analysis instrument and module for performing biochemical analysis, and method for operating an anal

专利摘要:
module for performing biochemical analysis, analysis instrument for performing biochemical analysis, system, and methods for operating an analysis instrument for performing biochemical analysis and for a module for performing biochemical analysis - an analysis instrument comprises several modules connected together over a network of data, each module comprising an operable analysis apparatus for performing biochemical analysis of a sample. Each module comprises a control unit that controls the operation of the analyzer. Control units are addressed to select an arbitrary number of modules to operate as a cluster to perform a common biochemical analysis. control units communicate over the data network, repeatedly during the performance of common biochemical analysis, to determine the operation of each module's analyzer required to meet overall performance targets, with performance derived from the output data produced by the modules. . The arrangement of the instrument as interacting modules in this way provides a scaled analysis instrument.
公开号:BR112012013074B1
申请号:R112012013074
申请日:2010-12-01
公开日:2018-09-18
发明作者:Clive Gavin Brown；James Peter Willcocks
申请人:Oxford Nanopore Technologies Limited；
IPC主号:

专利说明:

(54) Title: ANALYSIS INSTRUMENT AND MODULE TO PERFORM BIOCHEMICAL ANALYSIS, AND, METHOD TO OPERATE AN ANALYSIS INSTRUMENT TO PERFORM BIOCHEMICAL ANALYSIS (51) Int.CI .: C12Q 1/68; G01N 33/487; G06F 19/20 (30) Unionist Priority: 10/1/2010 GB 1016614.8, 12/1/2009 US 61/265488, 12/31/2009 GB 0922743.0 (73) Holder (s): OXFORD NANOPORE TECHNOLOGIES LIMITED (72) Inventor (s): CLIVE GAVIN BROWN; JAMES PETER WILLCOCKS (85) National Phase Start Date: 05/30/2012 “ANALYSIS INSTRUMENT AND MODULE TO PERFORM BIOCHEMICAL ANALYSIS, AND, METHOD TO OPERATE AN ANALYSIS INSTRUMENT TO PERFORM BIOCHEMICAL ANALYSIS.”
First and second aspects of the present invention concern instruments for performing biochemical analysis of a sample, for example polynucleotide sequencing and / or biochemical analysis using nanopores, which produce output data from several parallel channels representing the results of biochemical analyzes. The third aspect of the present invention concerns the performance of biochemical analysis of a sample using nanopores, for example, sequencing of polynucleotides.
Considering the first and second aspects of the present invention, there are many types of analyzes that produce output data from several parallel channels. Instruments for performing such a biochemical analysis in an automated manner are known and provide efficiencies in obtaining large amounts of output data that are inherent in biochemical analysis.
Merely by way of example, one such type of biochemical analysis that produces output data from several parallel channels is DNA sequencing. Conventional DNA sequencing instruments, and laboratory instrumentation in general, are based on a model where an instrument operates as a stand-alone device. Typically, instruments perform a measurement task in finite time with a predefined realization criterion. We can describe this design model as "monolithic".
DNA sequencing, as an example, is an inherently high-yield laboratory technique.
Experiments cover a wide variety of data sizes and durations and the data produced is very complex, heterogeneous and requires intensive downstream processing. The nature of research around DNA sequencing makes it difficult to treat the core of the analysis, the instrument system, as a black-box measuring device. There is a growing need for scaled systems for DNA sequencing, capable of scaling up and down. This is triggered by a recent market demand to sequence more things, different things, and all cheaper, faster and more effective. Sequencing systems must then also be able to accommodate heterogeneous work flows and are capable of piping samples of varying types and sizes according to use cases. This is desirably done efficiently and economically. Measurement artifacts associated with the substrate, or how it has been prepared, should not trigger efficient processing on an instrument leading to lost reagents or low redundant time. Institutes that can operate industry efficiently based on sequencing processes will dominate high-yield, low-cost applications. However, desires are difficult to achieve.
Current monolithic DNA sequencing instruments are difficult to scale to analyze at different scales. The instruments cannot be designed to serve very large industry operations, while at the same time being accessible to unqualified laboratory personnel with smaller projects. Scaling for current DNA sequencing instruments usually comes from increasing the amount of data they can produce in one run, which is a single analysis performed by an instrument. However, modularity and flexibility are limited and in order to achieve them, the user has to resort to breaking the substrate, making the substrates individually addressable by adding labels, and breaking the reaction chambers of the sequencers. In both cases, artifacts are introduced and there are intrinsic limits on how much scale of modularity can be achieved without a complete redesign of the instrument itself. In other words, the basic design of the instrument has a construction source limit that hinders its ability to handle the demands of real-world work flows.
In many DNA sequencing instruments, individual filaments or clonally amplified colonies of limited lengths of DNA are located on a surface or in a sphere. This surface / sphere arrangement is usually in a flow cell that allows reagents to be passed through them thus applying chemistry of various types that allow the DNA to be decoded. The biochemical analysis process within most instruments uses cyclic chemistry step by step, followed by an imaging stage to detect the incorporation, annealing or removal of chemically labeled fluorescent probes that allow the DNA under study to be decoded.
During the basic identification stages, in most systems a high resolution imaging device takes the image of the total flow cell surface as a sequential series of tilted sets of images. In some technologies, a single region is converted to an image very quickly by detecting chemical cycles in real time as a basis are incorporated asynchronously.
Generally, in the case of sequential imaging of systems based on synchronous chemistry, the total imaging stage takes a significant amount of time and generally has to complete a current number of chemical cycles, or current run time, before the user can take the data and analyze it, thus judging whether the experiment has been successful and yielding sufficient useful information. Generally, just by following the analysis, the user can decide if the experiment has been successful, and if so, then a new total analysis run has to be performed, and this is repeated until sufficient data of the required quality has been collected. In most cases, each run has a fixed cost derived from the price of the reagents. That is why the price of success is difficult to determine beforehand as is the time to result.
For many instruments, an execution takes at least several days or even weeks with a significant chance of failure by the instrument during the experiment, usually causing truncation or even complete data loss. High outputs per run can be achieved by packing more DNA molecules into the flow cell, however this tends to increase the time to take the images, depending on device resolution and speed / sensitivity, with limited improvement lately in net yield. For example, the company Heliocos Biosicneces sells an instrument referred to as the Heliscope that has fragments of 600-800M of DNA linked to two flow cells, and the company Alumina puts on the market an instrument referred to as a Genome Analyzer with fragments of 80 -100M of DNA. By comparison, it takes about 6 hours to incorporate and image a new base in each strand in the Heliscope compared to 1-2 hours per base in the Genome Analyzer. Thus, the two instruments are each better suited to tasks at different scales.
These providers of such instrumentation have realized that users do not necessarily want large data output in a sample as this substantially reduces modularity, flexibility and utility, and thus typically physically divides the surface area into individually addressable sections (eg 8 subchannels, or 'lanes', in the flow cell for the Genome Analyzer, 25 subchannels per flow cell for the Heliscope, to allow the user to measure more than one sample per flow cell, albeit in the output data reduced concurrently by Such an area will still produce at least 250 Mb of DNA sequence, thus generating a large oversample of a sample containing small genomes, for example a typical 0.5 Mb bacterium would be covered at least 500 times. the inefficiency of using the instrumentation and reagents, both in terms of time and cost to the user.
For the user, an additional problem experienced with existing instrumentation is that no matter how few DNA strands / strands / samples are required to be sequenced, throughput is linked to the measurement time cycle across the total flow cell surface. Current instruments have only one processing unit (the camera / flow cell surface) and cannot divide the task of measuring each sample sufficiently to give the desired output to the user.
An additional problem for the user is that he must pay for the time of the processing unit by depreciating the costs of the instrument in advance, as well as the costs of the reagents across the total surface in order to achieve its result, without knowing in advance whether success is guaranteed in an execution.
A specific example of a composition problem is that bases are not added evenly during the biochemical analysis process to each available fragment (some fragments will happen to have a disproportionate amount of A's over C's for example, consisting of repeating homopolymers), and they are not always measured with uniform accuracy (agglomerate lag, out-of-focus areas in the flow cell, enzyme / polymerase breakdown, background signal construction). This means that some areas of the flow cell will generate more data than others, but the nature of the single processing unit means that it cannot adapt so much to maximize those areas that are generating high quality and useful information, or focus on areas that are failing to deliver enough data.
In short, existing systems run for a defined period of time and then cost, but produce information for a fixed number of bases for the user in varying measurement quality. The net result for the user is of great inefficiency in time and cost when performing different DNA sequencing experiments given in the range of applications of interest to the user. This is particularly when the user is trying to analyze, in parallel, multiple samples within a project in a given class of sequencing device.
Although a DNA sequencing instrument has been discussed as an example for illustration, difficulties of a similar nature can be found in design instruments for a wide range of biochemical analysis that produces large amounts of output data from several parallel channels.
The first and second aspects of the present invention seek to alleviate some of these problems in the design of an instrument to perform biochemical analysis.
Considering the third aspect of the present invention, in recent years there has been considerable development of biochemical analysis of a sample using nanopores. A nanopore is a small hole in an electrically insulating layer and can be formed, for example, by pores of protein or channels introduced into an amphiphilic membrane. The nanopores can allow a flow of ions to travel through the amphiphilic membrane, modulated by the nanopore based on an analyte interaction, thus allowing the nanopore to provide biochemical analysis. Several types of nanopore and analyzer to use they have been developed for a range of types of biochemical analysis. An example of commercial interest is to use nanopores for sequencing polynucleotides such as DNA. An example of an analysis device for performing biochemical analysis of a sample using nanopores is disclosed in W02009 / 077734.
So nanopores offer the potential for a platform for biochemical analysis on a commercial scale. However, in such a context it would be desirable to provide efficient handling of the samples in the apparatus in order to maximize yield and minimize costs of carrying out biochemical analyzes.
According to a first aspect of the present invention, an analysis instrument is provided to perform biochemical analysis, the instrument comprising several modules, each module comprising an analysis apparatus that is operable to perform biochemical analysis of a sample, the module being arranged for produce output data from at least one channel representing the results of the biochemical analysis, the operation of the module being controllable in a way that varies its performance, the analysis instrument still comprising a control system that is arranged to accept input by selecting an arbitrary number of the modules as a cluster to perform a common biochemical analysis and to accept input representing global performance targets with respect to the common biochemical analysis, the control system being arranged to control the operation of the cluster modules to perform the common biochemical analysis, and in that the control system is arranged to determine , at least once during the performance of the common biochemical analysis, performance measurements of each module of the output data produced by the modules, and the control system is arranged to vary the control of the operation of the modules of the cluster based on the determined measurements of the performance of all modules and the overall performance targets, and / or arranged (b) to take remedial action in response to the overall performance targets not being achievable based on the determined measurements of the performance of all modules.
Instead of the user having a single instrument, similar to the monolithic instruments existing in the case of DNA sequencing, the user has a parallelized group of modules at his disposal and is able to group any number of such modules into a larger instrument that can perform an analysis common biochemistry. Thus, the instrument is physically parallelized in the sense that it comprises several modules, each comprising an analysis apparatus that is operable to perform biochemical analysis of a sample. The modules can, but are not required to be, identical. In this way, a common biochemical analysis can be performed through an arbitrary number of such modules. This provides dimensioning in which the number of modules can be selected which is suitable to carry out the biochemical analysis which can in general require different amounts of resource depending on its nature. The size and utility of the cluster is a function of the arbitrary number of individual modules that are selected. The design of the modules and the encapsulated functionality allows them to be dimensioned linearly as a single operating unit with reference to an external control system or entry door computer. This design provides efficiency gains, because an appropriate number of modules can be selected for the task at hand, thus freeing up other modules for other tasks.
An arbitrary number of such physical modules can be executed, addressed and treated as a single logic device. However the size and utility of the logic device is a function of the arbitrary number of individual modules the user has incorporated into the set (or 'cluster'). Equally importantly, an individual module can be addressed by a user (or software) and operated as a stand-alone unit, performing the same core tasks as the assembly but in isolation. No further modifications to the modules are required in order to run them individually or in large groups.
Additionally, efficiency gains are achieved in addition to those resulting purely from scaling the number of modules, because the operation of individual modules can also intelligently be stopped. This takes advantage of the ability to independently control the analysis devices for each module, as follows. Performance measurements for each module are determined from the output data produced by the modules. These performance measurements are used as a basis for controlling the operation of the modules to find global performance targets adjusted by input, eg user input or stored data with respect to the biochemical analysis being performed. Such performance targets and measurements can be the time to produce output data, the amount of output data, and / or the amount of output data. This determination is carried out at least once, or preferably repeatedly, or even continuously, during the performance of the common biochemical analysis.
The control of the operation of the analysis device of the individual modules can be varied based on the performance measurements for the cluster of modules to find the overall performance targets. In general, the performance of each module can vary based on numerous factors, and so this control of the operation of each module allows the overall performance of the instrument to be managed to find the global performance targets. This produces efficiency gains, because better use is made of the individual modules in the cluster.
Alternatively or in addition, remedial action can be taken in response to global performance targets that are not achievable. A variety of remedial action is possible, for example by increasing the number of modules by performing the common biochemical analysis, producing outputs to notify a user, or even stopping the biochemical analysis. This produces efficiency gains, because better use is made of the individual modules in the cluster. For example, using additional modules allows you to find targets that would otherwise be missed, or by stopping the analysis frees up the modules for another biochemical analysis.
For example, the instrument can measure the quantity and quality of output data in real time, and provides dynamic flexibility to respond and adapt global performance targets set by the user to maximize cost and time efficiencies. Such an instrument could then vary the performance of the biochemical analysis in any of the modules, as needed. Examples of such parameters that can be controlled include: the temperature of the analyzer; parameters of biochemical analysis, eg fluidic, optical, electrical parameters; or sampling characteristics of the output data. Examples of electrical parameters are trend voltage and current. Examples of fluidic parameters are flow rate, sample addition, sample removal, temporary storage change, reagent addition or removal, nanopore addition or removal, bilayer placement and system update. Examples of the sampling characteristics are sample rate, amplifier reset time and amplifier settings such as bandwidth, gain, integrator capacitance. Variation of these and other parameters allow performance to be varied, for example by changing the quantity, quality and rate of the output data. This is, for example, possible to end the analysis when enough data has been collected, or to focus on samples within the experiment that have yet to produce enough data, while releasing sample sources that have already produced enough data according to requirements. user experiments.
For example, in the case that the biochemical analysis is sequencing of a polynucleotide in the sample, the instrument can be operated in numerous different ways, for example: until a defined number of bases has been sequenced; until a particular sequence is detected, eg, pathogen detection in a large background, detection of cancer mutation in plasma DNA, for very long periods of time to allow measurement of very rare amounts of polynucleotide; or providing an analysis pipeline at optimal performance without a user guide.
Such a modular and intelligent sequencing instrument allows to radically reshape work flows to provide efficient piping of experiments and samples. Work flows can be optimized in terms of priority, time, cost and overall result. This gives a significant efficiency gain over traditional monolithic instruments.
Additionally according to the first aspect of the invention, a single module in isolation can be provided, which is capable of connection to other modules to form such a biochemical analysis apparatus, or a corresponding method of operating an analysis apparatus can be provided .
Advantageously, the modules are capable of connecting to a data network to allow connection over the network together, for example on a non-hierarchical basis. This allows the control system to take advantage of the data network to facilitate communication and control.
Although the control system can be implemented in an independent device that is connected to the network, advantageously, the control system comprises a control unit in each module that is operable to control the operation of that module. In this case, the control units can be addressed over the data network to provide said input by selecting an arbitrary number of modules to operate as a cluster to perform a common biochemical analysis and said user input representing global performance targets with respect to common biochemical analyzes. For example, this can be achieved by the control units being arranged to present a user interface over the data network to a computer connected to it, for example using a browser. Then, the control units of the cluster modules control the operation of their respective modules to perform the common biochemical analysis.
Such a division of the control system in the modules' control units allows the modules themselves to be addressed and operated as a single instrument, simply by connecting the modules to the network. Large groups of modules can be managed to provide biochemical analysis interfaces of any number of more simply because the network interface allows a single command to simultaneously issue to a cluster. Similarly, feedback and data from any cluster of modules can be grouped and logically formatted and addressed like the output of a single module. This operating efficiency can manifest itself as a pipeline and can have a positive impact on the upstream sample preparation and downstream analysis of the output data. So the overall workflow of a laboratory, from the substrate for analysis, can be done more efficiently regardless of how complex or heterogeneous the substrate or analysis has to be. The provision of control units in the modules also means that an individual module has the ability to be addressed and operated in a stand-alone unit, performing the same core tasks as the chipboard but in isolation. Thus, no additional modifications to the modules are required in order to run them individually or in large groups.
The respective control units of the cluster modules can be arranged to derive performance measures with respect to their respective module from the output data produced by their respective module, and to communicate performance measurements over the data network to form the basis of the decision in additional control. Deriving performance measurements locally on the modules, it is only necessary to divide the performance measurements to implement the control. This facilitates control and reduces bottlenecks in data flows as performance measurements require significantly less data than output data.
The control units of the cluster modules can be arranged to communicate over the data network to make a decision while still controlling operation. This has the advantage that the control system is implemented to provide control units in each of the modules. Thus a group of modules can be operated simply by connecting the modules to a data network, without the need for any additional control system to be provided.
Advantageously, the control system is arranged to determine local performance targets for each module based on the global performance targets and the control unit in each module is arranged to control the operation of that module based on its local performance target. In this way, the control system can vary the local performance targets, based on the determined performance measurements and the global performance targets, in order to vary the control of the operation of the cluster modules.
There are numerous ways to distribute the determination of local performance targets.
In a first implementation, this determination can be made in all control units, for example each control unit determining its local performance target. This provides load sharing of the processing performed by the control units, both to derive performance measurements and to determine the required operation. This also provides scaling of operation and management by avoiding a single connection point or bottleneck computer system.
In a second implementation, this determination can be performed on one (or a subset) of control units. This concentrates determining the local performance targets in a single control unit (or a subset of the control units in the cluster), which increases the processing load on that control unit, but can simplify the processing required to perform the determination.
In a third implementation, this determination can be carried out on a separate federation control unit also connected to the data network. This concentrates the determination of the local performance targets in a separate federation control unit, which decreases the processing load on the module control units. This is an expense of requiring an additional federation control unit but has advantages in simplifying the processing required to perform the determination.
The instrument can in general be to perform any type of biochemical analysis, for example analysis of a molecule in a sample, for example a polymer or more specifically a polynucleotide.
In an advantageous example, biochemical analysis is sequencing of a polynucleotide in the sample, so the output data includes sequence data representing a polynucleotide sequence.
In another advantageous example, the analyzer is capable of supporting multiple nanopores and is operable to perform biochemical analysis of a sample using the nanopores, for example using electrodes to generate an electrical signal through each nanopore case from which the data output are derived. In this case, biochemical analysis can again be sequencing of a polynucleotide, but nanopores can also be used to provide other types of biochemical analysis.
The second aspect of the present invention is specifically concerned with an instrument to perform biochemical analysis of a sample using nanopores where electrodes are used to generate an electrical signal through each nanopore and a signal processing circuit is used to generate output data from several parallel channels from the electrical signals. This type of instrument is known, for example, from W02009 / 077734. However, it remains desirable to optimize the efficiency of the instrument in the production of the output data.
According to the second aspect of the present invention, a module is provided to perform biochemical analysis, the module comprising:
an analysis apparatus that is capable of supporting several nanopores and being operable to perform biochemical analysis of a sample using the nanopores, the analysis apparatus comprising electrodes arranged to generate an electrical signal through each nanopore; and a signal processing circuit arranged to generate from electrical signals generated from said electrode output data from the various parallel channels representing the results of the biochemical analysis, the module being controllable in a way that varies its performance and still comprises an operable control unit to control the operation of the module on the basis of a performance target.
Such a module provides efficiency gains in the generation of output data from the biochemical analysis because the operation of the module is controlled based on the performance targets. Such performance targets and measurements can be the time to produce output data, the amount of output data, and / or the quality of output data.
The control unit can be arranged, at least once during the performance of the biochemical analysis, to determine performance measurements of the biochemical analysis and to vary the control of the module's operation based on the performance measurements to find the performance targets. This provides efficiency gains in the generation of the output data from the biochemical analysis because the operation of the module is intelligently controlled, as follows. The control unit determines performance measurements from output data produced by the module and varies the experimental parameters of the biochemical analysis based on the performance measurements to find performance targets. This determination and control can be performed repeatedly, or even continuously, during the biochemical analysis. Examples of the experimental parameters that can be varied include the temperature of the analysis apparatus, electrical parameters of the biochemical analysis, or sampling characteristics of the output data. Variation of these and other experimental parameters allow performance to be varied, for example by changing the quantity, quality and rate of the output data. In general, the performance of the module can vary based on numerous factors, and so this dynamic operational control allows the overall performance of the instrument to be managed efficiently to find targets. This produces efficiency gains.
For example, in the case that biochemical analysis is the sequencing of a polynucleotide in the sample, the instrument can be operated in numerous different ways, for example: up to a defined number of bases have been sequenced; until a particular sequence is detected, eg, pathogen detection between large bottom, detection of cancer mutation in the DNA plasma; for very long periods of time to allow measurement of very rare amounts of polynucleotide, or providing an analysis pipeline at optimal performance without a user guide.
US Application No. 61 / 170,729 discloses a method of detecting a physical phenomenon, the method comprises: providing a sensor device comprising a set of sensor elements including respective electrodes, each sensor element being arranged to emit an electrical signal at the electrode that it is dependent on the physical phenomenon with a performance that is variable; providing a detection circuit comprising a plurality of detection channels each capable of amplifying an electrical signal from one of the sensor elements, the number of sensor elements in the array being greater than the number of detection channels; providing a switching arrangement capable of selectively connecting the detection channels to the respective sensor elements; control the switching arrangement to selectively connect the detection channels to respective sensor elements that have acceptable performance based on the amplified electrical signals that are emitted from the detection channels. Optionally, the second aspect of the invention can exclude the method disclosed in U.S. Application No. 61 / 170,729.
A module according to the second aspect of the invention may optionally be able to operate as part of a cluster to make a common biochemical apparatus according to the first aspect of the invention.
The module can generally be used to perform any type of biochemical analysis using nanopores. In an advantageous example, biochemical analysis is sequencing a polynucleotide in the sample, so that output data includes sequence data representing a sequence of the polynucleotide.
According to the third aspect of the present invention, a module is provided for performing biochemical analysis, the module comprising an electronic unit and a cartridge that is removably connected to the electronic unit, in which the cartridge comprises:
a sensor device that is capable of supporting multiple nanopores and being operable to perform biochemical analysis of a sample using the nanopores, the sensor device comprising an electrode assembly through each nanopore;
at least one container for receiving a sample;
at least one reservoir for holding material to perform biochemical analysis, and a fluidic system configured to controllably supply a sample from at least one container and material from at least one reservoir to the sensor device; and the electronic unit contains a drive circuit and a signal processing circuit arranged to be connected to the electrode assembly through each nanopore when the cartridge is connected to the electronics unit, the drive circuit being configured to generate drive signals to perform the biochemical analysis and the signal processing circuit being arranged to generate output data representing the results of the biochemical analysis from electrical signals generated from the electrode assembly through each nanopore.
The module has a construction that encapsulates the components and material needed to perform the biochemical analysis in a cartridge separately from the electronics unit including a drive circuit and a signal processing circuit. In particular, the module incorporates the operable sensor device to perform biochemical analysis of a sample using nanopores with at least one reservoir to maintain the necessary material and a fluidic system that can supply the material to the sensor device, under proper control. The cartridge is removably attached to the electronics unit, thus allowing the cartridge to be replaced for further sample analysis. This allows for efficient performance of biochemical analysis.
Modalities of the present invention will now be described by means of a non-limiting example with reference to the accompanying drawings, in which:
Fig. 1 is a schematic view of a biochemical analysis instrument;
Fig. 2 is a perspective view of an instrument module;
Fig. 3 is a perspective view of a cartridge that is replaceable in the module;
Fig. 4 is a cross-sectional view of part of a cartridge sensor device;
Figs. 5 and 6 are top and bottom perspective views of the sensor device mounted on a PCB;
Fig. 7 is a perspective view of the module;
Fig. 8 is a schematic diagram of the electrical circuit of a module;
Fig. 9 is a schematic diagram of the control unit;
Fig. 10 is a diagram of a detection channel;
Fig. 11 is a perspective view from above of a cartridge having an alternative construction;
Figs. 12 and 13 are seen in perspective from below the cartridge of Fig. 11, showing a cavity plate, connected and separated, respectively;
Fig. 14 is a sectional perspective view of part of the cavity plate;
Figs. 15 and 16 are seen in perspective from above and below respectively of a valve assembly incorporating a valve;
Fig. 17 is a cross-sectional view through the valve assembly;
Fig. 18 is a partial plan view from above of a valve assembly body around a valve stator;
Fig. 19 is a plan view from below of a valve rotor;
Fig. 20 is a partial cross-sectional view of the valve assembly body and a cavity of the cavity plate;
Fig. 21 is a plan view from below of a second plate of the valve assembly;
Fig. 22 is a perspective view of the valve assembly including a motor; and
Fig. 23 is a flow chart of the instrument control process.
An instrument will first be described for performing biochemical analysis using nanopores in the form of protein pores supported on an amphiphilic membrane, but this is not limiting the invention.
Instrument 1 is formed of a plurality of modules 2 which are each connected to a data network 3. In this example, network 3 is formed as a conventional local area network by each module 2 being connected by a cable 4 to a switch of network 5. In general, modules 2 can be connected to any type of data network, including wireless networks, wide area networks and the internet.
Connected to network 3, it can also be a storage device 6 of any type, for example a NAS, and an external computer 7 which is used to address modules 2 and can be a conventional computer having an HTTP browser.
Due to the network configuration of instrument 1, any number of modules 2 can be provided in a given location, depending on local requirements, for example from a small number of modules 2 or even a single module 2 in a research facility. small scale to a large margin of modules 2 in a commercial sequencing center. Similarly, modules 2 do not need to be physically close and then instrument 1 can be formed from modules 2 that are distributed in different locations, up to different countries.
An individual module 2 will now be described.
As shown in Fig. 2, module 2 has a cartridge 10 which is replaceable in housing 11 of module 2. The cartridge 10 forms an analysis apparatus to perform a biochemical analysis as will now be described. The cartridge 10 has two alternative constructions shown in Figs. 3 and 10.
The cartridge 10 comprises a body 37 formed for example of molded plastic. The body 37 of the cartridge 10 mounts a sensor device 14 which is an apparatus as described in detail in WO 2009/077734 which is incorporated herein by reference. Without limitation to generalize the teaching here, the sensor device 14 has a construction as shown in the cross section in Fig. 4 comprising a body 20 in which a plurality of cavities 21 are formed, each being a recess having a cavity electrode 22 arranged therein. . A large number of wells 21 are provided to optimize the data collection rate. In general, it can have any number of wells 21, although only a few of the wells 21 are shown in Fig. 4. In one example, the number of wells is 256 or 1024, but it can be one, two or three orders of magnitude more. The body 20 is covered by a lid 23 which extends over the body 20 and is hollow to define a chamber 24 in which each of the cavities 21 opens. A common electrode 25 is disposed within the chamber 23.
The sensor device 14 is prepared to form an amphiphilic membrane 26, such as a lipid bilayer, through each well 21 and to insert nanopores that are protein pores into the amphiphilic membrane 26. This preparation is achieved using the techniques and materials described in detail in WO 2009/077734, but can be summarized as follows. Aqueous solution is introduced into chamber 24 to form amphiphilic membrane 26 through each well 21 separating aqueous solution in well 21 from the remaining volume of the aqueous solution in chamber 24. Protein pores are supplied in the aqueous solution, for example being introduced into the aqueous solution before or after it is introduced into chamber 24 or deposited on an internal surface of chamber 24. The protein pores spontaneously insert from the aqueous solution into amphiphilic membranes 26.
A protein pore is an example of a nanopore and can be used to perform a biochemical analysis, as follows. With respect to any given cavity 21, when an amphiphilic membrane 26 has been formed and a pore of protein is inserted into it, cavity 21 is capable of being used as a sensor element to detect interactions between molecular entities and the pore of protein that they are stochastic physical events because the electrical output signal through the amphiphilic membrane 26 is dependent on those interactions in which the interactions cause characteristic changes there. For example, there will typically be interactions between the protein pore and a particular molecular entity (analyte) that modulates the flow of ions through the pore, creating a characteristic change in the current flow through the pore. The molecular entity can be a molecule or part of a molecule, for example a base of DNA. Thus, the interaction appears as a characteristic event in the electrical signal through the protein pore in each amphiphilic membrane 26.
More details on the nature of the Mea sensor device and the biochemical analysis performed on it are set out below until the end of this description.
The electrical signals can be detected as signals between the electrodes in the cavity 22 and the common electrode 25, and can subsequently be analyzed to produce output data representing the results of the biochemical analysis. Separate electrical signals are derived from the protein pores in the amphiphilic membranes 26 in different cavities 21, each resulting in a different channel of the output data.
A wide range of types of biochemical analysis can be performed. Such a biochemical analysis is polynucleotide sequencing. In this case, the electrical signal is modulated differently for each different base, allowing its discrimination.
The body 37 of the cartridge 10 encapsulates the components and material necessary to carry out the biochemical analysis and is capable of preparing the sensor device 14 automatically. For this purpose, cartridge 10 assembles reservoirs 30 containing sufficient volumes of the necessary materials, such as buffer solutions, lipids, protein pores (in solution), pretreatment (if required), and sample, as well as many 'updates' of the analysis are possible. Thus, cartridge 10 is totally self-contaminated in all reagents and other materials required for biochemical analysis are present and can be used for sample preparation. The cartridge 10 mounts a waste reservoir 35 for disposing waste products from the sensor device 14, the waste reservoir 35 being shown in Fig. 11 but below the body 37 in the construction of Fig. 3 and then not visible in Fig. 3.
The body 37 of the cartridge 10 also mounts a fluidic system 31 to supply the fluids from the reservoirs 30 to the sensor device 14. The fluidic system 31 includes supply channels 32 and inlet pumps 33 for pumping fluids from the reservoirs 30 to the sensor device. sensor 14. The fluidic system also includes an outlet pump 34 for pumping fluids out of the sensor device 14 through an inlet channel 36 connected to the waste reservoir 35 for fluid disposal. Pumps 33 and 34 can be syringe pumps depending on the volume and flow rate required (for example as supplied by Hamilton
Company, Via Crusch 8, Bonaduz, GR, Switzerland CH-7402).
The fluidic system also includes a selector valve 45 arranged in the supply channels 32 between inlet pumps 33 connected to reservoirs 30 and outlet pump 34. Selector valve 45 selectively connects sensor device 14 to reservoirs 30 or the reservoir of waste 35. Waste container 35 is open to the atmosphere.
One of the reservoirs 30 holds the lipid and the fluid system 31 supplies the lipid to the sensor device 14 in the same manner as the other materials. As an alternative to supplying the lipid, the delivery channels 32 of the fluidic system 31 can pass in the sensor device 14 through a lipid assembly holding lipid so that the fluid flowing into the sensor device 14 acquires lipid and introduces it into the sensor device 14.
The pumps 33 and 34 can thus be operated to control the flow of fluids to prepare the sensor device 14 to form an amphiphilic membrane 26 through each well 21 and to insert nanopores that are protein pores into the amphiphilic membrane 26, as discussed above .
In the construction of Fig. 3, the body 37 of the cartridge 10 mounts a container 44 to receive a sample. In use, the sample is introduced into container 44 before loading cartridge 10 into module 2. After preparation of sensor device 14, fluidic system 31 is controlled to deliver the sample from container 44 to sensor device 14 for perform biochemical analysis.
In the construction of Fig. 11, the cartridge 10 is capable of receiving a plurality of samples as follows. As shown in Fig. 12, the body 37 of the cartridge 10 is arranged to allow connection of a cavity plate 100. In particular, the body 37 has a pair of clips 101 protruding from the bottom and to which a cavity plate 100 can be turned on by pressing the cavity plate 100 against the clips 101 in the direction of the arrows in Fig. 13.
As shown in Fig. 14, the cavity plate 100 is of standard construction and forms a plurality of cavities 102 opening a flat top surface 103 of the cavity plate 100. In this example, the cavity plate 100 has cavities 96, but in general can have any number of wells 102. Wells 102 are used as containers for receiving respective samples. In use, samples are inserted into the respective wells 102 before connecting the well plate 102 to the cartridge 10 and before loading the cartridge 10 into the module 2. The well plate 102 can be filled with samples using techniques of parallel handling to the base known plate that are intrinsically efficient. As the cavity plate 100 is a separate element from the body 37 of the cartridge 10 and is easily filled before connection, it facilitates the filling of the cavities 102. More generally, similar advantages can be achieved by replacing the cavity plate 100 with any other type of element. container comprising a plurality of containers which can be cavities or closed containers.
After introducing samples, the cavity plate 100 is attached to the cartridge 10 with the flat top surface 102 against the body 37, to encapsulate the cavity plate 100 in the cartridge 10.
Subsequently, cartridge 10 is loaded into module 2.
The fluidic system 31 is configured to selectively supply samples from cavities 102 to sensor device 14, using a valve 110 which is a rotary valve and will now be described.
The valve 110 is formed in a valve assembly 111 illustrated in Figs. 15 to 21 which is incorporated into the body 37 of the cartridge 10.
The valve 110 comprises a stator 112 and a rotor 113. Stator 112 is provided in a body 120 formed by a first plate
121, a second plate 122 and a third plate that are fixed together by interfacing contact surfaces 124 between the first and second plates 121 and 122 and interfacing contact surfaces 125 between the first and second plates 122 and 123.
The rotor 113 is rotatably mounted on the stator 112 for rotation on the rotational geometry axis R. A bearing for the rotational assembly is provided by the rotor 113 comprising a short bearing pin 114 which is mounted in a bearing recess 115 formed in the stator 112. In particular, the short bearing pin 114 has a length chosen to provide clarity between the end of the short bearing pin 114 and the first sheet 121. Around the recess of the bearing 115, the second sheet 122 has an annular relief 126 that it points towards the first sheet 121 and the stator 113, and the second sheet 123 having a circular opening 127 into which the annular relief 126 fits.
In addition, the bearing for rotational assembly is provided by rotor 113 comprising a disk 116 having a cylindrical outer surface 117 which is mounted on an annular wall 118 formed in stator 112 and protruding from it, in particular from the third plate 123 outside the circular opening 127. Alternatively, there may be a clearance space between disk 116 and annular wall 118.
Stator 112 and rotor 113 have contact surfaces 130 that are annular and extend perpendicular to the rotational geometry axis R, being provided as follows. The contact surface 130 of the rotor 113 is formed by a lower surface of the disc 116 that extends perpendicular to the rotational geometry axis R both overlapping the annular relief 126 of the second plate 122 and overlapping the third plate 123 outside the opening 127. Thus the surface of contact 130 of stator 112 is formed by the adjacent parts of the upper surface of the annular relief 126 of the second plate 122 and the upper surface of the third plate 123, which are level with each other.
Sealing the contact surfaces interfacing 130 of stator 112 and rotor 113 is facilitated by applying a load between stator 112 and rotor 113 along the rotational geometry axis R. This is achieved by a request arrangement arranged as follows to order the rotor 113 against stator 112. A fixing ring 131 is connected to stator 113, in particular screwed to the annular wall 118. A disc spring 132 is arranged between and engages the fixing ring 131 and rotor 112. Disc spring 132 provides resilient solicitation between stator 112 and rotor 113, although it can be replaced by another type of resilient soliciting element.
The contact surface 130 of stator 112 is arranged as shown in Fig. 18 which is a plan view of stator 112 without the fixing ring 131. In particular, a plurality of inlet holes 133 are formed in the contact surface 130 of the stator 112 arranged in a circle around the rotational geometric axis R. The inlet holes 133 are evenly spread, except for a space in a lower position in Fig. 18. The inlet holes 133 are formed in particular on the upper surface of the annular relief 126 of the second plate 122, facing the contact surface 130 of the rotor 113.
Also, a collection chamber 134 is formed on the contact surface 130 of the stator 112. The collection chamber 134 is formed as a groove on the upper surface of the third plate 122, facing the contact surface 130 of the rotor 113. The collection chamber 134 extends outside the entry holes 133 in a circular ring around the rotational geometry axis R aligned angularly with the entry holes 133, which is with a space angularly aligned around the rotational geometry axis R with the space in the entry holes 133.
Stator 112 also includes an outlet orifice 135 in communication with the collection chamber 134 being formed on the bottom surface of the collection chamber 134.
The rotor 113 is provided with a passage 136 formed as a groove in the contact surface 130 of the rotor 113. The passage 136 extends radially from the position of the inlet holes 133 to the position of the collection chamber 135. Thus, the passage 136 it is capable of communicating with any of the input holes 133 depending on the rotational position of the rotor 113. Rotation of the rotor 113 allows different input holes 133 to be selected. As the collection chamber 134 is angularly aligned with the inlet holes 133, in all rotational positions where the passage 136 communicates with an inlet hole 133, the passage 136 also communicates with the collection chamber 134, thus connecting the inlet hole. selected 133 for outlet orifice 135. Therefore, rotor rotation 136 selectively connects individual inlet orifices 133 to outlet orifice 135.
When the rotor 133 is aligned with the space in the inlet holes 133 and the space in the collection chamber 134, the passage 136 is closed against the contact surface 130 of the stator 112, thus closing the valve 110. However, as an alternative, inlet orifices 133 can be brought together to omit the space so that the inlet orifices are arranged in a complete circular crown and valve 110 cannot be closed.
As an alternative to forming the collection chamber 134 on the contact surface 130 of the stator 112, a similar operation can be achieved by alternatively forming the collection chamber 134 as a groove in the contact surface 130 of the rotor 113 opening in the passage 136.
To provide positioning of the rotor 112, the contact surface 130 of stator 112 has a circular arrangement of holes 137 in the same pitch as the inlet holes 133, and the contact surface 130 of rotor 113 has pins 138 that fit into holes 137. The pins 138 can be pushed out of the holes 137 in the rotation of the rotor 112 but are aligned to maintain the rotational position of the rotor 112 in the rotational positions in steps that are each located in the passage 136 in communication with each respective entry hole 133, or in one of the rotational positions in steps to locate the passage 136 over the space in the inlet holes 133 and the space in the collection chamber 134.
The size of the valve 110 is minimized by arranging the inlet holes 133 as close as possible, but the same operation can be achieved by increasing the size of the space in the inlet holes 133 so that the inlet holes 133 extend around a smaller part of the circular crown. In this case, the collection chamber 134 can be correspondingly reduced in length to extend over a shorter part of the circular crown.
The body 120 defines channels connecting the cavities 102 of the cavity plate 100 to the inlet holes 133 as follows.
The first plate 121 is arranged on the underside of the cartridge 10 in the position where the cavity plate 100 is attached and has an arrangement of nozzles 140 protruding outwards and having the same spacing as the cavities 102 of the cavity plate 100 to align with them . As a result, when plate 100 is attached to cartridge 10, each nozzle 140 protrudes into a respective cavity, as shown in Fig. 20. Each nozzle 140 comprises a through hole 141 that extends through nozzle 140 and through the first plate 121 to the contact surface 124 of the first plate 121 to form part of a channel with respect to the cavity 102.
The nozzles 140 extend on the walls 102 for a sufficient distance that the end of the nozzle 140 is submerged below the surface of a sample 142 in the cavity 102. In this way, the sample 142 effectively seals the nozzle 140. This avoids the need for an airtight seal between the cavity plate 100 and the first plate 121.
The contact surface 124 of the second plate 122 is formed with a set of grooves 143 that form part of the channel with respect to each cavity 102. Each groove 143 communicates at one end with the through hole 141 that extends through the nozzle 140 and through the first plate 121. As shown in Fig. 20, grooves 143 extend from nozzles 140 to stator 122, in particular to annular shoulder 126 on the opposite side of second plate 122 from outlet holes 133. The rest of the channels are formed by through holes 144 extending through the shoulder 126 of the second plate 122 from a respective groove 144 in the contact surface 124 of the second plate 122 to a respective entry hole 133.
Body 120 also defines a channel connecting outlet port 135 as follows. The third plate 123 has a through hole 145, shown on the dotted line in Figure 17, which extends from the inlet hole 135 through the third plate 123 to the contact surface 125 of the third plate, forming part of the channel. The remainder of the channel is formed by a groove 146 on the contact surface 125 of the third plate 123 extending away from the through hole 145. As shown in Fig. 17, the groove 146 extends to a metering pump 147 operable to pump a sample a from a cavity 102 selected by the rotational position of valve 110 through valve 110 for sensor device 14.
The first, second and third plates 121-123 can be formed from any suitable material that provides sealing for defined channels between contact surfaces 124 and 125. Suitable materials include PMMA (poly (methyl methacrylate)), PC (polycarbonate) or COC (cyclic olefin copolymer). The first, second and third plates 121-123 can be sealed by any suitable technique, for example ultrasonic welding, laser welding or bonding. PMMA is particularly effective due to the ability to use PPMA broadcast links. The first, second and third plates 121-123 can be injection molded.
Similarly, rotor 113 can be formed from any suitable material that provides sealing and sufficiently low friction for rotation. A suitable material is PTFE (polytetrafluoroethylene) which can be machined with a section made of an elastomer (eg silicone) to provide compression. PTFE can lower the torque required for rotation and has good sealing properties. The elastomer allows the rotor 112 to be fixed but still to rotate. Alternatively, the rotor 113 can be made from a material that can be injection molded, for example, FEP (fluorinated ethylene propylene) or UHMWPE (ultra high molecular weight polyethylene).
Valve 110 is not limited to use on cartridge 10 and can be used in other applications. The valve 110 can be used to flow in the opposite direction to the inlet ports 133, the outlet port 135, more generally, the inlet ports 133 can be referred to as first orifices and the outlet port 135 can be referred to as a second port. Valve 110 is particularly suitable as a miniature element for handling low volumes of fluid, in which the inlet ports 133, the passage 136, the collection chamber 134 and the outlet port 135 have cross-sectional areas of no more than 10 mm, preferably not more than 1 mm.
The rotor 113 is driven by a motor 150 as shown in Fig. 22. The rotor 113 has a coupling element 152 protruding upward from the rotor 113 and on which a geometrical drive shaft 151 is fitted that mounts a gear wheel 153. Motor 151 has an output shaft 154 which assembles a gear profile 155 by engaging gear wheel 153 so that motor 150 drives rotation of the drive shaft 151 and then the rotor 113. The drive shaft 151 also mounts a coding wheel 156 whose position is detected by a sensor 157. The motor 150 is driven based on the output of sensor 157, allowing the rotor
113 to be rotated 5 around to select the desired entry hole 133.
The fluidic system 31 is controlled to perform biochemical analysis with respect to successive samples sequentially. The sensor device 14 is prepared and then the fluidic system 31 is controlled to deliver the sample from one of the wells 102 to the sensor device 14. After the biochemical analysis has been performed, the sensor device 14 is emptied and washed to clean the sample. Then the sensor device 14 is prepared again and the fluidic system 31 is controlled to supply the sample from the next cavity 102 by rotating the rotor 112 of the valve 110.
A specific example of the method of using cartridge 10 with the construction of Fig. 11 will now be described. The materials used are those described in detail in WO 2009/077734.
First, a pretreatment coating is applied to modify the body surface 20 of the sensor device 14 surrounding the cavities 21 to increase its affinity for amphiphilic molecules. The required volume pretreatment is a hydrophobic fluid, typically an organic substance, in an organic solvent it is extracted from a reservoir 30 and dispensed by an inlet pump 33 through the supply channels 32 to fill the chamber 24 covering the body 20 and the cavities 21.0 excess material is expelled in the waste tank 35.
Cartridge 10 can be used in various configurations to expel excess pretreatment. An example is to apply a gas flow with an inlet pump 33 through the supply channels 32 and chamber 24 to move fluid through the outlet channel 36 in the waste tank 35. Alternatively, pretreatment can be dispensed from the inlet pump 33 with gas behind the required volume and the excess expelled through chamber 24 in outlet channel 36 in the waste reservoir 35 in a single action. Gas flow is continued through chamber 24 to wash solvent vapor from the system until the final pretreatment coating is achieved. In further modification, this final step can be achieved more quickly by heating the gas flow or the body 20.
After application of the pretreatment coating an aqueous solution containing amphiphilic molecules, it is drained through the body 20 to cover the cavities 21. The required volume of aqueous solution is extracted from the appropriate reservoir 30 and dispensed by an inlet pump 33 by means of supply channels 32 to fill the chamber 24 covering the body 20 of the cavities 21.
Formation of the amphiphilic membrane 26 is formed with the amphiphilic molecules either directly or improved if a multipass technique is applied in which aqueous solution covers and uncovers the recess cavities 21 at least once before covering the cavities 21 for a final time. The aqueous solution containing amphiphilic molecules can be directly extracted from a reservoir 30 or in the alternative approach mentioned above formed by passing aqueous solution through the lipid assembly in the flow path of the supply channel 32 to the chamber 24.
In a first example, multiple passes of the solution air interface can be achieved by reversing the flow in chamber 24. Flow to and from reservoirs 30 is prevented by operation of selector valve 45 and operation of outlet pump 34 by extracting the amphiphilic molecule containing solution through the supply channels 32 from each chamber 24 and drawing air from the outlet channel 36 to the waste reservoir 35. The direction of the outlet pump 34 is reversed and the solution resumed through the wells filled with solution 21.
The formation of the amphiphilic membrane 26 can be observed by monitoring the resulting electrical signals through electrodes 22 and 25 when a potential is applied to the formation by introducing a resistive barrier and a decrease in the measured current. In the event that an amphiphilic membrane 26 fails to form, it is a simple matter to perform another pass of the aqueous solution air interface.
Alternatively, in a second example, multiple air solution interface passes can be achieved by flow in a single direction by including air pockets in the solution supply. In this second example, the aqueous solution containing amphiphilic molecules is extracted in an inlet pump 33 from the reservoir 30 and then with operation of non-return valves pumped into the supply channels 32. An air pocket can be formed by stopping the flow of aqueous solution of amphiphilic molecule changing the position of the selector valve 45 and requiring air volume inside the channel behind the solution from the waste reservoir 35 (as it is spruce to the atmosphere) by the action of another inlet pump 33. The selector valve 45 it is resumed to the previous position and still aqueous solution of amphiphilic molecule pumped forward. As the inlet pump 33 moves the solution forward through the supply channels 32 to the chamber 24 and through the outlet channel 36 in the waste reservoir 35, the stream of aqueous amphiphilic molecule solution including air pockets is passed over the cavities 21.0 process is repeated to achieve the desired number of passes.
Excessive amphiphilic molecules are removed from chamber 24 by washing aqueous buffer solution from reservoir 30 via an inlet pump 33. Multiple volumes of aqueous buffer solution passed through chamber 24 in outlet channel 36 to supply to the reservoir of waste 35.
Excessive amphiphilic molecules are removed from chamber 24 by washing aqueous buffer solution from reservoir 30 by means of an inlet pump 33. Multiple volumes of aqueous buffer solution passed through chamber 24 in outlet channel 36 to supply to the waste tank 35.
Preparation of the sensor device 14 is completed by draining the aqueous solution containing a membrane protein, for example alpha-hemolysin or a variant thereof, from a reservoir 30 by means of an inlet pump 33 in the chamber on layer 26 allowing the membrane protein to be inserted spontaneously into layer 26 of the amphiphilic molecules after a period of time.
In an alternative approach, membrane proteins can be stored dry. In this case, the aqueous solution can be directed to a second reservoir 30 containing the membrane protein in dry form from an appropriate reservoir 30 via an inlet pump 33 through the supply channels 32 by changing the position of the selector valve 45 used for rehydrate the membrane proteins before using an inlet pump 33 to drain the resulting solution in chamber 24 over layer 26.
The insertion process in layer 26 can be observed by monitoring the resulting electrical signals through electrodes 22 and 25 when a potential is applied insertion resulting in an increase in ionic conduction and an increase in the measured current.
When the insertion period is removed completely from the supply channels 32 and chamber 24 by washing the aqueous buffer solution from a reservoir 30 by means of an inlet pump 33. Multiple volumes of aqueous buffer solution passed through the chamber 24 in the supply channel. outlet 36 for supply to the waste tank 35.
Analysis of the samples contained in the cavity plate 100 can begin upon completion of the preparation of the sensor device 14. The rotary valve 110 is configured to allow fluid to contact the first inlet orifice 133.The selector valve 45 is positioned to stop the flow at from the fluid reservoirs 30 and the outlet pump 34 operated to extract the sample material from the sample cavity 102. The rotary valve 110 is repositioned to direct flow in the direction of the supply channels 32 and fills the chamber 24 to cover the membrane layers 26 of the sensor system. Upon completion of the analysis the selector valve 45 is positioned to allow flow of the aqueous buffer from the inlet pump 33 to wash the sample from the supply channels 32, the rotary valve 110 and the chamber 24 with multiple volumes of buffer through the supply channel. outlet 36 in the waste reservoir 35 to prevent contamination of the succeeding samples.
Selector valve 45 is positioned to stop flow from fluid reservoirs 30 and valve 110 is repositioned to form a fluid connection to the next sample well 102 in cavity plate 100. This process is repeated for all samples.
After all samples have been analyzed, both cartridge 10 can be disposed of. Alternatively, since the cavity plate 100 is a separate element, it can be removed, disposed and replaced with a new cavity plate 100 loaded with fresh samples. Such use of the cavity plate 100 as a disposable element allows reuse of the cartridge 10.
The sensor device 14 is formed on a chip that is mounted on a printed circuit board (PCB) 38 electrically connected to PCB 38. Electrical contacts from PCB 38 are arranged as an edge connector block to make electrical connection for the sensor device 14. When inserting the cartridge 10 in module 2, contact 39 makes an electrical connection to the rest of the electrical circuit in module 2 which is described below. Three alternative designs for sensor device 24 and PCB 38 are as follows.
In the first possible project shown in Figs. 5 and 6, the sensor device 14 is formed as disclosed in WO 2009/077734 as a set of electrodes modeled in the cavities made of silicon with cavities made in a suitable passivation layer on top of the silicon, with the electrical connections at the base of the silicon substrate using solder shock through PCB 38 connected to PCB 38. The PCB provides has an equivalent number of connections for two (or in general any number of) 40 specific application integrated circuits (ASICs) 40 connected in a similar manner to the opposite side of PCB 38. ASICs 40 include some components of the electrical circuit of module 2 described above. ASICs 40 may include components of the processing circuit to process electrical signals from sensor device 14, for example an amplifier, a sampling circuit and an analog to digital converter (ADC) to provide a digital output. The digital output is provided from contracts 39 to allow the digital output to leave the sensor device 14 using a suitable interface, for example low voltage differential signaling (LVDS).
Alternatively, the output signal can be provided in an analogue amplified form with ADC provided inside the module. ASICs 40 may also include some components of the control circuits for example accepting power and control commands through the contacts in order to adjust and monitor the operating parameters, including for example current measurement sample rate (1 Hz to 100 Hz ), integration capacitors, bit resolution, applied trend voltage.
The second possible design is to form the sensor device 14 as a simple electrode assembly chip made of silicon, mounted on PCB 38 and wired to contacts 39. This connection can then interface in the electrical circuit, either as a series of discrete channels, or using an appropriate ASIC. Such an ASIC can be a conventional electronic reading chip, for example, as provided by FLIR systems (eg FLIR ISC 9717) as an arranged electrode measurement device.
The third possible design is to manufacture the sensor device 14 and ASIC 40 as a device that is then mounted on PCB 38.
The configuration of module 2 will now be described with reference to Fig. 7 showing module 2 with housing 11 removed to show the physical layout. Module 2 includes an internal card 50 and an embedded computer 51 connected together by a PCI data acquisition module 52, which together provides an electrical circuit described below. The inner plate 50 makes contact with the contacts 39 of the cartridge 10 on insertion in the module 2.
Embedded computer 51 may be a conventional computer, including a processing unit and a storage unit. Embedded computer 51 includes a network interface 53 that allows module 2 to connect to network 3, thus turning module 2 into a stand-alone network device while still providing 'hooks' to allow many modules 2 to be run, managed and controlled as a cluster, as described below. For example, the built-in computer 51 can run a reduced operating system (eg, LINUX) and applications to perform the various functions described below. Complete development kits for such embedded systems are commercially available.
Module 2 includes a loading mechanism 54 to automatically load and eject cartridge 10 to and from module 2. The loading mechanism 54 can be, for example, a proprietary mechanism driven by a high precision step motor.
Module 2 also includes a microcontroller 58 and an FPGA 72 mounted on the internal board 50 that controls various components of module 2 as described below.
Module 2 also includes fluidic drive unit 60 which is mounted on internal plate 40 and controls fluidic system 31.
The module 2 also comprises a thermal control element 42 arranged to control the temperature of the cartridge 10 and the sensor device 14 in particular. Thermal control element 42 can be, for example, a Peltier thermal control, such as a 32 Watt Single Stage Thermoelectric Module (for example as supplied by Ferrotec Corp, 33 Constitution Drive, Bedford NH 03110 USA - part number 9500/071 / 060B). The thermal control element 42 can be mounted, for example, under the cartridge 10 and is not visible in Fig. 7. The thermal control element 42 can be considered as part of the analysis apparatus formed primarily by the cartridge 10 and can alternatively be mounted on cartridge 10.
Finally, module 2 includes a display 55 to display basic operational status information, a power supply 56 to supply power to the various components of module 2, and a radiator 57 to cool module 2.
The electrical circuit provided by the internal board 50 and the built-in computer 51 will now be described with reference to Figs. 8 and
9. The electrical circuit has two main functions, namely a signal processing function and a control function, so that it acts as both a signal processing circuit and as a control unit for module 2.
The signal processing function is distributed between the internal board 50 and the embedded computer 51 and is provided as follows.
The sensor device 14 is connected to a switch arrangement 62 formed in an ASIC 40 on PCB 38 of the cartridge 10 and controlled by the control interface for ASIC 40. Switch arrangement 62 is arranged to selectively connect the cavity electrodes 22 from the sensor device 14 to a respective contact to provide a detection channel 65 of the signal processing function, so a greater number of cavities 21 than the detection channels. Switching arrangement 62 is arranged and operated as described in detail in U.S. Application No. 61 / 170,729 which is incorporated herein by reference.
Alternatively switching arrangement 62 can be supplied and controlled separately from ASIC 40 as a stand-alone function block between sensor device 14 and detection channels 65, detection channels 65 being provided within a reading chip, for example as supplied by FLIR Systems, (eg FLIRISC 9717).
ASIC 40 provides a set of detection channels 65 each arranged as shown in Fig. 10 to amplify the electrical signal from one of the cavity electrodes 26. Detection channel 65 is then designed to amplify very small currents with sufficient resolution to detect the characteristic changes caused by the interaction of interest. Detection channel 65 is also designed with a high bandwidth to provide the resolution time required to detect each such interaction. These limits require sensitive and therefore expensive components.
Detection channel 65 includes a charge amplifier 66 which is arranged as an integration amplifier by means of a capacitor 67 being connected between a charge amplifier inversion input 66 and the charge amplifier output 66. The charge amplifier 66 integrates the current supplied in it from the cavity 21 to provide an output representative of the load supplied in the successive integration periods. As the integration periods are of fixed duration the output signal is representative of the current, which duration is short enough to provide sufficient resolution to monitor events occurring in the cavity 21 connected to it. The output of the load amplifier 66 is provided through a low-pass filter 68 and a programmable gain stage 69 for a hold sample stage 70 which is operable to sample the output signal of the load amplifier 66 and produces an output signal. sampled current. The output current signal is supplied to an ADC 71 to convert to a digital signal. The digital signals from each detection channel 65 are output from ASIC 40.
The digital signals emitted from the ASIC 40 are supplied via contacts 39 from PCB 38 of cartridge 2 to a field programmable port assembly (FPGA) 72 provided on the internal plate 50 of module 2. FGPA 72 includes arranged temporary storage to temporarily store digital signals from each detection channel 65 before supplying via PCI data acquisition module 52 to inserted computers 51.
In an alternative arrangement, the digital output of the detection is provided from a reading chip located on the internal plate 50 of module 2 and supplied to FGPA 72.
The built-in computer 51 is arranged to follow the processing of digital current signals from each detection channel 65 as follows. A PCI data acquisition module 52 controls the transfer of digital current signals from the FPGA 72 to the built-in computer 51 where it is stored as digital data.
Thus, the digital data stored in the embedded computer 51 is raw output data which is a signal data representing the electrical signal measured from each detection channel 65, which is the current measured by each cavity electrode 22 with respect to a nanopore in the amphiphilic membranes 26 of the corresponding cavity. The current of each nanopore is a measured electrical signal channel. This raw output data is processed by a processing module 73 that includes a pipe 74 with respect to each rod. The processing module 73 is implemented by software running on an embedded computer 51.
The nature of the signal processing performed on each pipe 74 of the processing module 73 is as follows. Piping 74 processes the raw output data representing the measured electrical signal to produce output data representing the results of the biochemical analysis with respect to the corresponding channel. As discussed above, interactions between the nanopore and the sample cause characteristic changes in the electrical current that are recognizable events. For example, an analyte passing through the nanopore can cause the electric current to decrease by a characteristic amount. Thus, piping 74 detects those events and generates output data that is an event data representing those events. Examples of such processing are disclosed in WO 2008/102120 which is incorporated herein by reference. The output data that is event data may in the simplest case represent only the fact that the event occurred, but more typically includes other information about the event, for example the magnitude and period of the event.
In addition, the pipeline can classify the event and the output data can represent the classification of the event. For example, the nanopore may have an interaction that differs in how different analytes in the sample cause different modulation of the electrical signal. In this case, piping 74 classifies the analyte based on the modulated electrical signal. An example of this is that the nanopore can have an interaction based on a polynucleotide in which each base modulates the electrical signal differently. For example, a base passing through the nanopore can cause the electrical current to decrease by an amount that is characteristic of the base. In this case, piping 74 classifies the event by identifying the base from the electrical signal modulation. In this way, biochemical analysis is the sequencing of a polynucleotide in the sample, and the resulting output data is a sequence data representing a polynucleotide sequence. This can be referred to as "called the base".
Piping 74 also produces output data which is the quality data representative of the quality of the output data that represents the results of the biochemical analysis. This may represent a probability of detecting and / or classifying the events being incorrect.
The output data can be represented in any suitable format. In the case of sequencing a polynucleotide, the output data that is sequence data and the quality data can be represented in the FASTQ format, which is a conventional text-based format for a nucleotide sequence and its associated quality count.
All output data is stored on the embedded computer 51 and some or all of the output data can also be transferred over network 3 and stored on storage device 6. Typically this includes at least the output data representing the event classification ( eg sequence data) and quality data, as this is a relatively small amount of data compared to the raw output data representing the measured electrical signal. Additionally and depending on the user's requirements, the output data that is event data and / or the raw data representing the electrical signals measured through each pore can also be transferred and stored.
Processing module 73 can also derive and store quality control metrics representing parameters of biochemical analysis itself.
Aspects of the signal processing performed by the pipeline 74 can be performed on the internal plate 50 before the data is transferred to the embedded computer 51. This approach is of particular use for large numbers of channels and the FPGA 72 may be particularly suitable for this type of assignment.
The control function that is arranged to control the operation of module 2 will now be described. The control function is distributed between the internal board 50 and built-in computer 561 and is provided as follows.
The control function includes a controller 58, for example a Cortex M3 Microcontroller, provided on the internal board 50. Controller 58 controls the operation of all components of the analyzer 13. Controller 58 is arranged to send, via standard protocols and through low level device actuators, control to pumps 33 and 34 of the fluidic system 31 and other prerequisites to read data. Status information is stored based on error codes derived from the triggers.
Controller 58 is itself controlled by a control module 80 which is implemented in the embedded computer 51 by software running on it. Control module 80 communicates with controller 58 through an RS232 interface 81.0 control module 80 controls controller 58 as follows so that it can operate together to constitute a control unit for module 2.
Controller 58 controls loading mechanism 54 to load and eject cartridge 10. Upon loading controller 58 detects that appropriate electrical contact is made between contacts 39 and inner plate 50.
Controller 58 controls fluidic drive unit 60 to control fluidic system 31 to prepare sensor device 14.
During this preparation, the control module 80 can monitor the output of electrical signals from the sensor device 14 to detect which preparation takes place correctly, for example using the analysis techniques disclosed in WO 2008/102120 which is incorporated here by reference, typically, the control module 80 will determine which of the cavities 2 are adjusted correctly to start a run. This can include detection of bilayer quality, electrode quality, pore occupation and even if the nanopore is active following the detection of a sample.
Based on this monitoring, controller 58 also controls switching controller 63 to cause switch arrangement 62 to connect sensing channels 65 to cavity electrodes 26 of cavities 22 of sensor device 14 that have acceptable performance, in the manner disclosed in detail in US Order No. 61/170729.
In the case of polynucleotide sequencing, control module 80 can also detect the presence and status of any modifications to nanopores that may be required in order to process and measure DNA, eg binding of exonuclease enzymes, cyclodextrin adapters.
The controller adjusts the experimental parameters that follow.
Controller 58 controls a trend voltage source 59 that supplies a trend voltage to common electrode 25. In this way, controller 58 controls the trend voltage across each nanopore. Controller 58 controls the thermal control element 42 to vary the temperature of the analyzer 13. Controller 58 controls the operation of the ASIC 40 to vary the sampling characteristics, for example the sampling rate, the integration period and period of resetting of capacitor 67, and the resolution of the resulting signal.
Controller 58 can perform the above control functions and other experimental parameters via the FPGA 72. In particular, control of the ASIC 40 is provided via the FPGA 72.
Once the sensor device 14 has been prepared correctly, then the controller 58 controls the analysis device 13 to introduce the sample and to perform the biochemical analysis. The biochemical analysis is then carried out with the result that electrical signals are emitted from the sensor device 13 and processor by the processing module 73 to produce output data representative of the analysis.
As further described below, control module 80 has global performance targets that are derived based on the input as discussed below. The local performance targets represent the desired performance for the operation of module 2. The performance targets can relate to any combination of: the time within which output data is produced; the amount of output data that is produced; or the quality of the output data that is produced, depending on the requirements for biochemical analysis.
During operation, the control module 80 determines, from the output data, measurements of the performance of the biochemical analysis, this being of the same nature as the local performance targets, eg the time within which the output data is produced; the amount of output data that is produced; or the quality of the output data that is produced. Based on performance measurements, control module 80 controls controller 58 to control analyzer 13 to find performance targets. This is done by starting and stopping operation of the analysis device and / or varying the operational parameters. To find local performance targets, controller 58 controls the following operational parameters that affect performance, in terms of data collection speed and quality:
1) the thermal control element 42 to vary the temperature of the analysis apparatus 13. This affects the biochemical analysis taking place in the sensor device 14, for example by changing the rate of movement of the molecules through the nanopore and / or the rate of processing by enzymes, for example in the case of sequencing the enzyme that feeds bases sequentially through the nanopore. Typically, increasing the temperature increases the data collection rate but decreases the quality, and vice versa.
2) the trend voltage source 59 to vary the trend voltage across each nano-pore. This is an electrical parameter of biochemical analysis that affects performance and can be varied to change speed and quality, or used for 'fine-tuning' a nanopore to focus on high quality measurement for a particular analyte.
3) the operation of ASIC 40 to vary the sampling characteristics, for example the sampling rate, the integration period and reset period of capacitor 67, and the resolution of the resulting signal. This affects the quantity and quality of the output data, typically, increasing the sample rate reduces the chance of missing real events, but increases noise causing poorer measurement quality of each observed event, and vice versa.
To find local performance targets, controller 58 also controls the operation of the analyzer 13, for example:
4) the trend voltage source 59 to vary the trend voltage across each nano-pore. This is an electrical parameter of biochemical analysis so it affects performance;
5) to control the switching arrangement 62 to change the nanopores whose electrical signals are provided to the detection channels 65;
6) to add more fluids, to add more nanopores to a working set of amphiphilic membranes 26 with none or a few nanopores present;
7) to add more samples if the sensor device 14 as a whole is making sufficient measurements;
8) to add a different sample if the measurement requirements for a sample have been met;
9) to apply a reversible trend potential to 'unblock' a nanopore in the case of zero current flow in an individual nanopore;
10) to reconfigure the analyzer 13, whether a global chip failure adjustment has been achieved, or if required before a new sample to be measured is introduced, or if a different type of nanopore is needed to measure the sample, applying sufficient trend potential to break all amphiphilic membranes 26 and then prepare the analyzer again 13.
In the case of sequencing polynucleotides, the analyzer 13 may contain DNA control embedded in real samples. This also allows for quality monitoring of the state of the individual nanopores. Derived data from the embedded control sample can also be used to adjust and refine the algorithms used to process the data originating from the real DNA samples proceeding in parallel.
The control module 80 can also control the signal processing function, for example to control the pipes 74 to perform varying degrees of data processing.
Control module 80 performs the determination of performance measurements and operation control repeatedly during the biochemical apparatus, typically continuously. In this way, the operation of a single module 2 can be optimized in real time with the result that module 2 is more efficiently used. When the control module 80 determines from performance measurements that the biochemical analysis has been completed, the control module 80 controls the controller 58 to stop the biochemical analysis and controls the loading mechanism 54 to eject the cartridge 2. The module 2 it is then ready for insertion of a new cartridge 2, which can be performed by an automated procedure as part of the overall workflow piping for an experiment or series of experiments being performed by the instrument to meet the user's global requirements.
In the manner described above, each module 2 is a stand-alone device that can perform a biochemical analysis independently of the other modules 2. It will now be discussed how a cluster of modules 2 are operated as a common instrument 1 to perform a common biochemical analysis. This is achieved by a cluster of modules 2 being connected together via network 3 via network interface 53. in an overview, module 2 connects network 3 as a self-aware network device following the “device” model widely used. Module 2 can then perform data and communication services. Settings and protocols are stored and run as part of control module 80. Each module 2 can operate as both a service for client and data, and as a server for data and services, for any other module 2. Thus, arbitrary number of modules 2 can be grouped together in a larger logical instrument 1.
Modules 2 can also communicate to share other information, such as dynamically determined calibration criteria, allowing consistent data quality for each module 2, or filtering rules for output data, shared output locations and conflict-free competitor data output at from the same substrate of the same name to a shared repository.
Each module 2 includes a network services module 82 that provides a graphical user interface (GUI) and a control / federation request programming interface (API).
The GUI is present through network 3 to the external computer 7 and displayed on it. For example, the GUI can be presented in HTTP on the standard HTTP port or in any other format allowing it to be viewed by a conventional browser. The user can see the displayed GUI and connect to this network service using standard protocols (eg HTTP) to use the GUI to provide user input to modules 2. The GUI can be a series of network pages that allow control of modules 2, input of parameters, shows states, data of graphs, etc. The user is able to see the status of module 2 that he has selected and sends the command through this interface. This same service runs all 2 modules and can be connected to it in the same way. The GUI can be replaced by any other suitable interface, for example a command line.
The API allows modules to interact with one another.
The GUI allows the user to address modules 2 to select an arbitrary number of modules 2 to operate as a cluster to perform common biochemical analysis. Each module has the GUI, so any module 2 can be accessed by a user and used to select multiple modules 2. This causes the API to send a single command to all modules in cluster 2 informing them that they are addressed. The modules 2 selected for the cluster are given an arbitrary and temporary label, referred to as a 'namespace', mnemonically identifying both control module 80 and user as a cluster doing the common biochemical analysis.
Additionally, the GUI allows the user to provide input representing global performance targets with respect to instrument 1. Alternatively, input representing global performance targets can be derived by instrument 1, for example being retrieved from the stored table of performance targets. global respect for different types of biochemical analysis.
Global performance targets are of the same nature as local performance targets, ie any combination of: the time within which output data is produced; the amount of output data that is produced; or the quality of output data that is produced, depending on the user's requirements for biochemical analysis. Overall performance targets must be fully defined, or some can be left undefined, for example a requirement to produce a certain amount of data of a certain quality is achieved by adjusting the quantity and quality of targets but leaving the target time unadjusted. For example, global module performance targets may be sufficient to acquire sufficient data to cover (or across the sample) the sample in question 20 times more, in a given period, saying 6 hours, and with a minimum required level of quality data, saying a minimum average error rate of less than one in a thousand across all measured bases.
Subsequently, cartridges 10 are prepared with aliquots of the sample to be analyzed and loaded into modules 2 of the agglomerate. This step can be performed by the user. Alternatively, this step can be automated to some extent, for example by module 2 having a sensor that provides for automated registration of cartridges 10. Then, a command is issued to modules 2 of the cluster instructing them to start the analysis.
In advanced systems, the preparation of the cartridges 10 with sample to be analyzed and / or the loading of the cartridges 10 in modules 2 can be automated.
In another alternative, cartridge 10 contains a mechanism to manage and process multiple samples in series, or time multiplexing, as for example with the construction shown in Fig. 11, using cavity plate 100 to store multiple samples to be processed by the chip of sensor 14 in series. In this case each module 2 controls the cartridge 10 loaded in it to process samples from a selected well 102. The software in module 2 is adjusted by the user, for example receiving user input, to be aware of which samples are in which wells 102. This adds a layer of information to the sample management. All other operations of the cluster remain the same, except that the coordination now takes into account that samples are being processed from a given cavity 102 on plate 100 instead of assuming that there is a sample mapping for each cartridge 2. Thus Coordination occurs at the level of samples per plate 100 instead of samples per cartridge 2. When a new cartridge 2 is inserted, the control module 80 references the sample-well table loaded by the user. This can also be accessed from a central database using an internal bar code provided on cartridge 2 as a search key (the plate and sample information having been associated with this cartridge by a user in the time that the well 100 was connected to the cartridge 2).
The cluster modules 2 are now 'aware' that they are cooperating and their control modules 80 communicate and interact as follows so that together they provide a control system for instrument 1 as a whole.
The control process is shown in Fig. 23.
In step SI, based on global performance targets 90, local performance targets 91 are determined for each module 2 in instrument 1 which together find global performance targets 90. Step SI is a global determination carried out for all modules 2 in the agglomerated. Initially, step SI is performed based on the overall performance saved 90 alone, although as discussed below, subsequently SI is also performed based on the performance measurements 93 of each module 2 in the cluster derived from the output data 92 of each module 2.
Step S2 is performed in a local control process with respect to each module 2 in the cluster, carried out based on the local performance targets 91 for that module 2. In Fig. 23, fourth such local control processes are shown by way of illustration , but in general it has the same number of local control processes as modules 2. The local performance targets 91 effectively indicate the operation that is required from each respective module 2, and in step S2, each module 2 is operated according to with the local performance targets 91 to provide the required operation, so that modules 2 together perform the common biochemical analysis.
Step S2 itself comprises the following steps.
In step S3, based on the local performance targets 91, the operation of the analysis device 13 is controlled in the manner described above, which is starting and stopping operation of the analysis device and / or varying the operational parameters.
Initially, step S3 is performed based on the overall 90 performance targets alone. However, once the operation has started, output data 92 is derived. As part of the local control process of step S2 with respect to each module 2, in step D3 measurements are derived from performance 93 from output data 94, as described above. So in the process of local control of step S2 with respect to each module 2, step S3 is performed based on the performance measurements 93, as well as the local performance target 91. In this way, the control of the operation of each module 2 is varied based on the performance measurements 91 that are currently being achieved by module 2. The control performed in step S3 is updated in this way by resuming the performance measurements 93 derived from output data 92 repeatedly, and typically continuously during the performance of the biochemical analysis.
In addition, at least once during the performance of the biochemical analysis, performance measurements 93 from all modules 2 in the cluster are resumed to step S1. Then, in step S1, based on the performance averages 93 from all modules 2 and the global performance targets 90, the local performance targets 91 are varied, if necessary to find the overall performance targets. The respective modules 2 are then operated in step S3 according to the updated local performance saved 91. Updating the local performance targets effectively indicates that the required operation from each respective module 2 has changed. Operation of modules 2 under control of control modules 80 according to an updated local performance target 91 varies the operation required of modules 2 to find global performance targets 90.
Such update of the SI step to vary the local performance targets, if necessary, is performed at least once, but is preferably performed repeatedly, preferably periodically, and preferably with an interval that is much longer than the period of biochemical analysis, typically by at least one end magnitude, and much longer than the period in which the control of the operation of the modules in step S3 is updated, typically by at least one end magnitude. Increasing the frequency of the update improves the management of modules 2 but this is at the expense of taking up resources of the embedded computer 51 and network 3 and the improvement reduces as the interval approaches a characteristic interval for a biochemical analysis event. Typically the interval should be in the order of 1 to 5 minutes, but the management of modules 2 is still effective in longer intervals, it says in the order of hours. But even performing the update once during the biochemical advantage provides an advantage over a monolithic device.
In step Sl, when trying to adjust or update the local performance targets 91, it is possible that the required operation will not be achieved, this is because the local performance targets 91 of the modules 2 required to find the global performance targets 90 are not reachable. To address this, control modules 80 are arranged to determine if this is the case and to take remedial action. A variety of remedial actions are possible.
One type of remedial action is to increase the number of modules 2 in the cluster used to perform the common biochemical analysis.
This allows the global performance target 90 to be found. To achieve this, control units 80 can produce output by notifying a user. In response, the user can use the GUI to address one or more additional modules 2 to form part of the cluster to fit those modules 2 in the same way as the original modules, including inserting a sample into cartridge 10 and loading the cartridge into each one or more additional modules 2.
Alternatively any of these steps can be automated.
Another type of remedial action is to control modules 2 of the cluster to stop biochemical analysis completely. This frees up modules 2 for another biochemical analysis as the global performance target cannot be found.
The SI and S3 decision making steps can be an execution of any suitable computational method. The simplicity of the approach is to use a search table, stored in the embedded computer 51, for contingencies to be performed in scenario data. For example, such a scenario may be an inability to meet a certain set of performance criteria because of an under realization node, for which the action may be for the other nodes to increase their rate of data acquisition. Direct programmatic logic can be used to analyze the data and derive the decision, coded in the software. Other more complex methods may include the distorted recognition of certain patterns in the data and the generation of a response, eg through a trained neutral network.
It will now be discussed where various steps of the control process shown in Fig. 23 are implemented.
Step S2 is a local control process with respect to each module 2 that is performed based on the local performance targets 91 for that module 2 and involves calculating performance measurements 93 from the output data 92. Thus the control module 80 of each module 2 advantageously performs the local control process of step S2 in compliance with its own module. In this way, the control of the operation in step S3 and the determination of the performance measurements 93 can be carried out locally in module 2 without the need to transmit any data over the network. This assists in dimensioning the control process with the number of modules 2. Each module performs the local control process of step S2 independently, so that any number of modules 2 can be included in the cluster without an increase in the load on data transfer through of network 3 being necessary to implement the local control process of step S2. This also effectively divides the process loading of step S2 between modules 2 as each control module 80 performs its own processing.
In principle, step S3 or step S4 can be implemented with respect to one or more modules 2 extremly, which is inside a different module 2 or an additional computer connected to network 3. To perform step S4 extremly, it may be necessary to transmit derivative through from network 3 the output data from which the performance measurements are. Similarly, to perform step S3 extremly, it may be necessary to transmit derivative measurements and control signals to module 2 via network 3. This would increase the load on the network, especially as control is varied in step S3 frequently. For any practical implementation of network 3 and external processing, this could create bottlenecks, in terms of both or both data transfer and processing. Such bottlenecks may reduce the design by effectively limiting the number of modules 2 that can be incorporated into a cluster.
There is an increased degree of flexibility where the SI step is implemented. Step S1 does not require performance measurements 94 of all modules to be taken into account and as a result there must be some data transfer over network 2 so that step S1 can be performed based on performance measurements 94. However , the amount of data needed to be transmitted is relatively small, with performance measurements 94 and messages to implement negotiation between control modules 80. This requires a significantly smaller amount of data than the actual output data. For example, performance measurements simply represent the value of each measurement, which has only a handful, where the amount of output data that is given in sequence will be greater, the amount of output data that is event data is typically in the order of magnitude greater than the sequence data, and the amount of output data that represents the measured signal is typically in the order of magnitude greater than the event data. Additionally, it is noted that according to step SI it is updated in a much longer period than the period in which the control of the operation of the modules in step S3 is updated, the frequency at which data that needs to be transferred through network 3 is lower, which still causes the load on network 3 to be much lower than if step S2 was implemented extreme to modules 2.
In a first implementation, the processing of the SI step is divided between the control modules 80 of modules 2 in the cluster. In this case, the control modules 2 cooperate with each other to perform step SI to determine local performance targets 91 for each module 2 on instrument 1 that together meet the overall performance targets 90. This can be achieved by an iterative process. Each control module 80 derives its own proposed local performance targets and then communicates with the other 2 modules in the cluster. Upon receipt of the proposed local performance targets from all other modules 80, each control module 80 determines whether global performance targets are found and if necessary to review its own proposed local performance targets. This process is repeated until the local performance targets have agreed.
When SI step is performed initially, this occurs based on global performance targets 90 alone because no output data has yet been generated. When step SI is performed subsequently to update, if necessary, the local performance targets of each module 2, step SI is performed based on the performance measurements 94 derived by the control modules 80 of each module 2 with respect to that module 2. For To this end, control modules 80 communicate performance measurements 94 to each other via network 3. In this way, control modules 80 actively report performance measurements 94 to one another in order to complete the biochemical analysis more efficiently. Each module 2 can reach its own decision. Decisions can then be encoded in a search table present in each module 2. Each module 2 then transmits, through the network service, its decision to the other modules 2 so that each module 2 now stores a table of responses from other modules 2 proposed. Once this table has been assembled, simple majority voting can be applied to choose the proposed course of action if more than one is signaled.
Thus, the control module 80 of each module 2 is capable of carrying out the required computations and decision making without user input, but they are also collectively capable of doing the same in combination. They can also split individual internal decisions, and collectively make goal decisions, at a level above that, around the overall outcome. In this way, the control / federation API generates decisions by making modules 2 in the cluster in order to optimize a laboratory workflow.
In this way, the modules 2 in the cluster making instrument 1 produce output data from several channels from a common biochemical analysis. Optionally, modules can include a federation layer (not shown) to allow for consistent filtering, normalization and aggregation of the output data. In the case of sequencing of polynucleotides, modules 2 can be controlled to perform sequencing analysis together in combination on single high-throughput samples; so that each module 2 is equivalent to a subchannel or ‘track’ in an optical measurement DNA sequencing instrument based on a typical flow cell.
This first implementation helps to dimension the control process with the number of modules 2. Each module 2 contributes equally to step Sl, so that the processing load is divided equally and the processing load in a single module 2 is increased minimally by an increase in the number of modules 2 in the cluster. Increasing the number of modules 2 in the cluster merely increases the amount of data transmitted through the network in proportion to the number of modules 2. This will in principle eventually limit the cluster size for any practical network given 3, but the amount of data is relatively low, so in practice large numbers of modules can be accommodated.
As each module 2 participates in the decision making process in this first implementation, this divides the processing load and has the advantage that instrument 1 can be formed from any combination of modules 2 because they all have the capacity to make a decision. However, making a decision can be divided into different ways.
In a second implementation, the processing of step S1 is performed by the control unit 80 of only one of the modules 2 acting as a principal, or by the control units 80 of a subset of the modules 2, to make decisions on the local performance targets 91 of each module 2 in the cluster, based on the performance measurements 94 communicated from other modules 2. This still requires data representing the performance measurements to be transmitted through network 3, and increases the processing load in module 2 acting as main. Ideally, any module 2 has the ability to act as a principal, so that a principal is arbitrarily selected from which modules 2 are addressed as a cluster. Alternatively, only special modules 2 can act as a main, but this has the disadvantage of requiring the user to select one of modules 2 in each cluster that is addressed.
In a third implementation, the processing of step S1 is performed by an additional computer that is connected to network 3, such as the external computer 7 or a model 2 module that does not have an operational analysis device 13, to act as a unit of analysis. federation control to make decisions on local performance targets. In this case, the additional computer becomes part of the global control system and performance measurements are communicated from modules 2 to the additional computer to form the basis for making a decision. However, the requirement for a computer still properly programmed is itself a disadvantage in the sense that modules 2 in isolation are not sufficient to implement the control. On the other hand, this implementation reduces the processing requirement in the modules 2 themselves.
Another alternative is for additional nested levels of return are introduced in the control process shown in Fig. 23. In fig. 23, returns to performance measurements 94 at two levels, first at the level of the local control process of step S2 for a single module and second at the level of the cluster as a whole. Additional levels can be introduced by dividing modules 2 of the cluster into logical groups of modules 2 which are each subsets of the total number of modules 2 in the cluster. Achievement performance targets and measurements for each logic group are derived in the same way as local performance targets and performance measurements for an individual module 2 as described above. SI step of the control process shown in Fig. 23 is modified to include an additional level of return. That is, at the highest level, group performance targets are determined based on the overall performance targets and the performance measurements of each group. At the next level, in a separate group control process with respect to each group, the local performance saved from each module 2 in the determined group based on the group's performance targets and the performance measurements of each group 2 in the group. Similarly, performance measurements for the group as a whole are determined from performance measurements for each module 2 in the group. In general, any number of nested levels of return can be employed, for example by dividing groups into subgroups and so on.
In this case, the additional levels of return can be implemented using any of the implementations for step SI as described above.
This alternative increases the complexity of the control process, but has the advantage of allowing the control process to be adapted to the nature of the common biochemical analysis and / or to different network structures. The different levels of the control process can be implemented on different elements of instrument 1 and can be updated at different times, with consequent reductions in network load 3. This. For example, groups can be groups of modules 2 performing the same part of the common biochemical analysis that is advantageously controlled with reference to a group performance target for the whole group. Alternatively, groups can be groups of modules 2 that are connected to respective local networks that are interconnected, eg via the internet, in which case the data flow between local networks is reduced without impacting the control of any group connected to a local network.
The way in which modules 2 connect to network 3 and communicate on a non-hierarchical basis will now be discussed. Generally speaking, the interchange of status data between modules 2 to first make automated decision for performance management to be performed based on “eventual consistency” as a low update frequency is acceptable.
Modules 2 can identify each other using a service discovery protocol, for example Universal Plug and Play (UPnP) or Zeroconf (or Bonjour).
Metadata such as proposed local performance targets and performance measurements can be propagated using a variety of types of distributed database techniques such as CouchDB (HTTP, JSON), Tokyo Cabinet, or MemcacheDB.
Alternatively, metadata propagation and discovery can be achieved using message techniques such as network broadcasting, network multicast, The Spread Toolkit, ActiveMQ, RabbitMQ, or message queues in general.
One possible implementation is to use a perl script that runs in the publisher, subscriber or pub + sub mode to implement network broadcasting of signal packets using User Datagram Protocol (UDP), each flag packet containing encoded JSON data (javascript object notation plain text). Each module 2 acts as a node that broadcasts its own details and listens to the others. Received flag packets are decoded and incorporated into an internal memory data structure, such as hashing with a key in the module name. This has the advantage of simplicity, the flag packets containing in a very minimal hierarchical name (default host name), hierarchical time and system status & performance data. Then modules 2 retransmit their entire data structure including data received from other modules 2. Since UDP packets are uncertain and delivery of signal packets is not guaranteed this retransmission improves the likelihood that module 2 will receive data from other modules 2. As flag packets can include data for all modules 2 in the cluster, modules 2 never incorporate external data intended to be their own.
UDP packets are most efficient up to the maximum transmission unit (MTU) of the subnet. By default this is around -1500 bytes. Compression of the payload (eg using common gzip / LZW) can be useful to maintain transmission size under the MTU. With a fixed signaling frequency, as the number of modules 2 in a cluster increases, you have a much greater risk of network packet collisions and retransmission causing congestion and loss of bandwidth. This can be handled using a dynamic signaling frequency inversely proportional to the number of active modules 2.
The advantages of instrument 1 are that efficiency gains are achieved as compared to a monolithic instrument due to the modularization of the analysis apparatus 13 themselves and due to the operation of the individual modules 2 being intelligently paralyzed. The user has a parallelized group of modules 2 at his disposal and can group a cluster of any number of such modules into a larger instrument 1 to meet the requirements of the common biochemical analysis that is desired to perform. This dimensioning allows the performance of the biochemical analysis of a range of complexity without being limited by the capacity of a single instrument. Similarly, control of the operation of modules 2 optimizes their performance to find global targets. Both of these factors produce efficiency gains, because better use is made of individual modules 2, effectively releasing modules 2 to perform other tasks.
For example, a small number of modules 2 or even a single module 2 can be used for low-throughput applications and large clusters can be used for massively parallel applications such as large sequencing projects, eg sequencing a human genome.
This allows management of workflows that provide efficiency gains in the use of the equipment. In the specific case of sequencing, the resulting workflows overcome problems with current monolithic DNA sequencing instrumentation and meet the needs of users by performing large genome sequencing projects where high throughput is required, while also matching the needs of intermediate labs doing projects smaller but highly replicated or heterogeneous, or just smaller experiments.
Instrument 1 can be applied with a different number of modules 2 to perform a range of types of analysis, for example:
• Human Genome Re-sequencing Set.
• Low cover methylation or cancer rearrangement • A highly replicated short-reading experiment, such as gene expression.
• A molecule analysis using a small sample or mixed cell population.
Some specific examples of situations where efficiencies are obtained will now be described:
1) A user adjusts modules 2 often in a cluster to measure DNA from a single sample. The user adjusts the experiment so that 10 sample rates are added to each module 2 to provide the necessary sample material, and after selecting his preferred settings (eg time to perform, data quality, etc.) starting the experiment . A module 2 has a defective chip and is reposting very little data. The user was asked to perform the experiment in a certain time, so the other nine modules 2 in the cluster increase their sequencing rate, through automatic temperature manipulation to accelerate the processing speed of each nanopore, in order to find the target. Without this dynamic readjustment, the experiment would have concluded within the defined period, but it could have generated less data than expected by the user, potentially compromising its results and overall experimental result.
2) In another case, the user creates a cluster of 8 modules 2 to measure a single sample, again aliquoted through the 8 modules 2. Four of the eight modules 2 are reporting very little data quality and another 4 cannot compensate due to pre-specified performance parameters required by the user (eg output and measurement quality). Therefore, defective modules 2 end their executions and send an email to the operator with a report of what has been done and why, thus allowing the operator both to allow an update of nanopores on the same chips within the defective modules 2 with alternate rates of the sample minimal loss of time or cost to the user, or to load another set of four chips immediately, which will minimize any loss of time. In this example, defects can be detected earlier in the runs and additional chips can be loaded before the expected time for the sample to be made, thus saving the project. By comparison, if a user has performed the same experiment on the Illumina Genome Analyzer, and four of its eight 'clues' have failed causing poor data production, the user can just as much end the entire experiment earlier, losing all data generated through all tracks until the point in time, or allowing the execution to finish and only end with approximately half the expected amount of high quality data, but at the same cost and taking the same amount of time as a complete functional experiment.
3) As a continuation of the above scenario, another useful situation may occur. The user's laboratory in question only has eight modules 2 installed, and the defective four have been ejected. But another urgent project is in a 'queue' to be executed on the system. The operator can then make a decision to allow more time to carry out the original project on the remaining modules 2 and to use the free modules 2 to process the waiting projects as soon as possible. Thus resources can be globally adjusted to a laboratory priority.
4) A user wants to perform an experiment on a sample, or a set of samples, looking for a particular result in them. The user can then specify the experimental processing of the sample or samples to continue until a particular data (eg an exact DNA sequence motif) has been observed once, or a specific number of times. In particular, one data can be used as a marker or proxy for the likely overall success of the experiment once all adjusted data have been analyzed. For example, coverage of a certain level of a particular region of the genome is known, from previous sequencing runs using the same library of DNA fragments, to ensure full coverage (degree of oversampling) across the sample is sufficient to the study that the user requires. In a cluster of modules 2 such as a search can be divided across modules 2 and when sufficient data of the required type has been observed this can be used to adjust a stop condition for some or all of the participating modules 2. This time optimization and cost to achieve an experimental result cannot be realized in current DNA sequencing instrumentation.
5) A user has set a requirement for a cluster of modules 2 to analyze a DNA sample at a high prespecified quality. During the experiment, modules 2 collect data in a higher quantity than expected by the user, but not with sufficient high quality. In order to achieve the required end quality faster, modules 2 collectively adjust their analysis conditions to improve data quality, even if this is at the expense of the result (amount of data given has already been achieved). For example, by lowering the operating temperature, DNA bases move through each nanopore more slowly, on average, thus allowing more analysis time per base, which improves the quality of the base measurement, albeit at a slower data yield per nanopore. Alternatively, or in parallel, the rate at which current flowing through each nano-pore is measured can be changed, either by sampling faster or slower, which can improve particular aspects of data quality, depending on the signal for media profile and speed of the bases through the nanopore.
6) A module 2 in a cluster during an experiment experiences a catastrophic hardware failure, and is safely shut down without causing a loss of experimental data (nb all data generated by module 2 until the time of the failure is usable and has already passed in one dedicated storage area). All remaining modules 2 respond by increasing their experimental periods in order to meet the user's current needs for a required data output without user intervention. The system also sends an automated message to the manufacturer in order to replace the product. Minimal disruption to the user experiment and workflow has occurred.
In the case where a cartridge 2 is capable of processing multiple samples, as for example with the construction of Fig. 11, examples of global performance targets that can be found are as follows:
1) A sample is being processed on a plate 100 in a co-op cluster mode. The user specified that a certain amount of data is required. The sample exists on another plate 100 and is also being processed by another cluster node. Modules 2 coordinate as previously described.
2) The scenario as shown in 1 is followed but in this case the second sample on the second plate 100 is of low quality. Module 2 responds to the performance target by scanning the internally stored plate sample table to see if another instance of the sample exists on its plate 100 if so it then reconfigures its valve to use this sample instead of an exhausted one and coordination continues.
3) In another example, ten modules 2 are identical processing plates 100 of the sample and work through them. A user changes the priority of one of his / her samples that have not been processed. Some of the cluster's modules 2 now reconfigure their valves to move to the sample in order to deliver their data on time. The remaining modules 2 of the cluster remain in the original samples and speed up their processing rates by changing temperature.
4) In another example, a cluster of modules 2 is processing identical plates. Before they start they set their valves 110 to move through cavities 102 where they take a small amount of the sample and perform a short run. From this they then together, pre-calculate the probable quality and quantity of data and drawing from each sample (or cavity 102). They then, together, compute the optimal sequence in which to process the samples in order to deliver data of the required quality and quantity to their respective users on the line with present properties. If wells 102 are found to be empty, or samples are of very low quality to find the targets, the cluster notifies users that fresh plates need to be made with the failed samples prepared.
A key enabler is the ability of modules 2, individually and in combination, to decide a sufficient, and sometimes present, condition of para. This ensures that neither too little nor too much data of the required quality is generated. In this way complete occupancy of the systems can be achieved, and no 'negligent' data is produced in the case of excess. Not even an extra full run has to be done a posteriori in order to adjust for any deficiencies in output or quality. This general scheme allows samples and data to be efficiently channeled through the result of optimizing total sequencing workflow, quality and costs. For any high-end laboratory this can achieve several times improvement in efficiency over systems that operate fixed run times with fixed data yields, specifically if those data yields are not always predictable, as is usually the case.
It is noted that all the above operations are enabled and performed by the implementation of specific control divided within each module 2. It is also noted that modules 2 can be executed individually and some, but not all of the above scenarios can be enacted in module 2 Internal optimizations can be enacted, but optimizations across multiple modules 2 cannot.
The operation of the instrument in example (1) will now be described in more detail.
In this case, instrument 1 is used for DNA sequencing. This means detection of at least four possible analytes corresponding to bases G, C, A and T. Ten modules 2 are being used and they have been given the same sample to process. The user requires that 12 Giigabases (109) of data are required in 1 day where 100% of the recorded bases have a quality score of Q20 or higher (eg a base has less than 1 in 100 chances of being incorrect). The amount of data and the amount of data has been chosen to ensure that when the DNA sample is analyzed, the user is almost certain to be able to find genetic elements (eg mutations they are looking for). These criteria may have been derived from previous empirical experience or from some simulations.
The user has at least ten modules 2 in suitable locations and knows the network addresses of the embedded computer 51 within each module 2. The user prepares his DNA sample in an appropriate manner for a given experiment. If this is the sequencing of a Human genome, it must randomly shear the DNA sample using appropriate shelf equipment.
The user decided, based on the probable yield (data per unit of time) to use ten modules 2 for this sample. The sample is inserted into ten cartridges 10 that are loaded in modules 2. Modules 2 must automatically read a barcode or RFID on each cartridge 10 uniquely identifying the cartridge 10 and stores the cartridge ID 10.
Modules 2 identify other modules 2 in the cluster and send a contact socket and receive basic information about the other modules 2. This information is then displayed on the GUI. In this example the user can see the twenty modules 2 in his network, but is only interested in the ten with 10 cartridges loaded containing his sample. These are identified through the GUI by name, address, state, location, etc. all of which are collected from the underlying network services. Any module 2 can be used to manage any other module 2 in this way and no other computers are required. Thus any arbitrary number of modules 2 can be connected, managed and executed in a linearly scalable way without the bottleneck of working through a connection point system.
The user now addresses the ten modules 2 of interest through the GUI. A GUI element allows a name to be assigned (eg 'Human ”. The same GUI allows commands to be addressed only for this collection and for any data taken from this module 2 to be treated as an aggregate and independently from from any cluster of modules 2. The user can also enter other information about the sample under study directly or then link the entire process to an external database system.
Through the GUI, the user now calls the 'Human' cluster of modules 2 that they are about to execute until 12 Gigabases of Q20 + DNA sequence data have been collected. Modules 2 are also called that they are running the same sample. The control modules 80 of each module 2 order these commands, storing performance measurements as to how much data has been collected and what quality it is. Other metrics can be useful for different use cases. This control module 80 monitors the data and status of module 2 in real time or near real time and is capable of making decisions. In this case, the control module 80 has stored the fact that it belongs to a group called 'Human' and that the group as a whole has a cooperative target of 12Gb of data Q20. This can be stored internally simply as a table in the memory of this process showing the name of module 2, the data generated, the target and quality data, etc. or in a more permanent stage, such as Table 1.
Table 1:
Module 2 Group Target gruno Output Targetinternal Quality Timeexecution fh) 124.45.23.1 Human 12 Gb 1 Gb 1 Gb Q20 6 124.45.23.2 Human 12 Gb 0.4 Gb 1 Gb Q20 6 124.45.23.3 Human 12 Gb 1 Gb 1 Gb Q20 6 .....etc
As shown in Table 1, each module 2 in the 'Human' group divides its table (data structure). A standard part of its opera could be for radio to merge, through its internal network service_
82, a copy of this table for the other modules 2 at regular intervals thus synchronizing them. Each module 2 can then see the status of the other modules 2 and at any time can carry out a pre-programmed operation such as the aggregation of the 'Output' column and a comparison of the total of the 'Target Group' column. Another internal computation could allow the rate of data generation of a given quality to be interpolated versus the runtime column showing whether any individual module 2, or the sum of the outputs of module 2, is on target to meet the adjusted time requirement by the user. Each module 2 has these computations encoded in its control module 80 and each module 2 executes them periodically in its synchronized and divided state data table. A large number of such computations have been coded in control module 80 covering other user-cases than this simple example. After 6 hours it can be seen that the amount of data generated is not on the path to find the target and each module 2 is fully aware of this. One module 2 in particular appears to be performing poorly. This can be for any number of reasons, but diagnostic information on the board does not show any flaws.
Modules 2 now make a decision based on the information they have in order to find their targets, as discussed above. In this case, the chosen course of action from all modules 2 is to increase the output of function modules 2. The table was unanimous. Having aggregated this result internally, modules 2 must now calculate how much extra data is required to achieve the objective. Fully they already know when each of them is producing per unit of time, and has also obtained from other modules 2 how much they are generating. Using pre-coded logic associated with the chosen course of action (eg a software function) modules 2 now compute how much of their own output needs to be increased to find the target. In the simplest algorithm, each module 2 proposes a small increase of a certain percentage and transmits this to the other modules 2. Each module 2 then, using its internal cable, calculates what effect this has on the target aggregate and result. This process is repeated until all modules 2 show, through their internal tables, that the target can be reached. In a more sophisticated alternative, modules 2 with smaller output make proposed increments that are larger than those with good output, thus ‘sharing load’. Again the same data sharing, followed by shared computing, followed by sharing a result, followed by a community vote, is used to allow modules 2 to choose a coarse collection of the action.
In this example the internal table is now being updated so that some modules 2 (only three shown) have increased their local performance targets from 1 Gb per day to 1.4 Gb per day to compensate for the weaker ones, as shown in Table 21. Since no more changes the calculation shows that the total output for the group as a whole will find the target time and quality. The modules 2 have thus adjusted their internal logic, with a return from other modules 2, to find a collective target.
Table 2:
Module 2 Group Target group Output Internal target Quality Time toexecution (h) 124.45.23.1 Human 12 Gb 1 Gb 1.4 Gb Q20 6 124.45.23.2 Human 12 Gb 0.4 Gb 1 Gb Q20 6 124.45.23.3 Human 12 Gb 1 Gb 1.4 Gb Q20 6 .....etc
Having done these individual modules 2 you should now translate collective decision making into internal remedial action. The logic for doing this is encoded in control module 80. For example, temperature sequencing can be used to control the rate at which nucleotides are cleaved from the DNA strands and passed through the nanopore. This can slightly decrease the quality of the observed data (see below) if the temperature is raised too high, but the basic procedure described in the steps above should detect this and seek to correct for a decrease in quality. In this case, the remedial action is greater base performance. Control module 80 then sends a command, such as a suitable function call, called RPC, or by sending a formatted string down from a communication socket, to microcontroller 58 on internal board 50. This command instructs microcontroller 58 to change the temperature of the analyzer 13. This can be enacted by an additional command being sent to a device driver controlling the thermal control element 42. The 'adjusted' temperature of this component is increased by an increment, perhaps derived from from a table search, which is expected to increase the number of bases per unit time by the desired amount. The thermal control element 42 accounts for less cooling, and sensors on the cartridge plate 10 detecting the change in temperature to the desired level. This information, the recorded values, any error codes, etc. are transmitted back to the control module 80 which now records that the remedial action has been taken sufficiently.
Control module 80 all the way through being recorded and counting bases and quality scores from data as they are transferred from ASIC 40 and processed by processing module 73. This process continues and the internal tables are updated and the results transmitted to the other modules 2 in the group. All being well instrument 1 as a whole is now on track to deliver the overall performance target. If not then further action may be needed to be taken and other scenarios explored. These scenarios follow the same basic data flow, but would have specific logic encoded in software modules accessible by the control module 80. For example, if the actions here are unable to meet the time requirements and quality requirements after adjusting the temperature, the modules 2 can then decide to send a message to a user (logged in at run time) instructing that a number of extra modules 2 are required to find the targets. This allows the user to then do other tasks, perhaps idle, modules 2 and insert extra cartridges 10 with the same sample and, in the manner written above, add them to the cluster so that they can then participate in the collective operation.
The core method is to allow collective decision making through modules 2. Each of them has the ability to operate alone, but it can also share internal state data structures and keep them up to date. Modules 2, once aggregated and linked in a cluster of cooperation systems, can then execute a stored protocol that responds to and / or modifies this structure. As well as allowing inter-module 2 communication, this protocol triggers the execution of the pre-coded logic, executing at least one embedded computer, which allows modules 2 to modify their behavior and to coordinate the modification with other modules 2.
Modules 2 cooperate to perform a biochemical analysis that is common to modules 2 of instrument 1. The respective biochemical analysis carried out in each module 2 can be the same or different, being in general terms needing to be “common” only in the sense that the criterion overall performance can be adjusted for global analysis. A typical example is for the biochemical analysis performed on each module 2 to be the same analysis performed on different rates of the same sample, or on samples that are different but perhaps related in some way, for example sampled from a given population. Another typical example is for the biochemical analysis carried out in each module 2 to be different but related types of analysis carried out in different aliquots of the same sample, or in samples that are different but perhaps related.
More details on the nature of the biochemical analysis that can be performed are as follows. The following paragraphs refer to the numerous documents that are all incorporated by reference.
The analysis apparatus 13 described above can perform biochemical analysis using nanopores in the form of protein pores supported on an amphiphilic membrane 26.
The nature of the amphiphilic membrane 26 is as follows. For amphiphilic systems, membrane 26 is typically composed of lipid molecules or their analogs and can either naturally occur (eg phosphatidicoline) or synthetic (DPhPC, diphthanoylphosphatidylcholine). Analogous non-natural lipids can be used such as 1,2-dioleoyl-3-trimethylammonium 10 propane (DOTAP). Amphiphilic membranes can be comprised of a single species or a mixture of species. Additives such as fatty acid, fatty alcohols, cholesterol (or similar derivatives) can also be used to modulate membrane behavior. Amphiphilic membranes provide a high resistive barrier for the flow of ions through the membrane.
Additional details of the amphiphilic membranes that are applicable to the present invention are given in WO 2008/102121, WO 2008/102120, and WO 2009/077734.
In the analysis apparatus 13, the amphiphilic membrane 26 is formed through a cavity 22, but the analysis apparatus 13 can be adapted to support an amphiphilic membrane in other ways including the following. The formation of electrically addressable amphiphilic membranes can be achieved by a number of known techniques. These can be divided into membranes or bilayers that are incorporated into one or more electrodes and those that provide the divider between two or more electrodes.
Membranes connected to the electrode can be bilayers or monolayers of amphiphilic species and can use direct current measurements or impedance analysis, examples of which are disclosed in (Kohli et al. Biomacromolecules. 2006; 7 (12): 3327-35; Anderson et al., Langmuit. 2007; 23 (6): 2924-7; and WO-1997 / 02024. Membranes dividing two or more electrodes can be formed in a number of ways including but not limited to: bent (e.g. Montai et al., Proc Natl Acad Sei USA. 1972, 69 (12), 3561-3566), tip immersion (eg Coronado et al., Biophys. J. 1983, 43, 231-236); drops ( Holden et al., J Am Chem Soc. 2007; 129 (27): 8650-5; and Heron et al, Mol Biosyst. 2008; 4 (12): 1 191-208); supported by glass (e.g. WO 2008/042018); supported by gel (eg W02008 / 102120); encapsulated by gel (eg WO 2007/127327); and tied and supported porous (eg Schmitt et al., Biophys J 2006; 91 (6): 2163-71).
Nanopores are formed by protein pores or channels introduced into the amphiphilic membranes 26. The protein pores or channels can be proteins that are either natural or synthetic, examples being disclosed in WO-OO / 79257; WO-OO / 78668; US-5368712; WO1997 / 20203; and Holden et al., Nat Chem Biol.; 2 (6): 314-8)]. Natural pores and channels can include structures where the membrane-spanning portion of the protein comprises a beta-barrel, such as alpha-hemolysin (eg Song et al., Science. 1996; 274 (5294): 1859-66), OmpG (eg Chen et al., Proc Natl Acad Sei USA. 2008; 105 (17): 6272-7), OmpF (eg Schmitt et al., Biophys J. 2006; 91 (6): 2163 -71) or MsPA (e.g. Butler et al., Proc Natl Acad Sei US A. 2008; 105 (52): 20647-52). Alternatively, the membrane-spanning portion of the protein may consist of an alpha helix, such as a potassium channel (eg Holden et al., Nat Chem Biol.; 2 (6): 314-8), (Syeda et al., J Am Chem Soc. 2008; 130 (46): 15543-8)]. The pore or channel can be a naturally occurring protein that is modified either chemically or genetically to provide desired nanopore behavior. As an example of a pore of chemically modified protein it is given in WO 01/59453 and an example of a pore of genetically modified protein is given in WO 99/05167. Adapters can also be added to the system to provide greater control and more detection of targeted analyte, examples of which are disclosed in US 6,426,231, US 6,927,070; and WO 2009/044170.
The nanopores allow a flow of ions to travel through the amphiphilic membrane 26. The flow of ions is modulated by pore based on the interaction of analyte, thus allowing the nanopore to provide a biochemical analysis. There are many examples of such modulation being used as the basis for biochemical analysis, for example in US 6,426,231; US 6,927,070; US 6,426,231; US 6,927,070; WO 99/05167; WO 03/095669; WO 2007/057668; WO 1997020203; Clarke et al. Nat Nanotechnol. 2009; 4 (4): 265-270; and Stoddart et al., Proc Natl Acad Sei USA. 2009; 106 (19): 7702-7707.
The analyzer 13 can use nanopores for sequencing polynucleotides, including DNA and RNA, and including synthetic and naturally occurring polynucleotides. It can apply a variety of techniques that have been proposed to derive sequence information in a fast and cost effective manner, typically using measurement of changes in the electrical signal through a single nanopore as a single strand of DNA passes through the nanopore. Such techniques include, without limitation:
nanoporous assisted sequencing by hydrolysis; filament sequencing; and exonuclease nanopore sequencing (eg D.Branton et al, Nature Biotechnology 26 (10), i -8 (2009)). The technique may involve the polynucleotide passing through the nanopore as an intact (modified or unmodified) polymer, or broken down into the constituent nucleotide components or bases (for example using the techniques disclosed in: US-5,795,782; EP-1,956,367 ; US-6,015,714; US7,189,503; US-6,627,067; EP-1. 192,453; WO-89/03432; US-4,962,037; WO-2007/057668; International Application No. PCT / GB09 / 001690 (corresponding to British Order No. 0812693.0 and US Order No. 61/078687); and International Order No. PCT / GB09 / 001679 (corresponding to British Order No. 0812697. US Order No. 61/078695).
in general, the present invention can be applied to any apparatus providing the measurement of the nanopores by providing two electrodes, either one side of an insulating membrane, into which a nanopore is inserted. When immersed in an ionic solution, a potential bias between the electrodes will trigger ionic flow through the nanopore that can be measured as current in an external electrical circuit. This current changes as DNA passes through the nanopore, and with sufficient resolution, the constituent bases can be recognized from the changes, for example as disclosed in Clarke et al. Nat Nanotechnol. 2009; 4 (4): 265-270; International Order No. PCT / GB09 / 001690 (corresponding to British Order No. 0812693.0 and U.S. Order No. 61/078687); and D. Stoddart, PNAS doi 10.1073 / pnas.0901054106, April 10, 2009.
In addition, the present invention can be applied to any apparatus in which sets of nanopores measure the same sample by individually providing addressable electrodes on one side of each nanopore in the set connected to either a common electrode or an equivalent number of addressable electrodes in the sample on the other side . External circuitry can then perform DNA measurements passing through each and every nanopore in the array without synchronizing the addition of base to each nanopore in the array, eg each nanopore is free to process a single DNA strand independent of the whole another, for example as disclosed in US-2009/0167288; WO-2009/077734; and US Order No. 61 / 170,729. Having processed a filament, each nanopore is then also free to begin processing a subsequent filament.
An advantage of nanopore based analysis is that the measurement quality does not change over time for a fully functioning nanopore, eg the accuracy of base identification is the same at the beginning of the sequencing as at any point in the future, subject to expected experimental limitations. This allows each sensor to perform, at constant average quality, multiple analyzes in a sequential manner on the same sample or multiple samples over time.
In addition to polynucleotide sequencing, nanopores can be applied to a range other than other biochemical analyzes, including without limitation: diagnostics (eg Howorka et al., Nat Biotechnol. 2001; 19 (7): 636-9); protein detection eg Cheley et al., Chembiochem. 2006; 7 (12): 1923-7; and Shim et al., J Phys Chem B. 2008; 112 (28): 8354-60); drug molecule analysis (eg Kang et al., J Am Chem Soc. 2006; 128 (33): 10684-5); ion channel screening (eg Syeda et al., J Am Chem Soc. 2008 Nov 19; 130 (46): 15543-8), defense (eg Wu et al., J Am Chem Soc. 2008; 130 (21): 6813-9; and Guan et al., Chembiochem. 2005; 6 (10): 187581); and polymers (eg Gu et al., Biophys. J. 2000; 79, 1967-1975; Movileanu et al., Biophys. J. 2005; 89, 1030-1045; and Maglia et al., Proc Natl Acad Sei USA. USA 2008; 105, 19720-19725).
The present invention can also be applied to an analysis apparatus in which nanopores are supplied in solid-state membranes. In this case, the nanopore is a physical pore in a membrane formed from a solid material. Such membranes have many advantages over fluid or semiconductor layers, particularly with respect to stability and size. The concept was proposed by researchers at Harvard University for examining polymers, such as DNA (eg WO 00/79257; and WO 00/78668).
Once the work has expanded to include the following techniques that can be applied to the present invention: manufacturing methods (eg WO-03/003446; US-7,258,838; WO-2005/000732; WO2004 / 077503; WO- 2005/035437; WO-2005/061373); data acquisition and evaluation (eg WO-01/59684; WO-03/000920; WO-2005/017025; and WO2009 / 045472), incorporation of nanotubes (eg WO-2005/000739; WO2005 / 124888; WO-2007/084163); and the addition of molecular motors (e.g.
WO 2006/028508); the use of field-effect transistors or similar embedded within nanopore structures (eg US 6,413,792, US 7,001,792); the detection of fluorescent probes interacting with a nanopore or nanochannel (eg US 6,355,420; WO 98/35012); and lighting and detection of fluorescent probes being removed from their target substrates as they translocate a nanopore (eg US 2009/0029477). Even the use of mass spectrometry can be employed in the analysis apparatus, for example as a polymer of interest passes through a nanopore or channel and whose monomers are then cleaved and ionized sequentially analyzed using mass spectrometry.
The present invention can also be applied to an analysis apparatus that is arranged to perform polynucleotide sequencing using techniques other than nanopores, for example: using gradual cyclic chemistry, followed by an image formation stage to detect incorporation, annealing or removing chemically labeled fluorescent probes that allow the polynucleotide under study to be decoded; techniques that measure the activity of enzymes that fuse with DNA in real time, including the measurement of DNA polymerase activity in zero-mode waveguides (eg Levene et al., Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations, Science 299: 682-686; Eid et al., Real-Time DNA Sequencing from Single Polymerase Molecules, Science 323: 133-138; US-7,170,050; US-7,476,503); techniques that measure emissions of energy provided by the transfer of fluorescent emission between groups of suitable chemicals supplied in both bases of embedded DNA and polymerase (eg US 7,329,492), for example using activated quantum dots connecting to polymerases acting on DNA in which DNA bases are incorporated into a new synthesized strand containing fluorescent groups that are energized in the presence of such activated quantum dots; or techniques that use ion-sensitive FET’s to measure local changes in ions (eg pH) to infer chemical activity as DNA bases are incorporated into a new strand (eg WO 2008/076406).
The present invention can also be applied to an analysis apparatus that is arranged to perform other types of biochemical analysis other than a nanopore, some examples of which are as follows.
The present invention can also be applied to an analysis apparatus that is arranged to perform other types of biochemical analysis that does not use nanopores, including, but not limited to:
1) Ion channel screening;
2) DNA amplification in real time (PCR, RCA,
NASBA);
3) Enzyme activity by measuring reagent or product change, including
The. Glucose oxidase,
B. G-coupled protein receptors;
ç. Fluorescent protein gene activation;
4) Monitored Surface Plasma Resonance reactions, including kinetic binding of ligands to target molecules (eg proteins for chemical inhibitors);
5) DNA micro-sets for transcriptome analysis or identification of infectious disease;
6) Chips of antibody set to measure proteins in samples or solutions; or
7) Protein binding set chips monitoring kinetics of protein interactions with substrates, targets, ligands, etc. using electromagnetic or fluorescent readings.
In each case a variety of experimental parameters can be varied to meet the user's overall requirements for the experiment, including temperature, experiment time, reading sample rates, light intensity or degree of electrical potential, pH or ionic strength .
The analysis can be a biological or chemical assay, and can be used to carry out biomarker validation studies, clinical tests and high-throughput screening. These tests may involve performing chromatography (HPLC (high performance liquid chromatography), TLC (thin layer chromatography), FPLC (fast protein liquid chromatography, flash chromatography, with detection of analyte in the liquid eluent (by absorbance, fluorescence, radiometric methods , light scattering, particle analysis, mass spectrometry), or an immunoassay or using direct mass spectrometry (MALDI (matrix-assisted laser desorption ionization), APCI (atmospheric pressure chemical ionization), ESI (electrospray ionization) ) Quadrupole ionization (single and multiple), flight time, ion trap detection. Immunoassays include an ELISA (enzyme-linked immunosorbent assay), lateral flow assay, radioimmunoassay, magnetic immunoassay or immunofluorescent assay.
These tests and trials can be used in the context of: identification of fetal abnormalities such as Down's Syndrome, wide genome association studies, pharmacokinetic and pharmacodynamic investigations in all tissues and animals, drug testing in sport, testing for microorganisms in environmental matrices (sewage, polluted water, etc.), testing for hormones and growth factors in treated water and so on.
The analysis can be applied to biomarker validation studies. The present invention can allow very high numbers of samples to be analyzed quickly and easily. For example, the current process of biomarker discovery is hampered by the validation step, eg once a candidate marker has been found, a large number of samples must be examined in order to statistically confirm their altered levels in the tissues of interest. An assay must, however, be developed for each marker. The system of the present invention has a single reading for all analytes, for example DNA, RNA, protein or small molecule, reducing the stages of assay development.
The analysis should be applied to clinical tests and ELISA substitute. When a sample is subjected to testing at a hospital or clinic, the test procedure is very likely to involve either mass spectrometry or ELISA. Both of these can be supplanted by the system of the present invention. Development of suitable tests in the system of the invention will give great increase in the yield and savings in the time of sample preparation and handling. This will apply to large proteins so that growth factors, peptides such as insulin, or small molecules such as drugs of abuse or prescription drugs.
The analysis can be applied to high-throughput screening. Any quantitative screening can be performed on the system of the present invention. Thus, if an assay (for example a protease assay) that gives a peptide or smaller molecule as a product is currently used in high throughput screening, the present invention can increase throughput and reduce sample handling and preparation time.

权利要求:
Claims (32)
[1]
1. Analysis instrument to perform biochemical analysis, characterized by the fact that it comprises several modules, each module comprising an analysis device that is operable to perform biochemical analysis of a sample, the module being arranged to produce output data from at least one channel representing the results of the biochemical analysis, the operation of the module being controllable in a way that varies its performance, the analysis instrument still comprises a control system that is arranged to accept input by selecting an arbitrary number of modules as a cluster to perform a common biochemical analysis and to accept input representing global performance targets with respect to common biochemical analysis, the control system being arranged to control the operation of the cluster modules to perform the common biochemical analysis, where the control system is arranged to determine at least once during the performance of the biochemical analysis common, performance measurement of each module from output data produced by the modules, and the control system is arranged to vary the control of the operation of the modules in the cluster based on the determined measurements of the performance of all modules and overall performance targets, and / or arranged (b) to take remedial action in response to global performance targets not being achieved based on determined measurements of the performance of all modules.
[2]
2. Analysis instrument, according to claim 1, characterized by the fact that the modules are capable of connecting to a data network to allow connection together over the network.
[3]
3. Analysis instrument, according to claim 2, characterized by the fact that the control system comprises a control unit in each module that is operable to control the operation of the module.
[4]
4. Analysis instrument, according to claim 3, characterized by the fact that the control units are arranged to present on the data network of a computer connected to it, a user interface allowing a user to address the units of control to provide this input by selecting an arbitrary number of modules as a cluster and to provide this input representing global performance targets.
[5]
5. Analysis instrument, according to claim 3, characterized by the fact that the control system is arranged to determine local performance targets for each module based on the global performance targets and the control unit in each module is arranged to control the operation of that module based on its overall performance target.
[6]
6. Analysis instrument, according to claim 5, characterized by the fact that each control unit is arranged to vary the control of the module's operation based on the performance measurements determined for that module to find its local performance target.
[7]
7. Analysis instrument, according to claim 6, characterized by the fact that the respective control units of the modules of the cluster are arranged to determine said performance measurements with respect to their modules from output data produced by their respective modules.
[8]
8. Analysis instrument, according to claim 5, characterized by the fact that the control system is arranged to vary the local performance targets based on the determined performance measurements and the global performance targets, in order to vary the control of the operation of the cluster modules.
[9]
9. Analysis instrument, according to claim 8, characterized by the fact that the respective control units of the cluster modules are arranged to determine said performance measurements with respect to their respective module from the output data produced by their respective module, and to communicate these performance measurements over the data network.
[10]
10. Analysis instrument, according to claim 9, characterized by the fact that the control unit of at least one of the modules of the cluster is arranged to vary the local performance targets based on the determined measurements of the reported performance over the network of data.
[11]
11. Analysis instrument, according to claim 10, characterized by the fact that the control units of all modules of the cluster are arranged to cooperate to vary the local performance targets based on the determined measurements of performance reported on the network of data.
[12]
12. Analysis instrument, according to claim 11, characterized by the fact that the control units of the cluster modules are arranged to cooperate on a non-hierarchical basis.
[13]
13. Analysis instrument, according to claim 11, characterized by the fact that the control unit of each module in the cluster is arranged to vary the local performance target of the respective module, based on the performance measurements of all modules communicated over the data network from the other control units of the cluster modules.
[14]
14. Analysis instrument, according to claim 10, characterized by the fact that the control unit of one or a subset of modules is arranged to vary the local performance targets of all modules in the cluster, based on said measurements the performance of all modules communicated over the data network from the control units of the other modules, said one or said subset of the control units being arranged to communicate the varied local performance targets to the control units of the other modules in the cluster .
[15]
15. Analysis instrument, according to claim 10, characterized by the fact that the control system also comprises an association control unit connected to the network, the association control unit being arranged to vary the local performance targets of all modules in the cluster, based on said performance measurements of all modules communicated over the data network from the module control units, the association control unit being arranged to communicate varying local performance targets to the respective control units, the respective control units being arranged to vary the control of the operation of the modules of the cluster in response to it.
[16]
16. Analysis instrument according to any one of claims 1 to 15, characterized by the fact that measurements of performance and overall performance targets represent at least one of:
the time within which output data is produced; the amount of output data that is produced; or the quality of the output data that is produced.
[17]
17. Analysis instrument according to any one of claims 1 to 15, characterized by the fact that the control system is arranged to determine performance measurements of each module from the output data produced by the modules repeatedly during the biochemical analysis common.
[18]
18. Analysis instrument according to any of claims 1 to 15, characterized by the fact that the control system is both arranged (a) to vary the control of the operation of the modules of the chipboard based on the determined measurements of the performance of all modules and overall performance targets, and arranged (b) to take remedial action in response to global performance targets not being achievable based on determined measurements of the performance of all modules.
[19]
19. Analysis instrument, according to claim 18, characterized by the fact that said remedial action is to increase the number of modules performing this common biochemical analysis.
[20]
20. Analysis instrument, according to claim 19, characterized by the fact that said remedial action includes producing output to indicate to a user that modules are still required to perform this common biochemical analysis.
[21]
21. Analysis instrument according to any one of claims 1 to 15, characterized by the fact that biochemical analysis is the analysis of a molecule in the sample.
[22]
22. Analysis instrument according to claim 21, characterized by the fact that the molecule is a polymer.
[23]
23. Analysis instrument according to claim 22, characterized by the fact that the polymer is a polynucleotide.
[24]
24. Analysis instrument, according to claim 21, characterized by the fact that biochemical analysis is the sequencing of the polynucleotide, the output data including output sequence data representing a polynucleotide sequence.
[25]
25. Analysis instrument according to any one of claims 1 to 15, characterized by the fact that the analysis apparatus is capable of supporting several nanopores and is operable to perform biochemical analysis of a sample using the nanopores.
[26]
26. Analysis instrument according to claim 25, characterized by the fact that the analysis apparatus comprises electrodes arranged to generate an electrical signal through each nano-pore, the module comprising a signal processing circuit arranged to generate said data of output from electrical signals generated from said electrodes.
[27]
27. Analysis instrument, according to claim 26, characterized by the fact that the control system is arranged to vary the operation of the modules of the cluster varying at least one of:
the temperature of the analysis apparatus; electrical parameters of biochemical analysis; fluid parameters of the analysis apparatus; or sampling characteristics of the output data.
[28]
28. Analysis instrument, according to claim 25, characterized by the fact that the signal processing circuit is arranged to detect events occurring in the nanopores from electrical signals through each nanopore, and to generate output data representing those events.
[29]
29. Module to perform biochemical analysis, characterized by the fact that it is able to connect to other modules over a data network, the module comprising an analysis device that is operable to perform biochemical analysis of a sample, the module being arranged for produce output data from at least one channel representing the results of the biochemical analysis, the operation of the module being controllable in a way that varies its performance, the module comprising an operable control unit to control the operation of the module, the control unit being addressed over the data network to provide input by selecting the module as one of an arbitrary number of modules as a cluster to perform a common biochemical analysis and to provide input representing global performance targets with respect to the common biochemical analysis, the control unit being arranged in response to it to control the operation of the module to perform the common biochemical analysis m, the control unit being arranged to determine, at least once during the performance of the common biochemical analysis, measurements of the performance of the module from output data produced by the module and to communicate said performance measurements over the data network, the control unit being arranged to communicate over the data network with the control units of the other modules in the cluster in order to (a) vary the control of the operation of the modules in the cluster based on the determined measurements of the performance of all modules and the global performance targets, and / or (b) taking remedial action in response to the global performance targets not being achievable based on the determined performance measurements of all modules.
[30]
30. Module according to claim 29, characterized in that the control unit is arranged to receive performance measurements from other modules in the cluster communicated over the network, the control unit is arranged to determine and vary a performance target location for the module based on global performance targets and based on the module's performance measurements determined by the control unit and the performance measurements received from other modules in the cluster, and the control unit is arranged to vary the operation control of the module based on the performance measurements of the modules determined by the control unit to find the determined local performance target.
[31]
31. Method for operating an analysis instrument to perform biochemical analysis, the instrument comprising several modules, each module comprising an analysis apparatus that is operable to perform biochemical analysis of a sample, the module being arranged to produce output data from at least a channel representing the results of the biochemical analysis, the operation of the module being controllable in a way that varies its performance, the method, characterized by the fact that it comprises: selecting an arbitrary number of modules as a cluster to perform a common biochemical analysis;
enter global performance targets with respect to common biochemical analysis;
control the operation of the cluster modules to perform the common biochemical analysis; and determine at least once during the performance of the common biochemical analysis, performance measurements of each module from output data produced by the modules, and either or both of (a) varying the operation control of the cluster modules based on the determined measurements of the performance of all modules and the overall performance targets, and / or (b) take remedial action in response to the overall performance targets not being achievable based on the determined measurements of the performance of all modules.
[32]
32. Method according to claim 31, characterized in that the biochemical analysis is sequencing of a polynucleotide and in which the method is carried out until a defined number of bases has been sequenced or until a particular sequence is detected.
1/15 ΒΒ- ⁵
.2·
3/15

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法律状态:
2018-07-10| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2018-09-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/12/2010, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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