Patent Application
20210291190
Custom gene synthesis, or polynucleotide synthesis, provides a powerful tool for research in biology and medicine and for various biotechnology applications. Gene synthesis typically involves building specially designed oligonucleotides, preparing the oligonucleotides and various reagents in a mixture, and assembling the oligonucleotides using a thermocycler. There remains a need for enhancing the quality of the synthesized polynucleotide and reducing the time required to generate the polynucleotides.
The devices, methods, and systems provided herein address this need, and provide additional advantages as well. The devices, methods, and systems provided herein improve throughput, reduce cost, and provide simple user interface to facilitate collaboration in polynucleotide synthesis. The devices, methods, and systems may comprise a thermocycler comprising a plurality of individual chambers, where the temperature setting of an individual chamber are controlled independently from the temperature setting of another individual chamber, and a system comprising a machine learning for a polynucleotide synthesis for generating a recommendation of a design of experiment for a polynucleotide synthesis. The devices, methods, and systems provided herein can control parameters of individual wells and is small, modular, and easy to maintain. The devices, methods, and systems provided herein may be compatible with laboratory automation, mobile devices, and laboratory information management software (LIMS) to improve throughput, synthesis quality, and reproducibility. Such devices, methods, and systems may improve quality of the polynucleotide product and success of the polynucleotide synthesis by automation and traceability of steps to reduce human errors and by cloud connectivity for distribution of optimized protocols for polynucleotide synthesis. The devices, methods, and systems provided herein may improve the throughput by running multiple protocols with different steps in parallel and using modular architecture that makes it easy to scale. The devices, methods, and systems provided herein may reduce costs by using fewer instruments where each well serves as its own device and has an independent functionality from another well. The devices, methods, and systems provided herein may also reduce costs by reducing labor where the device can fit on a robot deck and is compatible with automated liquid handlers.
Provided herein are devices, system, and methods for thermocycling for polynucleotide synthesis, comprising a plurality of reaction chambers, wherein a temperature setting of an individual reaction chamber can be controlled independent from a temperature setting of another individual reaction chamber. In some embodiments, the device comprises a rotating baseplate, a motor, and a rotating actuator, wherein the plurality of reaction chambers is configured to rotate using the rotating baseplate, the motor, and the rotating actuator. In some embodiments, the device comprises a microprocessor, thermoelectric modules, thermistors (temperature sensors), a heated lid, a heat sink fan, and a power coupling assembly to provide control of the temperature setting of the individual reaction chambers. In some embodiments, the thermoelectric module comprises a thermoelectric cooler (TEC) board and a heat sink, wherein the TEC board comprises a plurality of thermal adhesive pads on a backing material. In some embodiments, the plurality of thermal adhesive pads are spaced apart in similarly as the plurality of reaction chambers. In some embodiments, the thermistor is in contact with an outer wall of the individual reaction chamber. In some embodiments, the device comprises a touchscreen display, a camera, or a microphone, or a combination thereof for interfacing with a user. In some embodiments, the device comprises an online database comprising an artificial intelligence, wherein the online database stores data from a plurality of polynucleotide synthesis reactions, wherein the data comprises reagent conditions, reaction chamber conditions, and quality scores of polynucleotide products. In some embodiments, the online database connects to a web-based application, wherein the web-based application takes an input from a user and displays an output to a user. In some embodiments, the device comprises 96 individual reaction chambers. In some embodiments, the temperature setting of the individual reaction chambers is controllable to at least 0.5° C. In some embodiments, the temperature setting of the individual reaction chambers is controllable to about 0.05° C. In some embodiments, the temperature setting of the individual reaction chambers has a temperature ramp at least 1° C./s. In some embodiments, the temperature setting of the individual reaction chambers has a temperature ramp of about 5° C./s. In some embodiments, the device interfaces with an online database comprising an artificial intelligence, wherein the artificial intelligence generates a report of a recommendation to synthesize a polynucleotide of interest having a high fidelity and wherein the recommendation comprises reagent conditions and reaction chamber conditions. In some embodiments, the generating of the report of the recommendation by a computing system comprises: determining, by artificial intelligence, reagents and reaction chamber conditions to include in the recommendation based on the data of reagents and the data of reaction chamber conditions in the artificial intelligence; and determining, by artificial intelligence, a sequence of the connections of the data of reagents and the data of reaction chamber conditions that provides a polynucleotide quality score above a threshold score as the recommendation. In some embodiments, the device interfaces with the online database by a touchscreen display, a camera, or a microphone, or a combination thereof. In some embodiments, the touchscreen display, the camera, or the microphone, or a combination thereof transmits an input from a user to the online database and displays the report to the user. In some embodiments, the user can modify the report using the touchscreen display, the camera, or the microphone, or a combination thereof before the report is executed by the device. In some embodiments, the online database connects to a web-based application, wherein the web-based application takes an input from a user and displays an output to a user. In some embodiments, the report is provided in a format compatible with the device. In some embodiments, the report provides assignments of a reaction mixture and a reaction chamber condition to a reaction chamber of the thermocycling device. In some embodiments, the method further comprising providing the data of reaction chamber conditions to the thermocycling device; and controlling the reaction chamber conditions of an individual reaction chamber of the thermocycling device after the individual reaction chamber is filled with a corresponding reaction mixture based on the report. In some embodiments, device comprises controlling reaction chamber conditions of a first subset of individual reaction chambers, wherein the first subset is less than the plurality of the reaction chambers, and generating an initial result. In some embodiments, the device comprises controlling reaction chamber conditions of a second subset of individual reaction chambers based on a second recommendation, wherein the second recommendation is generated using the initial result. In some embodiments, the device comprises a module for measuring the reaction chamber conditions of an individual reaction chamber during a run. In some embodiments, the device adjusts the reaction chamber conditions of an individual reaction chamber during the run based on the measured reaction chamber conditions. In some embodiments, the device adjusts the recommendation based on the measured reaction chamber conditions. In some embodiments, the device provides a report of the measured reaction chamber conditions.
Provided herein are computer-implemented methods of training an artificial intelligence for a chemical or biological reaction, comprising: obtaining data of a target product, data of reagents, data of reaction chamber conditions, and a quality score of a reaction product; applying, by a computing system, the data to an artificial intelligence; training, by the computing system, the artificial intelligence, wherein training comprises: assigning a connection between one of the data of reagents and one of the data of reaction chamber conditions; assigning a weight to the connection; generating a reward signal from the quality score of the reaction product; and updating the weight based on the reward signal. In some embodiments, training further comprises determining a sequence of connections that provides a reaction product quality score above a threshold score. In some embodiments, the reaction product is a polynucleotide.
Provided herein are computer-implemented methods of generating a recommendation of a design of experiment for a polynucleotide synthesis comprising: obtaining data of a target molecule; applying, by a computing system, the data to a trained artificial intelligence, wherein training of the artificial intelligence comprises: assigning a connection between one of data of reagents and one of data of reaction chamber conditions or between one of data of reagents and another of data of reagents; assigning a weight to the connection; generating a reward signal from a polynucleotide quality score; and updating the weight based on the reward signal; determining, by the computing system, a recommendation of a design of an experiment by the artificial intelligence, wherein determining comprises: determining reagents and reaction chamber conditions to include in the recommendation based on the data of reagents and the data of reaction chamber conditions in the artificial intelligence; and determining a sequence of the connections of the data of reagents and the data of reaction chamber conditions that provides a polynucleotide quality score above a threshold score as the recommendation; generating, by the computing system, a report of the recommendation, wherein the recommendation comprises the data of reagents and the data of reaction chamber conditions from the sequence. In some embodiments, the target molecule is a polynucleotide. In some embodiments, the artificial intelligence comprises machine learning. In some embodiments, the machine learning comprises reinforcement learning. In some embodiments, the reinforcement learning comprises heuristic optimization, wherein the heuristic optimization reduces a number of the sequences. In some embodiments, the methods comprise applying, by the computing system, a dimensionality reduction prior to step of applying, by a computing system, the data to a trained artificial intelligence, wherein training of the artificial intelligence. In some embodiments, the dimensionality reduction comprises clustering of data of oligonucleotides into groups, wherein the groups are based on similar experimental behaviors of the oligonucleotides. In some embodiments, the dimensionality reduction provides an improvement in mapping of sequences, wherein the improvement is characterized by improvement in polynucleotide quality score, computing time, or computing cost. In some embodiments, the data of reagents comprises data of a reagent identification, a reagent volume, and a reagent concentration. In some embodiments, the data of reagents comprises data of a sequence of a nucleic acid molecule, a volume of the nucleic acid molecule, and a concentration of the nucleic acid molecule. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the data of reaction chamber conditions comprises data of a target temperature, a temperature ramp rate, and a time duration at the target temperature. In some embodiments, the data of reaction chamber conditions comprises specified reaction chamber conditions provided before a run. In some embodiments, the data of reaction chamber conditions comprises measured reaction chamber conditions provided after a run. In some embodiments, the polynucleotide quality score provides a level of fidelity of a synthesized polynucleotide sequence compared to a targeted polynucleotide sequence. In some embodiments, training comprises updating a map of connections of the data of the reagents and the data of the reaction chamber conditions based on the data for the oligonucleotides to improve the polynucleotide quality score. In some embodiments, the report is provided in a format compatible with a thermocycling device for polynucleotide synthesis. In some embodiments, the report provides assignments of a reaction mixture and a reaction chamber condition to a reaction chamber of the thermocycling device. In some embodiments, the method further comprises providing the data of reaction chamber conditions to the thermocycling device; controlling the reaction chamber conditions of an individual reaction chamber of the thermocycling device after the individual reaction chamber is filled with a corresponding reaction mixture based on the report. In some embodiments, the device comprises a plurality of individual reaction chambers, wherein the reaction chamber conditions of the individual reaction chamber is capable of being controlled independently from another individual reaction chamber in the device. In some embodiments, the device comprises 96 individual reaction chambers. In some embodiments, the training takes place in an offline mode, wherein the computing system is not connected to a network. In some embodiments, the training takes place in an online mode, wherein the computing system is connected to a network. In some embodiments, the method further comprises communicating the report to a device for polynucleotide synthesis and providing the reaction chamber conditions to the device. In some embodiments, the computing system is within a remote server or an external database remote from a user. In some embodiments, the computing system is within a server or a database local to a user. In some embodiments, the data are obtained by optical character recognition, voice recognition, touchscreen display input, barcode scanning, or user-initiated data input. In some embodiments, the method comprises controlling reaction chamber conditions of a first subset of individual reaction chambers, wherein the first subset is less than the plurality of the reaction chambers, and generating an first result. In some embodiments, the method comprises controlling reaction chamber conditions of a second subset of individual reaction chambers based on a second recommendation, wherein the second recommendation is generated using the first result, and generating a second result. In some embodiments, the second result has a higher polynucleotide quality score than the first result. In some embodiments, the device comprises a module for measuring the reaction chamber conditions of an individual reaction chamber during a run. In some embodiments, the module measures measuring the reaction chamber conditions of an individual reaction chamber in real-time. In some embodiments, the device adjusts the reaction chamber conditions of an individual reaction chamber during the run based on the measured reaction chamber conditions. In some embodiments, the device adjusts the recommendation based on the measured reaction chamber conditions. In some embodiments, the method comprises measuring the deviation of the measured reaction chamber conditions from the specified reaction chamber conditions; correlating the comparison to the polynucleotide quality score; and adjusting the recommendation based on the correlation. In some embodiments, the device provides a report of the measured reaction chamber conditions.
Provided herein are systems, methods, and devices for training an artificial intelligence for a polynucleotide synthesis comprising: at least one processor; and a memory storing instructions that, when executed by the at least one processor, cause the system to perform: obtaining data of a sequence of a target polynucleotide, data of oligonucleotides, data of reagents, data of reaction chamber conditions, and a polynucleotide quality score; applying, by a computing system, the data to an artificial intelligence; training, by the computing system, the artificial intelligence, wherein training comprises: assigning a connection between one of the data of reagents and one of the data of reaction chamber conditions; assigning a weight to the connection; generating a reward signal from the polynucleotide quality score; and updating the weight based on the reward signal.
Provided herein are systems, methods, and devices for generating a recommendation of a design of experiment for a polynucleotide synthesis comprising: at least one processor; and a memory storing instructions that, when executed by the at least one processor, cause the system to perform: obtaining data of a sequence of a target polynucleotide and data of oligonucleotides; applying, by a computing system, the data to a trained artificial intelligence, wherein training of the artificial intelligence comprises: assigning a connection between one of data of reagents and one of data of reaction chamber conditions or between one of data of reagents and another of data of reagents; assigning a weight to the connection; generating a reward signal from a polynucleotide quality score; and updating the weight based on the reward signal; determining, by the computing system, a recommendation of a design of an experiment by the artificial intelligence, wherein determining comprises: determining reagents and reaction chamber conditions include in the recommendation based on the data of reagents and the data of reaction chamber conditions in the artificial intelligence; and determining a sequence of the connections of the data of reagents and the data of reaction chamber conditions that provides a polynucleotide quality score above a threshold score as the recommendation; and generating, by the computing system, a report of the recommendation, wherein the recommendation comprises the data of reagents and the data of reaction chamber conditions from the sequence. In some embodiments, the artificial intelligence comprises machine learning. In some embodiments, the machine learning comprises reinforcement learning. In some embodiments, the systems comprise applying, by the computing system, a dimensionality reduction prior to step of applying, by a computing system, the data to a trained artificial intelligence. In some embodiments, the polynucleotide quality score provides a level of fidelity of a synthesized polynucleotide sequence compared to a targeted polynucleotide sequence. In some embodiments, the training comprises updating a map of connections of the data of the reagents and the data of the reaction chamber conditions based on the data for the oligonucleotides to improve the polynucleotide quality score. In some embodiments, the report is provided in a format compatible with a thermocycling device for polynucleotide synthesis. In some embodiments, the report provides assignments of a reaction mixture and a reaction chamber condition to a reaction chamber of the thermocycling device. In some embodiments, the method further comprises providing the data of reaction chamber conditions to the thermocycling device; controlling the reaction chamber conditions of an individual reaction chamber of the thermocycling device after the individual reaction chamber is filled with a corresponding reaction mixture based on the report. In some embodiments, the device comprises a plurality of individual reaction chambers, wherein the reaction chamber conditions of the individual reaction chamber is capable of being controlled independently from another individual reaction chamber in the device. In some embodiments, the device comprises 96 individual reaction chambers.
Provided herein are methods, systems, and devices for polynucleotide synthesis, comprising: uploading data of a polynucleotide of interest to a computer-based application comprising an artificial intelligence using a computer; receiving a recommendation of a design of experiment, comprising determining, by artificial intelligence from the data of the polynucleotide of interest, reagents, reagent conditions, and reaction chamber conditions for polynucleotide synthesis to generate a polynucleotide with a high fidelity; and providing a recommendation of reagents, reagent conditions, and reaction chamber conditions for polynucleotide synthesis of the polynucleotide of interest; selecting reagent conditions and reaction chamber conditions from the recommendation to use in polynucleotide synthesis; optionally selecting reagent conditions and reaction chamber conditions not provided in the recommendation to use in polynucleotide synthesis, wherein selecting comprises choosing a reagent of interest, conditions for the reagent of interest, and reaction chamber conditions; preparing reaction mixtures from the selected reagent conditions; loading the reaction mixtures to the corresponding reaction chambers based on the recommendation; starting the thermocycling device to perform synthesis of polynucleotide of interest. In some embodiments, the method further comprises assessing the quality of the synthesized polynucleotide products and uploading the quality to the computer-based application. In some embodiments, the data of reagents comprises data of a reagent identification, a reagent volume, and a reagent concentration. In some embodiments, the data of reagents comprises data of a sequence of a nucleic acid molecule, a volume of the nucleic acid molecule, and a concentration of the nucleic acid molecule. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the data of reaction chamber conditions comprises data of a target temperature, a temperature ramp rate, and a time duration at the target temperature.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
Custom gene synthesis, or custom polynucleotide synthesis, provides a powerful tool for research in biology and medicine and for various biotechnology applications. Gene synthesis, or polynucleotide synthesis, typically involves building specially designed oligonucleotides, preparing the oligonucleotides and various reagents in a mixture, and assembling the oligonucleotides using a thermocycler to generate a polynucleotide or a gene. Once the mixture comprising the oligonucleotides and various reagents is placed inside the thermocycler, the mixture is exposed to a temperature profile that is iterated several times. An example of such polynucleotide synthesis is Gibson assembly. One hurdle in polynucleotide synthesis is that assembling complex polynucleotides from oligonucleotides, or nucleic acid molecules, often can be error prone. There is a need for improved methods for custom polynucleotide synthesis, including improving the quality of the synthesized polynucleotide and reducing the time required to generate the polynucleotides.
The quality of a polynucleotide generated by the polynucleotide synthesis may depend on production of the oligonucleotides, choice of reagents, concentrations of reagents or oligonucleotides, or temperature profiles that the mixture is subject to during thermocycling. The quality of the polynucleotide, or the success or failure of the polynucleotide synthesis, may be assessed by running the reacted mixture on an agarose gel or cloning the product into a plasmid and sequencing it.
Often, the custom polynucleotide molecules have errors and are not assembled properly. In order to generate the polynucleotide without an error, the entire polynucleotide synthesis process may be started over with adjustments in the synthesis conditions. The adjustments to new conditions are typically performed by an experienced person who is skilled in the art of custom polynucleotide assembly. The new adjusted conditions sometimes comprise at least one of adding a new reagent, changing a concentration of a reagent in the mixture, or changing the temperature conditions that the mixture is subjected to by the thermocycler. Often, the adjustments to the polynucleotide synthesis processes comprise changing the temperature and/or the length of time at a given temperature that the mixture is subject to by the thermocycler. Many commercially available thermocyclers are limited to less than 8 different temperature reaction chambers. This limits the number of simultaneous polynucleotide synthesis process conditions that can be tested at one time.
In the chaotic laboratory environment, it may be difficult to keep track of the numerous input factors that go into various experiments. These input factors are critical to assessing trends in experiments, but it can be a tedious process for a user to record the details of the step, especially in a research and development environment where intuitive decisions are highly valued. Experimental details can be recorded in laboratory notebooks, or digitally using spreadsheets or other software programs. However, in a lab environment, freeing one's hands from a pipette, or removing a glove to record experimental details may be often put aside until the end of the experiment. This may leave room for error in the records of the experiment.
The devices, methods, and systems provided herein may improve throughput, reduce cost, and provide simple user interface to facilitate collaboration in polynucleotide synthesis. The devices, methods, and systems may comprise a thermocycler comprising a plurality of individual chambers, where the temperature setting of an individual chamber are controlled independently from the temperature setting of another individual chamber, and a system comprising a machine learning for a polynucleotide synthesis for generating a recommendation of a design of experiment for a polynucleotide synthesis. The devices, methods, and systems provided herein can control parameters of individual wells and is small, modular, and easy to maintain. The devices, methods, and systems provided herein may be compatible with laboratory automation, mobile devices, and laboratory information management software (LIMS) to improve throughput, synthesis quality, and reproducibility. Such devices, methods, and systems may improve quality of the polynucleotide product and success of the polynucleotide synthesis by automation and traceability of steps to reduce human errors and by cloud connectivity for distribution of optimized protocols for polynucleotide synthesis. The devices, methods, and systems provided herein may improve the throughput by running multiple protocols with different steps in parallel and using modular architecture that makes it easy to scale. The devices, methods, and systems provided herein may reduce costs by using fewer instruments where each well serves as its own device and has an independent functionality from another well. The devices, methods, and systems provided herein may also reduce costs by reducing labor where the device can fit on a robot deck and is compatible with automated liquid handlers. As a result, such devices, methods, and systems can reduce capital cost, labor cost, space and energy consumption in a laboratory.
Disclosed herein are devices, methods, and systems for automated polynucleotide synthesis comprising a thermocycler device comprising a plurality of individual chambers, where the individual chamber have a capability to control its own temperature setting, and a machine learning for generating a recommendation of experimental conditions for a polynucleotide synthesis. Through practice of disclosure herein, a user inputs a desired polynucleotide to the system comprising the machine learning to generate a recommendation of a design of experiment for a polynucleotide synthesis, comprising experimental conditions. The user can prepare the reaction mixtures using recommended experimental conditions or using their own experimental conditions. The reaction mixtures can undergo polynucleotide assembly in the thermocycler device having a plurality of chambers, where an individual chamber and the reaction mixture in contact with the individual chamber have their own temperature setting. This allows for testing a variety of temperature conditions (i.e. temperatures and duration of the temperatures) for a single reaction mixture. Practice of some part of the disclosure herein achieves a more automated polynucleotide synthesis. Practice of some part of the disclosure herein achieves a more efficient polynucleotide synthesis with higher fidelity, or a more automated polynucleotide synthesis, by the use of the system comprising the machine learning and the device comprising a plurality of individual chambers with its own temperature control. Practice of some part of the disclosure herein provides a faster way to synthesize a polynucleotide having a higher fidelity and determine the conditions that synthesize the polynucleotide having the higher fidelity. In some cases, practice of some part of the disclosure herein reduces process time to less than one day to one week as compared to from around 2 weeks to 6 months.
Provided herein are devices, methods, and systems for thermocycling for polynucleotide synthesis, comprising a plurality of reaction chambers, wherein a temperature setting of an individual reaction chamber can be controlled independent from a temperature setting of another individual reaction chamber. The device disclosed herein may interface with an online database comprising an artificial intelligence, wherein the artificial intelligence generates a report of a recommendation to synthesize a polynucleotide of interest having a high fidelity and wherein the recommendation comprises reagent conditions and reaction chamber conditions. Alternatively or in combination, the device disclosed herein may interface with a web-based interface and an online database. In some embodiment, a user selects and save reagent conditions and reaction chamber conditions in the online database. The web-based interface may control the reaction chamber conditions of the device based on the user selection.
Provided herein are computer-implemented methods of training an artificial intelligence for a chemical or biological reaction, comprising: obtaining data of a target product, data of reagents, data of reaction chamber conditions, and a quality score of a reaction product; applying, by a computing system, the data to an artificial intelligence; training, by the computing system, the artificial intelligence, wherein training comprises: assigning a connection between one of the data of reagents and one of the data of reaction chamber conditions; assigning a weight to the connection; generating a reward signal from the quality score of the reaction product; and updating the weight based on the reward signal.
Provided herein are computer computer-implemented methods of generating a recommendation of a design of experiment for a polynucleotide synthesis comprising: obtaining data of a target molecule; applying, by a computing system, the data to a trained artificial intelligence, wherein training of the artificial intelligence comprises: assigning a connection between one of data of reagents and one of data of reaction chamber conditions or between one of data of reagents and another of data of reagents; assigning a weight to the connection; generating a reward signal from a polynucleotide quality score; and updating the weight based on the reward signal; determining, by the computing system, a recommendation of a design of an experiment by the artificial intelligence, wherein determining comprises: determining reagents and reaction chamber conditions to include in the recommendation based on the data of reagents and the data of reaction chamber conditions in the artificial intelligence; and determining a sequence of the connections of the data of reagents and the data of reaction chamber conditions that provides a polynucleotide quality score above a threshold score as the recommendation; generating, by the computing system, a report of the recommendation, wherein the recommendation comprises the data of reagents and the data of reaction chamber conditions from the sequence. The methods disclosed herein further comprises providing the data of reaction chamber conditions to the thermocycling device; and controlling the reaction chamber conditions of an individual reaction chamber of the thermocycling device after the individual reaction chamber is filled with a corresponding reaction mixture based on the report.
Provided herein are systems for training an artificial intelligence for a polynucleotide synthesis comprising: at least one processor; and a memory storing instructions that, when executed by the at least one processor, cause the system to perform: obtaining data of a sequence of a target polynucleotide, data of oligonucleotides, data of reagents, data of reaction chamber conditions, and a polynucleotide quality score; applying, by a computing system, the data to an artificial intelligence; training, by the computing system, the artificial intelligence, wherein training comprises: assigning a connection between one of the data of reagents and one of the data of reaction chamber conditions; assigning a weight to the connection; generating a reward signal from the polynucleotide quality score; and updating the weight based on the reward signal.
Provided herein are systems for generating a recommendation of a design of experiment for a polynucleotide synthesis comprising: at least one processor; and a memory storing instructions that, when executed by the at least one processor, cause the system to perform: obtaining data of a sequence of a target polynucleotide and data of oligonucleotides; applying, by a computing system, the data to a trained artificial intelligence, wherein training of the artificial intelligence comprises: assigning a connection between one of data of reagents and one of data of reaction chamber conditions or between one of data of reagents and another of data of reagents; assigning a weight to the connection; generating a reward signal from a polynucleotide quality score; and updating the weight based on the reward signal; determining, by the computing system, a recommendation of a design of an experiment by the artificial intelligence, wherein determining comprises: determining reagents and reaction chamber conditions include in the recommendation based on the data of reagents and the data of reaction chamber conditions in the artificial intelligence; and determining a sequence of the connections of the data of reagents and the data of reaction chamber conditions that provides a polynucleotide quality score above a threshold score as the recommendation; and generating, by the computing system, a report of the recommendation, wherein the recommendation comprises the data of reagents and the data of reaction chamber conditions from the sequence.
Provided herein are methods for polynucleotide synthesis, comprising: uploading data of a polynucleotide of interest to a computer-based application comprising an artificial intelligence using a computer; receiving a recommendation of a design of experiment, comprising determining, by artificial intelligence from the data of the polynucleotide of interest, reagents, reagent conditions, and reaction chamber conditions for polynucleotide synthesis to generate a polynucleotide with a high fidelity; and providing a recommendation of reagents, reagent conditions, and reaction chamber conditions for polynucleotide synthesis of the polynucleotide of interest; selecting reagent conditions and reaction chamber conditions from the recommendation to use in polynucleotide synthesis; optionally selecting reagent conditions and reaction chamber conditions not provided in the recommendation to use in polynucleotide synthesis, wherein selecting comprises choosing a reagent of interest, conditions for the reagent of interest, and reaction chamber conditions; preparing reaction mixtures from the selected reagent conditions; loading the reaction mixtures to the corresponding reaction chambers based on the recommendation; and starting the thermocycling device to perform synthesis of polynucleotide of interest. The methods disclosed herein further comprise assessing the quality of the synthesized polynucleotide products and uploading the quality to the computer-based application.
Disclosed herein is a thermocycling device having a plurality of reaction chambers, where the temperature setting of an individual chamber can be independently controlled. The device provides a user up to a plurality of reaction chambers with its own independent experimental conditions to assemble a polynucleotide. Alternatively or in combination, the device interfaces with an online database where specialized artificial intelligence (AI) algorithms automatically optimize the temperature conditions for each reaction chamber based upon the custom polynucleotide of interest and the reagents for the experiments. In some embodiments, the AI algorithm comprises machine learning (ML) algorithm. In some embodiments, the ML algorithm comprises reinforcement learning (RL) algorithm.
In an exemplary embodiment, the device serves as the main interface to the online database. The user can input any or all factors that are part of the experiment using manual input (via touchscreen display), machine readable barcode, optical character recognition or voice instruction. Alternatively or in combination, the user can input the factors affecting the experiment using a web-based application for the online database from a computer in order to plan the polynucleotide synthesis experiment. In some embodiments, the computer is separate from the device and is not connected directly to the thermocycling device. The computer may be connected to the device by internet. In some embodiments, the computer is connected to the thermocycling device. In some embodiments, the user interface is connected to the thermocycling device or a part of the thermocycling device. Once the experimental factors are selected, the machine learning algorithms search through the online database to select the optimal conditions to run the polynucleotide synthesis experiment. The web-based application can guide the user through the process of designing an experiment, including but not limited to input of polynucleotide of interest, selection of factors recommended by the online database, and addition of additional factors from the user. The chosen experimental conditions can be uploaded to the thermocycling device. Alternatively or in combination, the chosen experimental conditions can be provided to the user as a report, a display on a screen, or an audible verbal instruction through a speaker. The experimental conditions may comprise a type reagent to add, a volume of the reagent, a concentration of the reagent, and instructions on which individual reaction chamber to add the reagent.
The device can subject the loaded reaction mixture in individual reaction chamber to one of the optimized process conditions. The device can perform a plurality of individual experimental conditions in a single run as the temperature setting of the individual reaction chamber can be controlled independently.
In some embodiments, the devices, methods, and systems for polynucleotide synthesis comprises a single device capable of miniaturized liquid handling, multi-channel thermocycling, and polynucleotide sequencing. In some embodiments, the liquid handling component prepares multiple mixtures comprising different combinations of oligonucleotides, primers, and reagents at various concentrations. In some embodiments, the devices, methods, and systems for polynucleotide synthesis runs multiple temperature protocols based upon recommendations from the AI engine in parallel in multiple individual wells. In some embodiments, the resulting mixtures undergo through library preparation on the thermocycling and liquid handling components of the device to synthesize the target polynucleotides.
In some embodiments, at least one of the wells has resulting polynucleotide products having an average of 100% accuracy to the target polynucleotide. In some embodiments, at least one of the wells has resulting polynucleotide products having at least an average of 99% accuracy to the target polynucleotide. In some embodiments, at least one of the wells has resulting polynucleotide products having at least an average of 98% accuracy to the target polynucleotide.
The devices, systems, and methods described herein provide various advantages. In some embodiments, the devices, systems, and methods described herein allow for easier comparison of different protocols within a single run instead of using multiple thermocycler. In some embodiments, the devices, systems, and methods described herein allow for easier sharing and comparison of experimental protocols amongst the users. In some embodiments, the devices, systems, and methods described herein allow for saving of temperature protocols and input process data electronically that is easily accessible by the users and their collaborators.
The thermocycler device as disclosed herein comprises a rotating baseplate, a motor, a rotating actuator, a microprocessor, thermoelectric modules, thermistors (temperature sensors), a heated lid, a heat sink fan, a power coupling assembly, and a touchscreen display. Optionally, the device further comprises at least one of an online database, a camera, and a microphone. The thermocycling device is also referred herein as thermocycler or thermal cycler.
In some embodiments, the devices, methods, and systems provided herein comprise an integrated microfluidic workflow that monitor and control various process variables. In some embodiments, the various process variables include but are not limited to temperature, volume, time, optical density, or optical characteristics. In some embodiments, the device is a small, high throughput thermocycling device with multichannel control. In some embodiments, the device can combine with existing microfluidic liquid handling and quality control technology. In some embodiments, the device comprises sensors and control methods to regulate, monitor and control various process variables. In some embodiments, the devices, methods, and systems provided herein allow for flexible, reconfigurable workflows.
Provided herein are devices, methods, and systems having a capability to control the functionality of the individual wells independently where an individual well has the functionality of an independent instrument. The independently controlled functionality is achieved by embedded heating, cooling, and sensing mechanism for each individual well. In some embodiments, the devices, methods, and systems achieves 100° C. temperature difference between neighboring wells. In some embodiments, the devices, methods, and systems have temperature accuracy to 0.1° C.
In some embodiments, the devices, methods, and systems provided herein comprise an automated lid for automatic integration. In some embodiments, the devices, methods, and systems comprise samples and reagents that are barcoded and tracked by barcodes, where a barcode is specific to a sample or a reagent. In some embodiments, the devices, methods, and systems comprise a cloud-based or walk up operation.
An example of the disclosure provided herein is illustrated in
A detailed view of the core assembly mounted on top of the rotating base in an exemplary thermocycling device is shown in
Another example of a rotating actuator 8 for an exemplary thermocycling device is shown in
A detailed view of the power coupling assembly with the contacts in the energized position in an exemplary thermocycling device is shown in
A section view of well sleeve showing installation slot, thermistor, and thermoelectric module in an exemplary thermocycling device is shown in
In some embodiments, the well sleeve is a chamber or a reaction chamber. In some embodiments, a chamber or a reaction chamber is placed inside the well sleeve and the well sleeve surrounds the outside wall of the chamber or well. In some embodiments, the well sleeve comprises materials compatible with polynucleotide synthesis reaction. In some embodiments, the material for well sleeve comprises at least one of polyethylene, polypropylene, PTFE, plastics, and polymers. In some embodiments, the well sleeve comprises a heat conducting material. In some embodiments, the well sleeve comprise comprises heat conducting metal. In some embodiments, the well sleeve comprises aluminum. In some embodiments, the well sleeve comprises stainless steel. In some embodiments, the material for well sleeve is coated to provide a desired feature, including but not limited to increased hydrophilicity, hydrophobicity, low binding, etc.
In some embodiments, the individual well sleeve can hold various volumes. In some embodiments, the individual well sleeve holds at least 0.01 ml, at least 0.02 ml, at least 0.03 ml, at least 0.04 ml, at least 0.05 ml, at least 0.06 ml, at least 0.07 ml, at least 0.08 ml, at least 0.09 ml, at least 0.1 ml, at least 0.2 ml, at least 0.3 ml, at least 0.4 ml, at least 0.5 ml, at least 0.6 ml, at least 0.7 ml, at least 0.8 ml, at least 0.9 ml, or at least 1.0 ml. In some embodiments, the individual well sleeve holds about 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, or 1.0 ml. In some embodiments, the individual well sleeve holds no more than 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, or 1.0 ml.
An exploded view of core assembly showing component parts including cover, threaded well plate, thermoelectric PCB and thermistor PCB in an exemplary thermocycling device is shown in
Top and bottom views of the dynamic routing mechanism of the signal wires in an exemplary thermocycling device in a load positions and a run position is shown in
An example of high level system architecture for thermal portion of an exemplary thermocycling device is shown in
An example of a microprocessor architecture is shown in
An example of well sleeve temperature control architecture of an exemplary thermocycling device is shown in
In some embodiments, the thermocycling device comprises a thermoelectric cooler (TEC) board and heat sink to independently control the temperatures of individual chambers.
In an exemplary embodiment, the TEC board assembly is assembled by applying the thermal adhesive pad to the heat sink before placing the TEC board. The TEC board is then placed on to the base plate, and the adhesive sticker protecting the TEC board is removed. The TEC board assembly is mated with the well assembly and secured with screws. A thermistor board is placed onto the mated TEC board-well assembly, and thermal adhesive is applied to each thermistor to bond it to each well. A silicone pad is placed over the assembly. A fiberglass protective cover is further placed on top and secured with screws.
In some embodiments, the thermocycling device comprises a TEC board and well assembly to independently control the temperatures of individual chambers.
In an exemplary embodiment, the TEC board and well assembly is assembled by pressing the dowel pins 8 into the heat sink 1 to a predefined height. Apply heatsink adhesive 10 to heatsink 1 in the predefined location. Remove adhesive backing. Adhere TEC PBC 2 to heat sink 1 in a predefined orientation. Press evenly over the entire surface to ensure even adhesion. Attach TEC PCBA adhesive 9 to the top surface of each TEC of the TEC PCB. Leave adhesive backing in place. Assemble well holder 3, silicone mat 6 and all the individual wells 4 in an inverted orientation. Remove TEC adhesive backing from the TEC PCB and bond the PCB with heat sink to the components from the previous step while they remain inverted. Apply even pressure across entire surface area of heat sink bottom to ensure the TEC adhesive bonds to the well bottoms. While holding the bonded components from the previous step together, turn over and install the screws.
Alternatively or in combination, in some embodiments, the thermocycling device comprises a TEC board and well assembly to independently control the temperatures of individual chambers. In some embodiments, the thermistor active material is integrated into the individual well itself. In some embodiments, the integrated design circumvents the need to apply thermal adhesive and bond the thermistor to each well.
In some embodiments, the spot of thermal adhesive pads have a smaller area than the average diameter of a well of a standard multi-well plate. In some embodiments, the spot of thermal adhesive pads have a smaller area than the average diameter of a well of a standard multi-well plate by at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, the spot of thermal adhesive pads have a smaller area than the average diameter of a well of a standard multi-well plate by no more than 10%, 20%, 30%, 40%, or 50%. In some embodiments, the spot of thermal adhesive pads have a larger area than the average diameter of a well of a standard multi-well plate. In some embodiments, the spot of thermal adhesive pads have a larger area than the average diameter of a well of a standard multi-well plate by at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, the spot of thermal adhesive pads have a larger area than the average diameter of a well of a standard multi-well plate by no more than 10%, 20%, 30%, 40%, or 50%.
In some embodiments, a well sleeve has a larger area than the average diameter of a well of a standard multi-well plate. In some embodiments, a well sleeve has a larger area than the average diameter of a well of a standard multi-well plate by at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, a well sleeve has a larger area than the average diameter of a well of a standard multi-well plate by no more than 10%, 20%, 30%, 40%, or 50%. In some embodiments, the well sleeves in a well assembly have the same dimensions. In some embodiments, the well sleeves in a well assembly have different dimensions. In some embodiments, the well sleeves in a well assembly have more than one diameter. In some embodiments, the well sleeves in a well assembly have more than one height.
Provided herein are devices, systems, and methods to assess and calibrate the temperature control of individual wells. In some embodiments, a temperature assessment device is a calibration tool to assess and calibrate the temperature control of individual wells. In some embodiments, the temperature assessment device fits into a single individual well to calibrate the temperature control of the individual wells over time. In some embodiments, the temperature assessment device comprises a male version of an individual well. In some embodiments, the male version is substantially an inverse casting of the well space within the individual well. In some embodiments, the male version fits closely to the inner wall of the individual wall. In some embodiments, the temperature assessment device comprises a plurality of the male versions. In some embodiments, the temperature assessment device comprises a plurality of the male versions for a 96 well plate.
The temperature assessment device can use various code and steps to calibrate the temperature control of the individual wells. In some embodiments, the temperature assessment device uses a pseudo code. In some embodiments, the process is automated between the temperature assessment device and the thermocycler unit. In some embodiments, a user selects to calibrate on the thermocycler device. This directs the temperature assessment device to begin and start making set point of the temperature assessment device starting temperature. In each cycle, using feedback from the RTD of the temperature assessment device, the temperature assessment device controls the TEC amplifier until a steady state is reached at the set point. In some embodiments, the steady state is reached when the set point temperature varies less than 0.05° C. over a 10 second time interval. Once the steady state is reached, the temperature assessment device begins reading the measured temperatures from the TEC control board and saves the measured temperatures and the set point temperatures. The temperature assessment device begins increasing the set point by a predetermined increment and the cycle is repeated to set temperature, reach steady state, and take the measured temperature until the maximum temperature is reached. When the maximum temperature is reached, the thermocycler communicates to the temperature assessment device to cut the power to the TEC amplifier to cool down the temperature assessment device. Then, the measured temperature data are used to fit a fourth order polynomial for each of the wells. In some embodiments, the measured temperature data are used to fit to various non-linear relationships for each of the wells. Using the coefficients from the non-linear fit, the thermocycler calculates the actual temperature as compared to the measured temperatures from the TEC control board.
A block diagram demonstrating examples of user input methods for experimental runs and how the inputs interface to an online database is shown in
An example of a flowchart of the reinforcement learning process is shown in
The reinforcement learning process can be performed offline or online. In some embodiments, the offline learning facilitates learning from a large dataset or learning from data in batches. In some embodiments, online learning facilitates a more real-time feedback, where an experiment can be performed, feedback of the experimental results can be provided to the reinforcement learning algorithm, and the reinforcement learning algorithm can adapt based on the reward signal for the experimental results. In some embodiments, the online learning provides the capability to generate new data and control experiments. In some embodiments, the online learning may be more expensive per experiment because the online learning actuates the environment and uses reagents. In some embodiments, the online learning provides a link to design of experiments (DOE) as well as heuristic optimizations, which can reduce total search space of possible action sets and the processing time.
An example of a reinforcement learning for a positive or a good outcome is shown in
An example of a solution search space for the reinforcement learning process is shown in
The device, methods, and systems herein can provide a clearinghouse for polynucleotide synthesis recipe information. In some embodiments, the disclosure herein comprises a cloud hosted database of polynucleotide synthesis recipe execution and optimization information. In some embodiments, the reinforcement learning algorithm is able to capture knowledge of synthetic biologists and those skilled in the art and automate the adjustments those skilled in the art may make to improve the fidelity of synthesized polynucleotide. In some embodiments, the disclosure herein allows a user to search and see what oligonucleotides or reagents and/or under what conditions the oligonucleotides or reagents have been used by other users. In some embodiments, the disclosure herein provides a design of experiment framework for polynucleotide synthesis. In some embodiments, the design of experiment framework can recommend action sets of best practices based on user inputs and constraints. As the optimization engine builds knowledge, recommendations may be based on prior experimental learning information. In some embodiments, the disclosure herein provides a genetic algorithm for discovery.
Exemplary flowcharts of the reinforcement learning in providing recommended experimental conditions for a polynucleotide synthesis in a web-based application are shown in
A person of ordinary skill in the art will recognize many variations based on the teaching described herein as shown in
In some embodiments, a web-based interface controls the devices described herein. In some embodiments, the web-based interface comprises software to control the thermocycler from the web. In some embodiments, a user controls the software to control the thermocycler. In some embodiments, the web-based interface provides remote programming and monitoring of the device. In some embodiments, the web-based interface comprises a mobile application or a web application that controls one or more devices. In some embodiments, the web-based interface uses machine learning to improve the polynucleotide synthesis process over time with additional runs. In some embodiments, the web-based interface provides for sharing of protocols amongst users and devices. In some embodiments, the web-based interface comprises tools to create a temperature profile for an individual well that is distinct from a temperature profile for another well in the same well plate. In some embodiments, the web-based interface allows for automation of protocol selection and/or robotic operation. In some embodiments, the web-based interface is an application programming interface (API). In some embodiments, the API allows for easy integration with laboratory information management system and automation.
In some embodiments, the devices, methods, and systems provided herein comprise a central database that consolidates all workflows, process variables and performance metrics from users. In some embodiments, the devices, methods, and systems provided herein use machine learning and artificial intelligence to find trends in the data provided by the users.
In some embodiments, the temperature protocols and the input process data are stored in a centralized database. In some embodiments, the centralized database has temperature protocols and the input process data are from multiple users. In some embodiments, the centralized database facilitates sharing of data by one user to another user. The sharing of data facilitates scientific collaboration by sharing of experimental protocols and collaborative optimization of the protocols using PCR (DNA assembly, cloning, library preparation, etc.).
In some embodiments, the input process data are provided to the devices, methods, and systems for polynucleotide synthesis. In some embodiments, the input process data comprises oligonucleotide design, primer design, components, or reagents. In some embodiments, the oligonucleotides are designed by (digitally) breaking up the target polynucleotide into short segments of single stranded oligonucleotides at various points. The points at which they are “cut” is critical to DNA assembly. In some embodiments, the primers are designed such that once the polynucleotide has been assembled from the oligonucleotides, subsequent PCR can be performed. In some embodiments, the components and reagents are provided in polynucleotide assembly kits. In some embodiments, the components and reagents comprise buffers, polymerase and dNTP's. In some embodiments, the components and reagents further comprise additives such as magnesium, DMSO, EDTA.
In some embodiments, the devices, methods, and systems for polynucleotide synthesis provide an output data. In some embodiments, quantitative output metrics post-assembly comprise sequences and concentrations of the polynucleotide product. In some embodiments, the synthesis result, including but not limited to the polynucleotide quality score, quality, and accuracy, is combined with the input process data and uploaded to the online database. In some embodiments, the systems and methods provided herein interfaces with a LIMS system to save the input process data and output data. In some embodiments, the systems and methods provided herein uses a web interface to save the input process data and output data. In some embodiments, the devices, systems, and methods provided herein use AI to find trends in the data and to make process recommendations to the user.
The disclosure herein may improve upon the existing system by allowing the synthesis of polynucleotides or any other thermally assembled macromolecule in a high throughput approach. This approach can be combined with optimized process conditions that are specially prepared by machine learning algorithms that learn from an online database. The device is “high throughput,” because it can perform many different runs with different temperature conditions and reaction mixture conditions in parallel, which can save the time as compared to serial runs. The thermocycler device is “smart,” because it interacts with the user who enters the process input conditions for the polymeric molecule of interest for synthesis.
In various embodiments disclosed herein, the device is capable of controlling independently the temperature condition of individual reaction chambers. In some embodiments, the device has an externally mounted touchscreen display, camera and microphone, or a combination thereof that are used to interface with the user. When a user decides to begin an experimental run, they can input their experimental factors in a number of ways, including but not limited to manually via touchscreen, voice commands, by scanning reagent barcodes in front of the camera or by presenting hand written notes, product labels, etc. The optical character recognition system in the device can take this unstructured data and turn it into a set of structured input variables. Nonlimiting examples of these input variables include oligonucleotides used, polynucleotides to be made, reagents, concentrations, volumes etc.
Once the run input variable process is complete, the device can relay all the data up to the online database where a unique series of machine learning algorithms will combine the custom input factors for this experiment with the complete archive of anonymous input variables of past experiments from other users operating other devices and use past run quality data to develop a set of optimized experimental conditions for this users particular process. These optimized process conditions will be sent back down to the device and applied to each well in the device.
Once the optimized process are uploaded to the device and the user loads a 96 well plate containing the starting solutions for their experimental run, the run sequence can begin. The microprocessor on the device begins this process by sending a signal to the baseplate motor which starts to rotate a baseplate rotating actuator 7, 8 in
The core assembly of the devices and systems disclosed herein allows for individual thermal control of individual chambers. The device provides precise temperature control (i.e. about 0.05° C.) and fast ramp rates (up to 5° C./s) of individual chambers. In some embodiments, the device is capable of providing temperature control of at least about 0.01° C., 0.02° C., 0.03° C., 0.04° C., 0.05° C., 0.06° C., 0.07° C., 0.08° C., 0.09° C., 0.1° C., 0.2° C., 0.3° C., 0.4° C., or 0.5° C. In some embodiments, the device is capable of providing temperature control to at most about 0.01° C., 0.02° C., 0.03° C., 0.04° C., 0.05° C., 0.06° C., 0.07° C., 0.08° C., 0.09° C., 0.1° C., 0.2° C., 0.3° C., 0.4° C., or 0.5° C. In some embodiments, the device is capable of providing temperature control of about 0.01° C., 0.02° C., 0.03° C., 0.04° C., 0.05° C., 0.06° C., 0.07° C., 0.08° C., 0.09° C., 0.1° C., 0.2° C., 0.3° C., 0.4° C., or 0.5° C. In some embodiments, the device is capable of temperature accuracy of at least about 0.01° C., 0.02° C., 0.03° C., 0.04° C., 0.05° C., 0.06° C., 0.07° C., 0.08° C., 0.09° C., 0.1° C., 0.2° C., 0.3° C., 0.4° C., or 0.5° C. In some embodiments, the device is capable of temperature accuracy of about 0.1° C. In some embodiments, the device is capable of temperature accuracy of about 0.05° C. In some embodiments, the temperature ramp rate is the heating rate. In some embodiments, the temperature ramp rate is the cooling rate. In some embodiments, the temperature ramp rate is for an individual chamber. In some embodiments, the device is capable of providing temperatures ramp rates of at least about 1° C./s, 2° C./s, 3° C./s, 4° C./s, 5° C./s, 6° C./s, 7° C./s, 8° C./s, 9° C./s, or 10° C./s. In some embodiments, the device is capable of providing temperatures ramp rates of about 5° C./s. In some embodiments, the device is capable of providing temperatures ramp rates of about 4° C./s. In some embodiments, the device is capable of temperature difference between neighboring wells of at least about 10° C., 20° C., 30° C., 40° C., 50° C., 60° C., 70° C., 80° C., 90° C., 100° C., 110° C., 120° C., or 130° C. In some embodiments, the device is capable of temperature difference between neighboring wells of no more than about 50° C., 60° C., 70° C., 80° C., 90° C., 100° C., 110° C., 120° C., 130° C., 140° C., or 150° C. In some embodiments, the device is capable of temperature difference between neighboring wells is about 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., or 100° C. In some embodiments, the device is capable of temperature difference between neighboring wells is about 98° C. In some embodiments, the device is capable of temperature difference between neighboring wells is about 100° C. In some embodiments, the device has a temperature range of about 0° C.-100° C., 0° C.-95° C., 0° C.-90° C., −10° C.-100° C., or −20° C.-100° C.
The devices, methods, and systems provided herein use a temperature protocol to control the temperature setting on an individual well. In some embodiments, the temperature protocol is a time-dependent temperature setting. In some embodiments, the temperature protocol is a sequence of temperatures and temperature ramps over time, where each temperatures and temperature ramps are set for predetermined time frames. In some embodiments, the temperature protocol is chosen by the user. In some embodiments, the temperature protocol is automated. In some embodiments, the temperature protocol chosen based on the target polynucleotide. In some embodiments, the device uses a temperature protocol of an individual well that is independent of a temperature protocol for another well in the same well plate.
Many thermal cyclers currently on the market are manually actuated. This means that the user has to open and close the heated lid manually. The few automated solutions that are available on the market place the lid onto the well plate in one of two ways. These include placing the well plate into position and have a linear actuator move the well plate in one axis underneath the heated lid and then actuating a second linear actuator in another axis to “press” the heated lid onto the plate (U.S. Pat. No. 6,197,572). The other design “rotates” the lid into position above the well plate (U.S. Pat. No. 9,446,407 B2). The device listed in this disclosure operated by rotating the entire “core” assembly listed above underneath the heated lid and then actuating the heated lid so that it presses into the well plate. This is done by actuating the baseplate motor 7 which directly drives the baseplate rotating actuator 8 depicted in
The thermal cycler device disclosed herein runs at 80 A of power when every reaction chamber is heated. Since the core of the device is rotating, routing moving power cables may be more difficult. To solve this problem, a custom, spring loaded power coupling device was designed that is a larger version of what are known in the electronics industry as “pogo pins”. This can be seen in
When the core mechanism is rotated, the signal wires may rotate with it. To accomplish this, a “twisting” wire route was designed. This can be viewed when in the load position in
The device serves as a way for the user to enter experimental factors via touchscreen, voice, optical character recognition or barcode.
The process factors and the results of the process run are fed back to the online database and are anonymously grouped with the global user base who have adopted the device. This globally grouped data is used optimize the runs for all users of the device.
The method for assembling the well sleeves is unique in that the well sleeve has an external thread that is screwed into a well housing with 96 internal threads. Each sleeve is then torqued down with a special slotted tool until the bottom face contacts the top surface of the thermoelectric device with the right pressure.
The device herein may be manufactured by a variety of methods. In an exemplary approach, the core assembly portion of the invention is made first by:
The core assembly herein may be installed to the rotating baseplate by various methods. In an exemplary method, installation of core assembly with the rotating baseplate comprises:
In various embodiments disclosed herein, the user may use the device or the system herein on a laboratory bench. The user can either enter in a polynucleotide or a macromolecule of interest for synthesis via touchscreen or verbal instructions. Then, the system herein may prompt the user to choose any or all of the experimental factors determined by the machining learning algorithms in the online database. The system can interact with the user in providing information and prompting for input via camera, microphone or touchscreen display, or a combination thereof. Once all experimental factors have been decided by the user based on the recommendation by the online database, the user may initiate the experimental run. Based upon the experimental factors decided before the run, the instrument may retrieve the best set of process conditions based upon the machine learning algorithms in the online database. The system may run the individual process in the corresponding individual chamber.
Once the run is complete, the user may remove the well plate from the instrument and analyze the synthesized polynucleotides or macromolecules. The user may provide an objective quality metric associated of the synthesized polynucleotides or macromolecules synthesized in each chamber with its associated process condition. In some embodiments, for synthesized DNA, the quality metric can be based upon an agarose gel, sequencing, cell count, or other commonly used methods in the field. The device may prompt the user to upload the quality metric to the online database, where it may be pooled with other experimental factors and results.
The various embodiments disclosed herein is not limited to produce synthetic nucleic acid molecule. It is believed that the idea of using a laboratory instrument as an interactive device is novel and speech recognition and/or optical character recognition can be applied to any number of existing laboratory instruments including pipettes, liquid handling robots, plate cranes, plate sealers etc. The idea of optimizing experimental factors and/or process conditions may be applied to a multitude of instruments if such an instrument may interface with an online database. Further, the thermal portion of the device disclosed herein can be utilized in a high throughput device for solution synthesis, enzymology, proteolysis, or other similar approaches that can benefit from independent temperature control of individual reaction chambers.
The devices, methods, and systems disclosed herein provide an interactive, high throughput thermal processing system. The disclosures herein provide a system for recording experimental details via voice, barcode and optical character recognition. Disclosed herein is a system for providing a user-directed experimental recommendation based upon little experimental details of the desired end product. Disclosed herein is a pool of web-based user data that can be accessed by special machine learning algorithms to provide optimal process conditions.
The disclosures provided herein address various shortcomings in polynucleotide synthesis and work flow. For example, existing thermocyclers are not able to run 96 simultaneous process conditions with individual temperature conditions for individual chambers. In some instances, existing thermocyclers (and other lab equipment) are unable to gather experimental details from the user without direct input. In some cases, existing laboratory notebooks transcribe without interacting with an online database that contains pools of structured data to decide how the user provided unstructured data should be classified. In some instances, there are no centralized databases that contain global process data across an anonymous user set.
An exemplary work flow for polynucleotide synthesis comprises:
The devices, methods, and systems disclosed herein provide an online database or a cloud database of polynucleotide synthesis recipe execution and optimization information that acts as a clearinghouse. In some embodiments, the input for the online database comprises a FASTA file having information of the target polynucleotide of interest and/or a list of oligonucleotides for the polynucleotide of interest. Alternatively, the list of oligonucleotides for the polynucleotide of interest may be auto-calculate from polynucleotide of interest. In some embodiments, the recipe for the thermocycler is provided by the machine learning algorithm in the online database based on the input. In some embodiments, the recipe for the thermocycler comprises types of reagents, volumes of reagents, concentration of reagents, temperature process parameters, target temperature, ramp rate, and duration. In some embodiments, the recipe optionally comprises vendor for vendor quality assessment. In some embodiments, the output of the online database comprises quality of polynucleotide produced. In some embodiments, the quality of the polynucleotide can be assessed by client directly or via tools such as Oxford Nanopore. In some embodiments, any number of types of reagents, volumes of reagents, concentration of reagents, temperature process parameters, target temperature, ramp rate, and duration can be provided in the recommendation of a recipe for the thermocycler.
As the search space of possible recipes is infinite, the possible recipes are too large combinatorially to evaluate even when breaking value ranges down into discrete steps. As such, machine learning in online database can be a powerful tool in evaluating the possible recipes to generate a recommendation of optimal recipes. In some embodiments, the machine learning algorithm comprises intelligent reduction of the search space of parameters based on analyzing historical experiments and the resulting quality of the produced polynucleotides. This approach merges traditional Design of Experiments (DoE) with machine learning-based optimization of parameters. DoE provides information regarding selecting variable or step sizes to reduce the possible number of experiments. Machine learning may be used to auto-recommend recipes or parts of recipes that have been optimal in the past for similar polynucleotide fragments and bioparts (i.e. oligonucleotides). In some embodiments, if there is a partial match with previous experiment (reagent, volume/concentration, temperature, etc), the algorithm will do fuzzy/intelligent match to see if “similar” recipe has been executed in the past; and return the associated quality.
In some embodiments, reinforcement learning provides information on experiments performed with feedback for quality of synthesized products and facilitates determining optimal control strategy or recipe for high quality for given constraints. In some embodiments, the polynucleotide synthesis is represented as a recipe process having complex interactive components with nonlinear collective activities. In some embodiments, genetic algorithm can be used to generate random control recipes and reinforcement learning can be used to choose best rule. In some embodiments, an external state of these algorithms is modified by each action. In some embodiments, an action set comprises sequence of actions selected from an action list.
An exemplary approach of using online database for optimizing experimental conditions comprises various components. In some cases, a control/execution component comprises controls interactions with environment. In some cases, a reinforcement component distributes reward from environment to classifiers with optimal genetic quality output. In some cases, a discovery component exploits genetic algorithm to discover better recipes and improve existing ones according to fitness estimates. In some cases, slight tweaks to traditional reinforcement learning can be utilized, where instead of classification, numerical score output (genetic quality) is provided. This tweaked version of reinforcement learning may be a regression form of reinforcement learning.
The polynucleotide described herein can be of any of a variety of lengths. In some instances, the polynucleotide has a length of at least about 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1 megabase (Mb). In some instances, the polynucleotide has a length of about 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1 Mb. In some instances, the polynucleotide has a length of at most about 900 Mb, 800 Mb, 700 Mb, 600 Mb, 500 Mb, 400 Mb, 300 Mb, 200 Mb, 100 Mb, 90 Mb, 80 Mb, 70 Mb, 60 Mb, 50 Mb, 40 Mb, 30 Mb, 20 Mb, 10 Mb, 9 Mb, 8 Mb, 7 Mb, 6 Mb, 5 Mb, 4 Mb, 3 Mb, 2 Mb, 1 Mb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 400 kb, 300 kb, 200 kb, 100 kb, 90 kb, 80 kb, 70 kb, 60 kb, 50 kb, 40 kb, 30 kb, 20 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4 kb, 3 kb, 2 kb, or 1 kb.
The size of the oligonucleotides described herein may be any of a variety of lengths. In some cases, the oligonucleotides comprises nucleic acid molecules having a length of at least 2 bases, 5 bases, 10 bases, 15 bases, 20 bases, 25 bases, 30 bases, 35 bases, 40 bases, 45 bases, 50 bases, 75 bases, 100 bases, 125 bases, 150 bases, 175 bases, 200 bases, 225 bases, 250 bases, 275 bases, or 300 bases. In some cases, the oligonucleotides comprises nucleic acid molecules having a length of about 2 bases, 5 bases, 10 bases, 15 bases, 20 bases, 25 bases, 30 bases, 35 bases, 40 bases, 45 bases, 50 bases, 75 bases, 100 bases, 125 bases, 150 bases, 175 bases, 200 bases, 225 bases, 250 bases, 275 bases, or 300 bases. In some cases, the oligonucleotides comprises nucleic acid molecules having a length of at most about 300 bases, 275 bases, 250 bases, 225 bases, 200 bases, 175 bases, 150 bases, 125 bases, 100 bases, 75 bases, 50 bases, 45 bases, 40 bases, 35 bases, 30 bases, 25 bases, 20 bases, 15 bases, 10 bases, 5 bases, or 2 bases.
A synthesized polynucleotide with a high quality can have a high fidelity to the polynucleotide of interest. In some embodiments, a high fidelity refers to least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% match between the synthesized polynucleotide and the polynucleotide of interest. In some embodiments, a high fidelity refers to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% match between the synthesized polynucleotide and the polynucleotide of interest. In some embodiments, a high fidelity refers to 100% match between the synthesized polynucleotide and the polynucleotide of interest.
In some embodiments, the platforms, systems, media, and methods described herein include a digital processing device, or use of the same. In further embodiments, the digital processing device includes one or more hardware central processing units (CPUs) or general purpose graphics processing units (GPGPUs) that carry out the device's functions. In still further embodiments, the digital processing device further comprises an operating system configured to perform executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Internet such that it accesses the World Wide Web. In still further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.
In accordance with the description herein, suitable digital processing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those of skill in the art will recognize that many smartphones are suitable for use in the system described herein. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers include those with booklet, slate, and convertible configurations, known to those of skill in the art.
In some embodiments, the digital processing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device's hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. In some embodiments, the operating system is provided by cloud computing. Those of skill in the art will also recognize that suitable mobile smart phone operating systems include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®. Those of skill in the art will also recognize that suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in the art will also recognize that suitable video game console operating systems include, by way of non-limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.
In some embodiments, the device includes a storage and/or memory device. The storage and/or memory device is one or more physical apparatuses used to store data or programs on a temporary or permanent basis. In some embodiments, the device is volatile memory and requires power to maintain stored information. In some embodiments, the device is non-volatile memory and retains stored information when the digital processing device is not powered. In further embodiments, the non-volatile memory comprises flash memory. In some embodiments, the non-volatile memory comprises dynamic random-access memory (DRAM). In some embodiments, the non-volatile memory comprises ferroelectric random access memory (FRAM). In some embodiments, the non-volatile memory comprises phase-change random access memory (PRAM). In other embodiments, the device is a storage device including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage. In further embodiments, the storage and/or memory device is a combination of devices such as those disclosed herein.
In some embodiments, the digital processing device includes a display to send visual information to a user. In some embodiments, the display is a liquid crystal display (LCD). In further embodiments, the display is a thin film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light emitting diode (OLED) display. In various further embodiments, on OLED display is a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In yet other embodiments, the display is a head-mounted display in communication with the digital processing device, such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.
In some embodiments, the digital processing device includes an input device to receive information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus. In some embodiments, the input device is a touch screen or a multi-touch screen. In other embodiments, the input device is a microphone to capture voice or other sound input. In other embodiments, the input device is a video camera or other sensor to capture motion or visual input. In further embodiments, the input device is a Kinect, Leap Motion, or the like. In still further embodiments, the input device is a combination of devices such as those disclosed herein.
In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked digital processing device. In further embodiments, a computer readable storage medium is a tangible component of a digital processing device. In still further embodiments, a computer readable storage medium is optionally removable from a digital processing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.
In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable in the digital processing device's CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), data structures, and the like, that perform particular tasks or implement particular abstract data types. In light of the disclosure provided herein, those of skill in the art will recognize that a computer program may be written in various versions of various languages.
The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.
In some embodiments, a computer program includes a web application. In light of the disclosure provided herein, those of skill in the art will recognize that a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, by way of non-limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®. Those of skill in the art will also recognize that a web application, in various embodiments, is written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash® Actionscript, Javascript, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.
Referring to
In some embodiments, a computer program includes a mobile application provided to a mobile digital processing device. In some embodiments, the mobile application is provided to a mobile digital processing device at the time it is manufactured. In other embodiments, the mobile application is provided to a mobile digital processing device via the computer network described herein.
In view of the disclosure provided herein, a mobile application is created by techniques known to those of skill in the art using hardware, languages, and development environments known to the art. Those of skill in the art will recognize that mobile applications are written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, Java™, Javascript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.
Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.
Those of skill in the art will recognize that several commercial forums are available for distribution of mobile applications including, by way of non-limiting examples, Apple® App Store, Google® Play, Chrome Web Store, BlackBerry® App World, App Store for Palm devices, App Catalog for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, and Nintendo® DSi Shop.
In some embodiments, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Those of skill in the art will recognize that standalone applications are often compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable compiled applications.
In some embodiments, the computer program includes a web browser plug-in (e.g., extension, etc.). In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Makers of software applications support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. Those of skill in the art will be familiar with several web browser plug-ins including, Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®.
In view of the disclosure provided herein, those of skill in the art will recognize that several plug-in frameworks are available that enable development of plug-ins in various programming languages, including, by way of non-limiting examples, C++, Delphi, Java™, PHP, Python™, and VB .NET, or combinations thereof.
Web browsers (also called Internet browsers) are software applications, designed for use with network-connected digital processing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of non-limiting examples, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software® Opera®, and KDE Konqueror. In some embodiments, the web browser is a mobile web browser. Mobile web browsers (also called mircrobrowsers, mini-browsers, and wireless browsers) are designed for use on mobile digital processing devices including, by way of non-limiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting examples, Google® Android® browser, RIM BlackBerry® Browser, Apple® Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® for mobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web, Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSP™ browser.
In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and/or database modules, or use of the same. In view of the disclosure provided herein, software modules are created by techniques known to those of skill in the art using machines, software, and languages known to the art. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on cloud computing platforms. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.
In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases, or use of the same. In view of the disclosure provided herein, those of skill in the art will recognize that many databases are suitable for storage and retrieval of spotlight tour configuration information such as spotlight tour navigation steps, spotlight tour objects, spotlight tour shape properties, and spotlight tour controls data. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, and XML databases. Further non-limiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, and Sybase. In some embodiments, a database is internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing-based. In other embodiments, a database is based on one or more local computer storage devices.
The practice of some methods disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R. I. Freshney, ed. (2010)).
The terms “about” and “approximately” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about,” meaning within an acceptable error range for the particular value, should be assumed.
As used herein, the terms “polynucleotide”, “nucleic acid,” “oligonucleotide,” and “gene” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, isolated DNA of any sequence, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated RNA of any sequence, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Generally, oligonucleotides as used herein are shorter than polynucleotides.
The term “strand,” as used herein, refers to a nucleic acid made up of nucleotides covalently linked together by covalent bonds, e.g., phosphodiester bonds. In a cell, DNA usually exists in a double-stranded form, and as such, has two complementary strands of nucleic acid referred to herein as the “top” and “bottom” strands. In certain cases, complementary strands of a chromosomal region may be referred to as “plus” and “minus” strands, the “first” and “second” strands, the “coding” and “noncoding” strands, the “Watson” and “Crick” strands, or the “sense” and “antisense” strands. The assignment of a strand as being a top or bottom strand is arbitrary and does not imply any particular orientation, function or structure.
A polynucleotide may have a 5′ end and 3′ end, referring to the end-to-end chemical orientation of a single strand of polynucleotide or nucleic acid. In a single strand of linear DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide sugar-ring means that there generally exists a 5′ end which frequently contains a phosphate group attached to the 5′ carbon and a 3′ end which typically is unmodified from the ribose —OH substituent (hydroxyl group). In some cases, a polynucleotide may have a —OH substituent or a hydroxyl group at a 5′ end and —P group or phosphate group at a 3′ end. A phosphate group attached to the 5′-end permits ligation of two nucleotides, e.g., the covalent binding of a 5′-phosphate to the 3′-hydroxyl group of another nucleotide, to form a phosphodiester bond. Removal of the 5′-phosphate may inhibit or prevent ligation. The 3′-hydroxyl group is also important as it is joined to the 5′-phosphate in ligation.
The term “primer,” as used herein, generally refers to an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a nucleic acid molecule template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension reaction may be determined by the sequence of the template nucleic acid molecule. Usually primers are extended by a DNA polymerase. Sometimes primers are extended by a reverse transcriptase. Primers are generally of a length compatible with their use in synthesis of primer extension products, and usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges. Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 and so on, and any length between the stated ranges. In some embodiments, the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.
The terms “isolated” and “isolating,” with reference to a nucleic acid molecule or nucleic acid molecule complex generally refer to a preparation of the substance (e.g., nucleic acid molecule, nucleic acid molecule complex, extension products thereof) devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially obtained from (e.g., a biological sample, a sample reaction volume, e.g., a synthesis reaction volume, etc). For example, an isolated substance may be prepared using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis or in terms of a concentration, for example in terms of weight per volume of solution, molecules per volume of solution, or any other appropriate measure.
The term “gene synthesis,” as used herein, refers to polynucleotide synthesis or polynucleotide assembly. Polynucleotide synthesis refers to the process of covalently linking a nucleotide to another to another nucleotide, an oligonucleotide to another oligonucleotide, or a nucleotide to an oligonucleotide to generate a strand of nucleic acids, oligonucleotides, or polynucleotides.
As used herein, the terms “gene of interest” and “polynucleotide of interest” are used interchangeably. The terms mean that the sequence of the polynucleotide is known and chosen before synthesis or assembly of the polynucleotide product.
As used herein, the terms “well” and “chamber” are used interchangeably. A well refers to a container capable of holding reagents for the polynucleotide synthesis.
Provided herein is an exemplary device and system suitable for polynucleotide synthesis with the following instrument specification (Table 2). An exemplary device and system suitable for polynucleotide synthesis is shown in
Provided herein is an exemplary device for polynucleotide synthesis. The device comprises a 96 well thermocycler with individual well control. The device is automatable and is controlled via protocols stored on web-app.
Provided herein is an exemplary system comprising a recommendation engine for PCR optimization and design of experiment (DOE). The recommendation engine provides recommendation on which reagents at what concentrations and volumes to use in the synthesis protocol.
Provided herein is an exemplary device for polynucleotide synthesis. The device comprises a miniature thermocycler that is capable of thermal control and monitoring of individual wells. The device is small enough to be used on a bench top, modular as to include additional channels, and has a low cost. The device is a modular device that can be powered over USB. The device can be controlled remotely or by USB.
Provided herein is an exemplary system comprising a microfluidic liquid handling technology that is reconfigurable. The system comprises a liquid handling robot and has in-situ sensing capabilities to monitor conditions. The system can interface with a miniature thermocycler as described in Example 4. The system can be used to automatically monitor and control liquid handling workflows and variables to accommodate the desired biological product and application.
Provided herein is an exemplary method comprising chip-based quality control methods. The method comprises UV/Vis, electrophoresis, and next generation sequencing methods. Also provided herein is an exemplary device that is integrated with the system as described in Example 5 (MiniTherm and LiquiTherm) to form a fully reconfigurable, closed loop workflow (liquid handling, thermocycling and feedback). Also provided herein is an exemplary system using a refined recommendation engine with additional process variables, protocols, and applications.
Provided herein is an exemplary web-based interface that is used to control the devices, systems, and methods provided herein. The interface can be categorized into 3 categories: 1) onboarding, 2) data viewing and sharing, and 3) setting up and running experiments.
Provided herein is an exemplary web-based interface that is used to control the devices, systems, and methods provided herein.
Provided herein is an exemplary machine learning sequence. A FASTA file is entered to the computing system by a user. An exemplary input is the nucleotide sequence for green fluorescent protein (GFP) as shown below:
The computing system analyzes the input and compares the input to data from prior experiments. The computing system recommends factors and allows the user to select and/or add new factors. For example, the system recommends a temperature range for oligo annealing from 49° C. to 70° C. Then, the user manually changes the temperature range from the recommended range to a range from 48° C. to 72° C. The computing system recommends oligos for polynucleotide synthesis at a desired target length of 60 nucleotides. The user manually alters the desired target length to 63 nucleotides. The user informs the computing system the user input is complete. The following set of oligos for an oligo pool is generated by the computing system as shown in Table 3:
The computing system further recommends reagents and reaction chamber conditions. The reagents comprise a standard Taq PCR reaction (NEB M0273): 10× Standard Taq Reaction Buffer at 1×, dNTPs at 200 μM, 10 μM Forward Primer at 0.2 μM (ATGAGTAAAGGAGAAGAACTTTTCACTGG), Reverse Primer at 0.2 μM (TTATTTGTATAGTTCATCCATGCCA), Oligo pool at 5 nM each oligo, Taq DNA Polymerase at 1.25 units/50 μl, and nuclease-free water for balance of the reaction.
The reaction reagents are mixed to produce at least 96×25 μl reactions and distributed across a 96 well-plate placed into the device described herein. The computing system recommends the time and temperature profiles, where the first step is at 95° C. for 2 minutes, then a cycle that repeats 30 times (95° C. for 30 cycles, an annealing temperature chosen from a range from 48° C. to 72° C. in 0.25° C. increments, and an extension of 1 min at 72° C.), followed by a final extension of 72° C. for 5 minutes. The thermocycler device is loaded with the mixed reagents and programmed with the conditions for each reaction. The reactions, each individual wells comprising an individual reaction, are run on the device.
A sample of the reaction is visualized by agarose gel electrophoresis with ethidium bromide and a DNA size marker loaded for reference. The images are provided to the computing system to calculate size and quality of the reactions.
The completed reactions are additionally individually cloned into a Topo cloning vector (ThermoFisher TOPO™ TA Cloning™ Kit for Sequencing, with One Shot™ TOP10 Chemically Competent E. coli TOPO™ TA Cloning™ Kit Catalog number: K457501) such that the resulting amplicons are cloned into the vector. The TOPO cloning reactions are transformed into TOP10 cells and plated on agarose plates with ampicillin (Teknova LB Agar Plates, Ampicillin-100, 100 mm #L1004). For each original reaction, 96 individual colonies are picked and miniprepped by growth in LB liquid media with ampicillin (Teknova LB Broth, Ampicillin-100, 1 L #L8105) and column purified (Qiagen #27104 QIAprep Spin Miniprep Kit), and Sanger DNA sequenced with M13 forward, and M13 reverse primers (ThermoFisher BigDye™ Terminator v3.1 Cycle Sequencing Kit Catalog number: 4337455 and analyzed on an ABI 3130xl genetic analyzer to generate sequence traces).
The sequence traces are provided to the computing system to calculate identity and error rates to be used to calculate a quality reward score for analysis by the AI computing system. The process is optionally repeated for further refinement.
The quality reward score, reagents, oligonucleotides, and reaction conditions are saved and stored by the computing system and are analyzed to generate recommendations for reagent, oligonucleotide, and/or reaction condition in future runs. The AI-generated recommendation help to reduce numbers of reactions as compared to a run of reactions without the AI-generated recommendation when a set of reagents, oligonucleotides, and reaction conditions that resulted in a low quality score in previous runs can be suggested to be removed from the run by the AI or removed by the user.
Provided herein is an exemplary well plate block of the devices, systems, and methods described herein. The well plate block can control the temperature of individual wells of a well plate placed on the well plate block.
Provided herein in an exemplary method of control for the devices, systems, and methods described herein to communicate with the LIMS system. The method of control provides a way to control the device using external devices. The method of control uses a data structure that is of a standard format and is compatible for future updates and expansion of its capabilities. The method of control, also referred to as the Control Instructions, implements the specified conditions of the individual wells over time in seconds in JavaScript Object Notation (JSON). The method of control can use other file formats. The JSON object is provided to the device by at least one of direct transmission or remote transmission (e.g. over the air).
Over the Air: The device provides a way to connect to the Web Portal via an IoT messaging platform. A user is able to log into the online portal, select the device to be used, and send it a set of Control Instructions. The device is required to be connected to the internet. The device is required to be registered with an organization or a group that the user is a part of when the user uses the organization or group log-in information.
Stand Alone Solution: The device can be used without an internet connection and provides functionality to read the Control Instructions via a file on a USB Drive or other direct connection. The user navigates through menu options on the device to select and load the file.
An exemplary JSON structure for the Control Instructions is shown in
While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
The present application claims priority to U.S. Provisional Application Ser. No. 62/697,902, filed Jul. 13, 2018, and U.S. Provisional Application Ser. No. 62/858,948, filed Jun. 7, 2019, the contents of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/041485 | 7/11/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62858948 | Jun 2019 | US | |
| 62697902 | Jul 2018 | US |
