WO2018214816A1 - Procédé, dispositif et système de commande d'une réaction de séquençage - Google Patents

Procédé, dispositif et système de commande d'une réaction de séquençage Download PDF

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Publication number
WO2018214816A1
WO2018214816A1 PCT/CN2018/087458 CN2018087458W WO2018214816A1 WO 2018214816 A1 WO2018214816 A1 WO 2018214816A1 CN 2018087458 W CN2018087458 W CN 2018087458W WO 2018214816 A1 WO2018214816 A1 WO 2018214816A1
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Prior art keywords
port
sequencing reaction
reagent
reaction
sequencing
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PCT/CN2018/087458
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English (en)
Chinese (zh)
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吴平
颜钦
姜泽飞
周志良
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深圳市瀚海基因生物科技有限公司
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Publication of WO2018214816A1 publication Critical patent/WO2018214816A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors

Definitions

  • the invention relates to the field of sequence determination technology, in particular to a method and a sequence determination system and device for controlling a sequence determination reaction.
  • Sequencing includes the determination of nucleic acid sequences.
  • the current sequencing platforms on the market include a generation of sequencing platforms, second-generation sequencing platforms and three generations of sequencing platforms.
  • the platform for sequence sequencing based on biochemical reaction requires a biochemical reaction on the reaction device during the sequence determination.
  • a liquid route system is required to introduce different reagents together or sequentially onto a chip for reaction.
  • the liquid path system uses a valve body to switch input/output reagents.
  • the inventors have found that when each of a plurality of reagents is sequentially input and output by using a liquid path with a commercially available rotary valve, the influence of the residual of the pipeline is eliminated, and the next reagent always carries a certain amount. The last reagent affects the reaction by the next reagent.
  • the inventors made the present invention based on the following findings, assumptions, and tests for the resolution of the structure of the rotary valve.
  • Rotary valves currently on the market also known as injection valves, multi-position valves or rotary valves, are used as components for sample collection, liquid injection or flow path conversion.
  • the composition generally includes a stator and a rotor, and an effective seal can be formed by the tight combination of the stator and the rotor.
  • the rotary valve has a common port, the common port being a port through which different flow paths of liquid enter or exit, the common port being provided on the stator and/or the rotor, and having one or more other ports on the stator and/or rotor.
  • the connection between the rotor and the stator passage can be realized, thereby connecting the common port and other ports to achieve the function of selecting injection or splitting.
  • the general configuration/standard configuration of the rotary valve is a multi-pass selection type, that is, only one port is in communication with the common port during operation.
  • the communication of the common port and other ports generally requires communication through one or several common structures disposed on the rotor.
  • the common structure When there is liquid in the common structure, at least a part of the liquid in the common structure is inevitably brought to a place outside the common structure due to the rotation of the rotor, the relative movement of the sealing interface of the rotor and the stator connection, that is, in the flow path
  • it When converting, it will inevitably bring the liquid of the first-class road liquid with the first-class road, and if the flow path is reversed in the subsequent direction, the liquid of the next-class road mixed with the first-class liquid will be brought to the lower flow path.
  • the "common structure" referred to above is referred to as a communication groove.
  • embodiments of the present invention provide a method of controlling a sequence determination reaction, a sequence determination system, and a device for controlling a sequence determination reaction.
  • Embodiments of the present invention provide a method for controlling a sequence determination reaction, wherein the sequence determination reaction is performed on a reaction device, and the sequence measurement reaction is controlled by a sequence measurement system, and the sequence determination reaction includes a sequence of a sequencing reaction and a second sequencing reaction, the first sequencing reaction and the second sequencing reaction each comprise the following sequence of steps: base extension, image acquisition and excision, performing the base extension using a first reagent, The first reagent of the first sequencing reaction and the second sequencing reaction are different,
  • the ablation is performed using a second reagent comprising a fluid device comprising a valve body assembly and a drive assembly, the valve body assembly including a rotary valve including a connectable stator and rotor
  • the rotary valve has a common port, the stator has a plurality of ports, and the rotor has a communication groove through which the common port and one of the ports can communicate through the communication slot.
  • the plurality of ports include a first group of ports and a second group of ports respectively corresponding to the first sequencing reaction and the second sequencing reaction, the first group of ports including a first port and a second port, the first a port is connected to the first reagent of the first sequencing reaction, the second port is connected to the second reagent, and the second group of ports includes a third port and a fourth port, the first port, the second port, The third port and the fourth port are sequentially arranged in a preset rotation direction of the rotor, the third port is connected to the first reagent of the second sequencing reaction, and the fourth port is connected to the first port a reagent, the common port is connected to the reaction device, the method comprising the steps of: i) using the driving component to connect the first port and the common port, so that the first reagent of the first sequencing reaction is The rotary valve enters the reaction device to effect base extension of the first sequencing reaction; ii) the second port is communicated with the common port by the drive assembly, and the second rea
  • the communication tank is connected to the first port through the rotation of the rotor in one direction to input the first reagent of the first sequencing reaction, and then the rotation is switched.
  • Connecting the communication groove to the second port to input the second reagent enables the first or all of the first sequencing reaction in the communication groove and the sealing surface region of the communication groove to be brought between the rotor and the stator due to the rotation
  • a reagent is replaced by the second reagent, which greatly reduces the first reagent of the first sequencing reaction to be brought to the second sequencing reaction, and also prevents the first reagent of the second sequencing reaction from being brought into the first In the first reagent of the sequencing reaction, cross-contamination between different first reagents is avoided or greatly reduced.
  • This method is particularly suitable for sequencing reactions that require control of the sequential addition of different types of substrates or different substrate combinations in each round. This method relies on a simple device structure and controls the liquid in and out sequence, which can greatly reduce the different types of bottoms. Contamination between objects or between different combinations of substrates.
  • a sequence determining system controls a sequence determining reaction performed on a reaction device, the sequence determining reaction comprising a first sequencing reaction and a second sequencing reaction sequentially performed,
  • the first sequencing reaction and the second sequencing reaction each comprise the following sequence of steps: base extension, image acquisition and excision, performing the base extension using a first reagent, the first sequencing reaction and the second
  • the first reagent of the sequencing reaction is different, and the excision is performed
  • the sequencing system includes a control device and a fluid device, the control device is connected to the fluid device, and the fluid device comprises a valve body assembly and a driving assembly
  • the valve body assembly includes a rotary valve including a connectable stator and a rotor, the rotary valve having a common port, the stator having a plurality of ports, the rotor having a communication groove thereon, by rotating the rotor
  • the public port and one of the ports are connected through the communication slot, and the plurality of ports respectively correspond to the first a first set of ports and a second set of
  • the communication groove is connected to the first port through the rotation of the rotor in one direction to input the first reagent of the first sequencing reaction, and then the rotation is switched.
  • Connecting the communication groove to the second port to input the second reagent enables the first or all of the first sequencing reaction in the communication groove and the sealing surface region of the communication groove to be brought between the rotor and the stator due to the rotation
  • a reagent is replaced by the second reagent, which greatly reduces the first reagent of the first sequencing reaction to be brought to the second sequencing reaction, and also prevents the first reagent of the second sequencing reaction from being brought into the first In the first reagent of the sequencing reaction, cross-contamination between different first reagents is avoided or greatly reduced.
  • the system is particularly useful for sequencing reactions that require control of the sequential addition of different types of substrates or different substrate combinations in each round.
  • the system relies on a simple device structure and controls the liquid in and out sequence, which greatly reduces the number of different types of bottoms. Contamination between objects or between different combinations of substrates.
  • An apparatus for controlling a sequence determination reaction includes: a storage unit for storing data, the data including a computer executable program; a processor for executing the computer executable program, and an execution
  • the computer executable program includes the method of performing any of the above embodiments.
  • a computer readable storage medium for storing a program for execution by a computer, the method comprising executing the method of any of the above embodiments.
  • the computer readable storage medium may include read only memory, random access memory, magnetic or optical disks, and the like.
  • FIG. 1 is a schematic flow chart of a method for controlling a sequence determination reaction according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing a flow path structure of a sequence measurement system according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of another flow path structure of a sequence determination system according to an embodiment of the present invention.
  • FIG. 4 is a schematic view showing the relationship between a port of a rotary valve and a common port according to an embodiment of the present invention
  • FIG. 5 is a schematic view showing another relationship between a port of a rotary valve and a common port according to an embodiment of the present invention
  • FIG. 6 is a schematic view showing another relationship between a port of a rotary valve and a common port according to an embodiment of the present invention
  • Figure 7 is a functional block diagram of a sequence determination system according to an embodiment of the present invention.
  • first and second are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated.
  • features defining “first” or “second” may include one or more of the described features either explicitly or implicitly.
  • the meaning of "a plurality" is two or more unless specifically and specifically defined otherwise.
  • connection should be understood broadly, for example, it may be a fixed connection, a detachable connection, or an integral connection;
  • the mechanical connections may also be electrical connections or may communicate with each other; they may be directly connected or indirectly connected through an intermediate medium, and may be internal communication of two elements or an interaction relationship of two elements.
  • specific meanings of the above terms in the present invention can be understood on a case-by-case basis.
  • Sequence determination is the same as nucleic acid sequence determination, including DNA sequencing and/or RNA sequencing, including long fragment sequencing and/or short fragment sequencing.
  • the so-called “sequence determination reaction” is the same as the sequencing reaction.
  • 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.
  • sequencing reactions include extension reactions (base extension), information collection (photographing/image acquisition), and cleave.
  • nucleotide analog is a substrate, also known as a terminator, which is an analog of A, T, C, G and/or U, which can follow the principle of base complementation and a specific type of base.
  • the base pairing while being able to terminate the next nucleotide/substrate binding to the template strand.
  • an embodiment of the present invention provides a method for controlling a sequence measurement reaction.
  • the sequence determination reaction is performed on a reaction device 40, and the sequence measurement system is controlled by a sequence measurement system, and the sequence determination is performed.
  • the reaction includes a first sequencing reaction and a second sequencing reaction performed sequentially, and the first sequencing reaction and the second sequencing reaction each include the following sequential steps: base extension, image acquisition, and excision.
  • the first reagent is said to comprise at least one nucleotide analog, and the nucleotide analogs include analogs of the following nucleotides: adenine deoxynucleotide (A), thymine deoxynucleotide ( T), cytosine deoxynucleotides (C), guanine deoxynucleotides (G) and uracil deoxynucleotides (U).
  • A adenine deoxynucleotide
  • T thymine deoxynucleotide
  • C cytosine deoxynucleotides
  • G guanine deoxynucleotides
  • U uracil deoxynucleotides
  • the sequencing system includes a fluidic device 100 that includes a valve body assembly 29 and a drive assembly 50.
  • the valve body assembly 29 includes a rotary valve 70 including a communicable stator 81 having a common port 71, a plurality of ports on the stator 81, and a communication groove 72 on the rotor, which can be made common by rotating the rotor
  • the port 71 is connected to a port
  • the plurality of ports include a first group port and a second group port corresponding to the first sequencing reaction and the second sequencing reaction, respectively,
  • the first group port includes a first port 1 and a second port 2
  • the first Port 1 is connected to the first reagent of the first sequencing reaction
  • second port 2 is connected to the second reagent, and
  • the second group of ports includes the third port 3 and the fourth port 4, the first port 1, the second port 2, and the third port 3
  • the fourth port 4 is sequentially arranged in a predetermined rotation direction of the rotor, the third port 3 is connected to the first
  • the second port 2 and the common port 71 are connected by the driving assembly 50, and the second reagent is introduced into the reaction device 40 through the rotary valve 70 to realize the cutting of the first sequencing reaction;
  • the fourth port 4 and the common port 71 are communicated by the drive assembly 50, and the second reagent is introduced into the reaction device 40 via the rotary valve 70 to effect the ablation of the second sequencing reaction.
  • the communication groove 72 is communicated with the first port 1 by the rotation in one direction of the rotor to input the first of the first sequencing reaction.
  • the reagent is then rotationally switched so that the connection groove 72 communicates with the second port 2 to input the second reagent, and the sealing surface area 73 in the communication groove 72 and in the communication groove 72 that is brought between the rotor and the stator 81 by rotation can be brought.
  • the first reagent of all or most of the first sequencing reaction is replaced by the second reagent, which greatly reduces the first reagent of the first sequencing reaction to be brought to the second sequencing reaction, and also avoids the second sequencing reaction.
  • the first reagent is not carried into the first reagent of the first sequencing reaction, avoiding or greatly reducing cross-contamination between the different first reagents.
  • This method is particularly suitable for sequencing reactions that require control of the sequential addition of different types of substrates or different substrate combinations in each round. This method relies on a simple device structure and controls the liquid in and out sequence, which can greatly reduce the different types of bottoms. Contamination between objects or between different combinations of substrates.
  • the communication groove 72 communicates with the first port 1 and the common port 71, thereby causing the first port 1 to communicate with the reaction device 40, and the first sequencing reaction is first performed by the driving component 50.
  • the reagent enters the reaction device 40 via the rotary valve 70 for base extension of the first sequencing reaction.
  • the first reagent of the first sequencing reaction flows through the communication tank 72, the first reagent of the first sequencing reaction remains in the communication tank 72.
  • the rotor is rotated in a predetermined rotational direction by the drive assembly 50 to connect the communication groove 72 to the second port 2 and the common port 71, thereby allowing the second port 2 to communicate with the reaction device 40.
  • the second reagent is introduced into the reaction device 40 via the rotary valve 70 by the driving assembly 50 to perform the ablation of the first sequencing reaction.
  • the rotor is rotated in one direction to switch the communication groove 72 to the first port 1 to be connected to the second port 2, a part of the first reagent of the first sequencing reaction in the communication groove 72 remains in the rotor and the stator 81.
  • the sealing surface area 73 (as shown in FIG. 4), on the other hand, when the second reagent for cutting flows through the communication groove 72, the other part of the first reagent in the first sequencing reaction in the communication groove 72 Take it all or part of it.
  • the contamination caused by the first reagent of the first sequencing reaction to the first reagent of the second sequencing reaction can be greatly reduced, and the first reagent pair of the second sequencing reaction can be avoided.
  • the contamination caused by the first reagent of a sequencing reaction can be greatly reduced.
  • the reaction device 40 can be a chip with a plurality of channels disposed within the reaction device 40 to accommodate the sample.
  • the drive assembly 50 can include a motor and a pump that is coupled to the rotor and that is used to drive the rotor to rotate to communicate the communication slots 72 to different ports on the stator 81.
  • the pump is coupled to the reaction device 40 and is used to generate a negative pressure within the passage of the reaction device 40 to drive the reagent into the reaction device 40 and to draw the reagent away from the reaction device 40, and to stop generating a negative pressure to allow the reagent to remain in the reaction device. Biochemical reactions were carried out within 40.
  • the reagent pumped away by the pump can be pumped into the waste container 60 for recovery.
  • the predetermined rotational direction of the rotor is the clockwise direction shown in FIGS. 2 and 3. That is to say, the movement of the rotary valve 70 needs to be connected to the first port 1 when it is connected to the second port 2, and needs to be connected to the second port 2 or the like first when the third port 3 is to be connected.
  • the preset rotation direction of the rotor may be other directions, as shown in FIG. 2 and FIG. 3 in the counterclockwise direction, and the port may be re-selected from the plurality of ports according to the rotation direction, which is not specifically limited herein. .
  • the first port 1 and the second port 2 are adjacent, the second port 2 and The third port 3 is adjacent, and the third port 3 and the fourth port 4 are adjacent to each other, so that the cross-contamination between the first reagents can be avoided or greatly reduced, and the stroke of the rotor rotation is short, which facilitates rapid sequencing.
  • first port 1, the second port 2, the third port 3, and the fourth port 4 can also select other ports of the multiple ports, and only need to ensure the first port 1 and the second port 2
  • the third port 3 and the fourth 6 port 4 are arranged in order according to the preset rotation direction of the rotor.
  • the rotor is rotated in a predetermined rotational direction to sequentially complete the operation of different reagents entering the reaction device 40 via the rotary valve 70.
  • the DNA is sequenced
  • the reaction substrates are four nucleotide analogs of A, T, C, and G
  • at least three substrates of the four substrates carry at least one fluorescent label (fluorescent dye/luminescent group), when performing polymerization/base extension reaction, when the substrate is bound to the DNA template strand, under the excitation of a specific wavelength laser, fluorescence can be emitted, and the sequencing system is based on conversion and/or These light signals are collected as well as the sequence of addition of the various substrates, if any, to determine the DNA sequence.
  • fluorescent label fluorescent dye/luminescent group
  • the four substrates carry two fluorescent labels, two of which carry one fluorescent label, and the other two carry another fluorescent label
  • the first reagent of the first sequencing reaction is a reagent for two substrates
  • the two substrates in the first reagent carry different fluorescent labels
  • the first reagent in the second sequencing reaction is a reagent comprising two other substrates, two of the first reagents
  • the substrate also has different fluorescent labels
  • the first round of sequencing reaction includes a first sequencing reaction and a second sequencing reaction. After the first sequencing reaction is completed, the second sequencing reaction is performed, and after the second sequencing reaction is completed, the first sequencing reaction is performed, and the first sequencing reaction is performed. This is repeated.
  • the luminescent group on the reaction substrate (terminator) of the base extension of the first sequencing reaction is removed and then added.
  • the terminator of the base extension of the second sequencing reaction For example, in combination with the above example, after the first reagent of the first sequencing reaction is input into the reaction device 40, image acquisition of the reaction device 40 can be performed. After the image acquisition is completed, the luminescent group of the first reagent of the first sequencing reaction is excised and then the first reagent of the second sequencing reaction is added.
  • the plurality of ports are distributed in a circular shape and the common port 71 is concentrically disposed with the circle.
  • 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 groove 72 communicating with the corresponding port and the common port 71 when the rotor is rotated.
  • the communication slots 72 are linear. In this way, the flow path of the agent liquid in the communication tank 72 can be reduced, thereby achieving rapid sequencing.
  • the linear communication groove 72 can communicate the port and the common port 71 at both ends of the communication groove 72 in a short path.
  • the line shape is a straight line.
  • the valve body assembly includes a first valve 30, the second port 2 is coupled to the second reagent via a first valve 30, and the fourth port 4 is coupled to the second reagent via a first valve 30.
  • the second reagent can be provided to different ports, the pipeline connection is reduced, the liquid path in the sequence determination system is simplified, the problem is checked and maintained, and the industrial production is facilitated.
  • the first valve 30 includes a total port and a plurality of split ports. It can be understood that the number of the 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 two. The number of the ports of the first valve 30 is not less than two.
  • first valve 30 includes one total port and four ports, that is, one first valve 30 can provide a second reagent to up to four sequencing reactions.
  • the four ports of the first valve 30 are respectively connected to the second port 2, the fourth port 4, the fourteenth port 14 and the sixteenth port 16, and the total port connection of the first valve 30 is Second reagent. Therefore, when the communication groove 72 communicates with the common port 71 and the corresponding port, the drive assembly 50 drives the second reagent to flow through the first valve 30 and the rotary valve 70 into the reaction device 40.
  • the sequencing system includes an imaging device, the method comprising the steps of image acquisition using an imaging device.
  • the sequence determination system facilitates user image acquisition of samples within the reaction device 40.
  • the imaging device may include a light emitting device and a camera.
  • the illuminating device such as a laser
  • the group fluoresces and is photographed with a camera to collect fluorescence and form an image for sequence determination.
  • the second reagent is added to excise the luminescent group in the reaction device 40, and then the first reagent of the second sequencing reaction is added, and then the image acquisition of the second sequencing reaction is performed.
  • the third reagent is used for image acquisition
  • the first group of ports includes a first port 1, a fifth port 5, and a second port 2 arranged in a predetermined rotation direction
  • the second group of ports includes The third port 3, the sixth port 6, and the fourth port 4 are arranged in the order of rotation
  • the method includes: connecting the fifth port 5 and the common port 71 by using the driving component 50 after performing i) and before performing ii),
  • the third reagent is introduced into the reaction device 40 via the rotary valve 70 to effect image acquisition of the first sequencing reaction; after the iii) and before the iv), the sixth port 6 and the common port 71 are communicated by the drive assembly 50,
  • the third reagent enters the reaction device 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 reaction device 40 to be better captured after the base extension reaction.
  • the third reagent can reduce the photobleaching effect and/or anti-quenching of the sample, and can enable the luminescent group-containing substrate contained in the first reagent to be bonded to the template chain, and the luminescence is more stable under the excitation of the laser. Conducive to image acquisition, especially the acquisition of small molecules of a single molecule.
  • the sequence determination reaction includes the first sequencing reaction and the second sequencing reaction performed sequentially
  • the third port 3, the sixth port 6, and the fourth port 4 are sequentially arranged in a predetermined rotation direction.
  • the rotor is rotated in a predetermined rotational direction to sequentially complete the operation of different reagents entering the reaction device 40 via the rotary valve 70.
  • both the first sequencing reaction and the second sequencing reaction comprise the following sequence of steps: base extension, first wash, image acquisition, and excision.
  • the first reagent remaining in the communication channel 72 can be largely carried away by the first wash, further reducing cross-contamination of the first reagent of the different sequencing reactions.
  • the first wash is performed using a fourth reagent, the first set of ports including a first port 1, a seventh port 7, and a second port 2 arranged in a predetermined rotational direction, the second set of ports including pressing
  • the third port 3, the eighth port 8, and the fourth port 4 are sequentially arranged in a predetermined rotation direction
  • the method includes: connecting the seventh port 7 and the common port 71 by using the driving component 50 after performing i) and before performing ii)
  • the fourth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect a first wash of the first sequencing reaction; after the iii) and before the iv), the eighth port 8 is connected to the common port 71 by the drive assembly 50.
  • the fourth reagent is passed through the rotary valve 70 into the reaction device 40 to effect a first wash of the second sequencing reaction.
  • the first reagent of the first sequencing reaction when the rotor is rotated in a predetermined rotation direction to switch the communication groove 72 from the communication first port 1 to the seventh port 7, the first reagent of the first sequencing reaction remains mostly in the rotor and the stator.
  • the sealing surface area between 81 greatly reduces cross-contamination of the first reagent of the first sequencing reaction with the first reagent of the second sequencing reaction.
  • the fourth reagent is a cleaning agent that has no effect on the target sequencing reaction.
  • both the first sequencing reaction and the second sequencing reaction comprise the steps of performing the following sequence: base extension, first wash, second wash, image acquisition, and excision.
  • the first reagent remaining in the communication tank 72 can be largely carried away by the first washing, further reducing cross-contamination of the first reagent of different sequencing reactions, and the second washing step is to add a buffer.
  • the buffer is a solution which can maintain the liquid pH within a certain range to a certain extent, and is a weak acid, a weak base and/or a neutral solution.
  • the buffer is a solution that has no effect on the target sequencing reaction.
  • the first wash is performed using the fourth reagent
  • the second wash is performed using the fifth reagent
  • the first set of ports including the first port 1, the seventh port 7, and the ninth arranged in a predetermined rotational direction.
  • Port 9 and second port 2 the second group of ports includes a third port 3, an eighth port 8, a tenth port 10, and a fourth port 4 arranged in a predetermined rotation direction
  • the method comprising: performing i) after and performing Before the second washing, the seventh port 7 and the common port 71 are communicated by the driving assembly 50, so that the fourth reagent enters the reaction device 40 through the rotary valve 70 to realize the first washing of the first sequencing reaction;
  • the ninth port 9 and the common port 71 are communicated by the drive assembly 50, and the fifth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect a second washing of the first sequencing reaction. ;
  • the eighth port 8 and the common port 71 are communicated by the drive assembly 50, and the fourth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect the first washing of the second sequencing reaction. ;
  • the tenth port 10 and the common port 71 are communicated by the drive assembly 50, and the fifth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect a second washing of the second sequencing reaction. .
  • the first reagent of the first sequencing reaction when the rotor is rotated in a predetermined rotation direction to switch the communication groove 72 from the communication first port 1 to the seventh port 7, the first reagent of the first sequencing reaction remains mostly in the rotor and the stator. a sealing surface area between 81, further, when the rotor is rotated in a predetermined rotation direction to switch the communication groove 72 from the communication seventh port 7 to the ninth port 9, the first reagent of the first sequencing reaction remains further The area of the sealing surface between the rotor and the stator 81 further substantially reduces cross-contamination of the first reagent of the first sequencing reaction with the first reagent of the second sequencing reaction.
  • the fourth reagent is a buffer that has no effect on the target sequencing reaction and the fifth reagent has no effect on the target sequencing reaction.
  • both the first sequencing reaction and the second sequencing reaction comprise the steps of performing the following sequence: base extension, image acquisition, excision, and capping.
  • the so-called capping is primarily a group/bond exposed after removal of the protecting group.
  • the exposed group is a sulfhydryl group, and the sulfhydryl group can be protected from oxidation by capping, such as by the addition of an alkylating agent.
  • the sixth reagent is used for capping
  • the first group of ports includes a first port 1, a second port 2, and an eleventh port 11 arranged in a predetermined rotational direction
  • the second group of ports includes pressing
  • the third port 3, the fourth port 4, and the twelfth port 12 are sequentially arranged in a predetermined rotation direction
  • the method includes: using the drive assembly 50 to make the eleventh port 11 and the common port after performing ii) and before performing iii) 71 is connected to cause the sixth reagent to enter the reaction device 40 via the rotary valve 70 to achieve capping of the first sequencing reaction; after performing iv), the twelfth port 12 and the common port 71 are connected by the driving assembly 50, so that The six reagents enter the reaction unit 40 via the rotary valve 70 to effect capping of the second sequencing reaction.
  • the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, and a third sequencing reaction performed sequentially
  • the third sequencing reaction comprises the same steps as the first sequencing reaction or the second sequencing reaction, first The first reagents of the sequencing reaction, the second sequencing reaction and the third sequencing reaction are different
  • the plurality of ports further comprise a third group of ports corresponding to the third sequencing reaction
  • the third group of ports comprises the first order according to the preset rotation direction.
  • the thirteen port 13 and the fourteenth port 14, the first group port, the second group port and the third group port are arranged in a predetermined rotation direction, and the thirteenth port 13 is connected to the first reagent of the third sequencing reaction, the tenth The four port 14 is connected to the second reagent, and the method further comprises the steps of:
  • the thirteenth port 13 and the common port 71 are connected by the driving assembly 50, and the first reagent of the third sequencing reaction is introduced into the reaction device 40 through the rotary valve 70 to realize the alkali of the third sequencing reaction.
  • the fourteen port 14 and the common port 71 are communicated by the drive assembly 50, and the second reagent is introduced into the reaction device 40 via the rotary valve 70 to effect the ablation of the third sequencing reaction.
  • the method of controlling the sequencing reaction is more efficient. At the same time, this also greatly reduces the cross-contamination of the first reagent of the second sequencing reaction and the first reagent of the third sequencing reaction.
  • 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 to each other. Adjacent, the fourth port 4 and the thirteenth port 13 are adjacent, and the thirteenth port 13 and the fourteenth port 14 are adjacent, so that the rotation of the rotor is short, which facilitates rapid sequencing.
  • 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 can also select other ports of the plurality of ports, It is only necessary to ensure that 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.
  • the rotor rotates in a predetermined rotational direction of the rotor to sequentially complete the operation of different reagents entering the reaction device 40 via the rotary valve 70.
  • the third group of ports includes a thirteenth port 13 arranged in a predetermined rotation direction
  • the seventeenth port 17 and the fourteenth port 14 please refer to FIG. 2 and FIG. 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 a predetermined rotation direction.
  • the rotor rotates in a clockwise direction to sequentially complete the operation of different reagents entering the reaction device 40 via the rotary valve 70.
  • the seventeenth port 17 is connected to the third reagent.
  • the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction, and the steps of the third sequencing reaction and the fourth sequencing reaction are the same as the first step.
  • the sequencing reaction or the second sequencing reaction is the same, the first reagent of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction and the fourth sequencing reaction are different, and the plurality of ports further comprise a third corresponding to the third sequencing reaction.
  • the third group port includes a thirteenth port 13 and a fourteenth port 14 arranged in a predetermined rotation direction
  • the fourth group of ports includes a predetermined rotation direction
  • the fifteenth port 15 and the sixteenth port 16 are arranged, the first group port, the second group port, the third group port and the fourth group port are arranged in a preset rotation direction, and the thirteenth port 13 is connected to the third sequencing.
  • the first reagent of the reaction, the fourteenth port 14 is connected to the second reagent, the fifteenth port 15 is connected to the first reagent of the fourth sequencing reaction, and the sixteenth port 16 is connected to the second reagent.
  • the method further comprises the steps of:
  • the thirteenth port 13 and the common port 71 are connected by the driving assembly 50, and the first reagent of the third sequencing reaction is introduced into the reaction device 40 through the rotary valve 70 to realize the alkali of the third sequencing reaction.
  • the type of sequencing reaction can be increased, making the method of controlling the sequencing reaction more efficient.
  • this can also greatly reduce the cross-contamination of the first reagent of the second sequencing reaction and the first reagent of the third sequencing reaction, and greatly reduce the intersection of the first reagent of the third sequencing reaction and the first reagent of the fourth sequencing reaction. Pollution.
  • 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 to each other.
  • 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, the fifteenth port 15 and the tenth
  • the six ports are 16 adjacent, making the rotor rotate for a shorter stroke for quick sequencing.
  • 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 can also select other ports of multiple ports, only need to ensure first port 1, second port 2, third port 3, fourth port 4, thirteenth port 13, fourteenth port 14, fifteenth
  • the port 15 and the sixteenth port 16 may be arranged in order according to the preset rotation direction of the rotor.
  • the rotor rotates in a predetermined rotational direction of the rotor to sequentially complete the operation of different reagents entering the reaction device 40 via the rotary valve 70.
  • all four reaction substrates carry the same fluorescent label
  • the first reagents of the first, second, third, and fourth sequencing reactions are reagents containing one substrate, and one round of sequencing reactions.
  • the first sequencing reaction, the second sequencing reaction, the third sequencing reaction, and the fourth sequencing reaction are performed. After the first sequencing reaction is completed, the second sequencing reaction is performed, and after the second sequencing reaction is completed, the third sequencing reaction is performed, and the third sequencing reaction is completed. After the fourth sequencing reaction is performed, the first sequencing reaction is performed after the fourth sequencing reaction is completed, and the repeated steps are performed as follows.
  • the method is designed and controlled by a simple liquid path structure, so that the rotary valve always rotates in a rotation direction during the sequencing reaction, and the reagent cross-mixing between the sequencing reactions can be substantially avoided, such as the first and third, the second and the second.
  • the cross-mixing of reagents between the fourth sequencing reactions can greatly reduce the cross-mixing of reagents between adjacent sequencing reactions, such as first and second, second and third, third and fourth, fourth and first The reagents between the sequencing reactions cross.
  • the third group of ports includes a first order in a predetermined rotation direction.
  • the thirteenth port 13, the seventeenth port 17 and the fourteenth port 14, the fourth group of ports includes a fifteenth port 15, an eighteenth port 18 and a sixteenth port 16 arranged in a predetermined rotation direction, please combine 2 and 3, first port 1, fifth port 5, second port 2, third port 3, sixth port 6, fourth port 4, thirteenth port 13, seventeenth port 17, tenth
  • the four ports 14, the fifteenth port 15, the eighteenth port 18, and the sixteenth port 16 are sequentially arranged in a predetermined rotation direction.
  • the rotor rotates in a clockwise direction to sequentially complete the operation of different reagents entering the reaction device 40 via the rotary valve 70.
  • the seventeenth port 17 is connected to the third reagent, and the eighteenth port 18 is connected to the third reagent.
  • the valve body assembly 29 includes two rotary valves 70 and two first valves 30.
  • the reaction device 40 includes a first unit 41 and a second unit 42, and the first unit 41 is coupled thereto.
  • a common port 71 of the rotary valve 70, the second unit connection 42 is connected to the common port 71 of the other rotary valve 70, and the valve body assembly 29 includes a second valve 35, a third valve 36 and a fourth valve 37.
  • the second valve 35 Connecting two rotary valves 70 and a first reagent for the first sequencing reaction
  • the third valve 36 connects the two rotary valves 70 and the first reagent of the second sequencing reaction
  • the fourth valve 37 connects the second reagent and the two first valves 30.
  • Each first valve 30 is coupled to a second port 2 and a fourth port 4 of a rotary valve 70.
  • sequence determination reactions can be performed in the channel of the first unit 41 and the channel of the second unit 42, respectively, and the sequence determination reaction in the channel of the first unit 41 and the sequence determination reaction in the channel of the second unit 42 are staggered.
  • the non-synchronized, non-influenced ones thereby shortening the time for the sequence determination reaction.
  • the fluid device 100 transmits the first reagent of the first sequencing reaction for the reaction to the first unit 41, at this time, The same reagent is passed into the second unit 42 and vice versa.
  • the first reagent of the first sequencing reaction is delivered to the two first ports 1 of the two rotary valves 70 via the second valve 35, and the first reagent of the second sequencing reaction passes through the third valve 36.
  • Two second ports 3 are delivered to the two rotary valves 70, and the second reagent is delivered to the two first valves 30 via the fourth valve 37.
  • the second valve 35, the third valve 36, and the fourth valve 37 are all three-way valves.
  • the sequence determination reaction includes a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction, which are sequentially performed, and each sequencing reaction includes the following sequential steps: base Extension, first wash, second wash, image acquisition, ablation and capping.
  • the first group of ports includes 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 a predetermined rotational direction of the rotor.
  • the second group of ports includes 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 a predetermined rotational direction of the rotor.
  • the third group of ports includes the thirteenth port 13, the nineteenth port 19, the twentieth port 20, the seventeenth port 17, the fourteenth port 14, the first Twenty-one port 21.
  • the fourth group of ports includes a fifteenth port 15, a twenty-second port 22, a twenty-third port 23, an eighteenth port 18, and a sixteenth port 16 along a predetermined rotation direction of the rotor. And the twenty-fourth port 24.
  • 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 is ten
  • the sixth valve 51 connects the first reagent of the third sequencing reaction and the two thirteenth ports 13 of the two rotary valves 70
  • the seventh valve 52 connects the first reagent of the fourth sequencing reaction and the two fifteenth of the two rotary valves 70 Port 15.
  • the eighth valve 53 is connected to the third reagent and the two first valves 30, the ninth valve 54 is connected to the sixth reagent and the two first valves 30, and the tenth valve 55 is connected to the fourth reagent and the two first valves 30, tenth A 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 include a total port And two splits.
  • the seventh port 7, the eighth port 8, the nineteenth port 19 and the twenty-second port 22 are connected to the four ports of the same first valve 30, and the total port of the same first valve 30 is connected to the tenth valve 55.
  • a split port, the total port of the tenth valve 55 is connected to the fourth reagent.
  • the ninth port 9, the tenth port 10, the twentieth port 20 and the twenty-third port 23 are connected to the four ports of the same first valve 30, and the total port of the same first valve 30 is connected to the eleventh valve.
  • the total port of the eleventh valve 56 is 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 the four ports of the same first valve 30, and the total port of the same first valve 30 is connected to the eighth valve 53.
  • a split port, 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 the four ports of the same first valve 30, and the total port of the same first valve 30 is connected to the fourth valve 37.
  • a split port, 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-four port 24 are connected to the four ports of the same first valve 30, and the total port connection of the same first valve 30 is A split port of the nine valve 54 and a total port of the ninth valve 54 are connected to the sixth reagent.
  • 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 are all three-way valves, and the first valve 30 includes a total port and four ports.
  • the rotary valve 70 is a 28-port rotary valve. Therefore, in the embodiment shown in Fig. 2, the number of three-way valves is nine, and the number of first valves 30 is ten.
  • the reaction device 40 includes a first unit 41 and a second unit 42.
  • the first unit 41 is connected to a common port 71 of a rotary valve 70
  • the second unit 42 is connected to a common port 71 of the other rotary valve 70.
  • the drive assembly 50 includes an eight-row pump, four of the eight-row pumps are coupled to the first unit 41, and the other four pumps are coupled to the second unit 42 to produce a negative in the passages of the first unit 41 and the second unit 42, respectively. Pressure.
  • the use of the eight-row pump saves instrument installation space and saves costs, and reduces the use of solenoid valves and reduces the failure rate.
  • the eight-row pump can be a eight-row Thomas pump, and the eight-row Thomas pump has less noise and less vibration, enabling fast sequencing reactions.
  • the rotor of the rotary valve 70 completes the first washing, the second washing, and the first in a predetermined rotational direction (clockwise as shown in FIG. 2).
  • the process of three reagent addition, cleave and capping, the residual of the terminator of the first reagent in the rotary valve 70 is mostly defined in the sealing surface area between the rotor and the stator 81, and different sequencing reactions can be performed.
  • the first reagent cross-contamination is minimized.
  • the first reagent provided by the first sequencing reaction provides a base
  • the first reagent of the second sequencing reaction provides a base G
  • the first reagent of the third sequencing reaction provides The base is C
  • the base provided by the first reagent of the fourth sequencing reaction is T.
  • the second reagent is the reagent Cl for excision
  • the third reagent is the reagent I for image acquisition
  • the fourth reagent is the reagent R for the first washing
  • the fifth reagent is the reagent B for the second washing
  • the sixth reagent is the additive.
  • the bases provided by the first reagent of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction, and the fourth sequencing reaction may also be changed, for example, the first sequencing reaction The base provided by one reagent 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, and the first reagent of the fourth sequencing reaction The base provided is A or the like.
  • the first reagents A, T, C, G, the second reagent, the third reagent, and the sixth reagent can be placed in a refrigerated environment, and the fourth reagent and the fifth reagent can be placed in a room temperature environment.
  • the valve body assembly 29 includes a fifth valve 38
  • the reaction device 40 includes a first unit 41 and a second unit 42
  • the fifth valve 38 is coupled to the common port 71 and the first unit 41. And connecting the common port 71 and the second unit 42.
  • sequence determination reactions can be performed in the channel of the first unit 41 and the channel of the second unit 42, respectively, and the sequence determination reaction in the channel of the first unit 41 and the sequence determination reaction in the channel of the second unit 42 are staggered.
  • the non-synchronized, non-influenced ones thereby shortening the time for the sequence determination reaction.
  • the fluid device 100 delivers the first reagent for the first sequencing reaction to the first unit 41, at which time, The same reagent enters the second unit 42 and vice versa.
  • the fifth valve 38 can selectively communicate with the common port 71 and the first unit 41, or communicate with the common port 71 and the second unit 42, and when the fifth valve 38 communicates with the common port 71 and the first unit 41, the reagent can be
  • the first unit 41 is entered into the first unit 41 via the rotary valve 70 and the fifth valve 38 under the driving of the driving assembly 50.
  • the common port 71 and the second unit 42 are not in communication, and the reagent does not enter the second unit 42.
  • the reagent can drive the assembly 50 to drive through the rotary valve 70 and the fifth valve 38 into the second unit 42 for sequencing reaction.
  • the common port 71 and the first One unit 41 is not connected and the reagent does not enter the first unit 41.
  • the fifth valve 38 is a three-way rotary valve.
  • the sequence determination reaction includes a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction, which are sequentially performed, and each sequencing reaction includes the following sequential steps: base Extension, first wash, second wash, image acquisition, ablation and capping.
  • the first group of ports includes 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 a predetermined rotational direction of the rotor.
  • the second group of ports includes 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 a predetermined rotational direction of the rotor.
  • the third group of ports includes the thirteenth port 13, the nineteenth port 19, the twentieth port 20, the seventeenth port 17, the fourteenth port 14, the first Twenty-one port 21.
  • the fourth group of ports includes a fifteenth port 15, a twenty-second port 22, a twenty-third port 23, an eighteenth port 18, and a sixteenth port 16 along a predetermined rotation direction of the rotor. And the twenty-fourth port 24.
  • the valve body assembly 29 includes five first valves 30, and the first valve 30 includes a total port and four 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 four ports 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, the tenth port 10, the twentieth port 20 and the twenty-third port 23 are connected to the four ports 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, the sixth port 6, the seventeenth port 17, and the eighteenth port 18 are connected to the four ports 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 the four ports 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-four port 24 are connected to the four ports of the same first valve 30, and the total port connection of the same first valve 30 is Six reagents.
  • 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.
  • the drive assembly 50 includes an eight-row pump, four of the eight-row pumps are coupled to the first unit 41, and the other four pumps are coupled to the second unit 42 to produce a negative in the passages of the first unit 41 and the second unit 42, respectively. Pressure.
  • the use of the eight-row pump saves instrument installation space and saves costs, and reduces the use of solenoid valves and reduces the failure rate.
  • the eight-row pump can be a eight-row Thomas pump, and the eight-row Thomas pump has less noise and less vibration, enabling fast sequencing reactions.
  • the rotor of the rotary valve 70 completes the first washing, the second washing, and the first washing in a predetermined rotational direction (clockwise direction as shown in FIG. 3).
  • the process of three reagent addition, cleave and capping, the residual of the terminator of the first reagent in the rotary valve 70 is mostly defined in the sealing surface area between the rotor and the stator 81, and different sequencing reactions can be performed.
  • the first reagent cross-contamination is minimized.
  • a three-way rotary valve is used to effect switching of the two units.
  • the internal dead volume of the three-way rotary valve is almost zero, and there is no need to worry about cross-contamination of the reagent between the two units.
  • the embodiment of the invention can avoid the use of the three-way valve, reduce the number of joint connections, and reduce the cost.
  • a sequence determining system 300 controls a sequence determining reaction performed on a reaction device 40, the sequence determining reaction including a sequential first sequencing reaction and a second sequencing reaction, the first sequencing reaction and the second sequencing reaction each comprise the following sequence of steps: base extension, image acquisition and excision,
  • the sequence determination system 300 includes a control device 302 and a fluid device 100 that is 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 includes a rotary valve 70 including a communicable stator 81 having a common port 71, a plurality of ports on the stator 81, and a communication groove 72 on the rotor, which can be made common by rotating the rotor
  • the port 71 and one port are connected by a communication slot 72.
  • the plurality of ports include a first group of ports and a second group of ports respectively corresponding to the first sequencing reaction and the second sequencing reaction, and the first group of ports includes the first port 1 and the second port 2, the first port 1 is connected to the first reagent of the first sequencing reaction, the second port 2 is connected to the second reagent, and the second group of ports includes the third port 3 and the fourth port 4, the first port 1, the second port 2
  • the third port 3 and the fourth port 4 are sequentially arranged in a predetermined rotation direction of the rotor, the third port 3 is connected to the first reagent of the second sequencing reaction, the fourth port 4 is connected to the second reagent, and the common port 71 is connected to the reaction device 40.
  • the control device 302 is configured to:
  • the fourth port 4 and the common port 71 are communicated by the drive assembly 50, and the second reagent is introduced into the reaction device 40 via the rotary valve 70 to effect the ablation of the second sequencing reaction.
  • the communication groove 72 is connected to the first port 1 by the rotation in one direction of the rotor to input the first sequencing reaction.
  • a reagent, followed by rotational switching, causes the connecting groove 72 to communicate with the second port 2 to input a second reagent, and the sealing surface region in the communication groove 72 and in the communication groove 72 that is brought into rotation between the rotor and the stator 81 by rotation can be brought.
  • the first reagent of all or most of the first sequencing reaction of 73 is replaced by the second reagent, which greatly reduces the first reagent of the first sequencing reaction to be brought to the second sequencing reaction, and also avoids the second sequencing reaction.
  • the first reagent is not carried into the first reagent of the first sequencing reaction, avoiding or greatly reducing cross-contamination between the different first reagents.
  • the system 300 is particularly useful for sequencing reactions that require control of the sequential addition of different types of substrates or different substrate combinations in each round.
  • the system 300 relies on a simple device structure and controls the liquid in and out sequence, which greatly reduces the difference. Contamination between type substrates or between different substrate combinations.
  • the fluidic device 100 includes a fluid control unit that is coupled to a fluid control unit that electrically couples the valve body assembly 29 and the drive assembly 50 to control operation of the valve body assembly 29 and the drive assembly 50.
  • the fluid control unit can receive control signals from the control device 302 and control the valve body assembly 29, the drive assembly 50, and other components of the fluid device 100 in accordance with the control signals. As such, this enables a portion of the functionality of the control device 302 to be implemented by the fluid control unit, reducing the load on the control device 302.
  • the fluid control unit may be a device including a single chip microcomputer, a computer processor, or a central control processor to enable automatic operation of the fluid device 100 to improve efficiency.
  • the fluid control unit and control device 302 can be integrated into one component, module, or device to increase the integration of the sequencing system 300 and reduce cost.
  • control device 302 includes a unit that controls the fluid device, the unit that controls the fluid device electrically couples valve body assembly 29 and drive assembly 50 to control valve body assembly 29 and drive assembly 50 to operate.
  • the unit controlling the fluid device can 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 in accordance with the control signal. As such, this enables a portion of the functionality of the control device 302 to be implemented by the unit that controls the fluid device, reducing interference between the various 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, which can realize automatic operation of the fluid device 100 to improve efficiency.
  • control device 302 can also include other units, for example, control device 302 includes a unit that controls the imaging device.
  • the valve body assembly 29 includes a first valve, the second port 2 is coupled to the second reagent by a first valve, and the fourth port 4 is coupled to the second reagent by a first valve.
  • the sequence determination system 300 includes an imaging device 200 that is coupled to an imaging device 200 for image acquisition using the imaging device 200.
  • the third reagent is used for image acquisition
  • the first group of ports includes a first port 1, a fifth port 5, and a second port 2 arranged in a predetermined rotation direction
  • the second group of ports includes The third port 3, the sixth port 6, and the fourth port 4 are arranged in the order of rotation
  • the control device 302 is configured to: after the i) and before the ii), use the drive assembly 50 to make the fifth port 5 and the common port 71 is connected such that the third reagent enters the reaction device 40 via the rotary valve 70 to effect image acquisition of the first sequencing reaction; the sixth port 6 and the common port 71 are made by the drive assembly 50 after performing iii) and before performing iv) In communication, the third reagent enters the reaction device 40 via the rotary valve 70 to effect image acquisition of the second sequencing reaction.
  • both the first sequencing reaction and the second sequencing reaction comprise the following sequence of steps: base extension, first wash, image acquisition, and excision.
  • the first wash is performed using a fourth reagent, the first set of ports including a first port 1, a seventh port 7, and a second port 2 arranged in a predetermined rotational direction, the second set of ports including pressing
  • the third port 3, the eighth port 8 and the fourth port 4 are arranged in a predetermined rotation direction
  • the control device 302 is configured to: after the i) and before the ii), use the drive component 50 to make the seventh port 7 and the public
  • the port 71 is in communication such that the fourth reagent enters the reaction device 40 via the rotary valve 70 to effect a first wash of the first sequencing reaction; after the iii) and before the iv), the eighth port 8 and the public are utilized by the drive assembly 50
  • Port 71 is in communication such that the fourth reagent enters reaction unit 40 via rotary valve 70 to effect a first wash of the second sequencing reaction.
  • both the first sequencing reaction and the second sequencing reaction comprise the steps of performing the following sequence: base extension, first wash, second wash, image acquisition, and excision.
  • the first wash is performed using the fourth reagent
  • the second wash is performed using the fifth reagent
  • the first set of ports including the first port 1, the seventh port 7, and the ninth arranged in a predetermined rotational direction.
  • Port 9 and second port 2 the second group of ports includes a third port 3, an eighth port 8, a tenth port 10, and a fourth port 4, which are sequentially arranged in a predetermined rotation direction
  • the control device 302 is configured to: After the second washing, the seventh port 7 and the common port 71 are communicated by the driving assembly 50, and the fourth reagent is introduced into the reaction device 40 via the rotary valve 70 to achieve the first washing of the first sequencing reaction;
  • the ninth port 9 and the common port 71 are communicated by the drive assembly 50, and the fifth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect a second washing of the first sequencing reaction. ;
  • the eighth port 8 and the common port 71 are communicated by the drive assembly 50, and the fourth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect the first washing of the second sequencing reaction. ;
  • the tenth port 10 and the common port 71 are communicated by the drive assembly 50, and the fifth reagent is introduced into the reaction device 40 via the rotary valve 70 to effect a second washing of the second sequencing reaction. .
  • both the first sequencing reaction and the second sequencing reaction comprise the steps of performing the following sequence: base extension, image acquisition, excision, and capping.
  • the sixth reagent is used for capping
  • the first group of ports includes a first port 1, a second port 2, and an eleventh port 11 arranged in a predetermined rotational direction
  • the second group of ports includes pressing
  • the third port 3, the fourth port 4, and the twelfth port 12 are arranged in a predetermined rotation direction
  • the control device 302 is configured to: after the ii) and before the iii), make the eleventh port by using the driving component 50.
  • 11 is in communication with the common port 71 such that the sixth reagent enters the reaction device 40 via the rotary valve 70 to effect capping of the first sequencing reaction; after performing iv), the twelfth port 12 and the common port 71 are made by the drive assembly 50.
  • the sixth reagent enters the reaction device 40 via the rotary valve 70 to effect capping of the second sequencing reaction.
  • the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, and a third sequencing reaction performed sequentially, and the third sequencing reaction comprises the same steps 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, and the plurality of ports further include a third group of ports corresponding to the third sequencing reaction, and the third group of ports includes the first rotation direction according to a preset rotation direction.
  • the thirteenth port 13 and the fourteenth port 14, the first group port, the second group port and the third group port are arranged in a predetermined rotation direction, and the thirteenth port 13 is connected to the first reagent of the third sequencing reaction.
  • the fourteenth port 14 is connected to the second reagent, and the control device 302 is used to:
  • the thirteenth port 13 and the common port 71 are connected by the driving assembly 50, and the first reagent of the third sequencing reaction is introduced into the reaction device 40 through the rotary valve 70 to realize the alkali of the third sequencing reaction.
  • the fourteen port 14 and the common port 71 are communicated by the drive assembly 50, and the second reagent is introduced into the reaction device 40 via the rotary valve 70 to effect the ablation of the third sequencing reaction.
  • the sequencing reaction comprises a first sequencing reaction, a second sequencing reaction, a third sequencing reaction, and a fourth sequencing reaction, and the steps of the third sequencing reaction and the fourth sequencing reaction are the same as the first step.
  • the sequencing reaction or the second sequencing reaction is the same, the first reagent of the first sequencing reaction, the second sequencing reaction, the third sequencing reaction and the fourth sequencing reaction are different, and the plurality of ports further comprise a third corresponding to the third sequencing reaction.
  • the third group port includes a thirteenth port 13 and a fourteenth port 14 arranged in a predetermined rotation direction
  • the fourth group of ports includes a predetermined rotation direction
  • the fifteenth port 15 and the sixteenth port 16 are arranged, the first group port, the second group port, the third group port and the fourth group port are arranged in a preset rotation direction, and the thirteenth port 13 is connected to the third sequencing.
  • the first reagent of the reaction, the fourteenth port 14 is connected to the second reagent, the fifteenth port 15 is connected to the first reagent of the fourth sequencing reaction, the sixteenth port 16 is connected to the second reagent, and the control device 302 is used for:
  • the thirteenth port 13 and the common port 71 are connected by the driving assembly 50, and the first reagent of the third sequencing reaction is introduced into the reaction device 40 through the rotary valve 70 to realize the alkali of the third sequencing reaction.
  • the valve body assembly 29 includes two rotary valves 70 and two first valves
  • the reaction device 40 includes a first unit 41 and a second unit 42, the first unit 41 being connected to a common one of the rotary valves 70 Port 71, the second unit 42 is connected to a common port 71 of another rotary valve 70
  • the valve body assembly 29 includes a second valve 35, a third valve 36 and a fourth valve 37
  • the second valve 35 connects the two rotary valves 70 and a first reagent for sequencing reaction
  • a third valve 36 connects the two rotary valves 70 and a first reagent of the second sequencing reaction
  • a fourth valve 37 connects the second reagent and the two first valves, one for each first valve The second port 2 and the fourth port 4 of the rotary valve 70.
  • the valve body assembly 29 includes a fifth valve 38
  • the reaction device 40 includes a first unit 41 and a second unit 42
  • the fifth valve 38 connects the common port 71, the first unit 41, and the second unit 42.
  • an embodiment of the present invention provides a device 302 for controlling a sequence determination reaction, and the device 302 includes:
  • a storage device 304 configured to store data, where the data includes a computer executable program
  • the processor 306 is configured to execute a computer executable program, and the executing the computer executable program comprises the method of performing any of the above embodiments.
  • a computer readable storage medium for storing a program for execution by a computer, the program comprising the method of any of the above embodiments.
  • the computer readable storage medium may include read only memory, random access memory, magnetic or optical disks, and the like.
  • a "computer-readable storage medium” can be any apparatus that can contain, store, communicate, propagate, or transport a program for use in an instruction execution system, apparatus, or device, or in conjunction with such an instruction execution system, apparatus, or device.
  • each functional unit in each embodiment of the present invention may be integrated into one processing module, or each unit may exist physically separately, or two or more units may be integrated into one module.
  • the above integrated modules can be implemented in the form of hardware or in the form of software functional modules.
  • the integrated modules, if implemented in the form of software functional modules and sold or used as stand-alone products, may also be stored in a computer readable storage medium.

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Abstract

La présente invention concerne un procédé destiné à commander une réaction de séquençage, le procédé comprenant les étapes consistant : i) à permettre à un premier orifice de communiquer avec un orifice commun à l'aide d'un élément d'entraînement, de façon à permettre à un premier réactif d'une première réaction de séquençage d'entrer dans un dispositif de réaction par l'intermédiaire d'une vanne rotative et de réaliser une extension de base de la première réaction de séquençage; ii) à permettre à un deuxième orifice de communiquer avec l'orifice commun à l'aide d'un élément d'entraînement, de façon à permettre à un deuxième réactif d'entrer dans le dispositif de réaction par l'intermédiaire de la vanne rotative et de réaliser l'excision de la première réaction de séquençage; iii) à permettre à un troisième orifice de communiquer avec l'orifice commun à l'aide d'un élément d'entraînement, de façon à permettre à un premier réactif d'une deuxième réaction de séquençage d'entrer dans le dispositif de réaction par l'intermédiaire de la vanne rotative et de réaliser une extension de base de la deuxième réaction de séquençage; et iv) à permettre à un quatrième orifice de communiquer avec l'orifice commun à l'aide d'un élément d'entraînement, de façon à permettre à un deuxième réactif de la deuxième réaction de séquençage d'entrer dans le dispositif de réaction par l'intermédiaire de la vanne rotative et de réaliser l'excision de la deuxième réaction de séquençage. La présente invention concerne en outre un dispositif et un système de séquençage destinés à commander une réaction de séquençage.
PCT/CN2018/087458 2017-05-26 2018-05-18 Procédé, dispositif et système de commande d'une réaction de séquençage WO2018214816A1 (fr)

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EP1420197A1 (fr) * 2002-11-15 2004-05-19 Air Products And Chemicals, Inc. Soupape séquentielle rotative avec une plaque d'orifices flexible
CN105199949A (zh) * 2015-09-15 2015-12-30 深圳市瀚海基因生物科技有限公司 基因测序的流体控制装置
CN106635772A (zh) * 2015-11-03 2017-05-10 盛司潼 多通道测序反应小室及多通道测序反应装置
CN206553528U (zh) * 2017-02-22 2017-10-13 深圳市瀚海基因生物科技有限公司 序列测定控制系统

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