HK1254061A1 - Method, device, and system for controlling sequencing reaction - Google Patents

Description

Method, device and system for controlling sequencing reaction

Technical Field

The invention relates to the technical field of sequence determination, in particular to a method for controlling a sequence determination reaction, a sequence determination system and a sequence determination device.

Background

Sequencing, i.e., sequencing, includes determination of nucleic acid sequences. Sequencing platforms on the market at present comprise a first-generation sequencing platform, a second-generation sequencing platform and a third-generation sequencing platform.

In the sequence determination process, a biochemical reaction needs to be performed on a reaction device, for example, different reagents need to be introduced together or sequentially onto a chip for reaction by using a liquid path system. At present, in order to make a liquid path system in a platform compact and efficient, the liquid path system adopts a first valve to switch input/output reagents.

The first valve sold in the market at present inevitably has different degrees of reagent cross-contamination or mixing-in when different reagents are switched due to the structural characteristics of the first valve, namely, the reagent cross-contamination. Cross-contamination of reagents can affect the performance of reactions, and is especially fatal to reactions that require a small amount of reagents for their own reactions, such as single molecule sequencing.

Therefore, how to reduce or avoid cross contamination of reagents in the liquid path is a problem to be solved.

Disclosure of Invention

Embodiments of the present invention aim to at least solve one of the technical problems occurring in the related art or at least to provide a useful commercial choice. The inventors have made the present invention based on the following findings and assumptions of a split study of the structure of the first valve.

The first valves on the market today, also called sample valves, multi-position valves, rotary valves or rotary valves, are used as components for sample collection, liquid sampling or flow path switching, etc. The stator and the rotor are tightly combined to form an effective seal.

The first valve has a common port for the passage of liquid from or to different flow paths, the common port being provided in the stator and/or rotor, the stator and/or rotor having one or more further ports. Through the rotation of the rotor, the connection between the rotor and the stator passage can be realized, so that the public port and other ports are communicated, and the function of selecting sample introduction or shunting is achieved. The general/standard configuration of the first valve is a multi-port option, i.e. only one port communicates with the common port during operation.

The communication of the common port with the other ports typically needs to be through one or several common structures provided on the rotor. When liquid exists in the common structure, due to the rotation of the rotor and the relative movement of the sealing interface of the connection of the rotor and the stator, at least a part of liquid in the common structure is inevitably carried to a place outside the common structure, namely, when the flow path is switched, the next flow path liquid is inevitably carried with the liquid of the previous flow path, and then if the flow path is switched in the opposite direction, the next flow path liquid mixed with the liquid of the previous flow path is carried into the liquid of the lower flow path; thus, even if the amount of the cross-contamination is small, the cross-contamination is difficult to control and the influence is difficult to estimate.

The inventors have determined that the above unexpected influence on the sequence measurement results due to the incorporation of at least a part of the liquid in the common structure into the next flow path at the time of flow path switching, i.e., the next reaction process, i.e., the cross contamination of the reagent, is generated, based on the above analysis of the association of the respective parts of the device system and the above study of the separation of the structure of the first valve, while comparatively analyzing the manual results and the results of the on-machine measurement. Accordingly, embodiments of the present invention provide a method of controlling a sequencing reaction, a sequencing system, and a control device.

Embodiments of the present invention provide a method of controlling a sequencing reaction, the sequencing reaction including a first biochemical reaction performed on a reaction device using a first reagent, the sequencing reaction being controlled using a sequencing system. The sequencing system includes a fluidic device including a valve body assembly and a drive assembly. The valve body assembly comprises a first valve and a second valve, the first valve is connected with the reaction device, the first valve comprises a stator and a rotor which can be communicated, the first valve is provided with a common port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the common port and at least one port can be communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first port, and the second valve can be connected with the first port, the first reagent and/or a first buffer solution, the method comprises the following steps:

communicating the first port with the common port through the communication groove;

communicating the second valve with the first reagent and the first port;

using the driving assembly to enable the first reagent to enter the reaction device through the second valve and the first valve in sequence so as to perform the first biochemical reaction;

communicating the second valve with the first buffer and the first port prior to rotating the rotor;

flowing the first buffer solution through the second valve and the first valve in sequence using the drive assembly.

In the method, before the rotor rotates, the first buffer solution flows into the first valve, so that the liquid in the communicating groove is replaced by the first buffer solution before the rotor rotates, or the first buffer solution which has no influence on the target sequence determination reaction is used for replacing the first reagent in the communicating groove before the rotor of the first valve rotates, so that the original reagent in the communicating groove is prevented from being brought to other positions of the connecting interface of the stator and the rotor during the rotation of the rotor, and the risk of cross contamination when different reagents are switched is avoided.

A sequencing system according to an embodiment of the present invention controls a sequencing reaction including a first biochemical reaction performed on a reaction apparatus using a first reagent. The sequencing system comprises a control device and a fluid device, wherein the control device is connected with the fluid device, and the fluid device comprises a valve body assembly and a driving assembly. The valve body assembly comprises a first valve and a second valve, the first valve is connected with the reaction device, the first valve comprises a stator and a rotor which can be communicated, the first valve is provided with a common port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the common port and at least one port can be communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first port, the second valve can be connected with the first port, the first reagent and/or a first buffer solution, and the control device is used for:

communicating the first port with the common port through the communication groove;

communicating the second valve with the first reagent and the first port;

using the driving assembly to enable the first reagent to enter the reaction device through the second valve and the first valve in sequence so as to perform the first biochemical reaction;

communicating the second valve with the first buffer and the first port prior to rotating the rotor;

flowing the first buffer solution through the second valve and the first valve in sequence using the drive assembly.

In the sequencing system, before the rotor rotates, the first buffer solution flows into the first valve, so that the liquid in the communicating groove is replaced by the first buffer solution before the rotor rotates, or the first buffer solution which has no influence on the target sequencing reaction is used for replacing the first reagent in the communicating groove before the rotor of the first valve rotates, thereby avoiding the situation that the original reagent in the communicating groove is brought to other positions of the connecting interface of the stator and the rotor during the rotation of the rotor and further avoiding the risk of cross contamination when different reagents are switched.

A computer-readable storage medium of an embodiment of the present invention stores a program for execution by a computer, and executing the program includes performing the method of any of the above embodiments. The computer-readable storage medium may include: read-only memory, random access memory, magnetic or optical disk, and the like.

Additional aspects and advantages of embodiments 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 embodiments of the invention.

Drawings

The above and/or additional aspects and advantages of embodiments of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic flow diagram of a method of controlling a sequencing reaction according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of the structure of a sequencing system according to an embodiment of the present invention;

FIG. 3 is a schematic view of the relationship of the ports, communication grooves and common port of the first valve of an embodiment of the present invention;

FIG. 4 is a schematic structural view of a valve body assembly of an embodiment of the present invention;

FIG. 5 is a schematic structural diagram of a test platform according to an embodiment of the present invention;

FIG. 6 is a schematic illustration of a set of test data obtained by a test platform according to an embodiment of the present invention;

FIG. 7 is a schematic illustration of another set of test data obtained by the test platform according to an embodiment of the present invention;

FIG. 8 is a schematic comparison of different test data obtained from a test platform according to an embodiment of the present invention;

FIG. 9 is another schematic flow diagram of a method of controlling sequencing reactions according to an embodiment of the present invention;

FIG. 10 is a functional block diagram of a sequence measuring system according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, features defined as "first", "second", may explicitly or implicitly include one or more of the described features. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, "connected" is to be understood in a broad sense, e.g., fixedly, detachably or integrally connected; may be mechanically connected, may be electrically connected or may be in communication with each other; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The following disclosure provides many different embodiments or examples for implementing different features of the invention. To simplify the disclosure of the present invention, the components and settings of a specific example are described below. Furthermore, the present invention may repeat reference numerals and/or reference letters in the various examples, which have been repeated for purposes of simplicity and clarity and do not in themselves dictate a relationship between the various embodiments and/or configurations discussed.

The "sequencing" referred to in the embodiments of the present invention is similar to nucleic acid sequencing, including DNA sequencing and/or RNA sequencing, including long fragment sequencing and/or short fragment sequencing. The so-called "sequencing reaction" is the same as the sequencing reaction. Generally, in the determination of a nucleic acid sequence, one base or one specific type of base, which is selected from at least one of A, T, C, G and U, can be determined by one round of sequencing reaction. In sequencing reactions that are sequencing-by-synthesis and/or sequencing-by-ligation, a round of sequencing reactions is referred to as including extension reactions (base extension), information collection (photo/image capture), and radical excision (clean). The so-called "nucleotide analogs", i.e., substrates, also called terminators (terminators), are A, T, C, G and/or U analogs that are capable of pairing with a particular type of base following the base complementarity principle, while being capable of terminating the binding of the next nucleotide/substrate to the template strand.

Referring to fig. 1 and 2, the present invention provides a method for controlling a sequencing reaction, wherein the sequencing reaction comprises a first biochemical reaction, the first biochemical reaction is performed on a reaction device 40 by using a first reagent 11, and the sequencing reaction is controlled by using a sequencing system.

The sequencing system includes a fluidic device 100, the fluidic device 100 including a valve body assembly 10 and a drive assembly 50.

The valve body assembly 10 comprises a first valve 20 and a second valve 30, the first valve 20 is connected with a reaction device 40, the first valve 20 comprises a stator and a rotor which can be communicated, the first valve 20 is provided with a common port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove 21, the common port and at least one port can be communicated through the communication groove 21 by rotating the rotor, the plurality of ports comprise a first port 22, the second valve 30 can be connected with the first port 22, a first reagent 11 and/or a first buffer liquid 60, and the method comprises the following steps:

s11, communicating the first port 22 with the common port through the communication groove 21;

s12, connecting the first reagent 11 and the first port 22 to each other through the second valve 30;

s13, using the driving assembly 50 to make the first reagent 11 enter the reaction device 40 through the second valve 30 and the first valve 20 in sequence to perform the first biochemical reaction;

s14, before rotating the rotor, connecting the first buffer liquid 60 and the first port 22 to the second valve 30;

s15, the first buffer solution is flowed through the second valve 30 and the first valve 20 in sequence by the driving assembly 50.

In the above method, the first buffer solution 60 is flowed into the first valve 20 before the rotor is rotated, so that the liquid in the communicating groove 21 is replaced by the first buffer solution 60 before the rotor is rotated, or the first buffer solution 60 having no influence on the target sequence measurement reaction is used to replace the first reagent 11 in the communicating groove 21 before the rotor of the first valve 20 is rotated, thereby avoiding the original reagent in the communicating groove 21 from being brought to other positions of the connecting interface between the stator and the rotor during the rotation of the rotor, and further avoiding the risk of cross contamination when different reagents are switched.

Specifically, in certain embodiments, the reaction device 40 may be a chip, the reaction device 40 carrying a sample. Referring to FIG. 2, the reaction apparatus 40 includes a first unit 41 and a second unit 42, each of which includes a plurality of channels (channels), and is capable of performing different types of sequencing reactions in the channels of the first unit 41 and the channels of the second unit 42, respectively, wherein the sequencing reactions in the channels of the first unit 41 and the sequencing reactions in the channels of the second unit 42 are staggered, asynchronous, and non-interacting. For example, when a biochemical reaction is required on a sample in the first unit 41, the fluidic device 100 will deliver a reagent for the reaction to the first unit 41 without the same reagent entering the second unit 42, and vice versa.

In the embodiment of the present invention, referring to fig. 2, each unit is correspondingly connected with a first valve 20. Specifically, the common port of the first valve 20 communicates with the corresponding cell, so that the reagent output from the common port of the first valve 20 can enter the corresponding cell for biochemical reaction. In this way, the progress of sequencing can be accelerated.

In some embodiments, the sample to be sequenced, such as a DNA strand having a double-stranded or single-stranded structure, is immobilized on the surface of the channel of the first unit 41 and the second unit 42 of the reaction device 40 before the sequencing reaction is performed.

The first reagent 11 and the second valve 30, the first buffer solution 60 and the second valve 30, the port and the second valve 30, the second valve 30 and the first valve 20, and/or the first valve 20 and the reaction device 40 can be connected and communicated through hoses, so that the hoses can make the liquid path configuration more flexible.

In certain embodiments, the first valve 20 may be a multi-way valve. The second valve 30 can be a three-way valve, such as a three-way solenoid valve, in which a normally closed port and a normally open port of the three-way solenoid valve are connected to the reagent and the buffer to be added, respectively. In certain embodiments, the first valve 20 can be a rotary valve, thus, methods for controlling sequencing reactions have a wide range of applications.

In some embodiments, the common port is opened on the stator, the plurality of ports are disposed around the common port, and the common port is in corresponding communication with one end of the communication groove 21. In other embodiments, the common port is open at the rotor and is located at one end of the communication groove 21.

In the embodiment of the present invention, step S11 is performed before step S12, and in other embodiments, step S12 may be performed before step S11, or step S11 and step S12 may be performed simultaneously.

The buffer solution is a solution capable of maintaining the pH of the liquid in a specific range to some extent, and is a weak acid, a weak base and/or a neutral solution. In certain embodiments, the first buffer is a solution that does not affect the first biochemical reaction and/or other biochemical reactions of the sequencing reaction.

In general, the seal of the first valve 20 is basically sealed by the end face between the stator and the rotor, and the liquid chemical in the communication groove 21 remains on the seal surface between the stator and the rotor when the rotor rotates. As shown in fig. 3, when the first biochemical reaction is performed, the first reagent 11 enters the reaction apparatus 40 through the port 1 as the first port 22, the communication groove 21, and the common port 0. When other biochemical reactions are required, if the first reagent 11 in the communication groove 21 is not washed, when the rotary rotor rotates from the port 1 to the port 2, the first reagent 11 in the communication groove 21 remains on the area between the port 1 and the port 2 (e.g., the triangular area in fig. 3), and the remaining first reagent 11 contaminates other reagents entering through other ports with the rotation of the rotor. Therefore, referring to fig. 2 and 4, a three-way valve is connected to the outside of the port of the stator, and before the rotor is rotated, the three-way valve communicates the first buffer solution 11 with the first port 22, and the first buffer solution 60 flows through the second valve and the first valve 20 in sequence by the driving unit 50, so that the first reagent 11 remaining in the communicating groove 21 is washed, thereby greatly improving the cross contamination. It will be appreciated that in the present example, the second valve 30 may include one or more of the three-way valves V1-V8. The first port 22 may include one or more of the ports 1-8.

The following tests illustrate the cross-contamination performance before and after the improvement. In this test, the first valve 20 is illustrated as a rotary valve.

First, two types of commercially available rotary valves were selected and a test platform was constructed as shown in fig. 5, and the test platform was used to evaluate the cross-contamination performance of the two types of rotary valves (hereinafter referred to as rotary valve a1 and rotary valve B1). Referring to FIG. 4, the test is performed by selecting the adjacent ports 1, 2 and 8, the port 1 is connected to the fluorescence reagent 1, the port 2 and the port 8 are both buffers, the flow cell of the reaction device has two parallel channels A and B, and the test operation is as follows:

(1) selecting a channel A of a flow cell of a reaction device, enabling a fluorescent reagent 1 to flow through the channel A by using a driving assembly 50, then rotating a rotor clockwise to switch ports, enabling a communication groove 21 to be communicated with a port 2 and a common port 0, enabling excessive buffer solution to enter a rotary valve through the port 2 by using the driving assembly 50, and ensuring that all the fluorescent reagents 1 in a liquid path of the rotary valve including the common port 0 and the communication groove 21 are cleaned;

(2) switching the liquid path to a channel B of a flow cell of a reaction device, shooting the background of the channel B by using a single-molecule fluorescence detection system, and counting the number n1 of fluorescence points; then, the rotor is rotated anticlockwise, the communication groove 21 is communicated with the port 8 in a switching mode, namely the port 8 is communicated with the public port 0, a certain amount of buffer solution enters the rotary valve through the port 8 by utilizing the driving assembly 50 and flows to a channel B of a flow cell of the reaction device, a single-molecule fluorescence detection system is used for shooting the same area of the channel B, and the number n2 of fluorescence points is counted;

(3) since the buffer solution enters the rotary valve through the port 2 and the port 8 and no fluorescent spot exists, n2-n1 can be regarded as cross contamination caused by the process that the rotary valve is switched from the port 1 to the port 2 (clockwise) and then switched to the port 8 (anticlockwise), and n2-n1 is the increase of the fluorescent spot number which is certainly the fluorescent spot number detected by mixing the fluorescent reagent 1 into the buffer solution entering through the port 8, so that the value can evaluate the severity of the cross contamination when the rotary valve is switched.

Fig. 6 shows the original data of 8 sets of tests, and it can be seen that neither rotary valve a1 nor rotary valve B1 can avoid cross contamination of reagents, and since this contamination occurs when the rotor is rotated, even if the communication tank 21 is switched to port 2 to perform rotary valve cleaning and then switched to port 8 after the fluorescent reagent 1 is introduced, it is always unavoidable that the fluorescent reagent 1 is mixed with the buffer solution entering through port 8 to cause contamination, and therefore, the problem cannot be completely solved by the ordinary cleaning process. In fig. 6, in the histogram shown in the same test group, the left histogram indicates data of n1 before rotation, and the right histogram indicates data of n2 after rotation.

In the present embodiment, the cross-contamination can be improved by connecting the first buffer fluid 60 to the first port 22 through the second valve 30 and by sequentially passing the first buffer fluid 60 through the second valve 30 and the first valve 20 by the driving unit 50 before rotating the rotor. Specifically, referring to fig. 4, the second valve 30 is taken as a three-way solenoid valve for explanation. The normally closed and normally open ports of the three-way electromagnetic valve are respectively connected with a reagent and a buffer solution to be added, for example, the electromagnetic valve V1 is powered on (at the moment, the port 1 is communicated with the reagent 1), after the reagent 1 is introduced into the rotary valve by the driving component 50, the electromagnetic valve V1 is immediately closed (at the moment, the port 1 is communicated with the buffer solution), and the driving component 50 is utilized to clean the residual reagent 1 in the communicating tank 21 cleaned by a small amount of the buffer solution (the specific amount is determined according to the pipeline condition), so that after cleaning, the reagent 1 cannot remain on the end surface of the rotary valve when the rotor is rotated to switch different ports, although the residual buffer solution is the buffer solution, the buffer solution has no influence on the biochemical reaction, and the method can greatly reduce or avoid reagent cross contamination caused by the rotation.

Similarly, using a single molecule fluorescence detection system, the improved cross-contamination was assessed and the raw data is shown in FIG. 7. A comparison of n2-n1 before and after improvement is shown in FIG. 8. As can be seen from FIG. 8, compared with the prior rotary valves A1 and B1, the method of the present invention enables the cross contamination of the rotary valves to be significantly improved, and the method of the present invention eliminates the cross contamination of reagents from the source, and is very suitable for being applied to the occasions sensitive to trace cross contamination, such as a single-molecule gene sequencer system. In fig. 7, in the histogram shown by the same test number, the left histogram shows data of n1 before rotation, and the right histogram shows data of n2 after rotation. In FIG. 8, the left bar graph represents data for improved n2-n1, the middle bar graph represents data for n2-n1 for improved pre-rotary valve A1, and the right bar graph represents data for n2-n1 for improved pre-rotary valve B1 in the bar graphs shown in the same test group.

In some embodiments, referring to fig. 9, the sequencing reaction comprises a second biochemical reaction performed on the reaction device 40 using the second reagent 12, the valve assembly 10 comprises a third valve 31, the plurality of ports comprises a second port 23, the third valve 31 is connectable to the second port 23, the second reagent 12, and/or a second buffer, and the method comprises the steps of:

s16, rotating the rotor to make the communication groove 21 communicate the second port 23 and the common port;

s17, connecting the second reagent 12 and the second port 23 to the third valve 31;

s18, using the driving assembly 50 to make the second reagent 12 enter the reaction device 40 through the third valve 31 and the first valve 20 in sequence to perform the second biochemical reaction;

s19, before the rotor is rotated, communicating the second buffer solution and the second port 23 to the third valve 31;

s20, a second buffer solution is flowed through third valve 31 and first valve 20 in sequence using drive assembly 50.

Thus, the method of the embodiment of the invention can be applied to a plurality of different types of biochemical reactions in a sequence determination reaction, and the application range of the method of the embodiment of the invention is expanded.

Specifically, in an example of the present invention, please refer to FIG. 4, the second port 23 may comprise one or more of the ports 1-8, and the third valve 31 may comprise one or more of the three-way valves V1-V8. It should be noted that the second valve 30 and the third valve 31 should select different ones of the three-way valves V1-V8. The first port 22 and the second port 23 should select different ones of the ports 1-8.

The second buffer solution is a solution that does not affect the first biochemical reaction, and the first buffer solution 60 is a solution that does not affect the second biochemical reaction.

In the example of fig. 2 of the present invention, the second buffer and the first buffer 60 are the same buffer. Of course, the second buffer and the first buffer may alternatively be different buffers. In one example, the first buffer and the second buffer are the same buffer, and are "150 mM HEPES, 150mM NaCl, pH 7.0", and do not affect the sequencing reaction.

In certain embodiments, one of the ports 70 of the stator of the first valve 20 may be in communication with ambient air to facilitate the introduction of air to decontaminate the pipeline.

In the embodiment of the present invention, step S16 is performed before step S17, and in other embodiments, step S17 may be performed before step S16, or step S16 and step S17 may be performed simultaneously.

In certain embodiments, the first biochemical reaction comprises an extension reaction.

Specifically, the extension reaction is based on base complementarity, attaching a specific substrate to a sample to be sequenced, and determining the type of the bound substrate using a detectable group carried on the substrate to determine the sequence. In one example, the detectable group comprises a fluorophore that fluoresces under a laser of a particular wavelength.

In an embodiment of the present invention, the first reagent is referred to as a terminator reagent, i.e., a reaction substrate, including A, T, C and G base analogs, specifically, the base analogs, i.e., the terminator, have a structure of A/T/C/G-terminator-linking unit-luminescent group, i.e., the first reagent is referred to as a reagent comprising A-terminator-linking unit-luminescent group (hereinafter referred to as A reagent), a reagent comprising T-terminator-linking unit-luminescent group (hereinafter referred to as T reagent), a reagent comprising C-terminator-linking unit-luminescent group (hereinafter referred to as C reagent), and/or a reagent comprising G-terminator-linking unit-luminescent group (hereinafter referred to as G reagent). Wherein the terminating group is a photo-and/or chemically cleavable group, and the substrate is provided with a luminescent group by a linker.

In one embodiment, the four terminators have the same structure and the same color of the emitting groups when excited, and the four base analogs are contained in different reagent bottles. For sequencing, A, T, C and one of the G terminators were added in sequence, and each four terminator reactions was called a cycle. The reagent bottles containing different terminators are connected with the reaction device through a three-way valve and a first valve.

The process of adding the above-mentioned reagent in one example of the present invention will be described below with reference to FIG. 4.

In fig. 4, reagent 1 is reagent a, reagent 2 is reagent T, reagent 3 is reagent C, and reagent 4 is reagent G. When the extension reaction is carried out, the three-way valve V1 is electrified, the three-way valves V2-V8 are closed, the port 1 is communicated with the reagent A, the communication groove 21 is communicated with the port 1 and the common port 0, the driving assembly 50 enables the reagent A to enter the reaction device 40 through the three-way valve V1 and the first valve 20 for reaction, the three-way valve V1 is closed before the rotor is rotated, the port 1 is communicated with the buffer solution, and the driving assembly 50 enables the buffer solution to flow through the three-way valve V1 and the first valve 20. When the added reagent T, C, G and/or other reagents need to be replaced later, the rotor is rotated to enable the communication groove 21 to communicate the common port 0 and the corresponding port, and the process is carried out according to the process.

In certain embodiments, the second biochemical reaction comprises radical cleavage.

Specifically, when adding a terminator having a different structure to the reaction apparatus 40, the luminescent group on the terminator of the above structure is cleaved, and then a terminator of another structure is added. For example, referring to the above example, after the reagent A is added into the reaction device 40, a light-emitting device (e.g., a laser) can be used to emit an excitation light to the reaction device 40 to excite the luminescent group to emit fluorescence, and an image can be taken by an imaging device to collect the fluorescence and form an image for sequence determination. After the photographing is finished, the luminescent group of the reagent A needs to be cut off, and then other reagents need to be added. Further, in this example, the reagent 5 is a reagent for cleavage (hereinafter referred to as a cleavage reagent).

After the photographing is completed, when the excision reagent is added, the rotor is rotated to enable the communication groove 21 to communicate the port 5 with the common port 0, the three-way valve V5 is electrified, the three-way valves V1-V4 and V6-V8 are closed, the port 5 is communicated with the excision reagent, the cutting reagent is enabled to enter the reaction device 40 through the three-way valve V5 and the first valve 20 by the driving assembly 50 to carry out excision reaction, the three-way valve V5 is closed before the rotor is rotated, the port 5 is communicated with the buffer solution, and the buffer solution is enabled to flow through the three-way valve V5 and the first valve 20 by.

In certain embodiments, the extension reaction is performed using a ligase and/or a polymerase.

In certain embodiments, the second biochemical reaction comprises capping.

In particular, what is called capping is primarily the group/bond that is exposed after cleavage of the protecting group. In one example, the first biochemical reaction comprises an extension reaction and the second biochemical reaction comprises a radical cleavage, wherein after the cleavable group is cleaved optically and/or chemically, the exposed group is a thiol group that can be protected from oxidation by capping, e.g., by addition of an alkylating agent.

In this example, the reagent 6 is a reagent to be added for capping (hereinafter referred to as a capping reagent). When adding the capping reagent, the rotor is rotated to enable the communication groove 21 to communicate the port 6 with the common port 0, the three-way valve V6 is electrified, the three-way valves V1-V5 and V7-V8 are closed, the port 6 is communicated with the capping reagent, the driving assembly 50 enables the capping reagent to enter the reaction device 40 through the three-way valve V6 and the first valve 20 for capping reaction, the three-way valve V6 is closed before the rotor is rotated, the port 6 is communicated with the buffer solution, and the driving assembly 50 enables the buffer solution to flow through the three-way valve V6 and the first valve 20.

It should be noted that in some embodiments, the first reagent may comprise a reagent that does not affect the biochemical reaction in the sequencing reaction, and in this case, after the reagent enters the reaction device 40 through the second valve and the first valve 20 and before the rotor is rotated, it is not necessary to flow a wash solution or a buffer solution through the second valve and the first valve 20, so that the time for the sequencing reaction can be saved.

In certain embodiments, the drive assembly 50 includes a pump that communicates through the reaction device 40 to a common port.

Therefore, the driving of the reagent and the buffer can be realized by using the pump, and the control method is simple and easy to implement.

Specifically, in the present example, the pump includes a first pump 51 and a second pump 52, the first pump 51 communicates with the common port of one of the first valves 20 through the first unit 41, the second pump 52 communicates with the common port of the other first valve 20 through the second unit 42, the first reagent and the first buffer are sequentially introduced into the first unit 41 through the second valve 30 and the first valve 20 by the first pump 51, and the first reagent and the first buffer are sequentially introduced into the second unit 42 through the second valve 30 and the first valve 20 by the second pump 52.

In this way, the first pump 51 and the second pump 52 can be used to input the chemical liquid output by the first valve 20 to the first unit 41 and/or the second unit 42, respectively, which is convenient for operation.

Specifically, the first pump 51 and the second pump 52 are respectively piped to the first unit 41 and the second unit 42, for example, through hoses.

The first pump 51 is communicated with the common port of one of the first valves 20 through the first unit 41, the second pump 52 is communicated with the common port of the other first valve 20 through the second unit 42, when the first pump 51 works, the first pump 51 provides negative pressure to the first unit 41, so that the first unit 41 obtains the first reagent and/or other reagents (including buffer solution and/or other reagents) connected with the ports of the first valves 20 to carry out biochemical reaction and/or cleaning, and after the first unit 41 obtains the reagent solution, the first pump 51 stops providing the negative pressure.

What dose of liquid is admitted to the first unit 41 by the first pump 51 depends on: 1) which port the communication groove 21 communicates with; and 2) for that port (hereinafter referred to as a communication port) which communicates with the communication groove 21, a three-way valve connected to the communication port makes the communication port communicate with which agent liquid. For example, referring to fig. 4, when the communication groove 21 communicates with the port 1 and the three-way valve V1 connected to the port 1 communicates the port 1 with the reagent 1, the reagent 1 enters the first unit 41 through the three-way valve V1 and the first valve 20 when the first pump 51 supplies the negative pressure.

Similarly, the operation of the second pump 52 may refer to the operation of the first pump 51.

Further, in certain embodiments, the drive assembly 50 further includes a fourth valve 53, a fifth valve 54, and a waste bottle 55. The fourth valve 53 is piped between the first pump 51 and the first unit 41, while also being piped with a waste bottle 55. A fifth valve 54 is plumbed between the second pump 52 and the second unit 42, and also plumbed to a waste bottle 55.

The first pump 51 is connected to the first unit 41 or the waste liquid bottle 55 through the fourth valve 53, so that after the first pump 51 pumps the waste liquid in the first unit 41, which has completed the sequencing reaction, the waste liquid bottle 55 can be injected with the waste liquid, so that the first pump 51 performs the next negative pressure supply to the first unit 41 to perform the sequencing reaction. The fifth valve 54 is configured identically to the fourth valve 53, and will not be described in detail herein. In some examples, the fourth valve 53 and the fifth valve 54 may both be three-way valves.

In certain embodiments, the fluid device 100 includes a control unit that electrically connects the valve body assembly 10 and the drive assembly 50 to control the operation of the valve body assembly 10 and the drive assembly 50.

In this manner, automated control of the valve body assembly 10 and the drive assembly 50 may be achieved, thereby improving efficiency.

Specifically, in the present example, the control unit electrically connects the first, second, and third valves 20, 30, 31 and the drive assembly 50 to control the operation of the first, second, and third valves 20, 30, 31 and the drive assembly 50. The control unit can be a device comprising a single chip microcomputer, a computer processor, a central control processor and the like, and the control unit is used for controlling the first valve 20, the three-way valves V1-V8 and the driving assembly to operate, so that the fluid device 100 can automatically operate and the efficiency is improved.

In some embodiments, referring to fig. 2 and 4, the plurality of ports are distributed in a circle, and the common port is concentric with the circle.

Thus, the plurality of ports and common ports which are distributed in a circular shape are arranged concentrically with the circular shape, and the accuracy of communication between the communication groove 21 and the corresponding ports and common ports when the rotor is rotated is ensured.

In some embodiments, please refer to fig. 2 and fig. 4, the connecting groove 21 is linear. Thus, the flow path of the reagent solution in the communicating groove 21 can be reduced, and rapid sequencing can be ensured.

Specifically, the communication groove 21 having a linear shape can communicate the port and the common port at both ends of the communication groove 21 with a short path. In the present example, the line shape is a straight line shape.

Referring to fig. 10, a sequencing system 300 according to an embodiment of the present invention controls a sequencing reaction, which includes a first biochemical reaction performed on a reaction apparatus 40 using a first reagent 11.

The sequencing system 300 includes a control device 302 and the fluid device 100, the control device 302 being coupled to the fluid device 100, the fluid device 100 including the valve body assembly 10 and the drive assembly 50.

The valve body assembly 10 comprises a first valve 20 and a second valve 30, the first valve 20 is connected with the reaction device 40, the first valve 20 comprises a stator and a rotor which can be communicated, the first valve 20 is provided with a common port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove 21, the common port and at least one port can be communicated through the communication groove 21 by rotating the rotor, the plurality of ports comprise a first port 22, the second valve 30 can be connected with the first port 22, the first reagent 11 and/or the first buffer liquid 60, and the control device 302 is used for:

the first port 22 is made to communicate with the common port through the communication groove 21;

connecting the second valve 30 to the first reagent 11 and the first port 22;

using the driving assembly 50 to make the first reagent 11 enter the reaction device 40 through the second valve 30 and the first valve 20 in sequence to perform the first biochemical reaction;

prior to rotating the rotor, communicating the second valve 30 with the first buffer liquid 60 and the first port 22;

the first buffer solution 60 is flowed through the second valve 30 and the first valve 20 in sequence by the driving assembly 50.

In the above-described sequence measurement system 300, the first buffer solution 60 is flowed into the first valve 20 before the rotor is rotated, so that the liquid in the communication tank 21 is replaced with the first buffer solution 60 before the rotor is rotated, or the first reagent 11 in the communication tank 21 is replaced with the first buffer solution 60 having no influence on the target sequence measurement reaction before the rotor of the first valve 20 is rotated, thereby preventing the reagent originally in the communication tank 21 from being brought to another position of the connection interface between the stator and the rotor during the rotation of the rotor, and further avoiding the risk of cross contamination when different reagents are switched.

It should be noted that the explanation and explanation of the technical features and advantages of the method for controlling a sequence determination reaction in any of the above embodiments and examples are also applicable to the sequence determination system 300 of the present embodiment, and are not detailed here to avoid redundancy.

In some embodiments, the sequencing reaction comprises a second biochemical reaction performed on the reaction device 40 using the second reagent 12, the valve body assembly 10 comprises a third valve 31, the plurality of ports comprises a second port 23, the third valve 31 is connectable to the second port 23, the second reagent 12, and/or a second buffer, and the control device 302 is configured to:

rotating the rotor so that the communication groove 21 communicates the second port 23 and the common port;

communicating the second reagent 12 and the second port 23 to the third valve 31;

using the driving assembly 50 to make the second reagent 12 enter the reaction device 40 through the third valve 31 and the first valve 20 in sequence to perform the second biochemical reaction;

communicating a second buffer solution to third valve 31 and second port 23 prior to rotating the rotor;

a second buffer is flowed through third valve 31 and first valve 20 in sequence using drive assembly 50.

In certain embodiments, the first biochemical reaction comprises an extension reaction.

In certain embodiments, the second biochemical reaction comprises radical cleavage.

In certain embodiments, the extension reaction is performed using a ligase and/or a polymerase.

In certain embodiments, the second biochemical reaction comprises capping.

In certain embodiments, the drive assembly 50 includes a pump that communicates through the reaction device 40 to a common port.

In certain embodiments, the fluid device 100 includes a control unit, and the control device 302 is connected to the control unit, which electrically connects the valve body assembly 10 and the drive assembly 50 to control the operation of the valve body assembly 10 and the drive assembly 50.

Specifically, the control unit may receive control signals from the control device 302 and control the valve body assembly 10, the drive assembly 50, and other components of the fluid device 100 based on the control signals. In this way, part of the functions of the control device 302 can be implemented by the control unit, and the load on the control device 302 is reduced. In some embodiments, the control unit and control device 302 may be integrated into a single component, module or device to increase the integration of the sequencing system 300 and reduce costs.

In some embodiments, the plurality of ports are distributed in a circle, and the common port is arranged concentrically with the circle.

In some embodiments, the communication groove 21 has a linear shape.

Referring to fig. 10, an embodiment of the present invention provides an apparatus 302 for controlling a sequencing reaction, the apparatus 302 comprising:

a storage device 304 for storing data, the data comprising a computer executable program;

a processor 306 for executing a computer-executable program, the executing of the computer-executable program comprising performing the method of any of the above embodiments.

A computer-readable storage medium of an embodiment of the present invention stores a program for execution by a computer, and executing the program includes performing the method of any of the above embodiments. The computer-readable storage medium may include: read-only memory, random access memory, magnetic or optical disk, and the like.

In the description herein, references to the description of the terms "one embodiment," "certain embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The logic and/or steps represented in the flowcharts or otherwise described herein, such as an ordered listing of executable instructions that can be considered to implement logical functions, can be embodied in any computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, processor-containing system, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions. For the purposes of this description, a "computer-readable storage medium" can be any means that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

In addition, each functional unit in the embodiments of the present invention may be integrated into one processing module, or each unit may exist alone physically, or two or more units are integrated into one module. The integrated module can be realized in a hardware mode, and can also be realized in a software functional module mode. The integrated module, if implemented in the form of a software functional module and sold or used as a stand-alone product, may also be stored in a computer readable storage medium.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention.

Claims (9)

1. A method of controlling a sequencing reaction, the sequencing reaction comprising a first biochemical reaction carried out on a reaction device using a first reagent, the sequencing reaction controlled using a sequencing system,

the sequencing system includes a fluidic device including a valve body assembly and a drive assembly,

the valve body assembly comprises a first valve and a second valve, the first valve is connected with the reaction device, the first valve comprises a stator and a rotor which can be communicated, the first valve is provided with a common port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the common port and at least one port can be communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first port, and the second valve can be connected with the first port, the first reagent and/or a first buffer solution, the method comprises the following steps:

communicating the first port with the common port through the communication groove;

communicating the second valve with the first reagent and the first port;

using the driving assembly to enable the first reagent to enter the reaction device through the second valve and the first valve in sequence so as to perform the first biochemical reaction;

communicating the second valve with the first buffer and the first port prior to rotating the rotor;

flowing the first buffer solution through the second valve and the first valve in sequence using the drive assembly.

2. The method of claim 1, wherein the sequencing reaction comprises a second biochemical reaction performed on the reaction device using a second reagent, the valve body assembly comprises a third valve, the plurality of ports comprises a second port, and the third valve is connectable to the second port, the second reagent, and/or a second buffer, the method comprising the steps of:

rotating the rotor so that the communication groove communicates the second port and the common port;

communicating the third valve with the second reagent and the second port;

using the driving assembly to make the second reagent enter the reaction device through the third valve and the first valve in sequence so as to perform the second biochemical reaction;

communicating the third valve with the second buffer and the second port prior to rotating the rotor;

flowing the second buffer through the third valve and the first valve in sequence using the drive assembly.

3. The method of claim 1, wherein the first biochemical reaction comprises an extension reaction;

optionally, the second biochemical reaction comprises radical cleavage;

optionally, the extension reaction is performed using a ligase and/or a polymerase;

optionally, the second biochemical reaction comprises capping.

4. The method of claim 1, wherein the drive assembly comprises a pump in communication with the common port through the reaction device;

optionally, the fluid device comprises a control unit electrically connecting the valve body assembly and the drive assembly to control the operation of the valve body assembly and the drive assembly;

optionally, the plurality of ports are distributed in a circle, and the common port is arranged concentrically with the circle;

optionally, the communication groove is linear.

5. A sequencing system for controlling a sequencing reaction, wherein the sequencing reaction comprises a first biochemical reaction carried out on a reaction device using a first reagent,

the sequencing system comprises a control device and a fluid device, wherein the control device is connected with the fluid device, the fluid device comprises a valve body assembly and a driving assembly,

the valve body assembly comprises a first valve and a second valve, the first valve is connected with the reaction device, the first valve comprises a stator and a rotor which can be communicated, the first valve is provided with a common port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the common port and at least one port can be communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first port, the second valve can be connected with the first port, the first reagent and/or a first buffer solution, and the control device is used for:

communicating the first port with the common port through the communication groove;

communicating the second valve with the first reagent and the first port;

using the driving assembly to enable the first reagent to enter the reaction device through the second valve and the first valve in sequence so as to perform the first biochemical reaction;

communicating the second valve with the first buffer and the first port prior to rotating the rotor;

flowing the first buffer solution through the second valve and the first valve in sequence using the drive assembly.

6. The system of claim 5, wherein the sequencing reaction comprises a second biochemical reaction performed on the reaction device using a second reagent, the valve body assembly comprises a third valve, the plurality of ports comprises a second port, the third valve is connectable to the second port, the second reagent, and/or a second buffer, the control device is configured to:

rotating the rotor so that the communication groove communicates the second port and the common port;

communicating the third valve with the second reagent and the second port;

using the driving assembly to make the second reagent enter the reaction device through the third valve and the first valve in sequence so as to perform the second biochemical reaction;

communicating the third valve with the second buffer and the second port prior to rotating the rotor;

flowing the second buffer through the third valve and the first valve in sequence using the drive assembly.

7. The system of claim 5, wherein the first biochemical reaction comprises an extension reaction;

optionally, the second biochemical reaction comprises radical cleavage;

optionally, the extension reaction is performed using a ligase and/or a polymerase;

optionally, the second biochemical reaction comprises capping.

8. The system of claim 5, wherein the drive assembly comprises a pump in communication with the common port through the reaction device;

optionally, the fluid device comprises a control unit, the control device is connected with the control unit, and the control unit is electrically connected with the valve body assembly and the driving assembly to control the valve body assembly and the driving assembly to operate;

optionally, the plurality of ports are distributed in a circle, and the common port is arranged concentrically with the circle;

optionally, the communication groove is linear.

9. An apparatus for controlling sequencing reactions, comprising:

a storage unit for storing data, the data comprising a computer executable program;

a processor for executing the computer-executable program, execution of the computer-executable program comprising performing the method of any of claims 1-4.