CN108456748B

CN108456748B - Method, device and system for controlling sequence determination reaction

Info

Publication number: CN108456748B
Application number: CN201710098314.3A
Authority: CN
Inventors: 吴平; 颜钦; 姜泽飞; 周志良
Original assignee: Genemind Biosciences Co Ltd
Current assignee: Genemind Biosciences Co Ltd
Priority date: 2017-02-22
Filing date: 2017-02-22
Publication date: 2023-04-25
Anticipated expiration: 2037-02-22
Also published as: WO2018153377A1; CN108456748A

Abstract

The invention discloses a method for controlling a sequencing reaction, which utilizes a sequencing system to control the sequencing reaction. The sequencing system includes a fluid device in which a valve body assembly includes a first valve and a second valve, the first valve including a stator and a rotor, the first valve having a common port, the stator having a plurality of ports therein, the rotor having a communication slot therein, the method comprising the steps of: communicating the first port with the common port through the communication groove; communicating the second valve with the first reagent and the first port; enabling a first reagent to enter the reaction device through the second valve and the first valve in sequence by utilizing the driving component so as to carry out a first biochemical reaction; before rotating the rotor, placing the second valve in communication with the first buffer and the first port; the first buffer is sequentially flowed through the second valve and the first valve by the driving assembly. In the method, before the rotor is rotated, the first valve is cleaned by the first buffer solution, namely the first reagent in the first valve is replaced, so that the risk of cross contamination when the rotor switches different reagents can be avoided.

Description

Method, device and system for controlling sequence determination 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 the determination of nucleic acid sequences. The sequencing platforms currently on the market include a first generation sequencing platform, a second generation sequencing platform and a third generation sequencing platform.

In the sequence determination process using a platform for sequencing sequences based on biochemical reactions, the biochemical reactions need to be performed on a reaction device, for example, different reagents need to be introduced together or sequentially onto a chip by using a liquid path system to perform the reactions. Currently, to make the liquid path system in the platform compact and efficient, the liquid path system uses a first valve to switch the input/output reagent.

The first valve on the market at present inevitably has reagent cross or mixing in different degrees when different reagents are switched, namely reagent cross pollution, due to the structural characteristics of the first valve. Reagent cross-contamination can affect the progress of the reaction, especially for reactions requiring small amounts of reagents per se, such as single molecule sequencing, which can be fatal.

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

Disclosure of Invention

Embodiments of the present invention aim to solve at least one of the technical problems occurring in the related art or to provide at least one useful commercial choice. The present invention has been made based on the following findings of split studies of the structure of the first valve and the idea.

The first valve, also known as a sample valve, multi-position valve, rotary valve or rotary valve, is currently on the market as a component for sample collection, liquid sample injection, or flow path switching. The composition generally includes a stator and a rotor, and an effective seal can be formed by the close combination of the stator and the rotor.

The first valve has a common port, which is a port through which different flow paths of liquid enter or exit, the common port being provided on the stator and/or rotor with one or more other ports. Through the rotation of the rotor, the connection between the rotor and the stator passage can be realized, so that the common port and other ports are communicated, and the function of sampling or shunting is achieved. The general/standard configuration of the first valve is a multi-pass option, i.e. only one port communicates with the common port during operation.

Communication between the common port and the other ports generally requires communication through one or more common structures provided on the rotor. When there is liquid in the common structure, at least a part of liquid in the common structure is inevitably carried to a place outside the common structure due to the rotation of the rotor and the relative movement of the sealing interface of the rotor and the stator, that is, when the flow path is switched, the liquid with the previous flow path in the liquid of the next flow path is inevitably carried to the liquid of the lower flow path, and then if the flow path is switched in the opposite direction, the liquid of the next flow path mixed with the liquid of the previous flow path is carried to the liquid of the lower flow path; thus, even if a trace amount is mixed, the cross contamination is difficult to control and the influence is difficult to estimate.

The inventors, upon comparing and analyzing the manual results with the results of the on-machine measurement, repeatedly tested and reviewed the association of the respective components of the device system, and based on the above-described split study of the structure of the first valve, determined that the above-described cross contamination of reagents, which is a cause of adverse unexpected effects on the sequence measurement results due to incorporation of at least a part of the liquid in the common structure into the next flow path, i.e., the next reaction process, upon flow path switching. To this end, embodiments of the present invention provide a method of controlling a sequencing reaction, and a sequencing system and control device.

Embodiments of the present invention provide a method of controlling a sequencing reaction, the sequencing reaction comprising a first biochemical reaction performed on a reaction device using a first reagent, the sequencing reaction being controlled by a sequencing system. The sequencing system includes a fluid 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 public port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the public 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 the first buffer solution, and 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;

sequentially passing the first reagent through the second valve and the first valve into the reaction device using the drive assembly to perform the first biochemical reaction;

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

the first buffer is sequentially flowed through the second valve and the first valve using the driving assembly.

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

In one embodiment, a sequencing system controls a sequencing reaction that includes a first biochemical reaction that is performed on a reaction device using a first reagent. The sequencing system includes a control device and a fluid device, the control device being coupled to the fluid device, the fluid 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 public port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the public port and at least one port can be communicated with the communication groove through rotating the rotor, the plurality of ports comprise a first port, the second valve can be connected with the first port, a first reagent and/or a first buffer solution, and the control device is used for:

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

In the sequence measuring 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 sequence measuring reaction is utilized to replace the first reagent in the communicating groove before the rotor of the first valve rotates, the phenomenon that the original reagent in the communicating groove is brought to other positions of a connecting interface of the stator and the rotor in the rotating process of the rotor is avoided, and the risk of cross contamination during switching different reagents is avoided.

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

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

FIG. 1 is a schematic flow chart 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 diagram of the relationship of ports, communication slots and common ports of a first valve according to an embodiment of the present invention;

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

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

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

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

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

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

Fig. 10 is a functional block diagram of a sequencing system according to an embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary only for explaining the present invention and are not to be construed as limiting the present invention.

In the description of the present invention, it should 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 a relative importance or number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more of the described features. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

In the description of the present invention, it should be noted that "connected" is to be interpreted broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected, unless explicitly stated or defined otherwise; may be mechanically connected, may be electrically connected, or may be in communication with each other; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.

The following disclosure provides many different embodiments, or examples, for implementing different features of the invention. In order to simplify the present disclosure, components and arrangements of specific examples are described below. Furthermore, the present invention may repeat reference numerals and/or letters in the various examples, which are for the purpose of brevity and clarity, and which do not themselves indicate the relationship between the various embodiments and/or settings discussed.

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

Referring to fig. 1 and 2, an embodiment of the present invention provides a method for controlling a sequencing reaction, the sequencing reaction including a first biochemical reaction, the first biochemical reaction being performed on a reaction device 40 using a first reagent 11, the sequencing reaction being controlled by a sequencing system.

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

The valve body assembly 10 includes a first valve 20 and a second valve 30, the first valve 20 being connected to the reaction device 40, the first valve 20 including a stator and a rotor which are communicable, the first valve 20 having a common port, the stator having a plurality of ports thereon, the rotor having a communication groove 21 thereon, the common port and at least one port being communicable through the communication groove 21 by rotating the rotor, the plurality of ports including a first port 22, the second valve 30 being connectable to the first port 22, the first reagent 11 and/or the first buffer 60, the method comprising the steps of:

s11, enabling the first port 22 to be communicated with the common port through the communication groove 21;

s12, enabling the second valve 30 to be communicated with the first reagent 11 and the first port 22;

s13, enabling the first reagent 11 to sequentially enter the reaction device 40 through the second valve 30 and the first valve 20 by utilizing the driving assembly 50 so as to perform a first biochemical reaction;

S14, before rotating the rotor, enabling the second valve 30 to be communicated with the first buffer 60 and the first port 22;

s15, the first buffer solution is sequentially flowed through the second valve 30 and the first valve 20 by the driving unit 50.

In the above method, before the rotor is rotated, the first buffer solution 60 is flowed into the first valve 20, so that the liquid in the communication groove 21 is replaced by the first buffer solution 60 before the rotor is rotated, or, before the rotor of the first valve 20 is rotated, the first reagent 11 in the communication groove 21 is replaced by the first buffer solution 60 which has no influence on the target sequence measurement reaction, so that the original reagent in the communication groove 21 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 during the switching of different reagents is avoided.

Specifically, in certain embodiments, the reaction device 40 may be a chip, with 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) for 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 mutually independent. For example, when a biochemical reaction is desired on a sample on the first unit 41, the fluidic device 100 may deliver the reagent for the reaction to the first unit 41, in which case the same reagent is not allowed to enter the second unit 42, and vice versa.

In the embodiment of the present invention, referring to fig. 2, a first valve 20 is correspondingly connected to each unit. 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 to perform a biochemical reaction. In this way, the sequence determination process can be accelerated.

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

The first reagent 11 and the second valve 30, the first buffer 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 may be connected and connected by a hose, so that the hose may make the configuration of the liquid path more flexible.

In certain embodiments, the first valve 20 may employ a multi-way valve. The second valve 30 may be a three-way valve, such as a three-way solenoid valve, with the normally closed and normally open ports of the three-way solenoid valve being connected to the reagent and buffer, respectively, to be added. In certain embodiments, the first valve 20 may be a rotary valve, and thus, the methods of controlling the sequencing reaction are widely applicable.

In some embodiments, a common port is provided on the stator, a plurality of ports are provided around the common port, and the common port communicates with one end of the communication groove 21. In other embodiments, the common port is formed in the rotor at one end of the communication slot 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 a certain extent, and is a weak acid, weak base and/or 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 substantially an end face seal between the stator and the rotor, and when the rotor is rotated, the liquid agent in the communication groove 21 remains on the seal face between the stator and the rotor. As shown in fig. 3, in the first biochemical reaction, the first reagent 11 enters the reaction device 40 through the port 1 as the first port 22, the communication groove 21, and the common port 0. When the first reagent 11 in the communicating vessel 21 is not washed when other biochemical reactions are required, the first reagent 11 in the communicating vessel 21 remains on the area between the ports 1 and 2 (e.g., the triangle area in fig. 3) when the rotating rotor is rotated from the port 1 to the port 2, 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 is connected to the first buffer solution 11 and the first port 22, and the driving unit 50 is used to sequentially flow the first buffer solution 60 through the second valve and the first valve 20, so that the first reagent 11 remaining in the connecting tank 21 is cleaned, thereby greatly improving the cross contamination. It is understood 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 ports 1-8.

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

Firstly, two rotary valves existing in the market are selected, a test platform is built as shown in fig. 5, and the cross-contamination performance of the two rotary valves (hereinafter referred to as rotary valve A1 and rotary valve B1) is evaluated by using the test platform. Referring to fig. 4, immediately

adjacent ports

1,2 and 8 are selected as the test, port 1 is connected with fluorescent reagent 1, port 2 and port 8 are both buffers, the reaction device flow cell has two parallel channels a and B, and the test operation details are as follows:

(1) Selecting a channel A of a flow cell of a reaction device, enabling 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 public 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 fluorescent reagent 1 in a liquid path of the rotary valve including the public port 0 and the communication groove 21 is cleaned;

(2) Switching a liquid path to a channel B of a flow cell of a reaction device, firstly 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 switched to be communicated with the port 8, 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 using the driving assembly 50 and flows into a channel B of the reaction device flow cell, 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 spots exist, n2-n1 can be regarded as cross contamination caused by the process of switching the rotary valve from the port 1 to the port 2 (clockwise) and then to the port 8 (anticlockwise), and the increase of the number of the fluorescent spots n2-n1 is necessarily the number of the fluorescent spots detected by mixing the fluorescent reagent 1 into the buffer solution entering the rotary valve through the port 8, so that the severity of the cross contamination during the switching of the rotary valve can be estimated by the value.

As shown in fig. 6, which shows the raw data of the 8 sets of tests, it can be seen that cross contamination of the reagents cannot be avoided in either the rotary valve A1 or the rotary valve B1, and the common washing process cannot completely solve the problem because the contamination occurs when the rotor is rotated, and therefore, after the fluorescent reagent 1 is introduced, even if the communication groove 21 is switched to the port 2 for rotary valve washing and then to the port 8, the fluorescent reagent 1 is always prevented from being mixed into the buffer solution entering through the port 8. In fig. 6, in the bar graph shown in the same test group, the left bar graph indicates data of n1 before rotation, and the right bar graph indicates data of n2 after rotation.

In the embodiment of the present invention, the above-described cross-contamination situation can be improved by allowing the second valve 30 to communicate the first buffer 60 with the first port 22 before rotating the rotor, and allowing the first buffer 60 to sequentially flow through the second valve 30 and the first valve 20 by using the driving unit 50. Specifically, referring to fig. 4, the second valve 30 is exemplified as a three-way electromagnetic valve. The normally closed port and the normally open port 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 electrified (at this time, 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 closed by the vertical horse (at this time, the port 1 is communicated with the buffer solution), a small amount of buffer solution (a specific amount is determined according to the pipeline condition) is used for cleaning the residual reagent 1 in the communicating groove 21, so that after cleaning, the reagent 1 is not remained on the end face of the rotary valve when the rotary valve is rotated again to switch different ports, and the buffer solution is remained, but has no influence on biochemical reaction.

Similarly, the improved cross-contamination was evaluated using a single-molecule fluorescence detection system, and the raw data is shown in FIG. 7. The comparison of n2-n1 before and after improvement is shown in FIG. 8. As can be seen from fig. 8, compared with the previous rotary valves A1 and B1, the method of the embodiment of the present invention significantly improves the cross contamination of the rotary valves, and the method of the embodiment of the present invention eliminates the cross contamination of the reagents from the source, and is very suitable for applications that are very sensitive to micro cross contamination, such as a single molecule gene sequencer system. In fig. 7, in the bar graph shown in the same test number, the left bar graph indicates data of n1 before rotation, and the right bar graph indicates data of n2 after rotation. In fig. 8, in the bar graph shown in the same test group, the left bar graph shows the data of n2-n1 after improvement, the middle bar graph shows the data of n2-n1 of the rotary valve A1 before improvement, and the right bar graph shows the data of n2-n1 of the rotary valve B1 before improvement.

In certain embodiments, referring to fig. 9, the sequencing reaction comprises a second biochemical reaction performed on a reaction device 40 using a 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, the method comprising the steps of:

S16, rotating the rotor to enable the communication groove 21 to communicate the second port 23 and the common port;

s17, enabling the third valve 31 to be communicated with the second reagent 12 and the second port 23;

s18, enabling the second reagent 12 to sequentially enter the reaction device 40 through the third valve 31 and the first valve 20 by utilizing the driving assembly 50 so as to perform a second biochemical reaction;

s19, before rotating the rotor, communicating the second buffer solution with the second port 23 by the third valve 31;

s20, the second buffer solution is sequentially flowed through the third valve 31 and the first valve 20 by the driving unit 50.

Thus, the method of the embodiment of the invention can be applied to a plurality of different types of biochemical reactions required to be carried out in the sequencing reaction, and the application range of the method of the embodiment of the invention is enlarged.

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

The second buffer is a solution that does not affect the first biochemical reaction, and the first buffer 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 also be selected as different buffers. In one example, the first buffer and the second buffer are the same buffer, and are "150mM HEPES, 150mM NaCl, pH=7.0" buffers, and have no effect on the sequencing reaction.

In some 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 the line for the scavenger fluid.

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 complementation, attaching a specific substrate to the sample to be sequenced, and using the detectable groups on the substrate to determine the type of substrate bound thereto to determine the sequence. In one example, the detectable moiety comprises a fluorescent moiety that fluoresces under a laser of a particular wavelength.

In an embodiment of the present invention, the first reagent referred to as a terminator reagent, i.e., a reaction substrate, includes A, T, C and G base analogues, specifically, the base analogues referred to as a structure of A/T/C/G-terminator-linker-luminescent group, i.e., the first reagent referred to as a reagent containing A-terminator-linker-luminescent group (hereinafter referred to as A reagent), a reagent containing T-terminator-linker-luminescent group (hereinafter referred to as T reagent), a reagent containing C-terminator-linker-luminescent group (hereinafter referred to as C reagent), and/or a reagent containing G-terminator-linker-luminescent group (hereinafter referred to as G reagent). Wherein the terminating groups are photo and/or chemically cleavable groups, and the substrate is provided with luminescent groups by means of a linker.

In one specific example, the luminescent groups carried by the four terminators are of the same structure or emit the same color of detectable light when excited, and the four base analogues are contained in different reagent bottles, respectively. For sequence determination, one of A, T, C and G terminators was added sequentially, and every four terminator reactions were called one cycle. Reagent bottles containing different terminators are connected with the reaction device through a three-way valve and a first valve.

In the following, an example of the present invention will be described with reference to fig. 4.

In fig. 4, reagent 1 is a reagent, reagent 2 is a T reagent, reagent 3 is a C reagent, and reagent 4 is a G reagent. In the extension reaction, the three-way valve V1 is powered on, 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 component 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 buffer solution, and the driving component 50 enables the buffer solution to flow through the three-way valve V1 and the first valve 20. When the added T reagent, C reagent, G reagent and/or other reagents are to be replaced later, the rotor is rotated to allow the communication groove 21 to communicate with the common port 0 and the corresponding port, and the above-described process is performed.

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

Specifically, when adding a terminator of a different structure to the reaction apparatus 40, it is necessary to cut off a luminescent group on a terminator of a previous structure and then add a terminator of another structure. For example, in connection with the above example, when the reagent A is added to the reaction device 40, a light emitting device (e.g., a laser) may be used to emit excitation light to the reaction device 40 to excite the luminescent group to emit fluorescence, and an imaging device is used to photograph to collect the fluorescence and form an image for sequencing. After photographing is completed, other reagents are added after the luminous group of the reagent A is excised. Further, in this example, the reagent 5 is a reagent for excision (hereinafter referred to as excision reagent).

After photographing is completed, when the excision reagent is added, the rotor is rotated, the communication groove 21 is communicated with the port 5 and the public 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 drive assembly 50 enables the excision reagent to enter the reaction device 40 through the three-way valve V5 and the first valve 20 for excision reaction, before the rotor is rotated, the three-way valve V5 is closed, the port 5 is communicated with buffer solution, and the drive assembly 50 enables the buffer solution to flow through the three-way valve V5 and the first valve 20.

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

In certain embodiments, the second biochemical reaction comprises capping.

Specifically, the capping is referred to as mainly protecting the groups/bonds that are exposed after cleavage of the groups. In one example, the first biochemical reaction comprises an extension reaction and the second biochemical reaction comprises cleavage of a group, the group exposed after cleavage of the cleavable group by light and/or chemistry being a thiol group, the thiol group being protected from oxidation by capping, e.g. by addition of an alkylating agent.

In connection with the above example, further, in this example, the reagent 6 is a reagent added by capping (hereinafter referred to as capping reagent). When the capping reagent is added, the rotor is rotated, the communication groove 21 is communicated with the port 6 and 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 component 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, before the rotor is rotated, the three-way valve V6 is closed, the port 6 is communicated with buffer solution, and the driving component 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, and that there is no need to flow a buffer or a wash solution through the second valve and the first valve 20 after the reagent enters the reaction device 40 through the second valve and the first valve 20 and before the rotor is rotated, thus saving time in the sequencing reaction.

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

Therefore, the driving of the reagent and the buffer solution 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 dosage liquid output by the first valve 20 to the first unit 41 and/or the second unit 42, respectively, so that the operation is convenient.

Specifically, the first pump 51 and the second pump 52 are respectively connected to the first unit 41 and the second unit 42 by pipes, for example, by hoses.

The first pump 51 is connected to the common port of one of the first valves 20 through the first unit 41, the second pump 52 is connected to the common port of the other first valve 20 through the second unit 42, and in operation, the first pump 51 supplies negative pressure to the first unit 41 to enable the first unit 41 to obtain the first reagent and/or other reagents (including buffer solution and/or other reagents) connected to the ports of the first valves 20 to perform biochemical reaction and/or washing, and after the first unit 41 obtains the reagent solution, the first pump 51 stops supplying negative pressure.

What dosage is entered into the first unit 41 by the first pump 51 depends on: 1) The communication groove 21 communicates which port; and 2) for the port (hereinafter referred to as a communication port) communicating with the communication groove 21, a three-way valve connected to the communication port communicates with which agent liquid the communication port communicates. 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 negative pressure.

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

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

The first pump 51 communicates with the first unit 41 or the waste liquid bottle 55 via the fourth valve 53, so that after the first pump 51 pumps the waste liquid in which the sequencing reaction has been completed in the first unit 41, the waste liquid bottle 55 can be injected with the waste liquid, thereby causing the first pump 51 to perform 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 again here. In some examples, the fourth valve 53 and the fifth valve 54 may each be a three-way valve.

In certain embodiments, fluid device 100 includes a control unit that electrically connects valve body assembly 10 and drive assembly 50 to control operation of valve body assembly 10 and 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 valve 20, the second valve 30, the third valve 31, and the driving assembly 50 to control the operation of the first valve 20, the second valve 30, the third valve 31, and the driving assembly 50. The control unit may be a device including a single-chip microcomputer, a computer processor, or a central control processor, and the control unit is used to control the operation of the first valve 20, the three-way valves V1-V8, and the driving component, so as to realize the automatic operation of the fluid device 100, and improve the efficiency.

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

In this way, the concentric arrangement of the plurality of ports and the common port in a circular distribution with the circle ensures the accuracy of the communication groove 21 with the corresponding ports and common port when the rotor is rotated.

In some embodiments, referring to fig. 2 and 4, the communication groove 21 is linear. Thus, the flow path of the reagent liquid in the communicating groove 21 can be reduced, and further, the rapid sequencing can be ensured.

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

Referring to FIG. 10, a sequencing system 300 according to an embodiment of the present invention controls a sequencing reaction, including a first biochemical reaction, which is performed on a reaction device 40 using a first reagent 11.

The sequencing system 300 includes a control device 302 and a fluid device 100, the control device 302 being coupled to the fluid device 100, the fluid 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 being connected to the reaction device 40, the first valve 20 comprising a communicable stator and a rotor, the first valve 20 having a common port, the stator having a plurality of ports thereon, the rotor having a communication slot 21 thereon, the common port and at least one port being communicable through the communication slot 21 by rotating the rotor, the plurality of ports comprising a first port 22, the second valve 30 being connectable to the first port 22, the first reagent 11 and/or the first buffer 60, the control device 302 being adapted to:

Communicating the first port 22 with the common port through the communication groove 21;

placing the second valve 30 in communication with the first reagent 11 and the first port 22;

the first reagent 11 is sequentially introduced into the reaction device 40 through the second valve 30 and the first valve 20 by using the driving assembly 50 to perform a first biochemical reaction;

before rotating the rotor, the second valve 30 is put in communication with the first buffer 60 and the first port 22;

first buffer 60 is sequentially flowed through second valve 30 and first valve 20 by drive assembly 50.

In the sequencing system 300, before the rotor is rotated, the first buffer solution 60 is flowed into the first valve 20, so that the liquid in the communication groove 21 is replaced by the first buffer solution 60 before the rotor is rotated, or, before the rotor of the first valve 20 is rotated, the first reagent 11 in the communication groove 21 is replaced by the first buffer solution 60 which has no influence on the target sequencing reaction, so that the original reagent in the communication groove 21 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 during the switching of different reagents is avoided.

The technical features and advantages of the method for controlling a sequencing reaction in any of the above embodiments and examples are also applicable to the sequencing system 300 of the present embodiment, and are not further detailed herein to avoid redundancy.

In certain 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 comprising a third valve 31, the plurality of ports comprising a second port 23, the third valve 31 being connectable to the second port 23, the second reagent 12 and/or the second buffer, the control device 302 being configured to:

rotating the rotor to cause the communication groove 21 to communicate the second port 23 with the common port;

placing the third valve 31 in communication with the second reagent 12 and the second port 23;

the second reagent 12 is sequentially introduced into the reaction device 40 through the third valve 31 and the first valve 20 by using the driving assembly 50 to perform a second biochemical reaction;

before rotating the rotor, the third valve 31 is put in communication with the second buffer and the second port 23;

the second buffer is sequentially flowed through the third valve 31 and the first valve 20 by the driving assembly 50.

In certain embodiments, the second biochemical reaction comprises capping.

In certain embodiments, fluid device 100 includes a control unit to which control device 302 is coupled, the control unit being electrically coupled to valve body assembly 10 and drive assembly 50 to control operation of valve body assembly 10 and 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 executed by the control unit, and the load of the control device 302 can be reduced. In some embodiments, the control unit and control device 302 may be integrated into one component, module, or device to increase the integration of the sequencing system 300 and reduce cost.

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

In some embodiments, the communication groove 21 is linear.

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

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

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

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

In the description of the present specification, reference to the terms "one embodiment," "certain embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means 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 embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Logic and/or steps represented in the flowcharts or otherwise described herein, e.g., a ordered listing of executable instructions for implementing 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 each embodiment of the present invention may be integrated into one processing module, each unit may exist alone physically, or two or more units may be integrated into one module. The integrated modules may be implemented in hardware or in software functional modules. The integrated modules may also be stored in a computer readable storage medium if implemented in the form of software functional modules and sold or used as a stand-alone product.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A method for controlling a sequencing reaction, wherein the sequencing reaction comprises a first biochemical reaction performed on a reaction device using a first reagent, the sequencing reaction being controlled by a sequencing system,

The sequencing system includes a fluid device including a valve body assembly and a drive assembly,

the valve body assembly includes a first valve and a second valve, the first valve and the reaction device are connected, the first valve includes a stator and a rotor which can be communicated, the first valve has a common port, the stator has a plurality of ports, the rotor has a communication groove, the common port and at least one of the ports are communicated through the communication groove by rotating the rotor, the plurality of ports includes a first port, and the second valve connects the first port, the first reagent and the first buffer, the method includes the steps of:

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

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 comprising a third valve, the plurality of ports comprising a second port, the third valve connecting the second port, the second reagent, and a second buffer, the method comprising the steps of:

rotating the rotor to communicate the communication groove with the second port and the common port;

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

using the drive assembly to enable the second reagent to sequentially enter the reaction device through the third valve and the first valve 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;

the second buffer is sequentially flowed through the third valve and the first valve using the driving assembly.

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

4. The method of claim 2, wherein the second biochemical reaction comprises radical cleavage.

5. A method according to claim 3, wherein the extension reaction is carried out using a ligase and/or a polymerase.

6. The method of claim 4, wherein the second biochemical reaction comprises capping.

7. The method of claim 1, wherein the drive assembly comprises a pump that communicates with the common port through the reaction device.

8. The method of claim 1, wherein the fluid device comprises a control unit electrically connecting the valve body assembly and the drive assembly to control operation of the valve body assembly and the drive assembly.

9. The method of claim 1, wherein the plurality of ports are distributed in a circle, and the common port is disposed concentric with the circle.

10. The method of claim 1, wherein the communication channel is linear.

11. 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 public port, the stator is provided with a plurality of ports, the rotor is provided with a communication groove, the public port and at least one port are communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first port, the second valve is connected with the first port, the first reagent and the first buffer solution, and the control device is used for:

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

12. The system of claim 11, wherein the sequencing reaction comprises a second biochemical reaction performed on the reaction device using a second reagent, the valve body assembly comprising a third valve, the plurality of ports comprising a second port, the third valve connecting the second port, the second reagent, and a second buffer, the control device to:

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

13. The system of claim 11, wherein the first biochemical reaction comprises an extension reaction.

14. The system of claim 12, wherein the second biochemical reaction comprises radical excision.

15. The system of claim 13, wherein the extension reaction is performed using a ligase and/or a polymerase.

16. The system of claim 14, wherein the second biochemical reaction comprises capping.

17. The system of claim 11, wherein the drive assembly comprises a pump that communicates with the common port through the reaction device.

18. The system of claim 11, wherein the fluid device comprises a control unit, the control device coupled to the control unit, the control unit electrically coupled to the valve body assembly and the drive assembly to control operation of the valve body assembly and the drive assembly.

19. The system of claim 11, wherein the plurality of ports are distributed in a circle, and the common port is disposed concentric with the circle.

20. The system of claim 11, wherein the communication slot is linear.

21. An apparatus for controlling a sequencing reaction, comprising:

a storage unit configured to store data including a computer-executable program;

a processor for executing the computer executable program, the execution of the computer executable program comprising performing the method of any one of claims 1-10.