CN108949939B

CN108949939B - Method, device and system for controlling sequence determination reaction

Info

Publication number: CN108949939B
Application number: CN201710396131.XA
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: 2017-05-26
Filing date: 2017-05-26
Publication date: 2023-04-25
Anticipated expiration: 2037-05-26
Also published as: WO2018214816A1; CN108949939A

Abstract

The invention discloses a method for controlling a sequencing reaction, which comprises the following steps: i) The first port is communicated with the public port by utilizing the driving assembly, so that a first reagent of a first sequencing reaction enters the reaction device through the rotary valve to realize the base extension of the first sequencing reaction; ii) communicating the second port with the common port using a drive assembly to pass the second reagent through the rotary valve into the reaction device to effect excision of the first sequencing reaction; iii) The third port is communicated with the public port by utilizing the driving assembly, so that a first reagent of the second sequencing reaction enters the reaction device through the rotary valve to realize the base extension of the second sequencing reaction; iv) communicating the fourth port with the common port using a drive assembly to allow the second reagent to enter the reaction apparatus through the rotary valve to effect excision of the second sequencing reaction. The above method can avoid or greatly reduce cross-contamination between different first reagents. The invention also discloses a device for controlling the sequencing reaction and a sequencing system.

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. At present, in order to make a liquid path system in a platform compact and efficient, the liquid path system adopts a valve body to switch input/output reagents.

At present, more reagents are needed for biochemical reaction, and some reagent cross-overs, even trace cross-overs, may seriously affect the progress of the reaction or the result of the reaction, so how to avoid or reduce cross-contamination between different reagents becomes a problem to be solved.

Disclosure of Invention

The inventors found that when each of a plurality of reagents is sequentially inputted/outputted by using a liquid path with a commercially available rotary valve, the influence of the remaining pipe is eliminated, and the next reagent always has a certain amount of the former reagent, which affects the reaction depending on the next reagent. To solve this problem, the present inventors have made the present invention based on the following findings, ideas and experiments on the split study of the structure of a rotary valve.

Rotary valves, also known as sample valves, multi-position valves, or rotary valves, are currently on the market as components 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.

Typically, rotary valves have a common port, which is a port through which different flow paths of liquid may enter or exit, provided on a stator and/or rotor having one or more other ports thereon. 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 rotary valves is a multi-pass option, i.e. only one port communicates with a 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 the 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 where the rotor and the stator are connected, that is, when the flow path is switched, the liquid with the previous flow path in the next flow path liquid is inevitably carried to the liquid with the next flow path, and then when the flow path is switched in the opposite direction, the liquid with the previous flow path liquid mixed in the next flow path is carried to the liquid with the next flow path; thus, the cross contamination is difficult to control and the influence is difficult to predict. Herein, the above-mentioned "common structure" is referred to as a communication groove.

To this end, embodiments of the present invention provide a method and a sequence determination system for controlling a sequence determination reaction and an apparatus for controlling a sequence determination reaction.

The embodiment of the invention provides a method for controlling a sequencing reaction, wherein the sequencing reaction is carried out on a reaction device, the sequencing reaction is controlled by a sequencing system, the sequencing reaction comprises a first sequencing reaction and a second sequencing reaction which are sequentially carried out, and the first sequencing reaction and the second sequencing reaction comprise the following steps sequentially carried out: base extension, image acquisition and excision, said base extension being performed using a first reagent, said first reagent of said first sequencing reaction and said second sequencing reaction being different,

the excision is carried out with a second reagent, the sequencing system comprises a fluidic device comprising a valve body assembly and a driving assembly, the valve body assembly comprises a rotary valve, the rotary valve comprises a stator and a rotor which can be communicated, the rotary 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 one port can be communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first group of ports and a second group of ports which respectively correspond to the first sequencing reaction and the second sequencing reaction, the first group of ports comprises a first port and a second port, the first port is connected with the first reagent of the first sequencing reaction, the second port is connected with the second reagent, the second group of ports comprises a third port and a fourth port, the first port, the second port, the third port and the fourth port are sequentially arranged according to the preset rotation direction of the rotor, the fourth port is connected with the second port, the first port is connected with the second reagent, the common reagent comprises the steps of connecting: i) Communicating the first port with the common port using the drive assembly to allow a first reagent of the first sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the first sequencing reaction; ii) communicating the second port with the common port using the drive assembly, passing the second reagent through the rotary valve into the reaction device to effect excision of the first sequencing reaction;

iii) Communicating the third port with the common port using the drive assembly to allow the first reagent of the second sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the second sequencing reaction; iv) communicating the fourth port with the common port using the drive assembly to pass the second reagent through the rotary valve into the reaction device to effect excision of the second sequencing reaction.

In the method, before the first reagent of the second sequencing reaction enters the reaction device through the rotary valve, the first port is communicated with the communication groove through the rotation of one direction of the rotor to input the first reagent of the first sequencing reaction, then the rotation is switched to enable the communication groove to be communicated with the second port to input the second reagent, so that all or most of the first reagent of the first sequencing reaction in the communication groove and in the communication groove, which is brought into a sealing surface area between the rotor and the stator due to rotation, is replaced by the second reagent, the first reagent of the first sequencing reaction is greatly reduced from being brought into the second sequencing reaction, and meanwhile, the first reagent of the second sequencing reaction is prevented from being brought into the first reagent of the first sequencing reaction, and cross contamination among different first reagents is avoided or greatly reduced. The method is particularly suitable for sequencing reactions which require the sequential addition of different types of substrates or different combinations of substrates in each round, and can greatly reduce the pollution between different types of substrates or between different combinations of substrates by virtue of a simple device structure and the control of the liquid inlet and outlet sequence.

The sequencing reaction is controlled on a reaction device, the sequencing reaction comprises a first sequencing reaction and a second sequencing reaction which are sequentially carried out, and the first sequencing reaction and the second sequencing reaction comprise the following steps sequentially carried out: base extension, image acquisition and excision, said base extension being performed using a first reagent, said first reagent of said first sequencing reaction and said second sequencing reaction being different, said excision being performed using said excision, said sequencing system comprising a control device and a fluid device, said control device being connected to said fluid device, said fluid device comprising a valve body assembly and a drive assembly, said valve body assembly comprising a rotary valve, said rotary valve comprising a communicable stator and a rotor, said rotary valve having a common port, said stator having a plurality of ports, said rotor having a communication slot, said common port and one of said ports being communicable through said communication slot by rotation of said rotor, the plurality of ports comprise a first group of ports and a second group of ports corresponding to the first sequencing reaction and the second sequencing reaction respectively, the first group of ports comprise a first port and a second port, the first port is connected with a first reagent of the first sequencing reaction, the second port is connected with a second reagent, the second group of ports comprise a third port and a fourth port, the first port, the second port, the third port and the fourth port are sequentially arranged according to the preset rotation direction of a rotor, the third port is connected with the first reagent of the second sequencing reaction, the fourth port is connected with a second reagent, the common port is connected with the reaction device, and the control device is used for: i) Communicating the first port with the common port using the drive assembly to allow a first reagent of the first sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the first sequencing reaction; ii) communicating the second port with the common port using the drive assembly, passing the second reagent through the rotary valve into the reaction device to effect excision of the first sequencing reaction; iii) Communicating the third port with the common port using the drive assembly to allow the first reagent of the second sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the second sequencing reaction; iv) communicating the fourth port with the common port using the drive assembly to pass the second reagent through the rotary valve into the reaction device to effect excision of the second sequencing reaction.

In the system, before the first reagent of the second sequencing reaction enters the reaction device through the rotary valve, the first port is communicated with the communication groove through rotation of one direction of the rotor to input the first reagent of the first sequencing reaction, and then the second port is communicated with the communication groove through rotation switching to input the second reagent, so that all or most of the first reagent of the first sequencing reaction in the communication groove and in the communication groove, which is brought to a sealing surface area between the rotor and the stator due to rotation, is replaced by the second reagent, the first reagent of the first sequencing reaction is greatly reduced from being brought into the second sequencing reaction, and meanwhile, the first reagent of the second sequencing reaction is prevented from being brought into the first reagent of the first sequencing reaction, and cross contamination between different first reagents is avoided or greatly reduced. The system is particularly suitable for sequencing reactions requiring the sequential addition of different types of substrates or different combinations of substrates in each round, and can greatly reduce the pollution between different types of substrates or different combinations of substrates by virtue of a simple device structure and the control of the liquid inlet and outlet sequence.

An apparatus for controlling a sequencing reaction according to an embodiment of the present invention includes: a storage unit configured to store data including a computer-executable program; a processor configured to execute the computer-executable program, the execution of the computer-executable program including performing the method of any of the above embodiments.

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 flow path structure of a sequencer system according to an embodiment of the present invention;

FIG. 3 is another schematic flow path structure of a sequencing system according to an embodiment of the present invention;

FIG. 4 is a schematic diagram of the relationship of ports and common ports of a rotary valve according to an embodiment of the present invention;

FIG. 5 is another schematic diagram of the relationship of ports and common ports of a rotary valve according to an embodiment of the present invention;

FIG. 6 is a schematic diagram of yet another relationship between ports and a common port of a rotary valve according to an embodiment of the present invention;

fig. 7 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 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 sequencing reactions referred to include extension reactions (base extension), information collection (photograph/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, 2 and 3, an embodiment of the present invention provides a method for controlling a sequencing reaction, the sequencing reaction is performed on a reaction device 40, the sequencing reaction is controlled by a sequencing system, the sequencing reaction includes a first sequencing reaction and a second sequencing reaction that are sequentially performed, and each of the first sequencing reaction and the second sequencing reaction includes the following steps that are sequentially performed: base extension, image acquisition and excision.

The first reagent is used for base extension, the first reagent of the first sequencing reaction and the second sequencing reaction are different, and the second reagent is used for excision. The first reagent comprises at least one nucleotide analogue, including analogues of several nucleotides: adenine deoxynucleotide (A), thymine deoxynucleotide (T), cytosine deoxynucleotide (C), guanine deoxynucleotide (G) and uracil deoxynucleotide (U).

The sequencing system includes a fluid device 100, the fluid device 100 including a valve body assembly 29 and a drive assembly 50. The valve body assembly 29 comprises a rotary valve 70, the rotary valve 70 comprises a stator 81 and a rotor which can be communicated, the rotary valve 70 is provided with a common port 71, the stator 81 is provided with a plurality of ports, the rotor is provided with a communication groove 72, the common port 71 and one port can be communicated through rotating the rotor, the plurality of ports comprise a first group of ports and a second group of ports which respectively correspond to a first sequencing reaction and a second sequencing reaction, the first group of ports comprise a first port 1 and a second port 2, the first port 1 is connected with a first reagent of the first sequencing reaction, the second port 2 is connected with a second reagent, the second group of ports comprise a third port 3 and a fourth port 4, the first port 1, the second port 2, the third port 3 and the fourth port 4 are sequentially arranged according to the preset rotation direction of the rotor, the third port 3 is connected with the first reagent of the second sequencing reaction, the fourth port 4 is connected with the second reagent, and the common port 71 is connected with the reaction device 40, and the method comprises the steps of:

i) The first port 1 and the common port 71 are communicated by the drive assembly 50, and a first reagent of a first sequencing reaction enters the reaction device 40 through the rotary valve 70 to realize the base extension of the first sequencing reaction;

ii) communicating the second port 2 with the common port 71 using the drive assembly 50 to allow the second reagent to enter the reaction apparatus 40 through the rotary valve 70 to effect excision of the first sequencing reaction;

iii) The third port 3 and the common port 71 are communicated by the drive assembly 50, and the first reagent of the second sequencing reaction enters the reaction device 40 through the rotary valve 70 so as to realize the base extension of the second sequencing reaction;

iv) the fourth port 4 and the common port 71 are brought into communication by means of the drive assembly 50, and the second reagent is brought into the reaction device 40 via the rotary valve 70 to effect excision of the second sequencing reaction.

In the above method, before the first reagent of the second sequencing reaction is introduced into the reaction device 40 through the rotary valve 70, by rotating in one direction of the rotor to connect the communication groove 72 with the first port 1 to input the first reagent of the first sequencing reaction and then rotating to switch to connect the communication groove 72 with the second port 2 to input the second reagent, all or most of the first reagent of the first sequencing reaction in the communication groove 72 and in the communication groove 72 brought to the sealing surface region 73 between the rotor and the stator 81 due to the rotation can be replaced with the second reagent, and at the same time, the first reagent of the second sequencing reaction is prevented from being brought into the first reagent of the first sequencing reaction, and cross contamination between different first reagents is prevented or greatly reduced. The method is particularly suitable for sequencing reactions which require the sequential addition of different types of substrates or different combinations of substrates in each round, and can greatly reduce the pollution between different types of substrates or between different combinations of substrates by virtue of a simple device structure and the control of the liquid inlet and outlet sequence.

Specifically, when the first sequencing reaction is performed, the communication groove 72 communicates with the first port 1 and the common port 71, and the first port 1 is further communicated with the reaction device 40, and the first reagent for the first sequencing reaction is introduced into the reaction device 40 through the rotary valve 70 by the driving unit 50 to perform the first sequencing reaction. As the first reagent of the first sequencing reaction flows through the communication channel 72, the first reagent of the first sequencing reaction remains in the communication channel 72. Thereafter, the rotor is rotated in a preset rotation direction by the driving assembly 50 to communicate the communication groove 72 with the second port 2 and the common port 71, thereby communicating the second port 2 with the reaction device 40. In performing excision of the first sequencing reaction, the drive assembly 50 is used to cause excision of the first sequencing reaction by passing the second reagent through the rotary valve 70 into the reaction apparatus 40. On the one hand, when the rotor rotates in one direction to switch the communication groove 72 to the communication of the first port 1 to the communication of the second port 2, a part of the first reagent of the first sequencing reaction in the communication groove 72 remains in the sealing surface region 73 (region shown in fig. 4) between the rotor and the stator 81, and on the other hand, when the second reagent for excision flows through the communication groove 72, the other part of the first reagent of the first sequencing reaction in the communication groove 72 is carried away in whole or in part. Thus, by using a simple device and liquid input/output control, the pollution caused by bringing the first reagent of the first sequencing reaction to the first reagent of the second sequencing reaction can be greatly reduced, and the pollution caused by the first reagent of the second sequencing reaction to the first reagent of the first sequencing reaction can be avoided.

In some embodiments, the reaction device 40 may be a chip, and a plurality of channels (channels) are provided in the reaction device 40 to accommodate the sample. The drive assembly 50 may include a motor and a pump, the motor being coupled to the rotor and configured to drive the rotor in rotation such that the communication slots 72 communicate with different ports on the stator 81. The pump is connected to the reaction device 40 and is used to generate negative pressure in the channel of the reaction device 40 to drive the reagent into the reaction device 40 and suck the reagent out of the reaction device 40, and stop generating negative pressure to leave the reagent in the reaction device 40 for biochemical reaction. In addition, the pumped reagent may be pumped into the waste container 60 for recovery.

In the embodiment of the present invention, the preset rotation direction of the rotor is clockwise as shown in fig. 2 and 3. That is, the movement of the rotary valve 70 requires communication with the first port 1 before communication with the second port 2, communication with the second port 2 before communication with the third port 3, and the like. It will be appreciated that in other embodiments, the predetermined rotational direction of the rotor may be other directions, such as counterclockwise as shown in fig. 2 and 3, and the ports may be selected again from a plurality of ports according to the rotational direction, which is not particularly limited herein.

In some embodiments, referring to fig. 4, based on a rotary valve with multiple ports, the first port 1 and the second port 2 are adjacent, the second port 2 and the third port 3 are adjacent, and the third port 3 and the fourth port 4 are adjacent, in combination with the sequence of the steps involved in the sequencing reaction, the rotary stroke of the rotor is shorter while avoiding or greatly reducing cross-contamination between the first reagents, which facilitates rapid sequencing. It will be appreciated that in other embodiments, the first port 1, the second port 2, the third port 3 and the fourth port 4 may be other ports of the plurality of ports, which is only required to ensure that the first port 1, the second port 2, the third port 3 and the fourth port 4 are sequentially arranged according to the preset rotation direction of the rotor.

During the base extension, image acquisition and excision of the sequencing reaction, the rotor is rotated in a predetermined rotational direction to sequentially complete the entry of different reagents into the reaction apparatus 40 through the rotary valve 70.

In addition, in certain embodiments, DNA is sequenced, the reaction substrates are A, T, C and G four nucleotide analogs, at least three of the four substrates bear at least one fluorescent label (fluorescent dye/chromophore), and upon polymerization/base extension, the substrates fluoresce upon binding to the DNA template strand under excitation of a laser of a particular wavelength, and the sequencing system is based on converting and/or collecting these optical signals and the sequence of addition of the different substrates, if any, to determine the DNA sequence. In one specific example, four substrates are provided with two fluorescent labels, two of the substrates are provided with one fluorescent label, the other two are provided with another fluorescent label, the first reagent of the first sequencing reaction is a reagent containing two substrates, the two substrates in the first reagent are provided with different fluorescent labels, the first reagent of the second sequencing reaction is a reagent containing two other substrates, the two substrates in the first reagent are also provided with different fluorescent labels, and one round of sequencing reaction comprises a first sequencing reaction and a second sequencing reaction, the second sequencing reaction is performed after the first sequencing reaction is completed, and the first sequencing reaction is performed after the second sequencing reaction is completed, and the steps are repeated. When the reaction substrate for the base extension of the second sequencing reaction is controlled to flow into the reaction device 40, it is necessary to cleave the luminescent group on the base extension reaction substrate (terminator) of the first sequencing reaction and then add the terminator for the base extension of the second sequencing reaction. For example, in connection with the above example, after the first reagent of the first sequencing reaction is input into the reaction device 40, the image acquisition may be performed on the reaction device 40. After the image acquisition is completed, the luminescent group of the first reagent of the first sequencing reaction is required to be excised, and then the first reagent of the second sequencing reaction is added.

In some embodiments, referring to fig. 2 and 3, the plurality of ports are distributed in a circle, and the common port 71 is disposed concentrically with the circle. Thus, the concentric arrangement of the plurality of ports and common ports 71 in a circular distribution with the circular shape ensures the accuracy of the communication grooves 72 with the corresponding ports and common ports 71 when rotating the rotor.

In some embodiments, referring to fig. 2 and 3, the communication slot 72 is linear. Thus, the flow path of the reagent liquid in the communication groove 72 can be reduced, and further, the rapid sequencing can be ensured. Specifically, the communication groove 72 having a linear shape can communicate the port located at both ends of the communication groove 72 and the common port 71 in a short path. In the present example, the line is a straight line.

In certain embodiments, the valve body assembly includes a first valve 30, the second port 2 is connected to the second reagent through the first valve 30, and the fourth port 4 is connected to the second reagent through the first valve 30. Therefore, the second reagent can be provided for different ports, the pipeline connection is reduced, the liquid path in the sequence determination system is simplified, the problem investigation and maintenance are facilitated, and the industrial production is facilitated.

In the embodiment of the present invention, the first valve 30 includes one total port and a plurality of split ports, and it is understood that the number of split ports of the first valve 30 is not less than the number of sequencing reactions, for example, when the number of sequencing reactions is 2, the number of split ports of the first valve 30 is not less than 2.

For example, in the present example, the first valve 30 includes one total port and four partial ports, that is, one first valve 30 may provide the second reagent to up to four sequencing reactions. Specifically, in the present example, the four ports of the first valve 30 are connected to the second port 2, the fourth port 4, the fourteenth port 14 and the sixteenth port 16, respectively, and the total port of the first valve 30 is connected to the second reagent. Thus, when the communication channel 72 communicates with the common port 71 and the corresponding port, the drive assembly 50 drives the second reagent through the first valve 30 and the rotary valve 70 into the reaction device 40.

In certain embodiments, the sequencing system comprises an imaging device, the method comprising the steps of: image acquisition is performed using an imaging device. In this manner, the sequencing system can facilitate the user's image acquisition of the sample within the reaction device 40.

In particular, the imaging device may comprise a light emitting device and a camera. Taking the first sequencing reaction and the second sequencing reaction as an example, when the first reagent of the first sequencing reaction is added into the reaction device 40, a light emitting device (such as a laser) can be used to emit excitation light to the reaction device 40 so as to excite the luminescent group to emit fluorescence, and a camera is used to take a photograph to collect the fluorescence, and an image is formed to perform the sequence determination. After photographing is completed, a second reagent is added to cut off the luminescent group in the reaction device 40, then a first reagent for a second sequencing reaction is added, and then image acquisition of the second sequencing reaction is performed.

In some embodiments, image acquisition is performed using a third reagent, the first set of ports including a first port 1, a fifth port 5, and a second port 2 arranged in sequence in a preset rotational direction, the second set of ports including a third port 3, a sixth port 6, and a fourth port 4 arranged in sequence in the preset rotational direction, the method comprising: after performing i) and before performing ii), communicating the fifth port 5 with the common port 71 using the drive assembly 50, allowing a third reagent to enter the reaction device 40 via the rotary valve 70 to effect image acquisition of the first sequencing reaction; after iii) and before iv), the sixth port 6 and the common port 71 are brought into communication using the drive assembly 50, and a third reagent is introduced into the reaction apparatus 40 via the rotary valve 70 to effect image acquisition of the second sequencing reaction.

The presence of the third reagent allows the sample within the reaction device 40 to be better imaged after the base extension reaction. Specifically, the third reagent can reduce the photobleaching effect and/or anti-quenching effect of the sample, and can enable the substrate with the luminous group contained in the first reagent to be combined to the template chain, so that the luminescence is more stable under the excitation of laser, and the collection of images, in particular the collection of single molecule micro signals, is facilitated.

Specifically, in certain embodiments, when the sequencing reaction includes a first sequencing reaction and a second sequencing reaction that are sequentially performed, referring to fig. 2 and 3, the first port 1, the fifth port 5, the second port 2, the third port 3, the sixth port 6, and the fourth port 4 are sequentially arranged in a preset rotation direction. During the base extension, image acquisition and excision of the sequencing reaction, the rotor is rotated in a predetermined rotational direction to sequentially complete the entry of different reagents into the reaction apparatus 40 through the rotary valve 70.

In certain embodiments, the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: base extension, first wash, image acquisition and excision. In this manner, the first reagent remaining in the communication channel 72 can be mostly carried away by the first wash, further reducing cross-contamination of the first reagent by different sequencing reactions.

In certain embodiments, the first wash is performed with a fourth reagent, the first set of ports comprising a first port 1, a seventh port 7, and a second port 2 arranged in sequence in a preset direction of rotation, the second set of ports comprising a third port 3, an eighth port 8, and a fourth port 4 arranged in sequence in the preset direction of rotation, the method comprising: after performing i) and before performing ii), communicating the seventh port 7 with the common port 71 using the drive assembly 50, allowing a fourth reagent to enter the reaction apparatus 40 via the rotary valve 70 to effect a first wash of the first sequencing reaction; after iii) and before iv) the eighth port 8 and the common port 71 are brought into communication by means of the drive assembly 50 and a fourth reagent is brought into the reaction device 40 via the rotary valve 70 to effect a first wash of the second sequencing reaction.

For the first sequencing reaction, when the rotor rotates in the preset rotation direction to switch the communication groove 72 from communicating with the first port 1 to communicating with the seventh port 7, most of the first reagent of the first sequencing reaction remains in the sealing surface area between the rotor and the stator 81, so that cross contamination of the first reagent of the first sequencing reaction and the first reagent of the second sequencing reaction is greatly reduced.

It should be noted that the fourth reagent is a cleaning agent that has no effect on the target sequencing reaction.

In certain embodiments, the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: base extension, first wash, second wash, image acquisition and excision. Thus, the first reagent remaining in the communication channel 72 can be mostly carried away by the first wash, further reducing cross-contamination of the first reagent by different sequencing reactions, while the second wash step is to add a buffer solution, which is a solution that maintains the pH of the liquid to some extent within a specific range, that is a weak acid, weak base, and/or neutral solution. In certain embodiments, the buffer is a solution that does not affect the sequencing reaction of interest.

In certain embodiments, the first wash with the fourth reagent and the second wash with the fifth reagent, the first set of ports comprising the first port 1, the seventh port 7, the ninth port 9, and the second port 2 arranged in sequence in a preset direction of rotation, the second set of ports comprising the third port 3, the eighth port 8, the tenth port 10, and the fourth port 4 arranged in sequence in the preset direction of rotation, the method comprising: after performing i) and before performing the second wash, communicating the seventh port 7 with the common port 71 using the drive assembly 50, allowing the fourth reagent to enter the reaction apparatus 40 via the rotary valve 70 to effect a first wash of the first sequencing reaction;

After performing the first wash and before performing ii), communicating the ninth port 9 with the common port 71 using the drive assembly 50, allowing the fifth reagent to enter the reaction apparatus 40 via the rotary valve 70 to effect a second wash of the first sequencing reaction;

after iii) and before the second wash is performed, the eighth port 8 and the common port 71 are brought into communication by the drive assembly 50, and a fourth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect a first wash of the second sequencing reaction;

after the first wash is performed and before iv) the tenth port 10 and the common port 71 are brought into communication using the drive assembly 50 and a fifth reagent is introduced into the reaction apparatus 40 via the rotary valve 70 to effect a second wash of a second sequencing reaction.

For the first sequencing reaction, when the rotor rotates in the preset rotation direction to switch the communication groove 72 from communicating with the first port 1 to communicating with the seventh port 7, most of the first reagent of the first sequencing reaction remains in the sealing surface area between the rotor and the stator 81, and further, when the rotor rotates in the preset rotation direction to switch the communication groove 72 from communicating with the seventh port 7 to communicating with the ninth port 9, the first reagent of the first sequencing reaction remains in the sealing surface area between the rotor and the stator 81, so that cross contamination of the first reagent of the first sequencing reaction and the first reagent of the second sequencing reaction is greatly reduced.

In certain embodiments, the fourth reagent is a wash that has no effect on the target sequencing reaction and the fifth reagent is a buffer that has no effect on the target sequencing reaction.

In certain embodiments, the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: base extension, image acquisition, excision and 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 thiol group may be protected from oxidation by capping, such as by the addition of an alkylating agent, by cleaving the cleavable luminescent group by light and/or chemical cleavage, and exposing the group to the thiol group.

In certain embodiments, capping with a sixth reagent, the first set of ports comprising a first port 1, a second port 2, and an eleventh port 11 arranged in sequence in a preset direction of rotation, the second set of ports comprising a third port 3, a fourth port 4, and a twelfth port 12 arranged in sequence in the preset direction of rotation, the method comprising: after performing ii) and before performing iii), the eleventh port 11 and the common port 71 are brought into communication with a drive assembly 50, allowing a sixth reagent to enter the reaction device 40 via a rotary valve 70 to effect capping of the first sequencing reaction; after iv) is performed, the twelfth port 12 and the common port 71 are brought into communication using the drive assembly 50 and the sixth reagent is passed into the reaction device 40 through the rotary valve 70 to effect capping of the second sequencing reaction.

In certain embodiments, the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, and a third sequencing reaction that are sequentially performed, the third sequencing reaction comprising steps that are different from the first sequencing reaction or from the first reagent of the second sequencing reaction, the plurality of ports further comprising a third set of ports corresponding to the third sequencing reaction, the third set of ports comprising a thirteenth port 13 and a fourteenth port 14 that are sequentially arranged in a predetermined rotational direction, the first set of ports, the second set of ports, and the third set of ports being sequentially arranged in the predetermined rotational direction, the thirteenth port 13 being coupled to the first reagent of the third sequencing reaction, the fourteenth port 14 being coupled to the second reagent, the method further comprising the steps of:

v) after iv) the thirteenth port 13 and the common port 71 are brought into communication by the drive assembly 50, allowing the first reagent of the third sequencing reaction to enter the reaction device 40 via the rotary valve 70 to effect base extension of the third sequencing reaction;

vi) connecting the fourteenth port 14 to the common port 71 using the drive assembly 50, allowing the second reagent to enter the reaction apparatus 40 through the rotary valve 70 to effect excision of the third sequencing reaction.

Thus, the method for controlling the sequencing reaction is made more efficient. At the same time, cross-contamination of the first reagent of the second sequencing reaction and the first reagent of the third sequencing reaction can also be greatly reduced.

Specifically, in some embodiments, referring to fig. 5, the first port 1 is adjacent to the second port 2, the second port 2 is adjacent to the third port 3, the third port 3 is adjacent to the fourth port 4, the fourth port 4 is adjacent to the thirteenth port 13, and the thirteenth port 13 is adjacent to the fourteenth port 14, so that the rotation stroke of the rotor is short, which is convenient for rapid sequencing. It will be appreciated that in other embodiments, the first port 1, the second port 2, the third port 3, the fourth port 4, the thirteenth port 13 and the fourteenth port 14 may be other ports of a plurality of ports, so long as the first port 1, the second port 2, the third port 3, the fourth port 4, the thirteenth port 13 and the fourteenth port 14 are arranged in order according to the preset rotation direction of the rotor.

During the base extension, image acquisition and excision of the sequencing reaction, the rotor is rotated in a predetermined rotational direction of the rotor to sequentially complete the entry of different reagents into the reaction apparatus 40 through the rotary valve 70.

In certain embodiments, when the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, and a third sequencing reaction that are sequentially performed, the third set of ports comprises a thirteenth port 13, a seventeenth port 17, and a fourteenth port 14 that are sequentially arranged in a preset rotational direction, and referring to fig. 2 and 3, the first port 1, the fifth port 5, the second port 2, the third port 3, the sixth port 6, the fourth port 4, the thirteenth port 13, the seventeenth port 17, and the fourteenth port 14 are sequentially arranged in the preset rotational direction. Upon base extension, image acquisition and excision of the sequencing reaction, the rotor is rotated in a clockwise direction to sequentially complete the entry of the different reagents into the reaction device 40 through the rotary valve 70. Seventeenth port 17 is connected to a third reagent.

In certain embodiments, the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction that are sequentially performed, the third sequencing reaction and the fourth sequencing reaction comprise steps that are all identical to the first sequencing reaction or are all identical to the second sequencing reaction, the first reagents of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction, and the fourth sequencing reaction are all different, the plurality of ports further comprises a third set of ports corresponding to the third sequencing reaction and a fourth set of ports corresponding to the fourth sequencing reaction, the third set of ports comprises a thirteenth port 13 and a fourteenth port 14 that are sequentially arranged in a preset rotation direction, the fourth set of ports comprises a fifteenth port 15 and a sixteenth port 16 that are sequentially arranged in the preset rotation direction, the first set of ports, the second set of ports, the third set of ports, and the fourth set of ports are sequentially arranged in the preset rotation direction, the thirteenth port 13 is connected to a first reagent of the third sequencing reaction, the fourteenth port 14 is connected to a second reagent, the fifteenth port 15 is connected to a sixteenth reagent of the fourth sequencing reaction, and the method further comprises the steps of:

vi) communicating the fourteenth port 14 with the common port 71 using the drive assembly 50 to allow the second reagent to enter the reaction apparatus 40 through the rotary valve 70 to effect excision of the third sequencing reaction;

vii) communicating the fifteenth port 15 with the common port 71 using the drive assembly 50 to allow the first reagent of the fourth sequencing reaction to enter the reaction device 40 via the rotary valve 70 to effect base extension of the fourth sequencing reaction;

viii) the sixteenth port 16 and the common port 71 are brought into communication by means of the drive assembly 50, and the second reagent is brought into the reaction device 40 via the rotary valve 70 to effect excision of the fourth sequencing reaction.

Thus, the variety of sequencing reactions can be increased, making the method for controlling the sequencing reactions more efficient. At the same time, cross-contamination of the first reagent of the second sequencing reaction and the first reagent of the third sequencing reaction and cross-contamination of the first reagent of the third sequencing reaction and the first reagent of the fourth sequencing reaction can be greatly reduced.

Specifically, in certain embodiments, referring to fig. 6, the first port 1 and the second port 2 are adjacent, the second port 2 and the third port 3 are adjacent, the third port 3 and the fourth port 4 are adjacent, the fourth port 4 and the thirteenth port 13 are adjacent, the thirteenth port 13 and the fourteenth port 14 are adjacent, the fourteenth port 14 and the fifteenth port 15 are adjacent, and the fifteenth port 15 and the sixteenth port 16 are adjacent, so that the stroke of the rotor rotation is short, thereby facilitating rapid sequencing. It will be appreciated that in other embodiments, the first port 1, the second port 2, the third port 3, the fourth port 4, the thirteenth port 13, the fourteenth port 14, the fifteenth port 15 and the sixteenth port 16 may be other ports of a plurality of ports, so long as it is ensured that the first port 1, the second port 2, the third port 3, the fourth port 4, the thirteenth port 13, the fourteenth port 14, the fifteenth port 15 and the sixteenth port 16 are sequentially arranged in a preset rotation direction of the rotor.

In a specific example, the four reaction substrates are all provided with the same fluorescent label, the first reagent of the first, second, third and fourth sequencing reactions are reagents containing one substrate respectively, one round of sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, a third sequencing reaction and a fourth sequencing reaction, the second sequencing reaction is carried out after the first sequencing reaction is completed, the third sequencing reaction is carried out after the second sequencing reaction is completed, the fourth sequencing reaction is carried out after the third sequencing reaction is completed, and the first sequencing reaction is carried out after the fourth sequencing reaction is completed, so that the process is repeated.

The method ensures that the rotary valve always rotates according to one rotary direction in the sequencing reaction process through simple liquid path structural design and control, can basically avoid reagent cross mixing between the sequencing reactions at intervals, such as reagent cross mixing between the first sequencing reaction and the third sequencing reaction, the second sequencing reaction and the fourth sequencing reaction, and can greatly reduce reagent cross mixing between the adjacent sequencing reactions, such as reagent cross mixing between the first sequencing reaction and the second sequencing reaction, the third sequencing reaction, the fourth sequencing reaction and the first sequencing reaction.

In certain embodiments, when the sequencing reaction includes a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction that are sequentially performed, the third group of ports includes a thirteenth port 13, a seventeenth port 17, and a fourteenth port 14 that are sequentially arranged in a preset rotation direction, and the fourth group of ports includes a fifteenth port 15, an eighteenth port 18, and a sixteenth port 16 that are sequentially arranged in the preset rotation direction, referring to fig. 2 and 3, the first port 1, the fifth port 5, the second port 2, the third port 3, the sixth port 6, the fourth port 4, the thirteenth port 13, the seventeenth port 17, the fourteenth port 14, the fifteenth port 15, the eighteenth port 18, and the sixteenth port 16 are sequentially arranged in the preset rotation direction. Upon base extension, image acquisition and excision of the sequencing reaction, the rotor is rotated in a clockwise direction to sequentially complete the entry of the different reagents into the reaction device 40 through the rotary valve 70. Seventeenth port 17 is connected to the third reagent and eighteenth port 18 is connected to the third reagent.

In certain embodiments, referring to fig. 2, the valve body assembly 29 comprises two rotary valves 70 and two first valves 30, the reaction apparatus 40 comprises a first unit 41 and a second unit 42, the first unit 41 is connected to the common port 71 of one of the rotary valves 70, the second unit 42 is connected to the common port 71 of the other rotary valve 70, the 29 valve body assembly 29 comprises a second valve 35, a third valve 36 and a fourth valve 37, the second valve 35 is connected to the two rotary valves 70 and the first reagent of the first sequencing reaction, the third valve 36 is connected to the two rotary valves 70 and the first reagent of the second sequencing reaction, the fourth valve 37 is connected to the second reagent and the two first valves 30, and each first valve 30 is connected to the second port 2 and the fourth port 4 of one rotary valve 70.

In this way, different types of sequencing reactions can be performed in the channels of the first unit 41 and the channels of the second unit 42, respectively, and 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, unsynchronized, and do not affect each other, thereby shortening the time of the sequencing reactions. For example, when it is desired to base extend a first sequencing reaction on a sample on first unit 41, fluidic device 100 will deliver the first reagent of the first sequencing reaction for the reaction to first unit 41, at which time the same reagent will not be allowed to enter second unit 42 and vice versa.

Specifically, in the embodiment of the present invention, the first reagent of the first sequencing reaction is delivered to the two first ports 1 of the two rotary valves 70 through the second valve 35, the first reagent of the second sequencing reaction is delivered to the two third ports 3 of the two rotary valves 70 through the third valve 36, and the second reagent is delivered to the two first valves 30 through the fourth valve 37.

In some examples, the second valve 35, the third valve 36, and the fourth valve 37 are all three-way valves.

In the embodiment shown in fig. 2, the sequencing reactions comprise a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction that are performed sequentially, each sequencing reaction comprising the steps of, in order: base extension, first wash, second wash, image acquisition, excision and capping.

Corresponding to the first sequencing reaction, the first set of ports comprises a first port 1, a seventh port 7, a ninth port 9, a fifth port 5, a second port 2 and an eleventh port 11 along the preset rotation direction of the rotor. Corresponding to the second sequencing reaction, the second set of ports comprises a third port 3, an eighth port 8, a tenth port 10, a sixth port 6, a fourth port 4 and a twelfth port 12 along the preset rotation direction of the rotor. Corresponding to the third sequencing reaction, the third set of ports comprises a thirteenth port 13, a nineteenth port 19, a twentieth port 20, a seventeenth port 17, a fourteenth port 14, a twenty first port 21 along the preset rotational direction of the rotor. Corresponding to the fourth sequencing reaction, the fourth set of ports comprises a fifteenth port 15, a twenty-second port 22, a twenty-third port 23, an eighteenth port 18, a sixteenth port 16 and a twenty-fourth port 24 along the rotor preset rotation direction.

The valve body assembly 29 further includes a sixth valve 51, a seventh valve 52, an eighth valve 53, a ninth valve 54, a tenth valve 55, and an eleventh valve 56, the number of the first valves 30 being 10, the sixth valve 51 connecting the first reagent of the third sequencing reaction and the two thirteenth ports 13 of the two rotary valves 70, the seventh valve 52 connecting the first reagent of the fourth sequencing reaction and the two fifteenth ports 15 of the two rotary valves 70. The eighth valve 53 connects the third reagent and the two first valves 30, the ninth valve 54 connects the sixth reagent and the two first valves 30, the tenth valve 55 connects the fourth reagent and the two first valves 30, and the eleventh valve 56 connects the fifth reagent and the two first valves 30.

The second valve 35, the third valve 36, the fourth valve 37, the sixth valve 51, the seventh valve 52, the eighth valve 53, the ninth valve 54, the tenth valve 55 and the eleventh valve 56 each comprise one total port and two sub-ports.

The seventh port 7, the eighth port 8, the nineteenth port 19 and the twenty second port 22 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to one branch of the tenth valve 55, and the total port of the tenth valve 55 is connected to the fourth reagent.

The ninth port 9, tenth port 10, twentieth port 20 and twentieth port 23 are connected to 4 branches of the same first valve 30, the total port of the same first valve 30 being connected to one branch of the eleventh valve 56, the total port of the eleventh valve 56 being connected to the fifth reagent.

The fifth port 5, the sixth port 6, the seventeenth port 17 and the eighteenth port 18 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to one branch of the eighth valve 53, and the total port of the eighth valve 53 is connected to the third reagent.

The second port 2, the fourth port 4, the fourteenth port 14 and the sixteenth port 16 are connected to 4 branches of the same first valve 30, the total port of the same first valve 30 is connected to one branch of the fourth valve 37, and the total port of the fourth valve 37 is connected to the second reagent.

The eleventh port 11, the twelfth port 12, the twenty-first port 21 and the twenty-fourth port 24 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to one branch of the ninth valve 54, and the total port of the ninth valve 54 is connected to the sixth reagent.

The sixth valve 51, seventh valve 52, eighth valve 53, ninth valve 54, tenth valve 55 and eleventh valve 56 are three-way valves, the first valve 30 comprises a total port and 4 split ports, and the rotary valve 70 is a 28-port rotary valve. Thus, in the embodiment shown in fig. 2, the number of three-way valves is 9, and the number of first valves 30 is 10.

The reaction device 40 includes a first unit 41 and a second unit 42. The first unit 41 is connected to the common port 71 of one rotary valve 70 and the second unit 42 is connected to the common port 71 of the other rotary valve 70.

The driving assembly 50 includes eight pump banks, 4 of which are connected to the first unit 41 and the other 4 of which are connected to the second unit 42 to generate negative pressure in the passages of the first unit 41 and the second unit 42, respectively. The eight-row pump can save the installation space of the instrument and the cost, reduce the use of electromagnetic valves and reduce the failure rate. The eight-row pump can be an eight-row Thomas pump, the eight-row Thomas pump has smaller noise and smaller vibration, and rapid sequence determination reaction is realized.

After the first reagent required for base extension of each sequencing reaction is pumped in, the rotor of the rotary valve 70 will perform the first wash, second wash, third reagent addition, excision (clean) and capping (cap) processes in a predetermined rotational direction (clockwise as shown in FIG. 2), and the residual portion of the terminator of the first reagent in the rotary valve 70 is confined to the sealing surface area between the rotor and the stator 81, minimizing cross-contamination of the first reagent for different sequencing reactions.

In certain embodiments, the first reagent of the first sequencing reaction provides a base a, the first reagent of the second sequencing reaction provides a base G, the first reagent of the third sequencing reaction provides a base C, and the first reagent of the fourth sequencing reaction provides a base T. The second reagent is a reagent Cl for excision, the third reagent is a reagent I for image acquisition, the fourth reagent is a reagent R for first washing, the fifth reagent is a reagent B for second washing, and the sixth reagent is a reagent Ca for capping.

It will be appreciated that in other embodiments, the bases provided by the first, second, third and fourth reagents of the first, second, third and fourth sequencing reactions may be altered, e.g., the base provided by the first reagent of the first sequencing reaction is T, the base provided by the first reagent of the second sequencing reaction is G, the base provided by the first reagent of the third sequencing reaction is C, the base provided by the first reagent of the fourth sequencing reaction is A, etc.

Preferably, the first, second, third and sixth reagents A, T, C, G, third and sixth reagents can be placed in a refrigerated environment and the fourth and fifth reagents can be placed in a room temperature environment.

In certain embodiments, referring to fig. 3, valve body assembly 29 includes a fifth valve 38, reaction apparatus 40 includes a first unit 41 and a second unit 42, and fifth valve 38 connects common port 71 to first unit 41, and connects common port 71 to second unit 42.

In this way, different types of sequencing reactions can be performed in the channels of the first unit 41 and the channels of the second unit 42, respectively, and 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, unsynchronized, and do not affect each other, thereby shortening the time of the sequencing reactions. For example, where it is desired to base extend a first sequencing reaction on a sample on first unit 41, fluidic device 100 will deliver to first unit 41 a first reagent for the first sequencing reaction, at which time the same reagent will not be allowed to enter second unit 42 and vice versa.

Specifically, the fifth valve 38 may selectively communicate the common port 71 with the first unit 41, or communicate the common port 71 with the second unit 42, and when the fifth valve 38 communicates the common port 71 with the first unit 41, reagents may enter the first unit 41 through the rotary valve 70 and the fifth valve 38 to perform a sequencing reaction under the driving of the driving assembly 50, and at this time, the common port 71 is not in communication with the second unit 42, and reagents may not enter the second unit 42. When the fifth valve 38 is in communication with the common port 71 and the second unit 42, reagents may be driven by the drive assembly 50 through the rotary valve 70 and the fifth valve 38 into the second unit 42 for a sequencing reaction, at which time the common port 71 is not in communication with the first unit 41 and reagents do not enter the first unit 41.

In an embodiment of the present invention, the fifth valve 38 is a three-way rotary valve.

In the embodiment shown in fig. 3, the sequencing reactions comprise a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction that are performed sequentially, each sequencing reaction comprising the steps of, in order: base extension, first wash, second wash, image acquisition, excision and capping. Corresponding to the first sequencing reaction, the first set of ports comprises a first port 1, a seventh port 7, a ninth port 9, a fifth port 5, a second port 2 and an eleventh port 11 along the preset rotation direction of the rotor. Corresponding to the second sequencing reaction, the second set of ports comprises a third port 3, an eighth port 8, a tenth port 10, a sixth port 6, a fourth port 4 and a twelfth port 12 along the preset rotation direction of the rotor. Corresponding to the third sequencing reaction, the third set of ports comprises a thirteenth port 13, a nineteenth port 19, a twentieth port 20, a seventeenth port 17, a fourteenth port 14, a twenty first port 21 along the preset rotational direction of the rotor. Corresponding to the fourth sequencing reaction, the fourth set of ports comprises a fifteenth port 15, a twenty-second port 22, a twenty-third port 23, an eighteenth port 18, a sixteenth port 16 and a twenty-fourth port 24 along the rotor preset rotation direction.

The valve body assembly 29 includes 5 first valves 30, the first valves 30 including one total port and 4 split ports. The rotary valve 70 is a 28-port rotary valve.

The seventh port 7, the eighth port 8, the nineteenth port 19 and the twenty second port 22 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to the fourth reagent.

The ninth port 9, tenth port 10, twentieth port 20 and twentieth port 23 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to the fifth reagent.

The fifth port 5, sixth port 6, seventeenth port 17 and eighteenth port 18 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to the third reagent.

The second port 2, the fourth port 4, the fourteenth port 14 and the sixteenth port 16 are connected to 4 branches of the same first valve 30, and the total port of the same first valve 30 is connected to the second reagent.

The eleventh port 11, the twelfth port 12, the twenty-first port 21 and the twenty-fourth port 24 are connected to 4 split ports of the same first valve 30, and the total port of the same first valve 30 is connected to the sixth reagent.

The reaction device 40 includes a first unit 41 and a second unit 42. The first unit 41 is connected to one outlet of the fifth valve 38 and the second unit 42 is connected to the other outlet of the fifth valve 38.

After the first reagent required for base extension of each sequencing reaction is pumped in, the rotor of the rotary valve 70 will perform the first wash, second wash, third reagent addition, excision (clean) and capping (cap) processes in a predetermined rotational direction (clockwise as shown in FIG. 3), and the residual portion of the terminator of the first reagent in the rotary valve 70 is confined to the sealing surface area between the rotor and the stator 81, minimizing cross-contamination of the first reagent for different sequencing reactions.

In an embodiment of the invention, a three-way rotary valve is used to effect switching of the two units. Compared with a two-position three-way electromagnetic valve, the dead volume inside the three-way rotary valve is almost zero, and the cross contamination of reagents between two units is not needed to be worried about. The embodiment of the invention can avoid the use of the three-way valve, reduce the number of joint connection and reduce the cost.

It should be noted that in fig. 2 and 3, the piping lines of some valves to some ports and the piping lines of some valves to some reagents are not shown in order to avoid too many lines in the drawings to make the drawings unclear. It will be appreciated by those skilled in the art that the connection of the conduit wiring described above will be apparent from the above explanation of embodiments of the present invention.

Referring to fig. 7, a sequencing system 300 according to an embodiment of the present invention controls a sequencing reaction, the sequencing reaction being performed on a reaction device 40, the sequencing reaction comprising a first sequencing reaction and a second sequencing reaction that are sequentially performed, the first sequencing reaction and the second sequencing reaction each comprising the steps of, in order: base extension, image acquisition and excision,

the base extension is performed using a first reagent, the first reagent of the first sequencing reaction and the second sequencing reaction being different,

by means of the fact that the said excision is performed,

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 29 and a drive assembly 50,

The valve body assembly 29 comprises a rotary valve 70, the rotary valve 70 comprises a stator 81 and a rotor which can be communicated, the rotary valve 70 is provided with a common port 71, the stator 81 is provided with a plurality of ports, the rotor is provided with a communication groove 72, the common port 71 and one port can be communicated through the communication groove 72 by rotating the rotor, the plurality of ports comprise a first group of ports and a second group of ports which respectively correspond to a first sequencing reaction and a second sequencing reaction, the first group of ports comprise a first port 1 and a second port 2, the first port 1 is connected with a first reagent of the first sequencing reaction, the second port 2 is connected with a second reagent, the second group of ports comprise a third port 3 and a fourth port 4, the first port 1, the second port 2, the third port 3 and the fourth port 4 are sequentially arranged according to the preset rotation direction of the rotor, the third port 3 is connected with the first reagent of the second sequencing reaction, the fourth port 4 is connected with the second reagent, the common port 71 is connected with the reaction device 40, and the control device 302 is used for:

i) Communicating the first port 1 with the common port 71 using the drive assembly 50, allowing a first reagent of a first sequencing reaction to enter the reaction device 40 through the rotary valve 70 to effect base extension of the first sequencing reaction;

iii) Communicating the third port 3 with the common port 71 using the drive assembly 50 to allow the first reagent of the second sequencing reaction to enter the reaction device 40 through the rotary valve 70 to effect base extension of the second sequencing reaction;

In the system 300 described above, by rotating the communication groove 72 in one direction of the rotor to communicate the first port 1 to input the first reagent of the first sequencing reaction, and then rotating the switch to communicate the communication groove 72 to the second port 2 to input the second reagent, before the first reagent of the second sequencing reaction is caused to enter the reaction device 40 through the rotary valve 70, it is possible to replace all or most of the first reagent of the first sequencing reaction in the communication groove 72 and in the communication groove 72 that is brought between the rotor and the stator 81 by the second reagent, thereby greatly reducing the first reagent of the first sequencing reaction from being brought into the second sequencing reaction, and also avoiding the first reagent of the second sequencing reaction from being brought into the first reagent of the first sequencing reaction, and avoiding or greatly reducing cross contamination between different first reagents. The system 300 is particularly useful for sequencing reactions requiring control of sequential addition of different types of substrates or combinations of different substrates in each round, and the system 300 relies on a simple apparatus configuration and control of the liquid in and out sequence to greatly reduce contamination between different types of substrates or combinations of different substrates.

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, fluid device 100 includes a fluid control unit to which control device 302 is coupled, the fluid control unit electrically connecting valve body assembly 29 and drive assembly 50 to control operation of valve body assembly 29 and drive assembly 50.

Specifically, the fluid control unit may receive control signals from control device 302 and control valve body assembly 29, drive assembly 50, and other components of fluid device 100 based on the control signals. In this way, a part of the functions of the control device 302 can be performed by the fluid control unit, and the load of the control device 302 can be reduced. The fluid control unit may be a device including a single-chip microcomputer, a computer processor, or a central control processor, so as to implement automatic operation of the fluid device 100 and improve efficiency.

In some embodiments, the fluid control unit and control device 302 may be integrated into one component, module, or device to increase the integrity of the sequencing system 300 and reduce cost.

In certain embodiments, control device 302 includes a unit to control the fluid device that electrically connects valve body assembly 29 and drive assembly 50 to control the operation of valve body assembly 29 and drive assembly 50.

Specifically, the unit controlling the fluid device may receive an externally input control signal and control the valve body assembly 29, the drive assembly 50, and other components of the fluid device 100 according to the control signal. In this way, part of the functions of the control device 302 can be performed by the units controlling the fluid device, reducing interference between the units of the control device 302. The unit for controlling the fluid device may be a device including a single-chip microcomputer, a computer processor, or a central control processor, so that the fluid device 100 can automatically operate, and efficiency is improved.

In some embodiments, the control device 302 may also include other units, for example, the control device 302 includes a unit that controls the imaging device.

In certain embodiments, the valve body assembly 29 comprises a first valve through which the second port 2 is connected to the second reagent and the fourth port 4 is connected to the second reagent.

In some embodiments, the sequencing system 300 includes the imaging device 200, and the control device 302 is coupled to the imaging device 200, and the control device 302 is configured to perform image acquisition using the imaging device 200.

In some embodiments, the image capturing is performed using a third reagent, the first set of ports includes a first port 1, a fifth port 5, and a second port 2 sequentially arranged in a preset rotation direction, and the second set of ports includes a third port 3, a sixth port 6, and a fourth port 4 sequentially arranged in the preset rotation direction, and the control device 302 is configured to: after performing i) and before performing ii), communicating the fifth port 5 with the common port 71 using the drive assembly 50, allowing a third reagent to enter the reaction device 40 via the rotary valve 70 to effect image acquisition of the first sequencing reaction; after iii) and before iv), the sixth port 6 and the common port 71 are brought into communication using the drive assembly 50, and a third reagent is introduced into the reaction apparatus 40 via the rotary valve 70 to effect image acquisition of the second sequencing reaction.

In certain embodiments, the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: base extension, first wash, image acquisition and excision.

In certain embodiments, the first wash is performed with the fourth reagent, the first set of ports comprising a first port 1, a seventh port 7 and a second port 2 arranged in sequence in a preset direction of rotation, the second set of ports comprising a third port 3, an eighth port 8 and a fourth port 4 arranged in sequence in the preset direction of rotation, the control means 302 being adapted to: after performing i) and before performing ii), communicating the seventh port 7 with the common port 71 using the drive assembly 50, allowing a fourth reagent to enter the reaction apparatus 40 via the rotary valve 70 to effect a first wash of the first sequencing reaction; after iii) and before iv) the eighth port 8 and the common port 71 are brought into communication by means of the drive assembly 50 and a fourth reagent is brought into the reaction device 40 via the rotary valve 70 to effect a first wash of the second sequencing reaction.

In certain embodiments, the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: base extension, first wash, second wash, image acquisition and excision.

In some embodiments, the first wash is performed with a fourth reagent and the second wash is performed with a fifth reagent, the first set of ports comprising a first port 1, a seventh port 7, a ninth port 9 and a second port 2 arranged in sequence in a preset direction of rotation, the second set of ports comprising a third port 3, an eighth port 8, a tenth port 10 and a fourth port 4 arranged in sequence in the preset direction of rotation, the control means 302 being adapted to: after performing i) and before performing the second wash, communicating the seventh port 7 with the common port 71 using the drive assembly 50, allowing the fourth reagent to enter the reaction apparatus 40 via the rotary valve 70 to effect a first wash of the first sequencing reaction;

In certain embodiments, capping with a sixth reagent, the first set of ports comprising a first port 1, a second port 2 and an eleventh port 11 arranged in sequence in a preset direction of rotation, the second set of ports comprising a third port 3, a fourth port 4 and a twelfth port 12 arranged in sequence in the preset direction of rotation, the control means 302 being adapted to comprise: after performing ii) and before performing iii), the eleventh port 11 and the common port 71 are brought into communication with a drive assembly 50, allowing a sixth reagent to enter the reaction device 40 via a rotary valve 70 to effect capping of the first sequencing reaction; after iv) is performed, the twelfth port 12 and the common port 71 are brought into communication using the drive assembly 50 and the sixth reagent is passed into the reaction device 40 through the rotary valve 70 to effect capping of the second sequencing reaction.

In certain embodiments, the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, and a third sequencing reaction that are sequentially performed, the third sequencing reaction comprising steps that are either the same as the first sequencing reaction or the second sequencing reaction,

the first reagents of the first sequencing reaction, the second sequencing reaction and the third sequencing reaction are all different, the plurality of ports further comprise a third group of ports corresponding to the third sequencing reaction, the third group of ports comprises a thirteenth port 13 and a fourteenth port 14 which are sequentially arranged according to a preset rotation direction, the first group of ports, the second group of ports and the third group of ports are sequentially arranged according to the preset rotation direction, the thirteenth port 13 is connected with the first reagent of the third sequencing reaction, the fourteenth port 14 is connected with the second reagent, and the control device 302 is used for:

In certain embodiments, the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, a third sequencing reaction and a fourth sequencing reaction which are sequentially performed, the third sequencing reaction and the fourth sequencing reaction comprise steps which are identical to the first sequencing reaction or the second sequencing reaction, the first reagents of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction and the fourth sequencing reaction are different, the plurality of ports further comprises a third group of ports corresponding to the third sequencing reaction and a fourth group of ports corresponding to the fourth sequencing reaction, the third group of ports comprises a thirteenth port 13 and a fourteenth port 14 which are sequentially arranged according to a preset rotation direction, the fourth group of ports comprises a fifteenth port 15 and a sixteenth port 16 which are sequentially arranged according to the preset rotation direction, the first group of ports, the second group of ports, the third group of ports and the fourth group of ports are sequentially arranged according to the preset rotation direction, the thirteenth port 13 is connected with a first reagent of the third sequencing reaction, the fourteenth port 14 is connected with a second reagent, the fifteenth port 15 is connected with a fourth reagent of the fourth sequencing reaction, the sixteenth port 16 is connected with a fourth reagent for controlling the device 302:

In certain embodiments, the valve body assembly 29 comprises two rotary valves 70 and two first valves, the reaction apparatus 40 comprises a first unit 41 and a second unit 42, the first unit 41 is connected to the common port 71 of one of the rotary valves 70, the second unit 42 is connected to the common port 71 of the other rotary valve 70, the valve body assembly 29 comprises a second valve 35, a third valve 36 and a fourth valve 37, the second valve 35 is connected to the two rotary valves 70 and the first reagent of the first sequencing reaction, the third valve 36 is connected to the two rotary valves 70 and the first reagent of the second sequencing reaction, the fourth valve 37 is connected to the second reagent and the two first valves, and each first valve is connected to the second port 2 and the fourth port 4 of one rotary valve 70.

In certain embodiments, valve body assembly 29 includes a fifth valve 38, reaction device 40 includes a first unit 41 and a second unit 42, and fifth valve 38 connects common port 71, first unit 41, and second unit 42.

Referring to fig. 7, 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 of controlling a sequencing reaction, wherein the sequencing reaction is performed on a reaction device, the sequencing reaction is controlled by a sequencing system, the sequencing reaction comprises a first sequencing reaction and a second sequencing reaction that are performed sequentially, and the first sequencing reaction and the second sequencing reaction each comprise the steps performed sequentially: base extension, image acquisition and excision,

the excision is performed with a second agent,

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

the valve body assembly comprises a rotary valve, the rotary valve comprises a stator and a rotor which can be communicated, the rotary 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 one port can be communicated through the communication groove by rotating the rotor, the plurality of ports comprise a first group of ports and a second group of ports which respectively correspond to the first sequencing reaction and the second sequencing reaction, the first group of ports comprise a first port and a second port, the first port is connected with a first reagent of the first sequencing reaction, the second port is connected with a second reagent, the second group of ports comprise a third port and a fourth port, the first port, the second port, the third port and the fourth port are sequentially arranged according to the preset rotation direction of the rotor, the third port is connected with a first reagent of the second sequencing reaction, the fourth port is connected with a second reagent, and the common port is connected with the reaction device comprises the steps of:

i) Communicating the first port with the common port using the drive assembly to allow a first reagent of the first sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the first sequencing reaction;

ii) communicating the second port with the common port using the drive assembly, passing the second reagent through the rotary valve into the reaction device to effect excision of the first sequencing reaction;

iii) Communicating the third port with the common port using the drive assembly to allow the first reagent of the second sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the second sequencing reaction;

iv) communicating the fourth port with the common port using the drive assembly to pass the second reagent through the rotary valve into the reaction device to effect excision of the second sequencing reaction.

2. The method of claim 1, wherein the valve body assembly comprises a first valve, the second port is connected to the second reagent through the first valve, and the fourth port is connected to the second reagent through the first valve.

3. The method of claim 1, wherein the sequencing system comprises an imaging device, the method comprising the steps of: and carrying out image acquisition by using the imaging device.

4. A method according to claim 1 or 3, wherein the image acquisition is performed with a third reagent, the first set of ports comprising the first port, the fifth port and the second port arranged in sequence in the preset rotational direction, the second set of ports comprising the third port, the sixth port and the fourth port arranged in sequence in the preset rotational direction, the method comprising: after performing i) and before performing ii), communicating the fifth port and the common port with the drive assembly, passing the third reagent into the reaction device via the rotary valve to effect the image acquisition of the first sequencing reaction; after performing iii) and before performing iv), communicating the sixth port and the common port with the drive assembly, allowing the third reagent to enter the reaction device through the rotary valve to effect the image acquisition of the second sequencing reaction.

5. The method of claim 1 or 4, wherein the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: the base extension, first wash, the image acquisition and the excision.

6. The method of claim 5, wherein the first wash is performed with a fourth reagent, the first set of ports comprising the first port, a seventh port, and the second port sequentially arranged in the preset rotational direction, the second set of ports comprising the third port, an eighth port, and the fourth port sequentially arranged in the preset rotational direction, the method comprising: after performing i) and before performing ii), communicating the seventh port and the common port with the drive assembly, passing the fourth reagent into the reaction device through the rotary valve to effect the first wash of the first sequencing reaction; after performing iii) and before performing iv), communicating the eighth port and the common port with the drive assembly, passing the fourth reagent through the rotary valve into the reaction device to effect the first wash of the second sequencing reaction.

7. The method of claim 1, wherein the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: the base extension, first wash, second wash, the image acquisition and the excision.

8. The method of claim 7, wherein the first wash is performed with a fourth reagent and the second wash is performed with a fifth reagent, the first set of ports including the first port, seventh port, ninth port, and the second port sequentially arranged in the preset rotational direction, the second set of ports including the third port, eighth port, tenth port, and the fourth port sequentially arranged in the preset rotational direction, the method comprising: communicating the seventh port with the common port using the drive assembly after performing i) and before performing the second wash, passing the fourth reagent into the reaction apparatus via the rotary valve to effect the first wash of the first sequencing reaction;

communicating the ninth port with the common port using the drive assembly after the first wash is performed and before ii) passing the fifth reagent through the rotary valve into the reaction apparatus to effect the second wash of the first sequencing reaction;

After performing iii) and before performing the second wash, communicating the eighth port and the common port with the drive assembly, passing the fourth reagent through the rotary valve into the reaction device to effect the first wash of the second sequencing reaction;

after the first washing is performed and before iv) is performed, the tenth port and the common port are communicated using the drive assembly, and the fifth reagent is passed into the reaction device through the rotary valve to effect the second washing of the second sequencing reaction.

9. The method of claim 1, wherein the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: the base extension, the image acquisition, the excision and capping.

10. The method of claim 9, wherein the capping is performed with a sixth reagent, the first set of ports comprising the first port, the second port, and an eleventh port sequentially aligned in the preset rotational direction, the second set of ports comprising the third port, the fourth port, and a twelfth port sequentially aligned in the preset rotational direction, the method comprising: after performing ii) and before performing iii), communicating the eleventh port and the common port with the drive assembly, passing the sixth reagent through the rotary valve into the reaction device to effect the capping of the first sequencing reaction; after iv) is performed, the twelfth port and the common port are brought into communication using the drive assembly, and the sixth reagent is passed into the reaction device via the rotary valve to effect the capping of the second sequencing reaction.

11. The method of any one of claim 1 to 10, wherein the sequencing reaction comprises the first sequencing reaction, the second sequencing reaction and a third sequencing reaction that are performed sequentially, the third sequencing reaction comprising the steps of reacting with the first sequencing reaction or with the second sequencing reaction,

the first reagents of the first sequencing reaction, the second sequencing reaction and the third sequencing reaction are all different, the plurality of ports further comprises a third group of ports corresponding to the third sequencing reaction, the third group of ports comprises a thirteenth port and a fourteenth port which are sequentially arranged according to the preset rotation direction, the first group of ports, the second group of ports and the third group of ports are sequentially arranged according to the preset rotation direction, the thirteenth port is connected with the first reagent of the third sequencing reaction, and the fourteenth port is connected with the second reagent, and the method further comprises the steps of:

v) after iv) communicating the thirteenth port with the common port using the drive assembly, passing a first reagent of the third sequencing reaction through the rotary valve into the reaction device to effect base extension of the third sequencing reaction;

vi) communicating the fourteenth port with the common port using the drive assembly, allowing the second reagent to enter the reaction apparatus through the rotary valve to effect excision of the third sequencing reaction.

12. The method of any one of claims 1-10, wherein the sequencing reaction comprises the first sequencing reaction, the second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction that are sequentially performed, wherein the third sequencing reaction and the fourth sequencing reaction comprise steps that are either the same as the first sequencing reaction or the second sequencing reaction, wherein the first reagents of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction, and the fourth sequencing reaction are different, wherein the plurality of ports further comprises a third set of ports that correspond to the third sequencing reaction and a fourth set of ports that correspond to the fourth sequencing reaction, the third group of ports includes thirteenth and fourteenth ports sequentially arranged in the preset rotation direction, the fourth group of ports includes fifteenth and sixteenth ports sequentially arranged in the preset rotation direction, the first, second, third and fourth groups of ports sequentially arranged in the preset rotation direction, the thirteenth port is connected with a first reagent of the third sequencing reaction, the fourteenth port is connected with a second reagent, the fifteenth port is connected with a first reagent of the fourth sequencing reaction, and the sixteenth port is connected with the second reagent, the method further includes the steps of:

vi) communicating the fourteenth port with the common port using the drive assembly, passing the second reagent through the rotary valve into the reaction device to effect excision of the third sequencing reaction;

vii) communicating the fifteenth port with the common port using the drive assembly, allowing the first reagent of the fourth sequencing reaction to enter the reaction device through the rotary valve to effect base extension of the fourth sequencing reaction;

viii) communicating the sixteenth port with the common port using the drive assembly, passing the second reagent through the rotary valve into the reaction device to effect excision of the fourth sequencing reaction.

13. The method of any one of claims 1, or 3-12, wherein the valve body assembly comprises two of the rotary valves and two of the first valves, the reaction apparatus comprises a first unit that connects the common port of one of the rotary valves and a second unit that connects the common port of the other of the rotary valves, the valve body assembly comprises a second valve that connects the two rotary valves and the first reagent of the first sequencing reaction, a third valve that connects the two rotary valves and the first reagent of the second sequencing reaction, and a fourth valve that connects the second reagent and the two first valves, each of the first valves connecting the second port and the fourth port of one rotary valve.

14. The method of any one of claims 1, or 3-12, wherein the valve body assembly comprises a fifth valve, the reaction device comprising a first unit and a second unit, the fifth valve connecting the common port, the first unit, and the second unit.

15. A sequencing system for controlling a sequencing reaction, wherein the sequencing reaction is performed on a reaction device, the sequencing reaction comprising a first sequencing reaction and a second sequencing reaction that are performed sequentially, the first sequencing reaction and the second sequencing reaction each comprising the steps of, in order: base extension, image acquisition and excision,

by means of the fact that the said excision is performed,

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 rotary valve, the rotary valve comprises a stator and a rotor which can be communicated, the rotary 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 one port can be communicated through the communication groove by rotating the rotor, the ports comprise a first group of ports and a second group of ports which respectively correspond to the first sequencing reaction and the second sequencing reaction, the first group of ports comprise a first port and a second port, the first port is connected with a first reagent of the first sequencing reaction, the second port is connected with a second reagent, the second group of ports comprise a third port and a fourth port, the first port, the second port, the third port and the fourth port are sequentially arranged according to the preset rotation direction of the rotor, the third port is connected with a first reagent of the second sequencing reaction, the fourth port is connected with a second reagent, and the public port is connected with the reaction device is used for controlling the reaction device:

16. The system of claim 15, wherein the valve body assembly comprises a first valve, the second port is coupled to the second reagent through the first valve, and the fourth port is coupled to the second reagent through the first valve.

17. The system of claim 15, wherein the image acquisition is performed with a third reagent, the first set of ports comprising the first port, the fifth port, and the second port sequentially arranged in the preset rotational direction, the second set of ports comprising the third port, the sixth port, and the fourth port sequentially arranged in the preset rotational direction, the control device configured to: after performing i) and before performing ii), communicating the fifth port and the common port with the drive assembly, passing the third reagent into the reaction device via the rotary valve to effect the image acquisition of the first sequencing reaction; after performing iii) and before performing iv), communicating the sixth port and the common port with the drive assembly, allowing the third reagent to enter the reaction device through the rotary valve to effect the image acquisition of the second sequencing reaction.

18. The system of claim 15 or 17, wherein the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: the base extension, first wash, the image acquisition and the excision.

19. The system of claim 18, wherein the first wash is performed with a fourth reagent, the first set of ports comprising the first port, a seventh port, and the second port sequentially aligned in the predetermined direction of rotation, the second set of ports comprising the third port, an eighth port, and the fourth port sequentially aligned in the predetermined direction of rotation, the control means for: after performing i) and before performing ii), communicating the seventh port and the common port with the drive assembly, passing the fourth reagent into the reaction device through the rotary valve to effect the first wash of the first sequencing reaction; after performing iii) and before performing iv), communicating the eighth port and the common port with the drive assembly, passing the fourth reagent through the rotary valve into the reaction device to effect the first wash of the second sequencing reaction.

20. The system of claim 15, wherein the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: the base extension, first wash, second wash, the image acquisition and the excision.

21. The system of claim 20, wherein the first wash is performed with a fourth reagent and the second wash is performed with a fifth reagent, the first set of ports including the first port, seventh port, ninth port, and the second port sequentially arranged in the preset rotational direction, the second set of ports including the third port, eighth port, tenth port, and the fourth port sequentially arranged in the preset rotational direction, the control means for: communicating the seventh port with the common port using the drive assembly after performing i) and before performing the second wash, passing the fourth reagent into the reaction apparatus via the rotary valve to effect the first wash of the first sequencing reaction;

22. The system of claim 15, wherein the first sequencing reaction and the second sequencing reaction each comprise the steps performed in the following order: the base extension, the image acquisition, the excision and capping.

23. The system of claim 22, wherein said capping is performed with a sixth reagent, said first set of ports comprising said first port, said second port and an eleventh port arranged in sequence in said predetermined direction of rotation, said second set of ports comprising said third port, said fourth port and a twelfth port arranged in sequence in said predetermined direction of rotation, said control means for comprising: after performing ii) and before performing iii), communicating the eleventh port and the common port with the drive assembly, passing the sixth reagent through the rotary valve into the reaction device to effect the capping of the first sequencing reaction; after iv) is performed, the twelfth port and the common port are brought into communication using the drive assembly, and the sixth reagent is passed into the reaction device via the rotary valve to effect the capping of the second sequencing reaction.

24. The system of any one of claims 15-23, wherein the sequencing reaction comprises the first sequencing reaction, the second sequencing reaction, and a third sequencing reaction that are performed sequentially, the third sequencing reaction comprising the steps of reacting with the first sequencing reaction or with the second sequencing reaction,

the first sequencing reaction, the second sequencing reaction and the first reagent of the third sequencing reaction are all different, the plurality of ports further comprise a third group of ports corresponding to the third sequencing reaction, the third group of ports comprise a thirteenth port and a fourteenth port which are sequentially arranged according to the preset rotation direction, the first group of ports, the second group of ports and the third group of ports are sequentially arranged according to the preset rotation direction, the thirteenth port is connected with the first reagent of the third sequencing reaction, the fourteenth port is connected with the second reagent, and the control device is used for:

25. The system of any one of claims 15-23, wherein the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction that are sequentially performed, the third sequencing reaction and the fourth sequencing reaction comprising steps that are either the same as the first sequencing reaction or the second sequencing reaction, the first reagents of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction, and the fourth sequencing reaction are different, the plurality of ports further comprises a third set of ports that correspond to the third sequencing reaction and a fourth set of ports that correspond to the fourth sequencing reaction, the third set of ports comprises a thirteenth port and a fourteenth port that are sequentially arranged in the predetermined rotational direction, the fourth set of ports comprises a fifteenth port and a sixteenth port that are sequentially arranged in the predetermined rotational direction, the first set of ports, a second set of ports, a third set of ports and a fourth port are sequentially arranged in the predetermined rotational direction, the thirteenth port is sequentially connected to the fourth reagent, the thirteenth port is sequentially connected to the thirteenth port is sequentially arranged in the predetermined rotational direction, and the thirteenth port is sequentially connected to the reagent is sequentially connected to the thirteenth port is sequentially arranged in the fourth port:

26. The system of any one of claims 15, or 17-25, wherein the valve body assembly comprises two of the rotary valves and two of the first valves, the reaction apparatus comprises a first unit that connects the common port of one of the rotary valves and a second unit that connects the common port of the other of the rotary valves, the valve body assembly comprises a second valve that connects the two rotary valves and the first reagent of the first sequencing reaction, a third valve that connects the two rotary valves and the first reagent of the second sequencing reaction, and a fourth valve that connects the second reagent and the two first valves, each of the first valves connecting the second port and the fourth port of one rotary valve.

27. The system of any one of claims 15, or 17-25, wherein the valve body assembly comprises a fifth valve, the reaction device comprising a first unit and a second unit, the fifth valve connecting the common port, the first unit, and the second unit.

28. 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 1-14.