

19. The method for continuous sequencing of multiple samples according to claim 12, characterized in that: The first library and / or the second library is a DNA library and / or an RNA library.

20. The continuous sequencing method according to claim 12, characterized in that: The first library is different from the second library, and the first library is derived from a first sample, and the second library is derived from a second sample.

21. The continuous sequencing method according to claim 12, characterized in that: After sequencing the second library, the method further includes: using the sequencing chip to sequence the third library to the Nth library, and performing a "sequencing-cleaning" process in sequence according to the order of the libraries until all the libraries to be tested have completed sequencing, wherein N is a positive integer greater than three, and the libraries are different from each other.

22. A sequencing kit, characterized in that: The kit comprises the uncoupling reagent composition according to any one of claims 1 to 11.

23. The kit according to claim 19, characterized in that The kit further comprises a sequencing buffer.

24. Use of the uncoupling reagent composition according to any one of claims 1 to 11 or the kit according to claim 22 or 23 in sequencing.

25. The use according to claim 24, characterized in that The sequencing is nanopore-based high-throughput sequencing.

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