Nucleic acid extraction and NGS library building functional island

Abstract

The utility model relates to the technical field of high-throughput sequencing, and discloses a nucleic acid extraction and NGS library building functional island which comprises an automatic integration table top, and a central control system, a first working system, a second working system and a transfer system which are arranged on the automatic integration table top, the first working system comprises an extraction area automatic liquid workstation, a full-automatic high-flux tissue grinder, a temperature control turnover instrument, an automatic centrifugal machine and an automatic nucleic acid extraction instrument, and is used for realizing automatic crushing, splitting decomposition, centrifugation, nucleic acid extraction and purification of plant samples; the second working system comprises a library building area automatic liquid workstation, an automatic PCR (Polymerase Chain Reaction) instrument, an automatic genotyping instrument and a full-automatic nucleic acid protein analysis platform, and is used for realizing NGS library construction, amplification and quality control; and the transfer system comprises a slide rail robot and a full-automatic mobile robot and is used for transferring the consumables. The utility model aims to realize the automatic operation of the whole process of plant sample nucleic acid extraction, NGS library construction, genetic typing and library quality control.

Description

TECHNICAL FIELD

[0001] The utility model relates to high throughput sequencing technical field especially relates to a nucleic acid extraction and NGS library construction function island. BACKGROUND

[0002] NGS is high throughput sequencing technology, and it is the revolutionary nucleic acid sequencing technology after the first generation sequencing, realizes rapid, efficient and low cost nucleic acid sequence analysis through parallel sequencing strategy, and has become the core tool in the fields of molecular biology, genomics, clinical medicine and the like.

[0003] In the field of plant sample nucleic acid extraction, NGS library construction, genotyping and library quality control, the prior art relies on manual operation, and the experiment personnel need to manually transfer samples between different equipment to complete the whole process from plant sample pretreatment to final library quality control. This mode has many manual operation links, and the accuracy and repeatability of experimental results are easily affected by operation errors and cross contamination. Moreover, the equipment is scattered, the sample flow efficiency is low, the labor cost is high and time-consuming under the demand of high throughput experiment. Although there are NGS library construction single machine devices on the market, there is a lack of effective integration and collaborative control between the detection devices, and the whole process automation closed loop cannot be realized. The existing single function automation devices lack collaboration, such as manual transfer of nucleic acid extraction to the library construction workstation, which is difficult to meet the technical needs of high efficiency, standardization and non-human intervention for plant nucleic acid research and NGS library construction. At the same time, in the aspect of full-process automation of large-scale plant gene element screening under the system of synthetic biology, the existing technology cannot meet the needs.

[0004] Therefore, a nucleic acid extraction and NGS library construction function island is needed to solve the above problems. UTILITY MODEL CONTENT

[0005] The utility model aims at providing a nucleic acid extraction and NGS library construction function island, realizing the integrated automation operation of the whole process of plant sample related experiments, improving the experimental efficiency and quality, and reducing the labor cost to solve the problems existing in the prior art.

[0006] In order to achieve the above object, the utility model provides the following scheme: the utility model provides a nucleic acid extraction and NGS library construction function island, comprising:

[0007] The central control system is used for global intelligent scheduling of various equipment;

[0008] The first working system comprises an extraction area automatic liquid workstation, a full-automatic high-throughput tissue grinder, a temperature control turnover instrument, an automatic centrifuge and an automatic nucleic acid extractor, and is used for realizing automatic crushing, lysis, centrifugation, nucleic acid extraction and purification of plant samples;

[0009] The second working system comprises an automatic liquid workstation in a library building area, an automatic PCR instrument, an automatic genotyping instrument, and a full-automatic nucleic acid protein analysis platform, and is used for realizing NGS library construction, amplification, and quality control.

[0010] The transport system comprises a sliding rail track robot and a full-automatic mobile robot, and the sliding rail track robot and the full-automatic mobile robot are used for being responsible for transport of consumables.

[0011] The automatic integrated platform is used as a bearing platform of the full-system equipment, and integrates a wiring slot, illumination, and an ultraviolet disinfection module.

[0012] Preferably, the automatic integrated platform is provided with an automatic transfer window, which is vertically arranged at a physical boundary between the extraction area and the library building area, and realizes non-exposure transport of samples between the extraction area and the library building area.

[0013] Preferably, the automatic liquid workstation in the extraction area comprises a 96-channel mechanical arm, a 360° rotary gripper, and not less than 5 independent temperature control incubation refrigeration modules, and is used for completing plant tissue lysis, magnetic bead nucleic acid extraction, and purification.

[0014] Preferably, the automatic liquid workstation in the extraction area further comprises a plurality of standardized platform positions, and the standardized platform positions comprise a standard platform position, a reagent slot refrigeration carrier, and a deep-well plate reagent refrigeration carrier.

[0015] Preferably, the full-automatic high-throughput tissue grinder comprises a low-temperature adapter and a liquid nitrogen low-temperature tank, the low-temperature adapter and the liquid nitrogen low-temperature tank are matched to maintain a set grinding environment, and a ground and crushed sample is transferred to the automatic liquid workstation in the extraction area by the sliding rail track robot.

[0016] Preferably, the automatic integrated platform is provided with a mobile robot interaction position, which is fixed between the extraction area and the library building area, and realizes connection of intra-island transport and extra-island transport.

[0017] Preferably, the automatic integrated platform is provided with an automatic consumable stack, which adopts a partition storage mode, and realizes automatic identification, inventory early warning, calling, and replenishment of consumables.

[0018] Preferably, the automatic integrated platform is provided with a top cover at a top end, and the top cover is provided with a high-efficiency filtration platform, which is used for maintaining a micro-positive pressure environment in the automatic integrated platform, and guaranteeing cleanliness of an experimental environment.

[0019] Preferably, not less than one sliding rail track robot is arranged in each of the extraction area and the library building area, and the sliding rail track robot moves along a sliding rail laid on the automatic integrated platform.

[0020] Preferably, the full-automatic mobile robot is used for cross-island transfer.

[0021] Compared with the prior art, the nucleic acid extraction and NGS library construction functional island has the following advantages and technical effects: under a synthetic biology system, the nucleic acid extraction and NGS library construction functional island can meet the demand for full-process automation of large-scale plant genetic element screening, and solves the problem that the prior art cannot meet the demand. The central control system is used for globally intelligent scheduling of various devices, realizes full-process automatic closed loop, avoids manual sample transfer between different devices, and reduces the influence of operation errors and cross contamination on the accuracy and repeatability of experimental results. The first working system is composed of an extraction area automatic liquid workstation, a full-automatic high-throughput tissue grinder, a temperature control turnover instrument, an automatic centrifuge and an automatic nucleic acid extractor, and can realize automatic crushing, lysis, centrifugation, nucleic acid extraction and purification of plant samples. The second working system is composed of a library construction area automatic liquid workstation, an automatic PCR instrument, an automatic genotyping instrument and a full-automatic nucleic acid protein analysis platform, and is used for realizing NGS library construction, amplification and quality control. The first working system and the second working system are jointly arranged, realize full-process automation from plant sample pretreatment to final library quality control, overcome the problem of insufficient cooperativity between existing single-function automatic devices, and meet the technical demand for high efficiency, standardization and non-human intervention of plant nucleic acid research and NGS library construction. The transfer system includes a sliding rail track robot and a full-automatic mobile robot, is responsible for consumable transfer, changes the condition of device dispersion and low sample flow efficiency, reduces labor cost and shortens experimental time under high-throughput experimental demand. The automatic integrated table top is a bearing platform of the full-system device, and integrates a wiring slot, illumination and ultraviolet disinfection modules. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor. In the drawings:

[0023] Fig. 1 It is a top view of the nucleic acid extraction and NGS library construction functional island of the present application;

[0024] Fig. 2 It is a schematic diagram of the automatic integrated table top of the present application;

[0025] In the figure: 01, extraction area automatic liquid workstation; 02, fully automatic high-throughput tissue grinder; 03, temperature-controlled turner; 04, automatic centrifuge; 05, automatic nucleic acid extractor; 06, automatic transfer window; 07, library building area automatic liquid workstation; 08, automatic film sealing machine and film tearing machine; 09, automatic multifunctional enzyme label instrument; 10, automatic genotyping instrument; 11, automatic PCR instrument; 12, fully automatic nucleic acid protein analysis platform; 13, automatic 4℃ refrigerator; 14, sliding rail track robot; 15, fully automatic mobile robot; 16, mobile robot interaction site; 17, automatic consumable stack; 18, high-efficiency filtration platform; 19, three-color light buzzer module; 20, automatic integrated countertop. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] In order to make the above-mentioned purposes, features and advantages of the present application more apparent and easy to understand, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0028] Referring to Figs. 1-2 The present embodiment provides a nucleic acid extraction and NGS library building functional island, comprising:

[0029] The central control system is used for globally intelligent scheduling of various devices;

[0030] The first working system comprises the extraction area automatic liquid workstation 01, the fully automatic high-throughput tissue grinder 02, the temperature-controlled turner 03, the automatic centrifuge 04 and the automatic nucleic acid extractor 05, and is used for realizing automatic crushing, lysis, centrifugation, nucleic acid extraction and purification of plant samples;

[0031] The second working system comprises the library building area automatic liquid workstation 07, the automatic PCR instrument 11, the automatic genotyping instrument 10 and the fully automatic nucleic acid protein analysis platform 12, and is used for realizing NGS library construction, amplification and quality control;

[0032] The transfer system comprises the sliding rail track robot 14 and the fully automatic mobile robot 15, and the sliding rail track robot 14 and the fully automatic mobile robot 15 are used for being responsible for consumable transfer;

[0033] The automatic integrated countertop 20 is used as a bearing platform of the full-system device, and integrates a wiring groove, lighting and ultraviolet disinfection modules.

[0034] The utility model discloses a nucleic acid extraction and NGS library construction function island, under the synthetic biology system, can satisfy the demand of large -scale plant gene element screening whole process automation, solve the problem that prior art cannot satisfy this demand. Central control system is used to carry out global intelligent scheduling to various equipment, realizes whole process automation closed loop, avoids manual transfer sample among different equipment, reduces the influence of operating error, cross -contamination to experimental result accuracy and repeatability. The first working system is composed of extraction area automation liquid workstation 01, full -automatic high -throughput organization grinder 02, temperature -controlled overturning instrument 03, automatic centrifuge 04 and automatic nucleic acid extractor 05, can realize the automatic breaking, lysis, centrifugal, nucleic acid extraction and purification of plant sample, the second working system is composed of the library construction area automation liquid workstation 07, automation PCR instrument 11, automation gene typing instrument 10 and full -automatic nucleic acid protein analysis platform 12, is used to realize NGS library construction, amplification and quality control, the first working system and the second working system are jointly arranged, realize the whole process automation from plant sample pretreatment to final library quality control, overcome the problem that the cooperation of existing single function automation equipment is insufficient, satisfy the technical demand of plant nucleic acid research and NGS library construction to high efficiency, standardization, no artificial intervention. The transfer system includes slide rail track robot 14 and full -automatic mobile robot 15, is responsible for consumable transfer, changes the condition that equipment is dispersed, sample flow efficiency is low, under the high -throughput experiment demand, reduces the labor cost, shortens the experiment time -consuming.

[0035] In an embodiment of the utility model, the automation integrated platform 20 is the bearing core of the whole island equipment, adopts the 304 stainless steel integrated structure, has the characteristics of corrosion resistance and easy cleaning. The bottom of the platform is provided with a hidden wiring slot, an independent separation channel of built-in power line and signal line, which avoids signal interference and makes the circuit not exposed; two kinds of auxiliary stages of pull-out type and rotary type are configured, which is convenient for experimental personnel to supply consumables and maintain equipment; at the same time, LED lighting lamp and 254nm ultraviolet lamp module are integrated, the coverage area of the ultraviolet lamp reaches 90% of the platform, the sterilization cycle can be set through the central control system, and automatic sterilization is started, so as to ensure the cleanliness of the experimental environment.

[0036] Further optimization scheme, the automation integrated platform 20 is provided with an automation transfer window 06, the automation transfer window 06 is vertically arranged at the physical demarcation of the extraction area and the library building area, and the sample is transferred without exposure between the extraction area and the library building area.The automation transfer window 06 is vertically arranged at the physical demarcation of the extraction area and the library building area, as the only sample transfer channel between the two areas, two independent air pressure areas are formed through the acrylic plate, specifically, the extraction area maintains 0.3kPa micro-positive pressure, and the library building area maintains normal pressure, to block aerosol cross-contamination from the air pressure gradient.

[0037] In an embodiment of the present application, the automation transfer window 06 is provided with a central turntable driven by electricity, and photoelectric sensors and isolation door linkage devices are arranged on both sides of the turntable; after the nucleic acid sample plate is placed in the turntable by the extraction area mechanical arm, the turntable is rotated by 180° and then transferred into the library building area, and the library building area mechanical arm performs the plate taking action again, to realize the whole process of non-exposure transfer.

[0038] Further optimization scheme, the extraction area automation liquid workstation 01 includes a 96-channel mechanical arm, a 360° rotary gripper and not less than 5 independent temperature control incubation refrigeration modules, for completing plant tissue lysis, magnetic bead method nucleic acid extraction and purification; the extraction area automation liquid workstation 01 further includes a plurality of standardized platform plate positions, and the standardized platform plate position includes a standard plate position, a reagent groove refrigeration carrier and a deep-well plate reagent refrigeration carrier.The extraction area automation liquid workstation 01 is fixed to the right central region of the extraction area, as the operation core of the extraction area, and is integrated with 25 standardized platform plate positions, carries a 96-channel mechanical arm, a 360-degree rotary gripper and five independent temperature control incubation refrigeration modules.Each part is connected in series with the integrated circuit system through the liquid supply pipeline and the liquid discharge pipeline in the workstation, the mechanical arm is combined with a multi-axis transmission mechanism and a grating positioning system, to realize high-precision spatial positioning of ±0.1mm, and can independently complete the liquid adding, liquid dividing and temperature control reaction whole process operation of plant sample pretreatment stage gDNA and total RNA extraction.

[0039] In an embodiment of the present application, the operation instruction of the extraction area automation liquid workstation 01 is issued by the central control system.

[0040] In an embodiment of the present application, the standardized platform plate position of the extraction area automation liquid workstation 01 includes a standard plate position, a reagent groove refrigeration carrier and a deep-well plate reagent refrigeration carrier.

[0041] Further optimization scheme, the full-automatic high-throughput tissue grinder 02 includes a low-temperature adapter and a liquid nitrogen cryogenic tank, the low-temperature adapter and the liquid nitrogen cryogenic tank cooperate to maintain a set grinding environment, and the sample after grinding and crushing is transferred to the extraction area automatic liquid workstation 01 by the slide rail track robot 14.The full-automatic high-throughput tissue grinder 02 is installed on the lower left side of the extraction area and connects with the transfer path of the slide rail track robot 14.It is equipped with 96-hole plate low-temperature adapter and liquid nitrogen cryogenic tank each 1 set, and the grinding environment below-80 DEG C is maintained through vacuum heat insulation liquid nitrogen circulating pipeline, and the plant tissue sample frozen by liquid nitrogen is ground and crushed with high throughput and no cross contamination;The sample plate after crushing is automatically transferred to the designated sample position of the extraction area automatic liquid workstation 01 by the slide rail track robot 14 according to the system instruction.

[0042] In an embodiment of the present application, the temperature control turnover instrument 03 is located in the extraction area, adjacent to the right sample turnover position of the full-automatic high-throughput tissue grinder 02, and is linked with the main control unit of the extraction area automatic liquid workstation 01 through the RS485 communication interface.

[0043] In an embodiment of the present application, the temperature control turnover instrument 03 is located in the extraction area, adjacent to the right sample turnover position of the full-automatic high-throughput tissue grinder 02, and is linked with the main control unit of the extraction area automatic liquid workstation 01 through the RS485 communication interface.

[0044] In an embodiment of the present application, after the operation is completed, the temperature control turnover instrument 03 sends a signal through the photoelectric sensor, and triggers the slide rail track robot 14 to dock and transfer the sample plate.

[0045] In an embodiment of the present application, the automatic centrifuge 04 is provided with two, one is installed on the upper side of the extraction area, and the main body of the device is sunk below the automatic integrated table 20, only the automatic opening and closing type opening above the centrifuge is exposed, and the transfer path of the slide rail track robot 14 is accurately docked;One is installed in the lower side of the library building area, also adopts the sinking table installation design, and the opening height matches the grabbing height of the slide rail track robot 14 in the library building area.

[0046] In an embodiment of the present application, the automatic centrifuge 04 adopts a work mode of partition configuration and on-demand scheduling, and both devices are provided with a wide temperature control module of-4 DEG C to 40 DEG C, and the secondary gripper of the mechanical arm is used to access the island equipment circulation network.

[0047] In an embodiment of the utility model, the highest rotating speed of extraction area automatic centrifuge 04 reaches 6200rpm, and the purification scene of medium and high centrifugal force demand in nucleic acid extraction is adapted; the highest rotating speed of library building area automatic centrifuge 04 is 5100rpm, and the conventional centrifugal operation in NGS library construction is adapted; the running state and load condition of both are fed back to the central control system in real time, and load balancing scheduling is realized by the system, so that equipment idling or congestion is avoided.

[0048] In an embodiment of the utility model, automatic nucleic acid extractor 05 is fixed to the side adjacent position of extraction area automatic liquid workstation 01, and forms a cooperative operation unit with the workstation through a mechanical arm transmission linkage mechanism.

[0049] In an embodiment of the utility model, automatic nucleic acid extractor 05 is configured with 96DW deep hole plate magnetic head, 96DW heat block and 96KF heat block, each one, based on the principle of magnetic bead extraction method, through the precise magnetic attraction-release action of the magnetic head and the room temperature-95 DEG C temperature control linkage of the heat block, the automatic extraction and purification of DNA and RNA are realized; the product plate after extraction is directly transported to the extraction area entrance of automatic transfer window 06 by the mechanical arm, and no intermediate link intervention is needed.

[0050] In an embodiment of the utility model, library building area automatic liquid workstation 07 is installed on the left side of the library building area, and the sample receiving end thereof is directly connected with the output end of automatic transfer window 06, which is the core operation equipment of the library building area.

[0051] In an embodiment of the utility model, library building area automatic liquid workstation 07 comprises 45 standardized table panel positions, is equipped with a set of 96-channel high-throughput mechanical arm, a set of 8-channel high-precision flexible mechanical arm, two sets of 360-degree rotary grab handles, six incubation refrigeration modules and two sets of magnetic force frames; each functional module is cooperatively controlled through the control system of library building area automatic liquid workstation 07, the 96-channel mechanical arm is responsible for high-throughput liquid addition, the 8-channel mechanical arm is responsible for precise dispensing with the minimum volume of 1 mu L, and the whole-step reaction of WGS, RNA-seq and DAP-seq experimental processes can be compatible.

[0052] In an embodiment of the utility model, the operation parameters of library building area automatic liquid workstation 07 are automatically matched by the central control system according to the experimental scheme.

[0053] In an embodiment of the utility model, the standardized table panel positions of library building area automatic liquid workstation 07 comprise standard panel positions, PCR refrigeration panel positions, deep hole plate refrigeration panel positions, shallow hole plate refrigeration panel positions, oscillation plate positions and temperature control oscillation plate positions.

[0054] In an embodiment of the utility model, automatic sealing film machine and tear film machine 08 are configured with 4 sets, 2 sets in extraction area, 1 set is installed to full automatic high throughput tissue grinder 02 right side, is used to seal sample board after grinding, 1 set is installed to the interactive mouth of extraction area automatic centrifuge 04 top, is used to tear film of sample board before centrifugation. Library building area is 2 sets, 1 set is installed to the right side of library building area automatic liquid workstation 07, and automatic sealing film of viscous sealing film can be realized without heating, and the normal temperature reaction plate in library construction is adapted, 1 set is installed to the upper layer of PCR instrument, and is used to tear film of reaction plate before amplification.

[0055] In an embodiment of the utility model, all automatic sealing film machines and tear film machines 08 are connected through internal automatic conveying belt and mechanical docking mechanism of hole plate circulation system.

[0056] In an embodiment of the utility model, automatic sealing film machine and tear film machine 08 include automatic sealing film machine and tear film machine two kinds, and full automatic heat sealing film machine adopts 120 DEG C-180 DEG C adjustable hot pressure sealing film technology, tear film machine adopts flexible silica gel stripping mechanism, can effectively avoid hole plate liquid leakage and sample cross contamination, and action trigger signal and front and rear end equipment form interlocking control.

[0057] In an embodiment of the utility model, automatic genotyping instrument 10 adopts real-time fluorescent quantitative PCR instrument structure, is installed to the lower layer of automatic multifunctional enzyme marker 09 carrier, and is connected through standardization sample transfer track and product output end of library building area liquid workstation.

[0058] In an embodiment of the utility model, automatic genotyping instrument 10 detects nucleic acid concentration through SYBR Green I fluorescent dye method, completes quantitative quality control of library, and also supports SNP site genotyping detection;Detection result and corresponding sample information of library are stored through central control system binding, and standardization detection report can be directly exported.

[0059] In an embodiment of the utility model, automatic PCR instrument 11 is composed of 1 96 channel PCR instrument and 1 384 channel PCR instrument, and the two are installed side by side in the lower layer of library building area tear film machine carrier, and are connected through Ethernet interface and central control system, and are connected with hole plate circulation system and form linkage control. Two devices support the automatic switching of 96 hole plate and 384 hole plate, can be flexibly scheduled according to the experimental throughput demand, meet the nucleic acid amplification demand of different scales;Sample board after amplification is automatically transported back to library building area automatic liquid workstation 07 by slide rail track robot 14, and next step library construction operation is executed.

[0060] In an embodiment of the utility model, full -automatic nucleic acid protein analysis platform 12 is located at the right side of automatic gene typing appearance 10, links together through capillary electrophoresis pipeline and automatic sampling mechanism, and the transfer path of sample end and build base area slide rail track robot 14 is butted. The equipment passes through loading prefabricated adhesive card clamp reagent box, completes capillary electrophoresis separation of nucleic acid fragment, laser induced fluorescence detection and data analysis, and independently generates the analysis report containing the report of fragment length, purity, concentration, and the report is synchronous to central control system for experimental personnel to call.

[0061] In an embodiment of the utility model, automatic multi -functional enzyme label appearance 09 is still provided on automatic integrated platform 20, and automatic multi -functional enzyme label appearance 09 is fixed on the upper layer of the carrier of automatic gene typing appearance 10 in build base area, and communicates in real time with quality control module of central control system through RS232 data bus.

[0062] In an embodiment of the utility model, automatic multi -functional enzyme label appearance 09 integrates host computer optical filter module, grating module each 1 suit, and is equipped with 96 channel synchronous detection module, can carry out automatic detection to the ultraviolet absorbance of nucleic acid sample A260, A280 and A230, and detection data is uploaded to central control system in real time, and nucleic acid concentration and purity analysis report are automatically generated by system, and are used as the core data basis of library quality control.

[0063] In an embodiment of the utility model, automatic integrated platform 20 is still provided with automatic 4 DEG C refrigerator 13, and automatic 4 DEG C refrigerator 13 is installed in the lower right side of build base area, and is connected with sample circulation network of build base area through mechanical arm track.

[0064] In an embodiment of the utility model, automatic 4 DEG C refrigerator 13 is configured with 3 layered automatic carriers inside, each carrier is built-in photoelectric positioning sensor, and 24 standard sample plates can be stably preserved at a time;The movement of carrier and sample access action are all controlled by central control system, realize the automatic access of sample plate and real -time management of inventory, satisfy the short -term refrigeration demand of library sample.

[0065] Further optimization scheme, the mobile robot interaction site 16 is arranged on the automatic integration table 20, and the mobile robot interaction site 16 is fixed between the extraction area and the library building area, so as to realize the connection of island-in and island-out transfer. The mobile robot interaction site 16 is fixed to the upper edge region adjacent to the extraction area and the library building area, as a connection node of island-in and island-out transfer, and can realize precise docking with the full-automatic mobile robot 15. The mobile robot interaction site 16 is connected with the full-automatic mobile robot 15 and the slide rail track robot 14 through a standardized industrial Ethernet interface, a signal conversion module is arranged in the mobile robot interaction site 16 to realize cross-device communication protocol compatibility, a consumable transfer network of “island-in slide rail transfer-interaction site connection-full-automatic mobile robot cross-island transfer” is constructed, and consumable delivery and supply work between the island and other automatic islands is completed, so as to ensure the continuity of multi-island cooperation.

[0066] Further optimization scheme, the slide rail track robot 14 is arranged in the extraction area and the library building area, and the number of the slide rail track robots 14 is not less than one. The slide rail track robot 14 moves along the slide rail arranged on the automatic integration table 20. The slide rail track robot 14 selects a 6-axis mechanical arm as an island-in transfer core, and at least one slide rail track robot 14 is arranged in the center of the extraction area and the library building area and arranged along the high-precision slide rail. The mechanical arm adopts a 6-axis joint design and has a 360-degree full-space grabbing capability, and realizes positioning accuracy of ±0.05 mm through a servo motor and a ball screw transmission mechanism; photoelectric induction switches are arranged at the docking sites of the devices, so as to ensure the accuracy of sample plate grabbing and placing, and the slide rail track robot 14 is mainly responsible for the automatic conduction and transfer of island-in consumables and sample plates between devices, and the action instruction is automatically issued by the central control system according to the experimental process.

[0067] Further optimization scheme, the full-automatic mobile robot 15 is used for cross-island transfer. The full-automatic mobile robot 15 is a core device for cross-island transfer, and is arranged in a special full-automatic mobile robot channel outside the island body, and is provided with a 6-axis joint mechanical arm and an intelligent electric claw. The intelligent electric claw is provided with a pressure sensor, can adapt to different specifications of consumables such as 24-hole plates and 384-hole plates, realizes rapid clamping and stable adaptation, and is provided with a double laser radar sensing system. The double laser radar sensing system can monitor environmental obstacles in a range of 360° in real time, triggers an audible and light alarm and pauses the action immediately when obstacles are encountered, resumes operation after the obstacles are removed, and guarantees the safety and reliability of cross-island transfer.

[0068] Further optimization scheme, the automatic integration countertop 20 is provided with an automatic consumable stack 17, which adopts a partition storage mode to realize automatic identification, inventory warning, calling and replenishment of consumables. The automatic consumable stack 17 adopts a partition storage mode, one extraction area 1 is installed on the right side of the extraction area automatic liquid workstation 01, and one library building area is installed on the right side of the library building area automatic liquid workstation 07. The automatic consumable stack 17 is internally integrated with special storage units for multiple types of consumables such as deep well plates, shallow well plates and pipette tips, and is provided with a self-scanning device that can automatically identify the type and inventory quantity of consumables; through the Internet of Things communication module and the central control system, the data is interconnected, and the collaborative work of the full-automatic mobile robot and the sliding rail mechanical arm is realized, realizing the automatic identification, inventory warning, accurate calling and replenishment of consumables.

[0069] Further optimization scheme, the top of the automatic integration countertop 20 is provided with a top cover, and the top cover is provided with a high-efficiency filtering platform 18 for maintaining a micro-positive pressure environment in the automatic integration countertop 20 and ensuring the cleanliness of the experimental environment. The high-efficiency filtering platform 18 is integrally installed on the top cover on the top of the island body, covering the entire space above the extraction area and the library building area; the high-efficiency filtering platform 18 is provided with four sets of HEPA H14 high-efficiency air filters connected with the island space environment control system through a positive pressure air supply system, and the air supply amount reaches 1000m³ / h, which can maintain a micro-positive pressure environment of 0.5kPa in the island, so that the air cleanliness reaches the hundred-level standard, effectively blocks the invasion of dust and microorganisms in the external environment, and ensures the cleanliness of the experimental environment.

[0070] In an embodiment of the present application, the three-color light buzzer module 19 is installed around the edge of the top of the island body and is connected with the alarm unit of the central control system through a signal cable. The three-color light buzzer module 19 is provided with blue, green and red indicator lights and a buzzer component, and the state display accurately corresponds to the experimental process.

[0071] In an embodiment of the present application, the three-color light buzzer module 19 displays blue with a brightness of 500cd / m² when the experiment is static or paused; the three-color light buzzer module 19 displays green when the experiment is normally running; and the three-color light buzzer module 19 displays red when the experiment has an error, and simultaneously triggers a 85dB buzzer alarm, which directly indicates the running state of the equipment and guides the experimental personnel to intervene and handle in time.

[0072] The running process of the device is as follows:

[0073] The technical scheme of the present application takes the central control system as the core, realizes the cooperation between devices through the sliding rail track robot 14 and the full-automatic mobile robot 15, and completes the full-process automatic operation in the order of "nucleic acid extraction→genotyping→library construction→library quality control".

[0074] The core process of the extraction area is magnetic bead method nucleic acid extraction

[0075] Sample pre-treatment and reagent preparation: After the experiment starts, the extraction area automatic liquid workstation 01 first completes reagent preparation, adds plant tissue lysate, chloroform and other lysing reagents to the sample plate, simultaneously dispenses WashBuffer, elution Buffer and other extraction reagents, and places DNase enzyme, magnetic beads and other reagents that need to be stored at low temperature in the workstation incubation refrigeration module to maintain 4°C storage, ensuring biological activity. The slide rail track robot 14 synchronously transfers the sample plate to the automatic film sealing and film tearing machine 08 to complete sealing, avoiding reagent evaporation and cross contamination.

[0076] Tissue crushing and nucleic acid release: The sealed sample plate is transferred by the slide rail track robot 14 to the fully automatic high-throughput tissue grinder 02 to complete plant tissue grinding and crushing under the low-temperature environment of-80°C liquid nitrogen; then it is flipped and mixed by the temperature-controlled flipper 03 for ±180° to make the tissue fully contact with the lysate and release the nucleic acid; finally it is transferred to the extraction area automatic centrifuge 04 to centrifuge at 6200 rpm for 20 min to separate the nucleic acid from the tissue residue.

[0077] Magnetic bead method nucleic acid purification: After centrifugation is completed, the sample plate is transferred back to the extraction area automatic liquid workstation 01, and the 96-channel mechanical arm of the workstation precisely sucks the supernatant and adds magnetic beads to form a magnetic bead-nucleic acid complex by using the specific adsorption of magnetic beads to nucleic acid; then the sample plate and the dispensed reagent plate are transferred together to the automatic nucleic acid extractor 05 to complete the automatic process of “binding (5 min at room temperature) → washing (Wash Buffer for 3 times) → de-enzyme (DNase enzyme to digest RNA / DNA) → elution (elution Buffer for elution at room temperature)” according to the preset program, and finally high-purity nucleic acid is obtained.

[0078] Cross-zone transfer and concentration pre-adjustment: After nucleic acid extraction is completed, the sample plate is transferred to the library construction area through the isolation turntable of the automatic transfer window 06 to avoid aerosol pollution. The library construction area automatic liquid workstation 07 takes 5 μL of nucleic acid sample and dilutes it 20 times with TE buffer, and transfers it to the automatic multifunctional enzyme marker 09 to detect the A260 / A280 ratio and concentration; the central control system automatically analyzes the data and issues the concentration homogenization parameters to the workstation, which completes the precise dilution of the sample, so that all samples are unified into a standard system of 14.3 ng / μL and 50 μL. The remaining stock solution is transferred to the automatic 4°C refrigerator 13 for cold storage.

[0079] Quality control and detection in parallel: Genotyping

[0080] The automated liquid workstation 07 in the library building area takes 1 μL of the nucleic acid sample after concentration homogenization, automatically prepares the genotyping reaction system according to the proportion of “2x Probe Mix 5 μL + SNP special primer 0.14 μL + nucleic acid sample 1 μL + water 4 μL”, and after the reaction plate is sealed by the automatic sealing and tearing machine 08, it is transferred to the automatic genotyping instrument 10 by the sliding rail track robot 14, and real-time fluorescent quantitative PCR is performed by the fluorescence probe method, and two core tasks are completed at the same time: one is to verify the amplification activity of the nucleic acid sample, and the other is to realize the genotyping detection of the SNP site, and the detection data is uploaded to the central control system in real time for archiving.

[0081] Core process of library building area: NGS library construction

[0082] Nucleic acid fragmentation: The automated liquid workstation 07 in the library building area takes 35 μL of the nucleic acid sample after concentration homogenization, adds fragmentation enzyme and reaction buffer, and prepares a 50 μL fragmentation reaction system; the sample plate is transferred to the automatic PCR instrument 11, and the nucleic acid is broken into target fragments of about 300 bp at 37°C for 15 min and at 65°C for 30 min, and then the reaction is terminated and stored at 4°C. End repair and A tailing are completed at the same time of fragmentation.

[0083] Adapter ligation: After the reaction plate is prepared in the adapter ligation reaction system in the automated liquid workstation 07 in the library building area, it is transferred to the automatic PCR instrument 11 again, and the reaction is terminated and stored at 4°C after reaction at 20°C for 15 min; after returning to the workstation, the unconnected free adapters are removed by using the built-in magnetic stand to adsorb the magnetic beads, and the preliminary purification is completed.

[0084] Library amplification and purification: The automated liquid workstation 07 in the library building area takes the purified adapter ligation product, adds PCR primers, Taq enzyme and amplification buffer, and prepares a 50 μL amplification reaction system; the sample plate is transferred to the automatic PCR instrument 11, and the library amplification is completed according to the program of “95°C pre-denaturation 3 min→(98°C denaturation 20 s + 60°C annealing 15 s + 72°C extension 30 s) x 3 cycles→72°C final extension 5 min”; after the amplification product returns to the workstation, secondary magnetic bead purification is performed by the magnetic stand to remove primer dimers and non-specific products, and a high-quality NGS library is obtained.

[0085] Double verification process: library quality control

[0086] Fragment quality verification: The automated liquid workstation 07 in the library building area takes 1 μL of the library sample diluted 20 times with deionized water, and transfers it to the automatic nucleic acid protein analysis platform 12; the equipment is loaded with a pre-made gel card holder, the nucleic acid fragments are separated by capillary electrophoresis, and after laser-induced fluorescence detection, a fragment distribution map is automatically generated to verify whether the library fragments are concentrated in the range of 200-500 bp.

[0087] Concentration accurate quantification: 1 muL library sample is taken from the workstation, diluted 10000 times, nucleic acid standard with known concentration gradient is added, and qPCR absolute quantification reaction system is prepared; the reaction plate is transported to the automatic genotyping instrument 10, the library concentration is calculated by the standard curve method, the melting curve and the quantification report are automatically generated, and it is ensured that the library concentration meets the sequencing requirements.

[0088] Full-process auxiliary support

[0089] Automatic management of consumables: the full-automatic mobile robot 15 is connected to the island through the mobile robot interaction site 16, and supplies consumables such as deep well plates, suction heads and PCR plates to the automatic consumable stack 17; the automatic consumable stack 17 updates the inventory in real time through the RFID scanner, and when it is detected that a certain type of consumable is insufficient, it automatically sends a warning to the central control system, and simultaneously links with the slide rail track robot 14 to realize accurate retrieval of consumables.

[0090] Environment and state monitoring: the high-efficiency filtration platform 18 continuously maintains 0.5kPa micro-positive pressure and hundred-level cleanliness inside the island to avoid external pollution; the three-color light buzzer module 19 dynamically feeds back the running state - blue represents standby, green represents normal operation, and red light + buzzer alarm corresponds to equipment failure, insufficient reagents and other abnormal conditions, guiding personnel to quickly handle.

[0091] Device bearing and maintenance: all devices are installed on the automatic integrated table 20, hidden wiring grooves separate power lines and signal lines to avoid interference; the pull-out stage and the rotary stage facilitate the replenishment of bulk consumables and can also quickly expose the equipment maintenance interface; after the experiment is completed every day, the central control system automatically starts the ultraviolet lamp module to disinfect the table for 30 minutes, ensuring the cleanliness of the experimental environment the next day.

[0092] In the description of the utility model, it is understood that the orientation or positional relationship indicated by the terms "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like is the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the utility model, and does not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the utility model.

[0093] The above-described embodiments are only preferred modes of the utility model, and do not limit the scope of the utility model, and various modifications and improvements to the technical solutions of the utility model made by those skilled in the art without departing from the design spirit of the utility model should fall within the protection scope of the utility model claim.

Claims

1. A nucleic acid extraction and NGS library preparation functional island, characterized in that, The utility model relates to a kind of automatic nucleic acid extraction and purification system, including: Central control system, for global intelligent scheduling of various devices; First working system, including extraction area automation liquid workstation (01), full-automatic high-throughput tissue grinder (02), temperature control turnover instrument (03), automatic centrifuge (04) and automatic nucleic acid extractor (05), for realizing the automatic breaking, lysis, centrifugation, nucleic acid extraction and purification of plant sample; Second working system, including library building area automation liquid workstation (07), automatic PCR instrument (11), automatic genotyping instrument (10) and full-automatic nucleic acid protein analysis platform (12), for realizing NGS library construction, amplification and quality control; Transport system, including slide rail track robot (14) and full-automatic mobile robot (15), the slide rail track robot (14) and the full-automatic mobile robot (15) are responsible for consumable transport; Automatic integrated platform (20) is used as the bearing platform of full-system equipment, and integrates wiring slot, lighting and ultraviolet disinfection module; Automatic transfer window (06) is arranged on the automatic integrated platform (20), and the automatic transfer window (06) is vertically arranged at the physical boundary between extraction area and library building area, to realize the non-exposure transport of sample between the extraction area and the library building area; The extraction area automation liquid workstation (01) includes 96 channels mechanical arm, 360 ° rotary grab hand and not less than 5 independent temperature control incubation refrigeration module, for completing plant tissue lysis, magnetic bead method nucleic acid extraction and purification;The full-automatic high-throughput tissue grinder (02) includes low-temperature adapter and liquid nitrogen cryogenic tank, and the low-temperature adapter and the liquid nitrogen cryogenic tank cooperate to maintain the set grinding environment, and the sample after grinding and breaking is transferred to the extraction area automation liquid workstation (01) by the slide rail track robot (14).

2. The nucleic acid extraction and NGS library preparation functional island of claim 1, wherein: The extraction area automation liquid workstation (01) further includes a plurality of standardized platform panels, and the standardized platform panels include standard panel, reagent tank refrigeration carrier and deep-well plate reagent refrigeration carrier.

3. The nucleic acid extraction and NGS library preparation functional island of claim 1, wherein: Mobile robot interaction site (16) is arranged on the automatic integrated platform (20), and the mobile robot interaction site (16) is fixed between the extraction area and the library building area, to realize the connection of island-in and island-out transport.

4. The nucleic acid extraction and NGS library preparation functional island of claim 1, wherein: Automatic consumable stack (17) is arranged on the automatic integrated platform (20), and the automatic consumable stack (17) adopts partition storage mode, to realize automatic identification, inventory early warning, calling and supply of consumables.

5. The nucleic acid extraction and NGS library preparation functional island of claim 1, wherein: Top cover is arranged at the top of the automatic integrated platform (20), and high-efficiency filtration platform (18) is arranged on the top cover, to maintain the micro-positive pressure environment in the automatic integrated platform (20), and guarantee the cleanliness of experimental environment.

6. The nucleic acid extraction and NGS library preparation functional island of claim 1, wherein: More than one slide rail track robot (14) is arranged in the extraction area and the library building area, and the slide rail track robot (14) moves along the slide rail laid on the automatic integrated platform (20).

7. The nucleic acid extraction and NGS library preparation functional island of claim 1, wherein: The full-automatic mobile robot (15) is used for cross-island transport.

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