CN114410459A

CN114410459A - Gene sequencing device and sequencing method

Info

Publication number: CN114410459A
Application number: CN202210028085.9A
Authority: CN
Inventors: 林清进; 史蒂夫·德雷尔; 何荺; 伊戈尔·伊万诺夫; 田晖; 徐堃; 杨景灏
Original assignee: Anxuyuan Biotechnology Shenzhen Co ltd; Shenzhen Research Institute Tsinghua University
Current assignee: Anxuyuan Biotechnology Shenzhen Co ltd; Shenzhen Research Institute Tsinghua University
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-04-29

Abstract

The invention discloses a gene sequencing device and a sequencing method. This scheme is through solitary liquid way control unit to the first liquid way of storage unit and the chip that receives to and receive the second liquid way of chip and pump body and waste liquid recovery unit to control, set up an solitary liquid way control unit and pump body and recovery unit lug connection simultaneously, not through the third liquid way that receives the chip, and control it through liquid way control unit, thereby the washing work after the prewashing before the automatic completion chip is tested and the test is accomplished, compare in current gene sequencing device and have higher degree of automation, manual treatment's operation has been reduced to a great extent, avoid too much manual intervention to the influence that detects the accuracy nature, the technical requirement that further reduced the detection.

Description

Gene sequencing device and sequencing method

Technical Field

The application relates to the technical field of sequencing, in particular to a gene sequencing device and a sequencing method.

Background

The gene sequencing is a novel gene detection technology, and can analyze and determine gene sequences from blood, saliva, hair or mucous membranes, and predict the possibility of suffering from various diseases, individual behavior characteristics, behavior rationality and the like. The gene sequencing technology can lock the individual pathological gene and intervene or treat in advance. The gene sequencing technology is developed to the present, the corresponding gene sequencer also undergoes the change of covering the ground, the flux, the sequencing accuracy, the sequencing period, the reading length and the like are continuously promoted, the application is more and more extensive, and the most key part of the gene sequencing is mainly completed by a gene sequencing chip. In recent years, most of gene sequencing research is conducted around the development of gene chips, and gene sequencers for clinical or research use are mainly fluorescence detection sequencers or electrochemical detection sequencers, and in the testing process, professional technicians are required to perform complicated manual operations, so that the technical requirements are relatively high. While there have been attempts to reduce some manual operations by making changes to the structure of the sequencer, the improvement is less and it is necessary to reduce manual processing to a greater extent to improve the accuracy of automation.

Disclosure of Invention

The present application is directed to solving at least one of the problems in the prior art. Therefore, the application provides a gene sequencing device capable of reducing manual pretreatment to a greater extent.

In a first aspect of the present application, there is provided a gene sequencing apparatus comprising:

a storage unit for storing a reagent;

the device comprises a fixing unit, a sample injection unit and a solution cavity, wherein the fixing unit is used for fixing a chip to be tested, the chip to be tested comprises the sample injection unit and the solution cavity, the sample injection unit is used for inputting sample liquid into the solution cavity, and the solution cavity provides a space for the sample liquid to perform sequencing reaction;

the pump body and the waste liquid recovery unit are used for providing negative pressure and recovering the generated waste liquid;

a first liquid path, a second liquid path, a third liquid path and a liquid path control unit;

the first liquid path is used for conveying the reagent in the storage unit to the solution cavity;

the second liquid path is used for conveying the effluent of the solution cavity to the pump body and the waste liquid recovery unit;

the third liquid path is used for directly conveying the reagent in the liquid path control unit to the pump body and the waste liquid recovery unit;

the liquid path control unit is used for controlling the on-off of the first liquid path, the second liquid path and the third liquid path.

The gene sequencing device according to the embodiment of the application has at least the following beneficial effects:

this scheme is through solitary liquid way control unit to the first liquid way of storage unit and the chip that receives to and receive the second liquid way of chip and pump body and waste liquid recovery unit to control, set up an solitary liquid way control unit and pump body and recovery unit lug connection's third liquid way simultaneously, and control these liquid ways through liquid way control unit, thereby the washing work after automatic completion chip prewashing before the test and test completion, compare in current gene sequencing device has higher degree of automation, the operation of manual processing has been reduced to bigger degree ground, avoid too much manual intervention to detect the influence of accuracy nature, the technical requirement of detection has further been reduced.

In some embodiments of the present application, the pump body and the waste liquid recovery unit include:

a pump body for providing a negative pressure;

the waste liquid bottle is used for recovering the generated waste liquid;

the fourth liquid path is connected with the pump body and the waste liquid bottle and is connected with the second liquid path and the third liquid path;

and the pump body controller is used for controlling the on-off of the fourth liquid path and adjusting the flow direction in the pump body and the waste liquid recovery unit.

The fourth liquid path is connected with the second liquid path and the third liquid path, so that the pump body can utilize the negative pressure of the pump body to circulate the reagent and the sample liquid along the first liquid path and the third liquid path, and the operations of cleaning or reaction and the like are completed.

In some embodiments of the present application, the pump body controller includes a pump body control valve disposed on a fourth fluid path, a portion of the fourth fluid path between the pump body and the pump body control valve being in communication with the second fluid path and the third fluid path. The specific part in the fourth liquid path is selected to be communicated with the second liquid path and the third liquid path, so that the pump body control valve can be opened to communicate the pump body with the waste liquid bottle, and when other liquid paths are closed, the sample liquid or the reagent entering the pump body is directly discharged into the waste liquid bottle. It will be appreciated that where negative pressure is provided for reagent or sample fluid flow, the pump controller is located downstream of the reagent or sample fluid along the normal flow path. And by constructing in this way, the number of pipelines in the device is reduced, and the cost is reduced.

In some embodiments of the present application, the fluid path control unit includes:

the first control valve is used for controlling the on-off of the first liquid path;

the second control valve is used for controlling the on-off of the second liquid path;

and the third control valve is used for controlling the on-off of the third liquid path.

In the liquid path control unit, the on-off control of the first, second and third liquid paths is completed through three independent control valves, so that the control is simpler and more convenient.

In some embodiments of the present application, since the second and third fluid paths controlled by the second and third control valves both lead to the pump body and the waste liquid recovery unit, the second and third fluid paths may share a pipe at the end close to the pump body and the waste liquid recovery unit downstream in the flow direction thereof.

In some embodiments of the present application, the storage unit comprises a plurality of reagent bottles for holding different reagents; the liquid path control unit further comprises a plurality of reagent control valves, the reagent control valves correspond to the reagent bottles one to one and are used for controlling the connection and disconnection of the reagent bottles and the first liquid path and the third liquid path.

In some embodiments of the present application, the fluid path control unit further includes a reagent circulation line, the plurality of reagent control valves are connected in parallel to one end of the reagent circulation line, and the first control valve and the third control valve are connected in parallel to the other end of the reagent circulation line.

In some embodiments of the present application, the plurality of reagent bottles includes a buffer reagent bottle and a pure water reagent bottle. The pure water includes, but is not limited to, distilled water, deionized water, RO water, ultrapure water, and the like.

In some embodiments of the present application, the sample injection unit includes a plurality of sample injection ports, and the sample solution enters the solution cavity of the chip under test from the sample injection ports for reaction.

In some embodiments of the present application, the sample injection unit includes a plurality of sample injection ports, and different types of sample liquids enter the solution cavity of the chip under test through different sample injection ports for reaction.

In some embodiments of this application, gene sequencing device still includes actuating mechanism, and actuating mechanism is located on removing the seat including removing the seat, is fixed with the fixed unit that receives the chip, removes the seat and drives and receive the chip and remove to improve the degree of automation of sequencing.

In some embodiments of the present application, the driving mechanism further includes a supporting frame, a motor, and a positioning device, the moving seat is disposed on the supporting frame, a rotating shaft of the motor is connected to the linkage device of the moving seat, so as to control the moving seat to move on the supporting frame, and the positioning device positions a relative position of the moving seat on the supporting frame.

In some embodiments of the present application, the motor is a stepper motor.

In some embodiments of the present application, the gene sequencing apparatus further comprises a heater for maintaining the chip under test at a set temperature. The set temperature is the specific temperature required by normal running of the sequencing reaction in the solution cavity of the chip to be tested, and the stable running of the sequencing reaction is ensured by controlling the temperature.

In some embodiments of the present application, the heater is disposed below the fixing unit, and can be in full contact with the chip to be tested, so as to effectively maintain the reaction temperature and ensure efficient sequencing reaction.

In some embodiments of the present application, the gene sequencing apparatus further comprises an identification unit for identifying the reagent and/or the test chip.

In some embodiments of the application, the identification unit is a code scanning module, and specifically can be a code scanning module capable of identifying, for example, a two-dimensional code or a bar code, and identifies the reagent and/or the identification code on the chip to be detected, and then transmits the identification code to the main board control unit, so that the normal operation of the whole sequencing process is ensured, and the error in the manual reading process is avoided.

In some embodiments of the present application, the identification unit has a USB or serial interface, and is disposed on an outer surface of the gene sequencing apparatus.

In some embodiments of the present application, the control motherboard unit is a multilayer circuit board, and includes a single chip, a temperature sensor control circuit, an input/output interface, a valve control circuit, a temperature control circuit, a pump control circuit, a dc motor control circuit, a stepping motor control circuit, and the like.

In some embodiments of the present application, the temperature sensor is used to monitor the temperature inside the chip under test, ensuring that the temperature of the heater is maintained in a normal state.

In a second aspect of the present application, there is provided a sequencing method using the above gene sequencing apparatus, comprising performing the following tests before sequencing:

testing the fitting degree: detecting the fitting degree between the tested chip and the fixing unit;

wetting and wet testing: after the storage unit outputs buffer solution to the solution cavity for lubrication, detecting whether the nanopore of the chip to be detected is filled with the buffer solution;

film formation and film formation test: after the sample introduction unit inputs lipid sample liquid into the solution cavity, the storage unit outputs buffer liquid to the solution cavity for thinning and washing, and the thinning and washing are repeated for a plurality of times to detect whether a lipid covering layer is formed on the surface of the nanopore;

and (3) perforation and perforation test: and after the sample injection unit inputs sample liquid to be detected into the solution cavity, detecting whether the lipid covering layer is perforated or not and forming a nanopore.

In some embodiments of the present application, after the above-described pre-sequencing test is completed and passed, the specific method of sequencing is: and after the oligonucleotide sample liquid is input into the solution cavity by the sample injection unit, detecting the electrochemical signal of the nanopore.

In some embodiments of the present application, the lipid-like liquid is an organic solution of lipids.

In some embodiments of the present application, the organic solvent of the lipid sample solution includes a higher aliphatic hydrocarbon, such as a saturated aliphatic hydrocarbon having 8 or more carbon atoms, further such as a linear or branched alkane having 8 to 20 carbon atoms, such as a higher alkane such as tridecane, hexadecane, and octane.

In some embodiments herein, the volume ratio of higher alkane to octane is 1: (1-3).

In some embodiments of the present application, the lipid is selected from at least one of diphytanoylphosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, dioleoyl phosphatidylmethyl ester, dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin.

In some embodiments of the present application, the concentration of the lipid in the lipid sample solution is 1-100 mg/mL, further 5-50 mg/mL, 5-20 mg/mL, or about 10 mg/mL.

In some embodiments of the present application, the lipid-like liquid further comprises cholesterol.

In some embodiments of the present application, the total concentration of lipid and cholesterol in the lipid-like liquid is 1-100 mg/mL.

In some embodiments of the present application, the molar ratio of lipid to cholesterol is about 1: 1.

in some embodiments of the present application, the buffer is a buffered solution of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and a chloride salt.

In some embodiments of the present application, the chloride salt is potassium chloride.

In some embodiments of the present application, the molar ratio of HEPES to chloride salt is 1: (5-20).

In some embodiments of the present application, the buffer is a mixture of 0.3M KCl and 0.02M HEPES (pH7.5), and the lipid-like solution is an equal volume of 10mg/mL of a mixture of tridecane and octane of 1, 2-di-o-phytanyl-sn-glycero-3-phosphocholine phospholipid (DOPHPC).

In some embodiments of the present application, the lipid-like solution is a 10mg/mL equal volume mixture of hexadecane and octane of 1, 2-di-o-phytanyl-sn-glycerol-3-phosphocholine phospholipid (DOPhPc).

In some embodiments of the present application, the lipid-like solution is DOPhPc at 10mg/mL and cholesterol in a 1: volume ratio of hexadecane to octane of 1: 3, and (b).

In some embodiments of the present application, the buffer is a mixture of 0.15M KCl and 0.02M HEPES (ph 7.5).

In some embodiments of the present application, the oligonucleotides in the oligonucleotide sample are comprised of nucleotide analogs.

In some embodiments of the present application, the fit test determines the fit between the chip under test and the fixing unit by detecting electrical signals between the fixing unit and the chip under test.

In some embodiments of this application, four corners that correspond in the fixed unit and receive the chip are provided with elastic component (for example spring), receive four azimuths that chip and elastic component correspond and be provided with the stitch, can realize electric connection with the socket that corresponds on the control mainboard, to control mainboard transmission signal of telecommunication. When the pressing force of the elastic component reaches a preset value, namely the preset fit degree between the tested chip and the control mainboard is reached, the tested chip transmits an electric signal to the control panel, and the control mainboard detects that the electric signal can be read normally; if the electrical signal cannot be read normally, it indicates that the adhesion between the tested chip and the fixing unit is in problem.

In some embodiments of the present application, the thinning and rinsing in the film formation test includes a first cover layer thinning, a second cover layer thinning, a third cover layer thinning, and a fourth cover layer thinning, and a capacitance value between two sides of the sample liquid cover layer is detected after the fourth cover layer thinning, and whether the lipid cover layer is formed is determined according to a magnitude of the capacitance value. Wherein the first, second, third and fourth cover layer thinning differ in the buffer input speed.

Additional aspects and advantages of the present application 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 the present application.

Drawings

FIG. 1 is a schematic diagram showing the overall structure of a gene sequencing apparatus in one embodiment of the present application.

FIG. 2 is a partial schematic view of the gene sequencing apparatus shown in FIG. 1 of the present application.

FIGS. 3 to 4 are flowcharts of sequencing using the gene sequencing apparatus of the present application.

Reference numerals: the device comprises a storage unit 110, a first reagent bottle 111, a second reagent bottle 112, a reagent bottle holder 113, a fixing unit 120, a chip 130 to be tested, a solution cavity 131, a solution cavity input end 131-1, a solution cavity output end 131-2, a first sample inlet 132-1, a second sample inlet 132-2, a third sample inlet 132-3, a first sample inlet control valve 133-1, a second sample inlet control valve 133-2, a third sample inlet control valve 133-3, a first sample inlet channel 134-1, a second sample inlet channel 134-2, a third sample inlet channel 134-3, a fourth sample inlet 135, a chip outlet 136, a solution cavity output control valve 137, a first segment 137-1 of a solution cavity output flow channel, a second segment 137-2 of a solution cavity output flow channel, a chip input and output direct control valve 138, a first segment 138-1 of a chip input and output flow channel, a second segment 138-2 of a chip output flow channel, a first segment 137-2, a second segment, a chip input and a second segment, A chip input/output channel section 138-2, a reagent channel 139, a liquid path control unit 140, a first control valve 141, a second control valve 142, a third control valve 143, a first reagent control valve 144, a second reagent control valve 145, a first reagent input channel 146, a second reagent input channel 147, a reagent circulation pipeline 148, a first liquid path 151, a second liquid path 152, a third liquid path 153, a pump body and waste liquid recovery unit 160, a pump body 161, a waste liquid bottle 162, a pump body control valve 163, a fourth liquid path section 164-1, a fourth liquid path section 164-2, a control main board 200, an identification unit 210, a heater 220, a motor 230, a DC motor 240, a first sample injection control valve switch 241, a second sample injection control valve switch 242, a third sample injection control valve switch 243, a fourth sample injection control valve switch 244, a solution cavity output control valve switch 245, a driving mechanism 250, a bottom cover 310, a first sample injection control valve switch 241, a second sample injection control valve switch 242, a third sample injection control valve switch 242, a fourth sample injection control valve switch 244, a solution cavity output control valve switch 245, a driving mechanism 250, a driving mechanism, a, Rear cover 320, top cover 330, front cover 340, right cover 350, personal computer 410, display 420, keyboard/mouse 430, router 440, server 450.

Detailed Description

The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.

The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.

In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," 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 present application. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Referring to fig. 1, the present application provides a gene sequencing apparatus including a storage unit 110, a fixing unit 120, a pump body and waste liquid recovery unit 160, a liquid path control unit 140, and a plurality of liquid paths. The storage unit 110 is used to store reagents. The fixing unit 120 is used for fixing the chip 130 to be tested, the chip 130 to be tested includes a sample introduction unit and a solution cavity 131, the sample introduction unit is used for inputting sample liquid into the solution cavity 131, and the solution cavity 131 provides a space for the sample liquid to perform a sequencing reaction. The pump body and recovery unit 160 is used for providing negative pressure, so that the reagent, the sample liquid and the like entering the liquid path flow under the action of the negative pressure, and meanwhile, the pump body and recovery unit 160 is also used for recovering waste liquid generated after the reagent and the sample liquid flow and react. The plurality of liquid paths include a first liquid path 151, a second liquid path 152, and a third liquid path 153, wherein the first liquid path 151 is used for conveying the reagent in the storage unit 110 to the solution chamber 131, the second liquid path 152 is used for conveying the effluent liquid from the solution chamber 131 to the pump body and the effluent liquid recovery unit 160, and the third liquid path 153 is used for directly conveying the reagent in the liquid path control unit 140 to the pump body and the effluent liquid recovery unit 160 without passing through the chip 120 to be tested. It can be understood that, since the liquid path control unit 140 controls the on/off of the first liquid path 151, wherein a plurality of pipelines are involved, the third liquid path 153 connects the internal pipelines with the pump body and the waste liquid recovery unit 160, so as to avoid the problem that the liquid possibly existing in the liquid path control unit 140 cannot be completely discharged under the negative pressure effect due to the long path in the subsequent cleaning process. The liquid path control unit 140 is configured to control on/off of the first liquid path 151, the second liquid path 152, and the third liquid path 153, and the on/off meaning includes controlling on/off of the liquid paths. It can be understood that due to the difference of the flow sequence of the plurality of liquid paths, the liquid path control unit 140 can independently control the on/off of any one of the plurality of liquid paths without affecting the other liquid paths.

In some embodiments, the pump and waste fluid recovery unit 160 includes a pump 161, a waste fluid bottle 162, a pump controller, and a fourth fluid path. The pump body 161 is used to provide negative pressure for the gene sequencing apparatus, so that the reagents, sample liquids, etc. entering the liquid path flow in a preset sequence under the action of the negative pressure. The waste liquid bottle 162 is used for collecting waste liquid generated after flowing and reacting, which flows into the pump body and the waste liquid recovery unit 160. The fourth liquid path connects the pump body 161 and the waste liquid bottle 162, and is connected to the second liquid path 152 and the third liquid path 153. The pump body controller controls the on-off of the fourth fluid path, so that the flow direction in the pump body and the waste fluid recovery unit 160 can be adjusted, the flow direction refers to the flow direction of fluid entering the pump body and the waste fluid recovery unit 160, and the flow direction of the fluid including air, reagents, sample liquid and waste fluid entering the pump body and the waste fluid recovery unit 160 during the preparation stage of pretreatment, the experiment process and the cleaning after the experiment is completed is directly sucked into the pump body 161 or into the waste fluid bottle 162 under the negative pressure effect. In some of these embodiments, the pump body controller includes a pump body control valve 163, and the pump body control valve 163 is disposed on the fourth fluid path to divide the fourth fluid path into a fourth fluid path segment 164-1 and a fourth fluid path segment 164-2 between the pump body 161 and the pump body control valve 163, and the fourth fluid path segment 164-1 is in communication with the second fluid path 152 and the third fluid path 153.

In some of these specific embodiments, the fluid path control unit 140 includes a first control valve 141, a second control valve 142, and a third control valve 143. The first control valve 141 controls the on/off of the first fluid path 151; when the first liquid path 151 is connected, the reagent in the storage unit 110 can be transferred to the solution chamber 131, and when the first liquid path 151 is disconnected, the reagent in the storage unit 110 cannot be transferred to the solution chamber 131. The second control valve 142 controls the on-off of the second liquid path 152; when the second liquid path 152 is connected, the output liquid from the solution chamber 131 can be transferred to the pump body and the waste liquid recovery unit 160, and when the second liquid path 152 is disconnected, the output liquid from the solution chamber 131 cannot be transferred to the pump body and the waste liquid recovery unit 160. The third control valve 143 controls the on/off of the third fluid passage 153; when the third fluid path 153 is connected, the reagent in the storage unit 110 can be directly transported to the pump and the waste liquid recovery unit 160 without passing through the chip 130 to be tested, and when the third fluid path 153 is disconnected, the reagent in the storage unit 110 cannot be directly transported to the pump and the waste liquid recovery unit 160 without passing through the chip 130 to be tested. The liquid path control unit 140 controls the on/off of the first liquid path 151, the second liquid path 152 and the third liquid path 153 through three separate control valves, which is simpler and more convenient. In some embodiments, since the second and third

liquid paths

152 and 153 controlled by the second and third control valves 142 and 143 lead to the pump body and the waste liquid recovery unit, the second and third

liquid paths

152 and 153 may share a pipe at the downstream end of the flow direction near the pump body and the waste liquid recovery unit 160, that is, the second and third control valves 142 and 143 and the pump body and the waste liquid recovery unit 160 are connected by a three-way pipe, the pump body and the waste liquid recovery unit are connected by the three-way pipe by connecting the second control valve 142 to the second liquid path 152 and connecting the chip outlet 136, and connecting the third control valve 143 to the internal liquid path of the liquid path control unit 140 and connecting the internal liquid path to the storage unit 110. Further connected between the second control valve 142, the third control valve 143 and the pump body control valve 163 through a three-way flow pipe. Here, the downstream is a lower direction in which the reagent or the sample solution flows, starting from the first reagent bottle 111 or the second reagent bottle 112, and ending at the pump body and the waste liquid recovery unit 160.

In some embodiments, the storage unit 110 includes a plurality of reagent bottles, and the plurality of reagent bottles contain different reagents. Meanwhile, the liquid path control unit 140 further includes a plurality of reagent control valves, which are in one-to-one correspondence with the plurality of reagent bottles and are used for controlling the on/off of the plurality of reagent bottles with the first liquid path 151 and the third liquid path 153. In some embodiments, the storage unit 110 includes a first reagent bottle 111 and a second reagent bottle 112, the first reagent bottle 111 and the second reagent bottle 112 contain different reagents, for example, a buffer solution in the first reagent bottle 111, and pure water in the second reagent bottle 112. The buffer may be of the type that is optionally capable of being used for pre-washing of the chip under test, and in some preferred embodiments includes chloride ion and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES). Preferably, the chloride ion may be present in the form of potassium chloride. Further, the molar ratio of HEPES to chloride salt in the buffer was 1: (5 to 20), for example, the buffer solution is a mixture of 0.3M KCl and 0.02M HEPES, a mixture of 0.15M KCl and 0.02M HEPES (pH7.5), or the like. The pure water may be at least one of distilled water, deionized water, RO water, ultrapure water, and the like. In some embodiments, the fluid path control unit 140 further comprises a first reagent control valve 144 and a second reagent control valve 145, the first reagent bottle 111 and the first reagent control valve 144 are connected by a first reagent input channel 146, and the second reagent bottle 112 and the second reagent control valve 145 are connected by a second reagent input channel 147. In some embodiments, the first control valve 141 and the third control valve 143 are connected to the first reagent control valve 144 and the second reagent control valve 145 through separate channels, and thus are connected to the first fluid path 151 and the third fluid path 153. In some preferred embodiments, the fluid path control unit further comprises a reagent flow line 148, the first reagent control valve 144 and the second reagent control valve 145 are connected in parallel at one end of the reagent flow line 148, and the first control valve 141 and the third control valve 143 are connected in parallel at the other end of the reagent flow line 148.

Referring to FIG. 1 in conjunction with FIG. 2, in some embodiments, the gene sequencing apparatus further comprises an identification unit 210, a heater 220. The identification unit 210 is used for identifying the reagent in the storage unit 110 and/or the chip under test 130. The heater 220 is used to maintain the chip 130 under test at a set temperature, wherein the set temperature may be a specific temperature required for the sequencing reaction to normally proceed in the solution chamber 131 of the chip 130 under test, and the temperature is controlled to ensure the sequencing reaction to proceed stably. In some specific embodiments, the identification unit 210 is a code scanning module, and may specifically be a code scanning module capable of identifying, for example, a two-dimensional code or a bar code, and by identifying the reagent and/or the identification code on the chip 130 under test, the normal operation of the whole sequencing process is ensured, and errors in the manual reading process are avoided. In some embodiments, the storage unit 110 further includes a reagent bottle holder 113, and a plurality of reagent bottles, such as the first reagent bottle 111 and the second reagent bottle 112, are mounted on the reagent bottle holder 113. Meanwhile, the recognition unit 210 may also be mounted on the reagent bottle holder 113. In some embodiments, the heater 220 is disposed below the fixing unit 120, and can be in sufficient contact with the chip 130 to be tested, so as to effectively maintain the reaction temperature and ensure efficient sequencing reaction.

In some embodiments, the gene sequencing apparatus further comprises a driving mechanism 250, and the relative movement of the tested chip 130 in the gene sequencing apparatus is adjusted by the driving mechanism 250, so as to improve the automation degree of sequencing. In some embodiments, the driving mechanism 250 includes a movable base on which the fixing unit 120 fixing the chip 130 under test is located, and the movable base drives the chip 130 under test to move. In some of these embodiments, the drive mechanism 250 further includes a motor 230, and the motor 230 may be a stepper motor. It is understood that the driving mechanism 250 may further include a positioning device, a sensor, etc. for the purpose of further improving the automatic positioning and close fitting of the chip 130 under test. In some embodiments, the fluid path control unit 140 may be mounted to the driving mechanism 250 to facilitate the switching and flow direction control of the fluid path.

In some embodiments, the gene sequencing apparatus further comprises a control board 200, and the control board 200 receives a sequencing program command executed by the personal computer 410 for controlling the actual operation of the gene sequencing apparatus. In some embodiments, the control board 200 is a multi-layer circuit board and includes a single chip, an input/output interface, a fixing unit 120, a valve control circuit, a vacuum pump control circuit, a dc motor control circuit, a code scanning module control circuit, a buffer, and the like. The fixing unit 120 is provided with a probe for connecting the chip 130 to be tested and the control motherboard 200, and transmitting an electrical signal to perform a test of the degree of adhesion. The driving mechanism 250 is driven by the motor 230 to control the movement of the fluid path and the dc motor 240 in the gene sequencing apparatus, and fix and closely attach to the tested chip 130 and the control motherboard 200.

In some embodiments, the control board 200 of the gene sequencing apparatus is connected to the pc 410 through USB2.0 and USB3.0 interfaces, the pc 410 is connected to the external keyboard/mouse 430 through the USB2.0 interface, the external display 420 through the HDMI interface, and the external router 440 through the LAN network interface, and the router 440 can be further connected to the external server 450.

In some embodiments, the gene sequencing apparatus further comprises a base 300, a bottom cover 310, a rear cover 320, an upper cover 330, a front cover 340, and a right cover 350, and the remaining components are mounted in the space defined by the components. Specifically, the base 300 is used for carrying the components of the gene sequencing device, including the control main board 200, the pump body 161, the pump body controller 163, the motor 230, and the like. In some embodiments, the waste bottle 162 is mounted on the right cap 350, the bottom cap 310, the rear cap 320, the front cap 340, the right cap 350, and the reagent bottle holder 113 are mounted on the base 330, and the upper cap 330 is assembled with the rear cap 320, the front cap 340, the right cap 350, and the reagent bottle holder 113.

In some embodiments, the dut 130 is a disposable material for testing gene samples or specimens. In some embodiments, the chip under test 130 comprises a sample injection unit, a solution chamber 131, a fourth sample injection port 135, and a chip outlet 136. The sample introduction unit comprises a sample introduction port, a sample introduction control valve and a sample introduction channel, in some specific embodiments, the sample introduction unit comprises a first sample introduction port 132-1, a second sample introduction port 132-2 and a third sample introduction port 132-3 which are sequentially arranged, the three sample introduction ports are respectively communicated with a corresponding first sample introduction channel 134-1, a corresponding second sample introduction channel 134-2 and a corresponding third sample introduction channel 134-3 through a corresponding first sample introduction control valve 133-1, a corresponding second sample introduction control valve 133-2 and a corresponding third sample introduction control valve 133-3, and the first sample introduction channel 134-1, the second sample introduction channel 134-2 and the corresponding third sample introduction channel 134-3 are mutually connected in parallel to a solution cavity input end 131-1 of the solution cavity 131. The solution chamber 131 comprises a solution chamber output end 131-2 communicated with the outside, in addition to the solution chamber input end 131-1, and the conventional sample introduction or reagent introduction sequence is that the sample enters the solution chamber 131 from the solution chamber input end 131-1 and then is discharged through the solution chamber output end 131-2. The fourth sample inlet 135 (i.e., VIN) and the chip outlet 136 (i.e., VOUT) include a flow path through the solution chamber 131 and a flow path not through the solution chamber 131. The flow path not passing through the solution chamber 131 includes a chip input/output flow path divided into a chip input/output flow path first section 138-1 and a chip input/output flow path second section 138-2 by a chip input/output straight-through control valve 138(VBPY control valve). The flow path passing through the solution cavity 131 passes through the solution cavity 131 from the fourth sample inlet 135 through the reagent flow channel 139 through the solution cavity input end 131-1, reaches the chip outlet 136 at the solution cavity output end 131-2 through the solution cavity output flow channel, and is divided into a solution cavity output flow channel section 137-1 and a solution cavity output flow channel section 137-2 through the solution cavity output control valve 137. The first liquid path 151 is connected to the solution cavity 131 of the tested chip 130 through the fourth sample inlet 135, and the second liquid path 152 is connected to the solution cavity 131 of the tested chip 130 through the chip outlet 136. In some embodiments, the first sample injection control valve 133-1, the second sample injection control valve 133-2, the third sample injection control valve 133-3, the solution chamber output control valve 137, and the chip input/output through control valve 138 are controlled by a first sample injection control valve switch 241, a second sample injection control valve switch 242, a third sample injection control valve switch 243, a fourth sample injection control valve switch 244, and a solution chamber output control valve switch 245 in the dc motor 240.

The application also provides a sequencing method applying the gene sequencing device, which comprises the following tests before sequencing:

testing the fitting degree: detecting the fit degree between the tested chip 130 and the fixing unit 120;

wetting and wet testing: after the storage unit 110 outputs the buffer solution to the solution cavity 131 for lubrication, whether the nanopore of the tested chip 130 is filled with the buffer solution is detected;

film formation and film formation test: after the sample introduction unit inputs lipid sample liquid into the solution cavity 131, the buffer liquid is output to the solution cavity 131 from the storage unit 110 for thinning and washing, and the thinning and washing are repeated for a plurality of times to detect whether a lipid covering layer is formed on the surface of the nanopore;

and (3) perforation and perforation test: and after the sample injection unit inputs sample liquid to be detected into the solution cavity, detecting whether the lipid covering layer is perforated or not.

In some specific embodiments, after the test before sequencing is completed and passed, the specific method for sequencing is: and after the oligonucleotide sample liquid is input into the solution cavity by the sample injection unit, detecting the electrochemical signal of the nanopore.

In some specific embodiments, the lipid sample solution is an organic solution of lipids, wherein the organic solvent of the lipid sample solution includes higher aliphatic hydrocarbons, such as saturated aliphatic hydrocarbons with carbon atoms of 8 or more, further such as linear or branched alkanes with carbon atoms of 8-20, for example tridecane and octane. Preferably, the volume ratio of tridecane to octane is 1: (1-3). In some of these embodiments, the lipid is selected from at least one of diphytanoylphosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, dioleoyl phosphatidylmethyl ester, dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin. In some embodiments, the concentration of the lipid in the lipid sample solution is 1-100 mg/mL, further 5-50 mg/mL, 5-20 mg/mL, or about 10 mg/mL. In some embodiments, the lipid-like liquid further comprises cholesterol, the total concentration of lipid and cholesterol in the lipid-like liquid is 1-100 mg/mL, and the molar ratio of lipid to cholesterol is about 1: 1. in some specific embodiments, the buffer is a buffered solution of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and a chloride salt, which may be potassium chloride. Preferably, the molar ratio of HEPES to chloride salt is 1: (5-20). In some specific embodiments, the buffer is a mixture of 0.3M KCl and 0.02M HEPES (pH7.5), and the lipid-like solution is a mixture of 10mg/mL of 1, 2-di-o-phytanyl-sn-glycerol-3-phosphocholine phospholipid (DOPHPC) tridecane and octane at equal volumes; or the lipid sample solution is 10mg/mL mixed solution of hexadecane and octane of 1, 2-di-o-phytanyl-sn-glycerol-3-phosphocholine phospholipid (DOPHPC) with equal volume; or the lipid sample solution is 10mg/mL DOPHPC and cholesterol in a molar ratio of 1: volume ratio of hexadecane to octane of 1: 3, and (b). In some embodiments, the buffer is a mixture of 0.15MKCl and 0.02M HEPES (pH 7.5). In some embodiments, the oligonucleotide in the oligonucleotide sample is comprised of dNTP analogs.

In some embodiments, the fit test determines the fit between the tested chip 130 and the fixing unit 120 by detecting electrical signals between the fixing unit 120 and the tested chip 130.

In some embodiments, the fixing unit 120 is provided with elastic elements (e.g., springs) corresponding to four corners of the chip 130 under test, and the chip 130 under test is provided with pins corresponding to the elastic elements in four directions, so as to be electrically connected to corresponding sockets on the control motherboard 200 and transmit electrical signals to the control motherboard 200. When the pressing force of the elastic component reaches a predetermined value, that is, a preset adhesion degree is achieved between the chip 130 to be tested and the control mainboard 200, the chip 130 to be tested transmits an electric signal to the control mainboard 200, and the control mainboard 200 can normally read the electric signal when detecting the electric signal; if the electrical signal cannot be read normally, it indicates that the adhesion between the tested chip 130 and the fixing unit 120 is bad.

Sequencing methods referring to FIGS. 3 and 4, in conjunction with FIG. 1, are further illustrated as follows:

referring to fig. 3, when the user operates the gene sequencing apparatus to perform sequencing, if the system is not started, the power is turned on first to start the apparatus first, and when the system is started, the initialization including the heater 220, the motor 230 (stepping motor), the dc motor 240, the pump body 161 (vacuum pump), the fluid circuit control unit 140, the identification unit 210 (code scanning module), and the pump body control valve 163 is automatically performed, and then the personal computer 410 is waited to be connected to perform further operations.

After the personal computer 410 is connected, if a new reagent bottle needs to be installed, the bar code on the reagent bottle is scanned by the code scanning module and then installed on the corresponding reagent bottle position on the reagent bottle rack 113; next, the barcode on the chip 130 to be tested is scanned by the barcode scanning module and then placed on the fixing unit 120 (socket of the chip to be tested), then the command is sent to the personal computer 410 through the keyboard/mouse 430 to execute the sequencing step, after the sequencing is finished, the personal computer 410 displays the sequencing result through the display 420, and stores the sequencing data in the computer hard disk, and if necessary, the router 440 can also upload the sequencing data report to the server 450 for subsequent data analysis or centralized management. And after the sequencing is finished, taking the tested chip 130 out of the gene sequencing device, if the sequencing is to be continued, putting a new tested chip on the gene sequencing device again, and if the sequencing is not to be continued, quitting the sequencing program and shutting down to finish the operation.

In some embodiments of the present application, referring to fig. 4, before the sequencing starts, the calibration parameters of the motor 230 (stepping motor) pre-stored in the system are read, the stepping motor is rotated according to the calibration parameters, and then the related parameters on the chip 130 to be tested are read, if the read parameters are normal, the chip 130 to be tested passes the fit test with the control motherboard 200, otherwise, the chip 230 to be tested is determined as a defective product, and the sequencing step is ended.

The specific detection of the parameters may be as follows:

testing the fitting degree: elastic components (e.g., springs) are disposed at four corners of the movable seat in the driving mechanism 250, and pins are disposed at four positions of the fixing unit 120, so that the tested chip 130 can be electrically connected to the control motherboard 200 to transmit electrical signals to the control motherboard 200. When the distance that removes the seat and push down reaches predetermined numerical value, reach predetermined laminating degree promptly between chip 130 and the control mainboard 200 that receives, receive chip 130 to transmit the signal of telecommunication to control mainboard 200, control mainboard 200 detects the signal of telecommunication and can normally read.

Wetting and wet testing: after the dut 130 passes the conformity test, a wet test is first performed on the dut 130, before the wet measurement is performed, air in the whole liquid path is exhausted, the pump body 161 and the corresponding valves are opened to allow the buffer solution to pass through the first reagent input channel 146 from the first reagent bottle 111, the first reagent control valve 144, the first control valve 141 from the reagent circulation pipeline 148, the fourth sample inlet 135 along the first liquid path 151, the chip input/output channel section 138-1 through the chip input/output through control valve 138 and the chip input/output channel section 138-2 through the chip outlet 136 along the second liquid path 152 through the second control valve 142 (the third control valve 143 is in a closed state), and finally the buffer solution enters the pump body 161 in the fourth liquid path section 164-1, and the air is exhausted from the whole liquid path and filled with the buffer solution through the whole circulation sequence, and then the wet measurement is performed.

In the wet test, the pump body 161 and the corresponding valves are first opened to allow the buffer solution to flow from the first reagent bottle 111 through the first reagent input channel 146 and through the first reagent control valve 144, through the first control valve 141 by the reagent circulation line 148, along the first fluid path 151 through the fourth sample inlet 135 into the chip 130 under test, through the reagent flow channel 139 through the solution chamber input end 131-1 into the solution chamber 131, through the solution chamber output end 131-2 through the solution chamber output flow channel segment 137-1 and the solution chamber output control valve 137 and the solution chamber output flow channel segment 137-2, along the second fluid path 152 through the second control valve 142 by the chip outlet 136 (the third control valve 143 is in a closed state), and finally into the pump body 161 in the fourth fluid path segment 164-1 to allow the buffer solution to flow through the solution chamber, then, a wet test is performed to detect whether a lipid coating is formed on the surface of the nanopore in the solution chamber 131 of the chip 130 under test. If the wet test is passed, the action of film formation is carried out, otherwise, the buffer solution is continuously added, and the wetting work is carried out again until the wet test is passed.

It should be noted that during the above-mentioned wet and wet test and the following preparation of sequencing, buffer solution, pure water and/or reagent will continuously enter the pump body 161, and if the pump body 161 is full or is not enough to perform the next operation, the buffer solution, pure water and/or reagent in the pump body needs to be discharged to the waste liquid bottle 162. Specifically, all valves in the liquid path control unit 140 are closed, the pump body control valve 163 of the pump body and the waste liquid recovery unit is opened, and the liquid in the pump body 161 is discharged to the waste liquid bottle 162 along the fourth liquid path segment 164-1 and the fourth liquid path segment 164-2.

Before the chip 130 to be tested is subjected to the wet testing, the parameters related to the film formation are set. The first sample injection control valve 133-1, the chip outlet 136, the second control valve 142, the pump body 161, and the like are opened, so that the first sample liquid enters the solution chamber 131 of the tested chip 130 from the first sample injection port 132-1 through the first sample injection channel 134-1 and the solution chamber input end 131-1.

After the tested chip 130 is filled with the first sample solution, the sequence step continues with the thinning operation. The sequencing program first opens the pump body 161 and corresponding valves to let the buffer solution flow from the first reagent bottle 111 through the first reagent input channel 146 and through the first reagent control valve 144, through the first control valve 141 by the reagent circulation line 148, along the first fluid path 151 through the fourth sample inlet 135 into the chip 130 under test, through the reagent flow channel 139 through the solution chamber input end 131-1 into the solution chamber 131, through the solution chamber output end 131-2 through the solution chamber output flow channel segment 137-1 and the solution chamber output control valve 137 and the solution chamber output flow channel segment 137-2, along the second fluid path 152 through the second control valve 142 by the chip outlet 136 (the third control valve 143 is in a closed state), and finally into the pump body 161 in the fourth fluid path segment 164-1, so that the buffer solution flows through the solution chamber 131 to perform a film-forming thinning operation on the tested chip 130. The film thinning operation may be performed multiple times, and the sequencing process may then perform the film test. It is understood that the first sample is the lipid-like fluid described above.

If the film forming test is passed, the action of advancing the specimen is continued in order to run the perforating action, otherwise the test step is ended.

After the film formation test is performed, the sequencing program opens the second sample injection control valve 133-2, the chip outlet 136, the second control valve 142, the pump body 161, and the like, so as to allow the second sample solution to enter the solution cavity 131 of the tested chip 130 from the second sample injection port 132-2 via the second sample injection channel 134-2 and the solution cavity input end 131-1, and then closes the second sample injection control valve switch 242 controlling the second sample injection control valve 133-2, the control chip outlet 136, the pump body 161, and the like, thereby performing the perforation and perforation test, detecting whether the lipid coating is perforated and forming the nanopore.

If the puncture test is passed, the action of proceeding to the third sample liquid is continued so as to run the nanopore sequencing action, otherwise the testing step is ended. Specifically, after the perforation test, the sequencing program opens the third sample inlet control valve 133-3, the chip outlet 136, the second control valve 142, the pump body 161, and the like, so as to allow the third sample liquid to enter the solution cavity 131 of the chip 130 under test from the third sample inlet 132-3 via the third sample inlet channel 134-3 and the solution cavity input end 131-1, and then closes the third sample inlet control valve switch 243 controlling the third sample inlet control valve 133-3, the control chip outlet 136, the pump body 16, and the like, so as to perform the subsequent nanopore gene sequencing action. It will be appreciated that the third sample, which may be a standard or test sample for a single-stranded nucleic acid molecule, may optionally be modified to provide a suitable speed of pore passage, e.g., an oligonucleotide sample formed from a nucleotide analogue such as dNTP, dNMP, etc.

Referring to fig. 1 and fig. 2, after the gene sequencing operation is completed, if the chip 130 to be tested is to be cleaned, the pump body 161 and the corresponding valves are first opened, so that pure water flows from the second reagent bottle 112 through the second reagent input channel 147, through the second reagent control valve 145, through the reagent circulation line 148, through the first control valve 141, into the first fluid path 151, through the fourth sample inlet 135, through the reagent flow channel 139, into the solution chamber 131, then through the solution chamber output flow channel segment 137-1 and the solution chamber output flow channel segment 137-2, through the chip outlet 136, into the second fluid path 152, through the second control valve 142, into the fourth fluid path segment 164-1, and into the pump body 161, so that pure water flows through the solution chamber 131, thereby completing the operation of cleaning the chip to be tested. At this point, if the next test is to be performed, the tested chip 130 is withdrawn from the gene sequencer, replaced with another chip, and the sequencing process is repeated.

Before the gene sequencer is shut down, the whole instrument needs to be cleaned, and liquid in the instrument needs to be emptied. At this time, the first liquid reagent bottle 111 is taken out from the reagent bottle holder 113, and then the vacuum pump and the corresponding valves are opened to let the residual buffer solution pass through the first reagent input channel 146, the first reagent control valve 144, the first control valve 141 by the reagent circulation pipeline 148, the fourth sample inlet 135 along the first liquid path 151, the chip input/output channel segment 138-1, the chip input/output through control valve 138, and the chip input/output channel segment 138-2, the chip outlet 136, the second control valve 142 along the second liquid path 152 (the third control valve 143 is in a closed state), and finally the buffer solution in the fourth liquid path segment 164-1 is completely emptied. Subsequently, the second reagent control valve 145 is opened to allow pure water to pass through the second reagent input channel 147 from the second reagent bottle 112 and pass through the second reagent control valve 145, pass through the first control valve 141 by the reagent circulation line 148, pass through the fourth sample inlet 135 along the first liquid path 151, pass through the chip input/output flow channel segment 138-1 via the chip input/output through control valve 138 and the chip input/output flow channel segment 138-2, pass through the second control valve 142 along the second liquid path 152 by the chip outlet 136 (the third control valve 143 is in a closed state), and finally enter the pump body 161 in the fourth liquid path segment 164-1, so that the whole liquid path is cleaned by pure water.

Then, the pure water reagent bottle is taken out from the reagent bottle holder, and then the pump body 161 and the corresponding valves are opened to let the residual pure water pass through the second reagent control valve 145 from the second reagent input channel 147, the first control valve 141 from the reagent circulation pipeline 148, the fourth sample inlet 135 along the first liquid path 151, the chip input/output channel segment 138-1 via the chip input/output through control valve 138 and the chip input/output channel segment 138-2 via the chip outlet 136 along the second liquid path 152 via the second control valve 142 (the third control valve 143 is in a closed state), and finally enter the pump body 161 in the fourth liquid path segment 164-1, so as to empty the pure water in the whole liquid path. Finally, the second reagent control valve 145 and the third control valve 143 are opened to allow air to enter the pump body 161 from the second reagent input channel 147 through the second reagent control valve 145, from the reagent circulation line 148 through the third control valve 143, from the third fluid path 153 and finally through the pump body control valve 163, thereby emptying the entire fluid path control unit 140 of any fluid that may be present in the various lines. At the end of the sequencing process, the tested chip 130 is withdrawn from the gene sequencer and then shut down to complete all sequencing operations.

It can be seen from the above explanation that, this scheme controls the first liquid way of storage unit and chip under test through solitary liquid way control unit to and chip under test and the second liquid way of pump body and waste liquid recovery unit, set up an solitary liquid way control unit and pump body and recovery unit lug connection simultaneously, not through the third liquid way of chip under test, and control it through liquid way control unit, thereby the washing work after the prewashing before the automation accomplishes the test and the test, compare in current gene sequencing device and have higher degree of automation, the operation of manual treatment has been reduced to a greater extent, avoid too much manual intervention to the influence of detection accuracy, further reduced the technical requirement that detects.

The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

Claims

1. A gene sequencing device, comprising:

a storage unit for storing a reagent;

2. The gene sequencing apparatus of claim 1, wherein the pump body and waste fluid recovery unit comprises:

a pump body for providing a negative pressure;

a waste liquid bottle for recovering the generated waste liquid;

3. The gene sequencing apparatus of claim 2, wherein the pump controller comprises a pump control valve disposed on the fourth fluid path, a portion of the fourth fluid path between the pump and the pump control valve being in communication with the second and third fluid paths.

4. The gene sequencing apparatus of claim 1, wherein the fluid path control unit comprises:

the first control valve controls the on-off of the first liquid path;

the second control valve controls the on-off of the second liquid path;

and the third control valve controls the on-off of the third liquid path.

5. The gene sequencing apparatus of claim 4, wherein the storage unit comprises a plurality of reagent bottles for holding different reagents; the liquid path control unit further comprises a plurality of reagent control valves, the reagent control valves correspond to the reagent bottles one to one and are used for controlling the connection and disconnection of the reagent bottles and the first liquid path and the third liquid path.

6. The gene sequencing apparatus of claim 5, wherein the fluid path control unit further comprises a reagent flow line, the plurality of reagent control valves are connected in parallel at one end of the reagent flow line, and the first control valve and the third control valve are connected in parallel at the other end of the reagent flow line.

7. The gene sequencing apparatus of claim 5, wherein the plurality of reagent vials comprises a buffer reagent vial and a pure water reagent vial.

8. A sequencing method using the gene sequencing apparatus of any one of claims 1 to 7, comprising performing the following tests prior to sequencing:

and (3) perforation and perforation test: and after the sample injection unit inputs the sample solution to be detected into the solution cavity, detecting whether the lipid covering layer is perforated or not.

9. The sequencing method of claim 8, wherein the lipid-like solution is an organic solution of lipids;

preferably, the organic solvent of the lipid-like liquid comprises higher alkane and octane, and the higher alkane is selected from any one of tridecane or hexadecane;

preferably, the volume ratio of the higher alkane to the octane is 1: (1-3);

preferably, the lipid is selected from at least one of dipalmitoylphosphatidylcholine, palmitoyl oleoylphosphatidylcholine, dioleoylphosphatidylmethyl ester, dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and sphingomyelin;

preferably, the lipid-like liquid further comprises cholesterol;

preferably, the molar ratio of said lipid to said cholesterol is about 1: 1.

10. the sequencing method of claim 8, wherein the buffer is a buffered solution of HEPES and chloride salt;

preferably, the molar ratio of the HEPES to the chloride salt is 1: (5-20).