Underwater sequencer

Abstract

The invention provides an underwater sequencer. The underwater sequencer comprises: a sample extraction device for extracting an underwater microorganism sample; an input port of the library building device is connected with an output port of the sample extraction device, and the library building device is used for building a gene library of the microorganism sample; an input port of the sequencing device is connected with an output port of the library building device, and the sequencing device is used for performing gene sequencing on the gene library. According to related requirements of underwater sequencing, the extraction device, the library building device and the sequencing device are integrated, nucleic acid extraction, library building and sequencing can be automatically carried out without human intervention, operation is convenient, and the integrated underwater sequencer further has the advantages of being small in size, low in power consumption and the like.

Description

Underwater sequencer Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and in particular to an underwater sequencer. Background Art

[0002] In the unique underwater environment, especially the selective pressures of the deep sea and the diverse and unique extreme environments, microorganisms evolve along different paths, deriving adaptive metabolic patterns and functional characteristics. Currently, people know very little about deep-sea microbial ecosystems, including their metabolic pathways, symbiotic relationships, community characteristics, evolutionary lineages, etc., which still need to be studied. In addition, deep-sea microorganisms are a valuable resource library. The various functional proteins and enzymes involved in their life activities have excellent properties such as high temperature and salt tolerance, and they have great scientific significance and industrial value.

[0003] Currently, functional research on deep-sea microorganisms primarily relies on deep-sea sampling and ship-based or shore-based analysis. During this process, drastic changes in hydrostatic pressure, temperature, and salinity can alter the survival of abundant, uncultured microbial communities underwater. Subsequent analyses of functional gene expression, protein activity, and abundance may deviate from the true picture. Therefore, conducting in situ deep-sea scientific experiments represents the most cutting-edge and advanced research technology, revolutionizing deep-sea scientific research and resource development and utilization.

[0004] Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects and provide an underwater sequencer with high degree of automation, easy operation, small size and low power consumption.

[0006] An embodiment of the present invention provides an underwater sequencer, comprising:

[0007] A sample extraction device for extracting underwater microbial samples;

[0008] a library building device, wherein the input port of the library building device is connected to the output port of the sample extraction device, and the library building device is used to build a gene library of the microbial sample;

[0009] A sequencing device, wherein the input port of the sequencing device is connected to the output port of the library building device, and the sequencing device is used to perform gene sequencing on the gene library. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG1 is a schematic diagram of a module of an underwater sequencer provided by an exemplary embodiment of the present invention;

[0011] FIG2 is a schematic structural diagram of an underwater sequencer provided by an exemplary embodiment of the present invention;

[0012] FIG3 is a schematic structural diagram of a library building device provided by an exemplary embodiment of the present invention;

[0013] FIG4 is a schematic structural diagram of a microfluidic chip of a sample purification module provided by an exemplary embodiment of the present invention;

[0014] FIG5 is a schematic structural diagram of a microfluidic stop valve provided by an exemplary embodiment of the present invention;

[0015] FIG6 is a schematic structural diagram of another microfluidic stop valve provided by an exemplary embodiment of the present invention;

[0016] FIG7 is a schematic structural diagram of another purification module provided by an exemplary embodiment of the present invention;

[0017] FIG8 is a schematic structural diagram of an adsorption column provided by an exemplary embodiment of the present invention;

[0018] FIG9 is a schematic structural diagram of a volume-constant component provided by an exemplary embodiment of the present invention;

[0019] FIG10 is a schematic structural diagram of another constant volume component provided by an exemplary embodiment of the present invention;

[0020] FIG11 is a schematic structural diagram of a constant volume module provided by an exemplary embodiment of the present invention;

[0021] FIG12 is a schematic structural diagram of a PCR module provided by an exemplary embodiment of the present invention;

[0022] FIG13 is a schematic diagram of the assembled structure of an underwater sequencer provided by an exemplary embodiment of the present invention;

[0023] FIG14 is a schematic structural diagram of an underwater sequencer provided by an exemplary embodiment of the present invention;

[0024] FIG15 is an exploded view of an underwater sequencer provided by an exemplary embodiment of the present invention;

[0025] FIG16 is a layout diagram of an underwater sequencer provided by an exemplary embodiment of the present invention;

[0026] FIG17 is a schematic structural diagram of a sequencing device provided by an exemplary embodiment of the present invention;

[0027] FIG18 is a schematic structural diagram of another sequencing device provided by an exemplary embodiment of the present invention;

[0028] FIG19 is a schematic structural diagram of an electrical control system provided by an exemplary embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0030] Figure 1 is a schematic diagram of the modules of an underwater sequencer provided by an exemplary embodiment of the present invention. The underwater sequencer includes a sample extraction device 11, a library building device 12, and a sequencing device 13. The input port of the library building device 12 is connected to the output port of the sample extraction device 11, and the output port of the library building device 12 is connected to the input port of the sequencing device 13. Furthermore, the sample extraction device 11, the library building device 12, and the sequencing device 13 can be connected by a conduit.

[0031] The sample extraction device 11 is used to extract underwater microorganism samples.

[0032] The library building device 12 is used to build a gene library of microbial samples.

[0033] The sequencing device 13 is used to perform gene sequencing on the gene library.

[0034] The underwater sequencer of the embodiment of the present invention integrates an extraction device, a library construction device and a sequencing device to meet the relevant needs of underwater sequencing. It can automatically perform nucleic acid extraction, library construction, sequencing and other functions without human intervention. It is easy to operate, and the integrated underwater sequencer also has the advantages of small size and low power consumption.

[0035] The underwater sequencer of the embodiment of the present invention can realize in situ sequencing. The underwater sequencer is submerged underwater and started by a preset delayed start instruction or after receiving an external instruction. The sample extraction device extracts the underwater microbial sample, enriches the microbial sample, and treats it with a high-salt solution to obtain a corresponding nucleic acid sample mixture. The obtained nucleic acid sample mixture is provided to a library construction device to construct a gene library of the microbial sample, and then the sequencing device performs gene sequencing on the gene library. The entire sequencing process can be carried out underwater, realizing in situ species qualitative and quantitative identification.

[0036] The underwater sequencer of the present invention can be used in shallow waters to extract microbial samples for gene sequencing, or in deep-sea environments to extract microbial samples for gene sequencing. If the underwater sequencer of the present invention is used in deep-sea environments, it can be activated after the submersible platform sinks to the bottom and perform gene sequencing in the deep sea.

[0037] In one embodiment, referring to FIG2 , the sample extraction device includes a water inlet pipeline 11a, a sample transfer pipeline 11b, an enrichment sampler 11c and a sample liquid storage tube 11d; one end of the water inlet pipeline 11a serves as the input port of the sample extraction device 11, and the other end of the water inlet pipeline 11a is connected to the first feed port A of the enrichment sampler 11c; one end of the sample transfer pipeline 11b is connected to the discharge port B of the enrichment sampler 11c, and the other end of the sample transfer pipeline 11b is connected to the sample liquid storage tube 11d.

[0038] The water inlet pipe 11a is used to collect microbial samples to the enrichment sampler 11c.

[0039] The sample transport pipeline 11 b is used to transport the enriched microbial sample in the enrichment sampler 11 c to the sample liquid storage tube 11 d , so that the nucleic acid sample is provided to the library building device 12 by the sample liquid storage tube 11 d .

[0040] In one embodiment, the water inlet pipeline 11a includes a first connecting pipe 111a and a first pump 112a provided on the first connecting pipe 111a. The first pump 112a is used to pump the microbial sample into the enrichment sampler 11c.

[0041] The first pump 112a may be, but is not limited to, a peristaltic pump.

[0042] It can be understood that the first pump 112a pumps water containing the microbial sample into the enrichment sampler 11c through the first connecting pipe 111a, that is, the first pump 112a realizes pumping the microbial sample into the enrichment sampler 11c.

[0043] In one embodiment, the underwater sequencer also includes a controller (not shown in the figure), and the water inlet pipe 11a is also provided with a pressure gauge 113a, which is used to detect the pressure in the first connecting pipe 111a. The controller is used to start the first pump 112a when the pressure falls into a preset range, so that the water inlet pipe 11a can evenly and effectively collect microbial samples to the enrichment sampler 11c.

[0044] Among them, the preset range can be determined according to the pressure of the working environment of the underwater sequencer.

[0045] In one embodiment, the sample transport pipeline 11b includes a waste liquid collector 111b, a second connecting pipe 112b and a first three-way valve 113b, one end of the second connecting pipe 112b is connected to the discharge port B of the enrichment sampler 11c, the other end of the second connecting pipe 112b is connected to the first port of the first three-way valve 113b, the second port of the first three-way valve 113b is connected to the waste liquid collector 111b, and the third port of the first three-way valve 113b serves as the other end of the sample transport pipeline 11b, that is, the third port of the first three-way valve 113b is connected to the sample liquid storage tube 11d.

[0046] The first three-way valve 113b may be, but is not limited to, a two-position three-way valve.

[0047] By controlling the on / off state of the first three-way valve 113b, the discharge port B of the enrichment sampler 11c can be connected to the waste liquid collector 111b and / or the sample liquid storage tube 11d. When the discharge port B of the enrichment sampler 11c is connected to the sample liquid storage tube 11d, the electric push rod is used to transfer the enriched microbial sample in the enrichment sampler 11c to the sample liquid storage tube 11d through the sample transfer pipeline 11b. When the discharge port B of the enrichment sampler 11c is connected to the waste liquid collector 111b, the waste liquid generated in the enrichment sampler 11c can be transferred to the waste liquid collector 111b through the sample transfer pipeline 11b.

[0048] In one embodiment, the sample extraction device 11 also includes a flushing pipeline 11e, which includes a third connecting pipe 111e and a second three-way valve 112e and a second pump 113e provided on the third connecting pipe 111e. One end of the flushing pipeline 11e is connected to the second feed port C of the enrichment sampler 11c, and the other end of the flushing pipeline 11e is used to connect to a buffer supply source 114e. The buffer supply source 114e is used to provide buffer to the enrichment sampler 11c.

[0049] The second three-way valve 112e may be, but is not limited to, a two-position three-way valve. The second pump 113e may be, but is not limited to, a peristaltic pump.

[0050] In one embodiment, the enrichment sampler 11c includes a filter membrane 111c, and the sample extraction device 11 further includes an electric push rod 11f arranged opposite to the filter membrane 111c, and the electric push rod 11f is used to transfer the microbial sample enriched on the filter membrane 111c to the sample transfer pipeline 11b.

[0051] The extraction device 11 primarily collects microbial samples from underwater environments (e.g., seawater) and provides nucleic acid samples of appropriate concentration to the downstream (library building device). Seawater is oligotrophic, and the total number of microorganisms contained in it is limited. Microbial abundance is low, requiring large-scale enrichment to reach the required detection concentration. Furthermore, the seawater cannot be directly depressurized for subsequent processing.

[0052] In an embodiment of the present invention, in situ enrichment is achieved through the enrichment sampler 11c, and a large amount of microorganisms are enriched on the filter membrane of the enrichment sampler 11c. Through the action of the electric push rod 11f, the enriched microbial sample obtained after filtration is transferred to the sample liquid storage tube 11d through the sample transfer pipeline 11b. The sample liquid storage tube 11d is pre-stored with an appropriate amount of high-salt mixed solution. The microbial sample is lysed in the sample liquid storage tube 11d to obtain a nucleic acid sample mixed solution. The plunger pump included in the downstream library construction device pumps the nucleic acid sample mixed solution into the library construction device in batches to achieve the transfer and injection of the nucleic acid sample.

[0053] It should be noted that the type of pump included in the downstream library building device is not limited to a plunger pump. In practical applications, other types of pumps may also be used.

[0054] In one embodiment, 1 ml of the nucleic acid sample can be retained, for example, 1 ml of the nucleic acid sample mixture can be retained in the sample liquid storage tube 11d, instead of pumping all the nucleic acid sample mixture in the sample liquid storage tube 11d into the library construction device, to meet the requirements of the control group sequencing experiment on shore.

[0055] In one embodiment, the library construction device includes: a sample purification module, a constant volume module, a PCR module, a PCR purification module, and a sequencing adapter connection module. The input port of the sample purification module serves as the input port of the library construction device, the input port of the constant volume module is connected to the output port of the sample purification module, the input port of the PCR module is connected to the output port of the constant volume module, the input port of the PCR purification module is connected to the output port of the PCR module, the input port of the sequencing adapter connection module is connected to the output port of the PCR purification module, and the output port of the sequencing adapter connection module serves as the output port of the library construction device. Different modules are connected by conduits.

[0056] The sample purification module is used to purify the nucleic acid sample to obtain a first purified sample. Specifically, the sample purification module extracts the nucleic acid sequence from the nucleic acid sample and removes other impurities in the lysate to obtain the first purified sample.

[0057] The volume fixing module is used to perform volume fixing processing on the first purified sample to obtain a volume fixing sample.

[0058] The PCR module is used to amplify a fixed volume of sample to obtain an amplified sample. This PCR module can amplify nucleic acid samples, increase the amount of DNA sample, and meet the sequencing input requirements.

[0059] The PCR purification module is used to purify the amplified sample to obtain a purified sample. Specifically, the PCR purification module extracts the DNA sequence in the amplified sample and removes other impurities in the amplified sample to obtain a second purified sample.

[0060] The sequencing adapter ligation module is used to connect the second purified sample to the sequencing adapters required for sequencing and purify the purified sample after connection to the sequencing adapters to obtain the nucleic acid to be sequenced. Specifically, the sequencing adapter ligation module mixes the purified second purified sample with the various reagents of the sequencing adapter to a fixed volume, and then connects the sequencing adapter to the second purified sample to carry out the subsequent sequencing steps.

[0061] In the embodiment of the present invention, in response to the relevant needs of in situ library construction under underwater conditions, the library construction device integrates functions such as purification, volume determination, PCR amplification, adapter connection and adapter purification. It can automatically build the library without human intervention and is easy to operate.

[0062] In one embodiment, all or part of the sample purification module, volume control module, PCR module, PCR purification module and sequencing adapter connection module included in the library construction device are implemented by a microfluidic chip.

[0063] Taking the library construction device shown in Figure 3 as an example, the library construction device includes a microfluidic chip 600A for implementing the corresponding functions of the sample purification module, a microfluidic chip 300 for implementing the corresponding functions of the constant volume module, a microfluidic chip 500 for implementing the corresponding functions of the PCR module, a microfluidic chip 600B for implementing the corresponding functions of the PCR purification module, and a microfluidic chip 400 for implementing the corresponding functions of the sequencing adapter connection module.

[0064] The library building device may also include a sample pool 900. Sample pool 900 is used to receive samples discharged from the previous module. Sample pool 900 includes an inlet tube 910, a sample container 920, and an outlet tube 930. A sealing plug 921 is provided in sample container 920, and inlet tube 910 and outlet tube 930 are provided through sealing plug 921. Sample pool 900 connects the different modules into a whole, thereby improving overall library building efficiency.

[0065] In one embodiment, referring to FIG4 , a microfluidic chip 600A for implementing the corresponding functions of the sample purification module includes: a valve, a nucleic acid sample feed channel 622 , a first purification liquid feed channel, a first purified sample discharge channel 631 , a first waste channel 632 , and a first purification chamber 610 ;

[0066] The nucleic acid sample feed channel 622 and the first purification liquid feed channel are each connected to the input port of the first purification chamber 610 through a valve, and the first purified sample discharge channel 631 and the first waste channel 632 are each connected to the output port of the first purification chamber 610 through a valve.

[0067] Specifically, referring to Figure 4 , the nucleic acid sample feed channel 622 is connected to the input port of the first purification chamber 610 via a valve 642. When the valve 642 is opened, the nucleic acid sample feed channel 622 injects lysate into the first purification chamber 610. The first purified sample discharge channel 631 is connected to the output port of the first purification chamber 610 via a valve 651. When the valve 651 is opened, the purified first purified sample in the first purification chamber 610 is discharged through the first purified sample discharge channel 631. The first waste channel 632 is connected to the output port of the first purification chamber 610 via a valve 652. When the valve 652 is opened, the first waste channel 632 discharges waste generated by sample purification.

[0068] In one embodiment, the first purification liquid feed channel includes a first microbead channel 621, a first cleaning liquid channel 623, and a first eluent channel 624. The first microbead channel 621 is connected to the input port of the first purification chamber 610 via a valve 641. When the valve 641 is opened, the first microbead channel 621 injects a microbead phase (e.g., a magnetic bead phase) into the first purification chamber 610. The first cleaning liquid channel 623 is connected to the input port of the first purification chamber 610 via a valve 643. When the valve 643 is opened, the first cleaning liquid channel 623 injects a cleaning liquid into the first purification chamber 610 to clean the microbeads in the first purification chamber 610 and discharge the lysate and waste liquid from the microbead washing remaining in the first purification chamber 610 out of the first purification chamber 610. The first eluent channel 624 is connected to the input port of the first purification chamber 610 via a valve 644. When the valve 644 is opened, the first eluent channel 624 injects an eluent into the first purification chamber 610 to elute the nucleic acid sample attached to the microbeads to obtain a first purified sample.

[0069] In one embodiment, a column 611 is provided within the first purification chamber 610 to confine the microbeads. Optionally, the column 611 is located at the outlet of the first purification chamber 610. Thus, microbeads entering the first purification chamber 610 through the first microbead channel 621 are blocked by the column 611.

[0070] In one embodiment, all or part of the valves of the sample purification module are implemented using microfluidic stop valves. Referring to Figures 5 and 6, the sample purification module includes a first valve body layer 210, a first spacer layer 220, and a first fluid layer 230 arranged in a stacked manner; a first purification chamber 610 and a first stop position 231 are formed on the first fluid layer 230, and a through hole penetrating the first valve body layer 210 and the first spacer layer 220 forms a first channel 120a, which connects the first purification chamber 610 and the first stop position 231, and the first stop position 231 is located between the first channel 120a and the first purification chamber 610; the first valve The body layer 210 is also provided with a first gas valve through hole 1211 arranged opposite to the first stop position 231. The first gas valve through hole 1211 is used to pass gas to make the first spacer layer 220 compressed and deformed to fit the first stop position 231 (see Figure 6) to block the first channel 120a and the first purification chamber 610. At this time, the stop valve is in a closed state; when the gas is stopped from being passed into the first gas valve through hole 1211, there is a gap between the first spacer layer 220 and the first stop position 231 (see Figure 5), and the first channel 120a is connected to the first purification chamber 610. At this time, the stop valve is in an open state.

[0071] The first channel 120a shown in FIG. 5 and FIG. 6 may represent at least one of the following channels: a nucleic acid sample feeding channel, a first purified liquid feeding channel, a first purified sample discharging channel, and a first waste channel.

[0072] During the fabrication of the integrated microfluidic stop valve, the valve body layer 210 is templated using negative resist soft lithography, followed by casting and demolding of polydimethylsiloxane (PDMS) to form the valve body 1212. The spacer layer 220 is formed by spin-coating PDMS onto a silicon wafer and then curing it with heat. The fluid layer 230 is fabricated using negative resist soft lithography to create the first channel 120a and first purification chamber 610. Positive resist soft lithography is then used, followed by further heating to soften the resist, forming the stop position 231 with its curved cross-sectional features.

[0073] The working principle of the microfluidic chip 600A is further explained below with reference to FIG4 :

[0074] When the sample purification module is in use, valve 641 is opened, and the first microbead channel 621 introduces a microbead phase (e.g., a magnetic bead phase) into the first purification chamber 610. Under the blocking action of the column 611 in the first purification chamber 610, the microbeads in the microbead phase accumulate within the first purification chamber 610. Simultaneously, valve 652 is kept open and valve 651 is closed, allowing waste liquid to be discharged through the first waste channel 632. Subsequently, valve 641 is closed, valve 642 is opened, and a nucleic acid sample solution is introduced into the first purification chamber 610 through the nucleic acid sample feed channel 622. The microbeads in the first purification chamber 610 capture DNA sequences in the nucleic acid sample solution, and the waste liquid is discharged through the first waste channel 632. Subsequently, valve 642 is closed, valve 643 is opened, and a cleaning solution is introduced into the first purification chamber 610 through the first cleaning solution channel 623. The cleaning solution cleans the microbeads, and the lysate remaining in the first purification chamber 610 is discharged. After cleaning is completed, valve 652 is closed and valve 651 is opened. At the same time, valve 643 is closed and valve 644 is opened. The eluent is introduced into the first purification chamber 610 through the first eluent channel 624, and the first purified sample is discharged from the first purified sample discharge channel 631.

[0075] In one embodiment, the sample purification module is implemented using pipelines and containers. Referring to Figure 7 , the sample purification module includes: a valve, a one-way valve 750, a purification container (extraction unit) 720, a main feed pipeline 70, an air supply branch, a nucleic acid sample feed branch, a cleaning solution feed branch, an eluent feed branch, a purified sample discharge branch, and a waste branch. The air supply branch, nucleic acid sample feed branch, cleaning solution feed branch, and eluent feed branch are each connected to the main feed pipeline 70 via a valve; the output end of the main feed pipeline 70 is also connected to the feed port of the purification container 720 via a one-way valve 750; and the discharge port of the purification container 720 is connected to the purified sample discharge branch and the waste branch, respectively.

[0076] The nucleic acid sample feeding branch is provided with a sample container 710. The sample container 710 is used to contain a sample to be purified (nucleic acid sample).

[0077] In one embodiment, the sample container 710 is connected to the main feed line 70 via a first solenoid valve 741. The main feed line 70 is also provided with a suction assembly. When the first solenoid valve 741 is opened, the sample container 710 is connected to the main feed line 70, and the suction assembly 730 performs a liquid extraction operation to extract the sample from the sample container 710 into the purification container 720. In addition, a one-way valve 750 is provided between the suction assembly 730 and the purification container 720. The one-way valve 750 ensures that the suction assembly 730 can smoothly extract the sample from the sample container 710 into the purification container 720, preventing the liquid in the purification container 720 from flowing back.

[0078] The suction component 730 can be implemented by, but is not limited to, a syringe pump.

[0079] The cleaning liquid feed branch is equipped with a cleaning liquid container 760 for holding cleaning liquid. Cleaning liquid container 760 is connected to the main feed line 70 via a second solenoid valve 742. After the sample is pumped into the purification container 720, the first solenoid valve 741 is closed, the second solenoid valve 742 is opened, and the suction assembly 730 draws the cleaning liquid from the cleaning liquid container 760 into the purification container 720. After the liquid withdrawal operation, the second solenoid valve 742 is closed.

[0080] The air supply branch is provided with an air source, which may be, but is not limited to, an air pump. Air pump 770 is connected to main feed line 70 via third solenoid valve 743. After the cleaning liquid is pumped in, third solenoid valve 743 is opened, and air pump 770 is activated to introduce air into purification container 720 to dry the purification container 720.

[0081] The eluent feed branch is provided with an eluent container 780, which is connected to the main feed line 70 via a fourth solenoid valve 744. After the drying process is completed, the fourth solenoid valve 744 is opened, and the eluent in the eluent container 780 is pumped into the purification container 720 via the suction assembly 730 to elute the DNA sequences enriched on the extraction membrane in the purification container 720.

[0082] The waste branch is equipped with a waste liquid container 790, which is connected to the main feed line 70 via a fifth solenoid valve 745. During the extraction, cleaning, and drying processes, the fifth solenoid valve 745 remains open, allowing waste liquid and exhaust gas to flow into the waste liquid container 790. During the elution process, the fifth solenoid valve 745 remains closed, allowing the eluent to enter the next process module.

[0083] In one embodiment, an extraction filter membrane is provided in the purification container 720, and the extraction filter membrane is used to enrich and purify the sample to be purified.

[0084] In one embodiment, an adsorption column for purifying nucleic acid samples is provided in the purification container 720, as shown in FIG8 . The adsorption column includes two threaded channel joints and an adsorption membrane, and the adsorption membrane is provided between the two threaded channel joints. A step is provided between the two threaded pipe joints to form a chamber, and three or more layers of adsorption membranes with a diameter of 3-5 mm are confined in the chamber between the joints through the step. The number of layers of the adsorption membrane depends on the balance between the adsorption efficiency and the air pressure required to pass the liquid. The more membrane layers, the higher the adsorption efficiency, but at the same time the required air pressure may be too high. Optionally, the adsorption column uses three layers of 5 mm diameter membranes, which can take into account both the adsorption efficiency and the passing pressure, achieving an elution DNA recovery efficiency of approximately 50%, and the purification process takes 40 minutes. The pore size on the adsorption membrane can be, but is not limited to, 0.22 microns.

[0085] In one embodiment, the volume control module includes: a volume control component.

[0086] Figure 9 is a schematic structural diagram of a constant volume component according to an exemplary embodiment. As shown in Figure 9, the constant volume component includes a first constant volume chamber 911, a constant volume liquid feed channel 120 and a first exhaust channel 130a. The constant volume liquid feed channel 120 is connected to the first constant volume chamber 911 through a valve 121, and the valve 121 is used to control the on-off of the constant volume liquid feed channel 120 and the first constant volume chamber 911. The first exhaust channel 130a is connected to the first constant volume chamber 911 through a valve 131, and the valve 131 is used to control the on-off of the first exhaust channel 130a and the first constant volume chamber 911.

[0087] In one embodiment, the constant volume component includes at least two constant volume chambers, and each constant volume chamber is connected by a pipeline. The number of constant volume chambers can be set according to actual conditions, and can be 2 or 3. The constant volume chamber is connected to at least one constant volume liquid feed channel and / or at least one auxiliary liquid feed channel, and each constant volume liquid feed channel is connected to the input port of a constant volume chamber through a valve, and each auxiliary liquid feed channel is connected to the input port of a constant volume chamber through a valve.

[0088] In one embodiment, when the constant volume assembly includes at least two constant volume chambers, the output ports of all or some of the constant volume chambers are connected to an exhaust channel via a valve. The constant volume chamber connected to the constant volume liquid feed channel and the exhaust channel can introduce the constant volume liquid into the constant volume chamber through a cyclic pressure relief method; the constant volume chamber connected to the auxiliary liquid feed channel and the exhaust channel can also introduce the auxiliary liquid into the constant volume chamber through a cyclic pressure relief method.

[0089] Taking the three constant volume containers shown in Figure 10 as an example, the first constant volume chamber 911, the second constant volume chamber 112, and the third constant volume chamber 113 are connected in sequence. An auxiliary liquid feed channel 120b is further provided between the second constant volume chamber 112 and the first constant volume chamber 911. The auxiliary liquid feed channel 120b is connected to the second constant volume chamber 112 and is used to connect a liquid adding device to fill the first feed into the second constant volume chamber 112. A first control valve 121e is provided on the constant volume liquid feed channel 120b.

[0090] An auxiliary liquid feed channel 120c is provided between the third constant volume chamber 113 and the second constant volume chamber 112, and the auxiliary liquid feed channel 120c is connected to the third constant volume chamber 113, and is used to connect the liquid adding device to fill the second feed into the third constant volume chamber 113. A valve 121f is provided on the auxiliary liquid feed channel 120c. In addition, an exhaust channel 130b is provided on the side of the third constant volume chamber 113 away from the second constant volume chamber 112, and the exhaust channel 130b is connected to the second constant volume chamber 112 through the third constant volume chamber 113. A valve 121d is provided between the exhaust channel 130b and the third constant volume chamber 113, and the valve 121d is used to control the on-off of the second exhaust channel 130b and the third constant volume chamber 113.

[0091] In this way, in the process of realizing the first feed constant volume through the second constant volume chamber 112, the valve 121f is closed to block the auxiliary liquid feed channel 120c, and the exhaust channel 130b is used as the exhaust channel. In the process of realizing the second feed constant volume through the third constant volume chamber 113, the valve 121e is closed to block the auxiliary liquid feed channel 120b. And the exhaust channel 130b is also used as the exhaust channel. In other words, the second constant volume chamber 112 and the third constant volume chamber 113 share the exhaust channel 130b. In this way, the number of exhaust channels is reduced, thereby simplifying the structure of the constant volume component and reducing the module hardware manufacturing cost.

[0092] In one example, at least one row of columns 140 is provided in the first constant volume chamber 911, and the interval between adjacent columns 140 is less than or equal to a set distance. Due to the blocking effect of the columns 140 and the surface tension of the liquid, the first feed and the second feed can be evenly mixed after entering the first constant volume chamber 911. There is no specific limitation on the arrangement and number of the columns 140. For example, two rows of columns 140 are provided in the first constant volume chamber 911, and each row of columns 140 is arranged in an arc shape.

[0093] In one embodiment, the constant volume component is realized by a microfluidic chip, and the constant volume component includes a second valve body layer, a second spacer layer and a second fluid layer arranged in a stacked manner; a constant volume cavity and a second cutoff position are formed on the second fluid layer, and a through hole penetrating the second valve body layer and the second spacer layer forms a second channel, the second channel connects the constant volume cavity and the second cutoff position, and the second cutoff position is located between the second channel and the constant volume cavity; a second air valve through hole arranged opposite to the second cutoff position is also provided on the second valve body layer, and the second air valve through hole is used to pass gas so that the second spacer layer is compressed and deformed to fit the first cutoff position to block the second channel and the constant volume cavity; wherein the second channel includes at least one of the following: a purified sample feed channel, a constant volume liquid feed channel, and a constant volume sample discharge channel. All or part of the valves of the constant volume component are realized by microfluidic stop valves, and the specific implementation process is similar to the stop valve in the sample purification module, which will not be repeated here.

[0094] In one embodiment, the constant solution required for volume determination uses a PCR Mix phase, and the auxiliary liquid includes a spacer buffer phase and a binding buffer phase. Correspondingly, the constant solution feed channel includes a PCR Mix feed channel, and the auxiliary liquid feed channel includes a spacer buffer phase feed channel and a binding buffer phase feed channel. The constant volume process is further described below with reference to FIG11:

[0095] Valve 322 on PCR Mix feed channel 321 is opened, connecting PCR Mix feed channel 321 to constant volume chamber 320. Under the action of pressure, PCR Mix enters constant volume chamber 320 through PCR Mix feed channel 321. Valve 322 is closed, and valve 324 on PCR Mix exhaust channel 323 is opened to exhaust the gas in constant volume chamber 320. After the pressure reaches equilibrium with the external air pressure, valve 324 is closed and valve 322 is opened again. PCR Mix enters constant volume chamber 320 under the action of pressure. Valve 322 is closed and valve 324 is opened again. This process is repeated until the pressure in constant volume chamber 320 reaches equilibrium and constant volume chamber 320 is completely filled with PCR Mix. After the PCR Mix phase is prepared, the valve 312 on the purified sample feed channel 311 is opened, and the purified sample (liquid) enters the holding chamber 310 through the purified sample feed channel 311. By means of cyclic pressure relief, valve 312 and valve 314 on the feed and exhaust channel 313 are opened in sequence until the purified sample fills the holding chamber 310. Afterwards, valves 340 and 350 are opened, and by means of the same cyclic pressure relief, the purified sample is pushed into the PCR Mix phase in the constant volume chamber 320 and into the constant volume chamber 330 together to complete the constant volume. The mixing is completed by the surface tension of the liquid and the blocking effect of the columns in the constant volume chamber 330. After an appropriate incubation time, the mixing of the purified sample and the PCR Mix phase is completed to obtain a constant volume sample. Then, the valve 372 on the interval buffer phase feed channel 362 and the valve 371 on the binding buffer phase feed channel 361 are opened in sequence, and the interval buffer phase is injected into the constant volume chamber 330 through the interval buffer phase feed channel 362, and the binding buffer phase is injected into the constant volume chamber 330 through the binding buffer phase feed channel 361, so as to discharge the fixed volume sample from the fixed volume sample discharge channel 363 and enter the subsequent PCR part.

[0096] In one embodiment, all or part of the valves of the constant volume assembly are implemented using microfluidic stop valves. The structure and working principle of the microfluidic stop valve of the constant volume assembly are similar to those of the microfluidic stop valve in the sample purification module and will not be repeated here.

[0097] In one embodiment, the PCR module includes a temperature control device and a PCR device; the temperature control device is used to control the temperature of the PCR device to a first target temperature, so that a fixed volume of sample is amplified in the PCR device to obtain an amplified sample. The first target temperature is a temperature suitable for sample amplification.

[0098] FIG12 is a schematic diagram of a PCR device according to an exemplary embodiment. As shown in FIG12 , the PCR device includes: a sample inlet 510 , a denaturation zone pipeline 520 , a circulation zone pipeline 530 , and a sample outlet 540 .

[0099] Denaturation zone piping 520 is connected to the sample inlet 510. Circulation zone piping 530 is connected to the denaturation zone piping 520 and includes a plurality of sets of circulation unit piping 531. Sample outlet 540 is connected to circulation zone piping 530. The number of circulation unit piping 531 included in circulation zone piping 530 can be set according to actual conditions, for example, 35 sets of circulation unit piping 531.

[0100] The injection port 510 is used to introduce a fixed volume of sample.

[0101] The denaturation zone pipeline 520 is used to perform denaturation treatment on the fixed volume sample entering from the injection port 510, that is, to unwind the DNA double strands so that the DNA double strands are completely denatured and unwound into single strands.

[0102] The circulation zone pipeline 530 realizes cyclic amplification through several groups of circulation unit pipelines 531.

[0103] The sample outlet 540 is used to discharge the processed amplified sample as a sample for the subsequent PCR purification step.

[0104] Optionally, the circulation unit pipeline 531 includes a first pipeline 531a and a second pipeline 531b that are connected. As shown in Figure 12, the circulation unit pipeline 531 first extends along a set direction, and then bends 180° and extends in a direction opposite to the set direction. In this way, the processing path of the sample is greatly improved by several groups of circulation unit pipelines 531, and the amplification effect is optimized. Among them, in the circulation unit pipeline 531 connected to the deformation zone pipeline 520, the first pipeline 531a is specifically connected to the deformation zone pipeline 520. In addition, the processing temperature of the first pipeline 531a is higher than the processing temperature of the second pipeline 531b. Referring to Figure 12, the second pipeline 531b is distributed on both sides of the first pipeline 531a. Accordingly, the sample that has undergone high-temperature treatment in the denaturation zone pipeline 520 preferentially enters the high-temperature portion of the circulation unit pipeline 531 (i.e., the first pipeline 531a), and then enters the low-temperature portion of the circulation unit pipeline 531 (i.e., the second pipeline 531b). Furthermore, the first pipelines 531a of the two connected circulation unit pipelines 531 are connected. That is, the sample in the previous circulation unit pipeline 531 passes through the first pipeline 531a and enters the first pipeline 531a of the next circulation unit pipeline 531. The processing temperature range of the first pipeline 531a is 90°C-98°C, and the processing temperature range of the second pipeline 531b is 50°C-60°C.

[0105] Optionally, the circulation unit conduit 531 further includes a third conduit, the processing temperature of which is lower than that of the first conduit 531a and higher than that of the second conduit 531b. For example, the processing temperature of the third conduit is 70°C-80°C. Thus, the processing temperature distribution of the circulation unit conduit 531 is gradient-varied, optimizing the amplification effect.

[0106] Optionally, the circulation zone piping 530 further includes an extension zone piping 532, which is connected to the second piping 531b in the circulation unit piping 531 near the sample outlet 540. The extension zone piping 532 is used to receive samples that have passed through all of the circulation unit piping 531 and perform low-temperature base pairing on the samples. Furthermore, the extension zone piping 532 is connected to the sample outlet 540 to discharge the sample that has undergone low-temperature base pairing out of the sample outlet 540.

[0107] In one embodiment, the temperature control device includes a controller and a heat dissipation component and / or a heating component electrically connected to the controller; when the temperature of the PCR device is lower than the first target temperature, the controller is used to control the heating component to heat the PCR device; when the temperature of the PCR device is higher than the first target temperature, the controller is used to control the heat dissipation component to dissipate heat from the PCR device.

[0108] The temperature control device further includes a temperature probe, which detects the temperature of the PCR device; the heating component can be implemented by, but is not limited to, a heating plate or a heat transfer plate.

[0109] The temperature probe detects the temperature of the corresponding temperature zone in the PCR device, and the temperature information is transmitted back to the controller (which can be achieved through the temperature control board). The controller performs PID control on the temperature of the corresponding temperature zone, controls the heating plate to heat, and the heat transfer plate evenly transfers the temperature of the heating plate to the PCR device.

[0110] The number and deployment positions of the temperature probes can be set according to actual conditions. The deployment positions of the temperature probes are not limited to the first temperature measurement point 551, the second temperature measurement point 552, and the third temperature measurement point 553 shown in FIG12.

[0111] In one embodiment, the PCR purification module includes a valve, a second purification chamber, an amplified sample feed channel, a second purified liquid feed channel, a nucleic acid sample discharge channel, and a second waste channel; the amplified sample feed channel and the second purified liquid feed channel are each connected to the input port of the second purification chamber through a valve, and the purified sample discharge channel and the second waste channel are each connected to the output port of the second purification chamber through a valve.

[0112] In one embodiment, the second purified liquid feed channel includes a second microbead channel, a second cleaning liquid channel and a second eluent channel; the second microbead channel, the second cleaning liquid channel and the second eluent channel are each connected to the second purified liquid feed channel through a valve.

[0113] The specific implementation of the PCR purification module is similar to that of the sample purification module and will not be repeated here.

[0114] The sequencing adapter connection module is used to perform the sequencing adapter connection process and purification process. In one embodiment, the sequencing adapter connection module includes a sequencing adapter connection submodule and a sequencing adapter purification submodule.

[0115] The sequencing adapter connector module includes a volume-fixing component, which has a similar structure and working mechanism to the volume-fixing component included in the volume-fixing module and will not be described in detail here.

[0116] The sequencing adapter connector module is used to connect the necessary proteins to the sample to be sequenced, meeting the sample modification requirements for sequencing. The module includes a positive pressure gas source, a solenoid valve, a catheter, and a microfluidic device. The positive pressure of the gas source is controlled by turning the solenoid valve on and off. This positive pressure controls the opening and closing of the microvalves in the microfluidic device, thereby controlling the cutoff and flow of the reagent phase.

[0117] In one embodiment, the microfluidic device of the sequencing adapter connector module includes a purified sample holding chamber, an enzyme phase constant volume chamber, an enzyme Buffer phase constant volume chamber, a sequencing adapter constant volume chamber, and a sample mixing constant volume chamber before sequencing. When the PCR purification module performs the purification step, the sequencing adapter connector module can simultaneously complete the enzyme phase constant volume, the enzyme Buffer phase constant volume, and the sequencing adapter constant volume step by cyclically opening and closing the constant volume chamber inlet valve outlet valve. When the PCR purification module uses eluent to elute the DNA sequence on the microbead, the purified sample holding chamber of the sequencing adapter connector module is opened, the gas in the connecting conduit between the two modules is discharged, and finally the mixing and constant volume of the DNA sample and several other necessary reagents are completed using the mixing constant volume chamber. After completing constant volume, the sample tube between the sequencing adapter connector module and the sequencing adapter purification module is mixed with Binding Buffer as the sample for the subsequent sequencing adapter purification step.

[0118] The sequencing adapter purification submodule is used to extract DNA from samples ligated with sequencing adapters and discard any other components introduced during the sequencing adapter ligation step to prevent them from affecting subsequent sequencing steps. The sequencing adapter purification submodule consists of a positive pressure gas source, a solenoid valve, a catheter, and a microfluidic device. The positive pressure is controlled by turning the solenoid valve on and off. This on-off positive pressure controls the opening and closing of microvalves in the microfluidic device, thereby controlling the flow and shutoff of the reagent phase. This structure is similar to the sample purification module shown in Figure 4. First, microbeads (such as magnetic beads) dispersed with buffer are introduced into the sequencing adapter purification submodule, and the microbeads are captured by the pillar microstructure in the device cavity. Then, the DNA sample connected to the sequencing adapter is mixed with the binding buffer in the reagent chamber between the sequencing adapter connection module and the sequencing adapter purification module, and introduced into the sequencing adapter purification submodule, where the DNA is captured by the microbeads. After the DNA sample connected to the sequencing adapter is exhausted, the microbeads are cleaned by the cleaning solution, and the cleaning solution in the sequencing adapter purification device is blown dry with gas. Finally, the DNA enriched on the microbeads is eluted with the eluent and flows into the reagent chamber between the sequencing adapter purification module and the sequencing chip to mix with the sequencing buffer.

[0119] In one embodiment, the sequencing device comprises a miniature single-molecule sequencing system, which primarily consists of a sequencing module, a control motherboard, and a computing host. After the library construction device completes the construction of the single-molecule sequencing library, sequencing adapters are attached to one or both ends of the DNA molecule to be tested. The constructed library sample is then pushed forward and mixed with a preset volume of anchor sequences pumped from the storage chamber of the miniature single-molecule sequencing system. The anchor sequence can be prepared and injected into the storage chamber before launching, or stored as a dry powder in the storage chamber and dissolved and prepared underwater. After the library and anchor sequence are mixed, they are incubated at 25°C for a period of time. The sample is then transferred to a sequencing chip and incubated again for a period of time to complete sample loading and temperature equilibration. The sample on the sequencing chip is sequenced at a predetermined sequencing temperature. The sequencing process is fully automatically controlled by software, which monitors and intervenes in real time to ensure efficient and stable sequencing data output. Sequencing data can be stored in real time on an external solid-state drive. After the underwater mission is completed, the underwater sequencer is salvaged to the surface for subsequent microbial species identification or other analysis. In addition, if the underwater computer allows, species identification can also be completed directly underwater.

[0120] In one embodiment, the EM algorithm can be used, but is not limited to, to correct the raw alignment results obtained by performing biological species identification on the test data generated by the single-molecule sequencing system, and to achieve microbial species identification through operations such as traversal and deep filtering to improve the accuracy of microbial species identification. Specifically, first, it is necessary to set quality control standards for the test data based on the read length of the test data: for example, the read length range is [1300bp, 1600bp]; the test data with read lengths falling within the above read length range are determined as quality control test data and enter subsequent analysis; secondly, the quality control test data are aligned to the bacterial 16S rDNA database to obtain the raw alignment results; then, the raw alignment results are corrected using the EM algorithm, and preliminary species and abundance information is obtained through traversal calculation; finally, for any redundant species information that may exist within the same genus, the identification results of the previous step are further classified and merged to filter out any erroneous species and obtain more accurate relative abundance information for each true species.

[0121] In an embodiment of the present invention, in response to the demand for underwater in situ sequencing, a set of integrated devices (underwater sequencers) based on microfluidic library construction and single-molecule sequencing, which can be deployed underwater, are proposed. The underwater sequencer can realize full-process automation from nucleic acid extraction to library construction, sequencing and bioinformatics analysis underwater. Compared with the existing scheme that can only design primers based on known 16S sequences and obtain species information after qPCR reaction, the embodiment of the present invention can sequence unknown species and accurately determine the species by comparing with the database. In addition, the abundance of microbial communities can be accurately measured, which will be beneficial to the development of marine environmental ecological diversity research.

[0122] It should be noted that the single-molecule sequencing system is not limited to a specific technical path. Sequencers based on electrical, optical detection or hybrid measurement can be used as sequencing devices in this deep-sea sequencer system.

[0123] In one embodiment, as shown in Figures 13-15 , the underwater sequencer further includes a housing 811. The library building device 12 and sequencing device 13 are both located within the atmospheric-pressure chamber of the housing 811, and the sample extraction device 11 is located on a first end face of the housing 811. When the underwater sequencer is underwater, the end face of the housing 811 forms a high-pressure zone, meaning that the sample extraction device 11 operates within a high-pressure environment.

[0124] Based on considerations of compressive strength, watertightness, and corrosion resistance, the material of the outer shell 811 can be, but is not limited to, salt-resistant, high-strength materials such as aluminum alloy, stainless steel, and titanium alloy. The thickness of the outer shell 811 needs to take into account the underwater operating water pressure of the underwater sequencer. The water pressure per unit area at a depth of 1,500 meters is approximately 15 MPa. For safety redundancy, a 25% margin is usually reserved, that is, the design pressure is 20 MPa. The wall thickness is calculated based on the inner cavity size and material strength to determine strength and stability, and is verified through software simulation. For example, if high-strength aluminum alloy 7075 is used, a wall thickness of 15 mm is required for an outer shell 811 with a diameter of 180 mm and a length of 1,000 mm. The surface of the outer shell 811 can also be hard anodized to further enhance the compressive strength, watertightness, and corrosion resistance of the outer shell 811.

[0125] In one embodiment, the housing 811 may be cylindrical, and the library building device and the sequencing device are arranged in series in the cylindrical chamber.

[0126] In one embodiment, the underwater sequencer includes a first sealing end cap 812, which is disposed on a first end surface of a pressure-resistant housing 811. This first sealing end cap 812 is used to seal the housing 811, thereby forming a normal pressure chamber within the housing. The sample extraction device 11 is secured to the first sealing end cap 812.

[0127] The first sealing end cap 812 includes multiple high-pressure sealing rings. The enrichment sampler 11c enriches a large amount of microbial samples on the filter membrane 111c. Through the action of the electric push rod 11f, the nucleic acid sample is transferred through the multiple high-pressure sealing rings to the library building device 12 in the normal pressure chamber, thereby realizing the efficient transfer of samples from the underwater high-pressure environment to the library building device 12 in the normal pressure chamber.

[0128] In one embodiment, the underwater sequencer includes a second sealing end cap 813, which is disposed on a second end surface of the sealed housing 811. The second sealing end cap 813 is used to further seal the housing 811, so that a normal pressure chamber is formed in the housing 811.

[0129] It is understandable that the underwater sequencer may further include a connecting ring 816 for connecting the housing 811 and the first sealing end cover 812 and other components for implementing the underwater sequencer, which will not be described in detail here.

[0130] In one embodiment, referring to FIG16 , the pressure-resistant cylinder depicted in the figure represents the housing 811 of the underwater sequencer. The underwater sequencer also includes an electrical control system 815, an air supply system, a waste liquid recovery system, and a liquid supply system. The housing 811 comprises a first chamber section, a second chamber section, a third chamber section, and a fourth chamber section. The sample purification module included in the library construction apparatus 12 is located in the first chamber section, and the electrical control system 815 is located in the second chamber section. The air supply system, other modules of the library construction apparatus 12, the waste liquid recovery system, the sequencing apparatus 13, and the liquid supply system are located in the third chamber section. The PCR module included in the library construction apparatus is located in the fourth chamber section. Other modules are modules of the library construction apparatus other than the PCR module and the sample purification module.

[0131] The sample purification module in the first cavity section is realized by the pipelines and containers shown in FIG7 .

[0132] In an embodiment of the present invention, the components of the underwater sequencer are arranged in a modular building block structure across four chamber segments, based on their functions and accessory properties. For example, the partitioning of the second and third chamber segments prioritizes electro-hydraulic separation for protection. The PCR module in the fourth chamber segment is partitioned separately for heating and movement requirements. This modular building block structure enables the underwater sequencer to perform underwater sequencing more efficiently, maximizing the use of the cylindrical cavity, maintaining a compact structure, and reducing the overall volume and weight, thereby minimizing precious energy consumption during underwater activities. The overall cabin structure is designed in sections, taking into account local support and fixation during assembly and disassembly, facilitating overall disassembly, repair, and replacement. Each functional module can be easily disassembled and replaced, and can operate independently, facilitating fault location and repair.

[0133] In one embodiment, the sample purification module in the first cavity section can be implemented by the microfluidic chip shown in Figure 4, and the outlet of the first purified sample discharge channel 631 of the sample purification module can be connected to the inlet of the purified sample feed channel 311 of the constant volume module through a pipeline or other means. It should be noted that if the sample purification module is implemented by a microfluidic chip, the first cavity section can be omitted, and the microfluidic chip that implements the sample purification module is one of the microfluidic chip groups 827 shown in Figure 17 or Figure 18. In one embodiment, the PCR module can also be implemented by a microfluidic chip. It should be noted that if the PCR module is implemented by a microfluidic chip, the fourth cavity section can be omitted, and the microfluidic chip that implements the PCR module is one of the microfluidic chip groups 827 shown in Figure 17 or Figure 18.

[0134] In one embodiment, referring to FIG16 , the library construction device 12 and the waste liquid recovery system are centrally arranged in the third cavity section; the gas supply system, the sequencing device 13 and the liquid supply system are arranged on the periphery of the library construction device 12 and the waste liquid recovery system; and the liquid supply system is located between the waste liquid recovery system and the PCR module.

[0135] In one embodiment, referring to Figures 17 and 18, the underwater sequencer further includes components such as a gas valve group 821, a plunger pump 822, a liquid valve group 823, a high-pressure gas source 824, a pressure reducing valve 825, a low-pressure regulating valve group 826, and an adsorption column 829. The sequencing device 13 is specifically a single-molecule sequencing system 828. Figure 17 also shows a microfluidic chip group 827 including a PCR purification module and a sequencing adapter connection module. Figure 18 also shows a microfluidic chip group 827 including a constant volume module. The two microfluidic chip groups are respectively placed on both sides of the second layer of the third cavity section. It should be noted that the number of microfluidic chip groups can be set according to actual conditions. One group, two groups, or even more groups can be set.

[0136] When the microbial samples in the seawater sample, such as bacterial flora, are collected in the sample liquid storage tube 11d by the sample extraction device 11 and mixed with the lysate pre-stored in the sample liquid storage tube 11d, a nucleic acid sample is obtained. The nucleic acid sample in the liquid storage tube 11d is extracted into the sample purification module by the plunger pump contained in the sample purification module in an extraction manner for purification. After the purified nucleic acid sample enters the microfluidic chipset, the sequencing library is completed according to the steps of constant volume module, PCR, PCR purification, connector connection and connector purification. For the specific process, please refer to the above description. Among them, the plunger pump is controlled by an independent driver to achieve a flexible control effect.

[0137] Notably, for energy conservation reasons, the embodiments of the present invention utilize a pneumatic push method to propel reagents within the microfluidic chip. This results in virtually no gas loss and eliminates the need for active energy supply, making it ideally suited for the stringent energy constraints of underwater environments. Based on flow resistance calculations and reaction time requirements, the gas pressure for each specific library construction or sequencing step is typically 5-15 psi, sufficient to propel the reagents. Finally, the library products are fed into a single-molecule sequencing system for sequencing and identification.

[0138] In one embodiment, the underwater sequencer further includes a refrigeration unit and a temperature controller, both of which are located within the pressure-resistant housing. The temperature controller is configured to control the refrigeration unit to maintain the temperature within the pressure-resistant housing below a second target temperature. The second target temperature is a temperature suitable for operation of the sequencing device and the library construction device.

[0139] The number of cooling fins can be set according to actual conditions, and can be 1 or more. When multiple cooling fins are set, each cooling fin is dispersedly deployed in the normal pressure cavity.

[0140] In one embodiment, referring to FIG19 , the electrical control system integrates the main controller 832 of the underwater sequencer, the temperature controller 831, and other electrical components for implementing underwater sequencing, such as the relay 834, the hard disk 833, the sequencing main board included in the sequencing device, the sequencing temperature control module included in the PCR module, various pumps and their driver boards, etc. The main controller 832 controls the above-mentioned other electrical components to enable the underwater sequencer to perform orderly sequencing underwater.

[0141] Furthermore, the main controller 832, the thermostat 831, the relay 834 and the hard disk 833 can be dispersedly deployed on the metal layer plate, and the metal layer plate includes a first metal plate 835 and a second metal plate 836 arranged in a layer. The relay 834 and the solid-state hard disk 822 are respectively fixed on both sides of the first metal plate 835, and the thermostat 831 and the main controller 832 are respectively fixed on both sides of the second metal plate 836. The metal layer plate is also provided with an output terminal and / or an interface for connecting to external devices. The relay 834 is connected to the above-mentioned valve. One relay 834 can be connected to one valve or multiple valves. The main controller 832 controls the on-off of the valve by controlling the on-off of the relay 834. The hard disk 833 is used to store the operating data, test process data, test result data, etc. of the underwater tester.

[0142] In one embodiment, the underwater sequencer also includes various interfaces for connecting electrical components. For example, the overall device has two main external interfaces. One is the interface for connecting to the carrying platform, which provides power supply and data transmission and uses a watertight connector BH8M, a 24V&8A power supply, and an RS232 serial port. The other is the data export port, which uses a watertight connector MCBH8M and a network port.

[0143] The electrical components of the underwater sequencer are primarily divided into functional modules powered by 12V and 24V voltages. If the underwater sequencer is used in deep-sea environments, the main controller 832 communicates with the submersible platform's main control board via an RS232 serial port. The submersible platform's main control board controls the submersible platform's insertion of the underwater sequencer into the deep sea. Once the target depth for the underwater sequencer is determined, the submersible platform's main control board sends a command back to the main controller 832, triggering it to initiate sequencing operations. The 12V multi-channel interface powers the sequencing mainboard and thermostat. The 24V power is provided to the relay and plunger pump driver board.

[0144] In one embodiment, the underwater sequencer may also include a water leakage monitoring device, an insulation monitoring device, a voltage monitoring device, a current monitoring device, etc., to monitor various system status parameters such as water leakage, insulation, voltage, current, etc. in real time, and monitor whether the system has any abnormalities. When an abnormality is detected, the existing safety hazards are resolved in a timely manner.

[0145] The above-water and underwater process routes of the underwater sequencer provided by the embodiment of the present invention are further explained below.

[0146] Before launching the underwater sequencer, the gas cavity must be pre-filled. This is because the underwater sequencer contains numerous gas lines and cavities. Directly filling with high-pressure gas cylinders would waste significant compressed high-pressure gas. Therefore, before turning on the high-pressure gas cylinder, the underwater sequencer pre-fills the gas cavity using a one-way valve interface on shore, using alternative gas sources. This process significantly reduces gas consumption and minimizes the required high-pressure gas cylinder size.

[0147] The gas supply system of the underwater sequencer includes high-pressure gas cylinders, pressure regulating valves, and solenoid valve groups. High-pressure gas cylinders: The underwater sequencer requires a gas source for operation. High-pressure gas cylinders are used instead of air pumps in a cylindrical cavity with an inner diameter of 200 mm to avoid the impact of air pump vibration on the operation of the entire machine. High-pressure gas cylinders need to be equipped with corresponding high-pressure reducing valves and pressure regulating valves to meet the system's various air pressure requirements. Pressure regulating valve: The underwater sequencer operates in a closed cavity and requires three different pressure gases at the same time. A containerized non-overflow pressure regulating valve is selected to meet current usage requirements within the minimum volume occupied. Solenoid valve group: Considering the heating of the coil and the power consumption of the entire machine, an energy-saving solenoid valve was selected.

[0148] The fluid system includes a plunger pump and a solenoid valve. Plunger pump: The underwater sequencer requires precise injection of several different reagents. A compact, high-performance, long-life, maintenance-free plunger pump designed for precision liquid operations is selected. It requires less space, makes less noise, and can be pipetted in a single stroke from 1μL to 500μL. Solenoid valve: Considering the complexity of reagents and samples, the FFKM diaphragm material with the best compatibility is selected. It is compatible with strong acids and strong bases to meet the requirements of use. It has fast and silent switching, high back pressure sealing, excellent flushability and a 100% duty cycle, avoiding contamination caused by mixing between reagents and thus affecting the reagent results.

[0149] Stepper motor: Using a screw-type stepper motor can reduce the power consumption of the entire machine and reduce space occupation.

[0150] The operating procedures of the deep-sea in-situ instrument include installation of consumables, cabin sealing, installation and fixation, underwater operations, underwater sequencer recovery, data collection, and underwater sequencer cleaning.

[0151] 1. Installation of consumables

[0152] Before operation, internal piping is cleaned and decontaminated, and consumables are replaced or added. Consumables in the sample pretreatment module include filters and lysis buffers. Consumables in the downstream library construction and detection modules include library construction devices, sequencing devices, reagent kits, and waste liquid bottles. Pipeline exhaust is also performed. Reagent kits include sequencing reagents, library construction and purification reagents, adapter ligation reagents, and PCR library construction reagents.

[0153] 2. Cabin Packaging

[0154] After installing the consumables, check the seals and install the underwater sequencer, referring to Figures 13-16. Place a small amount of desiccant inside the housing 811 to remove moisture and prevent condensation after immersion, which could cause short circuits. The underwater sequencer's internal heat sink module must maintain effective contact with the inner surface of the housing 811 to dissipate heat.

[0155] 3. Installation and Fixing

[0156] The underwater sequencer is moved to the submersible platform and secured with a clamp. An external power supply is supplied via the watertight connector. The main controller 832 of the underwater sequencer confirms that the status is normal, completing the installation.

[0157] 4. Underwater Operations

[0158] There are two ways to start the underwater sequencer: one is to start it automatically with a delay, and the other is to start it after receiving a command. The second method requires an interface to communicate with the deep-sea platform.

[0159] 5. Underwater Sequencer Recovery

[0160] The underwater sequencer is recovered through a deep-sea diving platform, and the surface is rinsed after recovery. It can be disassembled and transferred to the laboratory for processing.

[0161] 6. Data Collection

[0162] Without disassembly, test data can be exported through interfaces located on the hatches of the first sealing end cap 812 and / or the second sealing end cap 813. These interfaces can be used, but are not limited to, serial RS232 and Ethernet ports. Data from the deep-diving platform control board and the single-molecule sequencing system can be stored and exported independently.

[0163] 7. Underwater Sequencer Cleaning

[0164] After dismantling the cabin, clean the internal pipes, remove the used consumables, and wait for the next use.

[0165] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An underwater sequencer, characterized in that: include: A sample extraction device for extracting underwater microbial samples; A library building device, the input port of which is connected to the output port of the sample extraction device, and the library building device is used to build a gene library of the microbial sample; A sequencing device, the input port of which is connected to the output port of the library building device, and the sequencing device is used to perform gene sequencing on the gene library.

2. The underwater sequencer according to claim 1, characterized in that: The sample extraction device comprises a water inlet pipeline, a sample transport pipeline, an enrichment sampler and a sample liquid storage tube; One end of the water inlet pipeline serves as the input port of the sample extraction device, and the other end of the water inlet pipeline is connected to the first feed port of the enrichment sampler; One end of the sample transport pipeline is connected to the discharge port of the enrichment sampler, and the other end of the sample transport pipeline is connected to the sample liquid storage tube; The water inlet pipeline is used to collect microbial samples to the enrichment sampler; The sample transport pipeline is used to transport the enriched microbial sample in the enrichment sampler to the sample liquid storage tube, so that the sample liquid storage tube provides the nucleic acid sample to the library building device.

3. The underwater sequencer according to claim 2, characterized in that: The library building device comprises: A sample purification module, the input port of which serves as the input port of the library building device, and the sample purification module is used to purify the nucleic acid sample to obtain a first purified sample; A volume fixing module, wherein the input port of the volume fixing module is connected to the output port of the sample purification module, and the volume fixing module is used to perform a volume fixing process on the first purified sample to obtain a volume fixing sample; A PCR module, wherein the input port of the PCR module is connected to the output port of the constant volume module, and the PCR module is used to amplify the constant volume sample to obtain an amplified sample; A PCR purification module, wherein the input port of the PCR purification module is connected to the output port of the PCR module, and the PCR purification module is used to purify the amplified sample to obtain a second purified sample; A sequencing adapter connection module, wherein the input port of the sequencing adapter connection module is connected to the output port of the PCR purification module, the output port of the sequencing adapter connection module serves as the output port of the library construction device, and the sequencing adapter connection module is used to connect the second purified sample to the sequencing adapter required for sequencing, and purify the second purified sample after connecting the sequencing head to obtain the nucleic acid to be sequenced.

4. The underwater sequencer according to claim 3, characterized in that: The PCR module includes a temperature control device and a PCR device; The temperature control device is used to control the temperature of the PCR device to a first target temperature, so that the fixed volume sample is amplified in the PCR device to obtain the amplified sample.

5. The underwater sequencer according to claim 4, characterized in that: The temperature control device includes a controller and a heat dissipation component and / or a heating component electrically connected to the controller; When the temperature of the PCR device is lower than the first target temperature, the controller is used to control the heating component to heat the PCR device; When the temperature of the PCR device is greater than the first target temperature, the controller is used to control the heat dissipation component to dissipate heat for the PCR device.

6. The underwater sequencer according to claim 3, characterized in that: The sample purification module comprises: a valve, a nucleic acid sample feeding channel, a first purification liquid feeding channel, a first purification sample discharging channel, a first waste channel and a first purification chamber; The nucleic acid sample feed channel and the first purification liquid feed channel are each connected to the input port of the first purification chamber through a valve, and the first purified sample discharge channel and the first waste channel are each connected to the output port of the first purification chamber through a valve.

7. The underwater sequencer according to claim 6, characterized in that: The first purification liquid feed channel includes a first microbead channel, a first cleaning liquid channel and a first eluent channel; The first microbead channel, the first cleaning liquid channel and the first eluent channel are each connected to the first purification chamber via a valve.

8. The underwater sequencer according to claim 3, characterized in that: The sample purification module comprises: a valve, a one-way valve, a purification container, a main feed pipeline, an air supply branch, a nucleic acid sample feed branch, a cleaning liquid feed branch, an eluent feed branch, a purified sample discharge branch, and a waste branch; The gas supply branch, the nucleic acid sample feed branch, the cleaning liquid feed branch, and the eluent feed branch are each connected to the main feed pipeline via a valve; The output end of the main feed pipeline is also connected to the feed port of the purification container through the one-way valve; The discharge port of the purification container is connected to the purified sample discharge branch and the waste branch respectively.

9. The underwater sequencer according to claim 8, characterized in that: An adsorption column for purifying the nucleic acid sample is arranged in the purification container. The adsorption column comprises two threaded channel joints and an adsorption membrane. The adsorption membrane is arranged between the two threaded channel joints.

10. The underwater sequencer according to claim 3, characterized in that: The volume-fixing module and / or the sequencing adapter connection module comprises: a volume-fixing component; The constant volume component comprises: a valve, a constant volume chamber, a purified sample feed channel, a constant volume liquid feed channel, an auxiliary liquid feed channel, and a constant volume sample discharge channel; The purified sample feed channel, the constant volume liquid feed channel and the auxiliary liquid feed channel are each connected to the input port of the constant volume chamber through a valve; the constant volume sample discharge channel is connected to the output port of the constant volume chamber through a valve.

11. The underwater sequencer according to claim 10, characterized in that: The number of the constant volume chambers is at least two, and the constant volume chambers are connected in sequence; the constant volume component also includes an exhaust channel; The constant volume chamber is connected to at least one constant volume liquid feed channel and / or at least one auxiliary liquid feed channel, and each constant volume liquid feed channel is connected to the input port of a constant volume chamber through a valve, and each auxiliary liquid feed channel is connected to the input port of a constant volume chamber through a valve; the output ports of all or part of the constant volume chambers are connected to an exhaust channel through a valve.

12. The underwater sequencer according to claim 10, characterized in that: At least one row of columns is arranged in the constant volume cavity, and the interval between adjacent columns is less than or equal to the set distance.

13. The underwater sequencer according to claim 4, characterized in that: The PCR device comprises an injection port for introducing a fixed volume of sample; a denaturation zone pipeline, connected to the injection port; a circulation zone pipeline, connected to the denaturation zone and including a plurality of groups of circulation unit pipelines; and The sample outlet is connected to the circulation zone pipeline and is used to discharge the amplified sample.

14. The underwater sequencer according to claim 3, characterized in that: The PCR purification module includes a valve, a second purification chamber, an amplification sample feeding channel, a second purification solution feeding channel, a nucleic acid sample discharging channel, and a second waste channel; The amplification sample feed channel and the second purification liquid feed channel are each connected to the input port of the second purification chamber through a valve, and the second purification sample discharge channel and the second waste channel are each connected to the output port of the second purification chamber through a valve.

15. The underwater sequencer according to claim 14, characterized in that: The second purified liquid feed channel includes a second microbead channel, a second cleaning liquid channel, and a second eluent channel; The second microbead channel, the second cleaning liquid channel and the second eluent channel are each connected to the second purified liquid feed channel via a valve.

16. The underwater sequencer according to claim 3, characterized in that: The sample purification module comprises a first valve body layer, a first spacer layer and a first fluid layer which are stacked; a first purification chamber and a first cut-off position are formed on the first fluid layer, and a through hole penetrating the first valve body layer and the first spacer layer forms a first channel, the first channel connects the first purification chamber and the first cut-off position, and the first cut-off position is located between the first channel and the first purification chamber; a first gas valve through hole which is arranged opposite to the first cut-off position is also provided on the first valve body layer, the first gas valve through hole is used to pass gas so that the first spacer layer is compressed and deformed to fit the first cut-off position, so as to block the first channel and the first purification chamber; wherein the first channel comprises at least one of the following: a nucleic acid sample feeding channel, a first purification liquid feeding channel, a first purified sample discharging channel, and a first waste channel; And / or, the constant volume component included in the constant volume module and / or the sequencing connector connection module includes a second valve body layer, a second spacer layer and a second fluid layer arranged in a stacked manner; a constant volume cavity and a second cut-off position are formed on the second fluid layer, and a through hole penetrating the second valve body layer and the second spacer layer forms a second channel, the second channel connects the constant volume cavity and the second cut-off position, and the second cut-off position is located between the second channel and the constant volume cavity; the second valve body layer is also provided with a second gas valve through hole arranged opposite to the second cut-off position, the second gas valve through hole is used to pass gas to make the second spacer layer deform under pressure and fit with the first cut-off position, so as to block the second channel and the constant volume cavity; wherein, the second channel includes at least one of the following: a purified sample feed channel, a constant volume liquid feed channel, and a constant volume sample discharge channel; And / or, the PCR purification module includes a third valve body layer, a third spacer layer and a third fluid layer which are stacked; a second purification chamber and a third cut-off position are formed on the third fluid layer, and a through hole penetrating the third valve body layer and the third spacer layer forms a third channel, the third channel connects the second purification chamber and the third cut-off position, and the third cut-off position is located between the third channel and the second purification chamber; a third gas valve through hole which is arranged opposite to the third cut-off position is also provided on the third valve body layer, and the third gas valve through hole is used to pass gas so that the third spacer layer is compressed and deformed to fit the third cut-off position, so as to block the third channel and the second purification chamber; wherein, the third channel includes at least one of the following: an amplification sample feed channel, a second purification liquid feed channel, a nucleic acid sample discharge channel, and a second waste channel.

17. The underwater sequencer according to any one of claims 1 to 16, characterized in that: The underwater sequencer also includes a shell, the library building device and the sequencing device are both arranged in the normal pressure chamber of the shell, and the sample extraction device is located on the first end surface of the shell.

18. The underwater sequencer according to claim 17, characterized in that: The underwater sequencer also includes an electrical control system, an air supply system, a waste liquid recovery system and a liquid supply system, and the housing includes a first cavity section, a second cavity section, a third cavity section and a fourth cavity section; The sample purification module included in the library construction device is located in the first chamber section, and the electrical control system is located in the second chamber section; the gas supply system, other modules of the library construction device, the waste liquid recovery system, the sequencing device and the liquid supply system are located in the third chamber section; the PCR module included in the library construction device is located in the fourth chamber section; wherein the other modules are other modules in the library construction device except the PCR module and the sample purification module.

19. The underwater sequencer according to claim 18, characterized in that: The library construction device and the waste liquid recovery system are centrally arranged in the third chamber section; the gas supply system, the sequencing device and the liquid supply system are arranged on the periphery of the library construction device and the waste liquid recovery system; and the liquid supply system is located between the waste liquid recovery system and the PCR module.

20. The underwater sequencer according to claim 17, characterized in that: The underwater sequencer further comprises a first sealing end cover, wherein the first sealing end cover is arranged on a first end surface of the housing; And / or, the underwater sequencer further includes a second sealing end cover, and the second sealing end cover is arranged on the second end surface of the housing.

21. The underwater sequencer according to claim 17, characterized in that: The underwater sequencer further includes a cooling plate and a temperature controller, and the cooling plate and the temperature controller are both located in the housing; The thermostat is used to control the refrigeration fins to cool so that the temperature inside the housing is lower than a second target temperature.

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