Reagent channel mixing system and method

Abstract

This disclosure relates to a reagent channel mixing system and method. An analytical system can operate on an analyte, which can be combined with a variety of reagents before being introduced into a flow cell. The instrument may include a volume into which the reagents to be combined with the analyte are aspirated one by one. The volume may be formed as a serpentine channel in a valve manifold associated with a pipette used for aspirating the reagents. The reagents can then be mixed by circulating a pump to move the reagents within the mixing volume or channel. For this purpose, the reagents can be aspirated from a receiver into the volume or channel, sprayed back into the receiver, and this process can be repeated to enhance mixing.

Description

[0001] This application is a divisional application of the application filed on December 21, 2017, with application number 201780038327.X, entitled "Reagent Channel Mixing System and Method".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Patent Application No. 15 / 841,102, filed December 13, 2017; to UK (GB) Patent Application No. 1704758.0, filed March 24, 2017; and to U.S. Patent Application No. 62 / 442,772, filed January 5, 2017, which claims priority to U.S. Patent Application No. 62 / 442,772, all of which are incorporated herein by reference in their entirety. Background Technology

[0004] Instruments have been developed and continue to evolve for sequencing molecules of interest (specifically, DNA, RNA, and other biological samples). Prior to sequencing, a sample of the molecules of interest is prepared to form a library or template. This library or template is mixed with reagents and ultimately introduced into a flow cell, where individual molecules attach to the sequencing sites and are amplified to enhance detectability. The sequencing operation then involves a cycle of repeating the following steps: binding molecules at the sites, labeling the bound components, imaging the components at the sites, and processing the resulting image data.

[0005] In such sequencing systems, a fluid system (or subsystem) provides the flow of matter (e.g., reagents) under the control of a control system, such as a programmed computer and appropriate interfaces.

[0006] Overview

[0007] Details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims.

[0008] In some embodiments, a system may be provided including a flow path to be fluidly connected to a flow cell to support a flow path of an analyte of interest in an analytical system. The system may further include: a selector valve fluidly coupled to the flow path to select among a plurality of reagent pipettes for analytical operation; a bypass line separate from the flow path, a portion of which serves as the mixing volume of the bypass line; a pump fluidly connected to the bypass line and displacing fluid via the bypass line during reagent premixing operations; and control circuitry operatively coupled to the selector valve and the pump, the control circuitry having one or more processors and a memory storing or to store machine-executable instructions, which are processed by the one or more processors. When multiple processors execute, they control one or more processors to: a) cause the selector valve to select among a set of reagent pipettes associated with the reagent to be premixed; b) cause the pump to draw fluid from each of the selected reagent pipettes and deliver the fluid drawn from the corresponding selected reagent pipette to the mixing volume one by one; c) cause the pump to dispense the drawn fluid from the mixing volume generated in (b) via the destination receiver pipette; and d) cause the pump to cyclically move the drawn fluid into and out of the mixing volume and through the destination receiver pipette to further mix the fluid.

[0009] In some of these embodiments, the mixing volume may include a serpentine channel or be in the form of a serpentine channel.

[0010] In some embodiments of the system, the system may further include a destination receiver positioned to receive fluid ejected via a destination receiver straw; the destination receiver may contain DNA to be sequenced.

[0011] In some implementations of this system, the pump may be an injection pump or include an injection pump.

[0012] In some embodiments of the system, the selector valve allows the pump to draw air into the mixing volume before delivering the fluid from the selected reagent pipette into the mixing volume.

[0013] In some embodiments of the system, the memory may store or will store additional machine-executable instructions that, when executed by the one or more processors, further control the one or more processors to cause the pump to cyclically move the pumped fluid into and out of the mixing volume, while maintaining a certain volume of air trapped between the pump and the fluid.

[0014] In some embodiments of the system, the memory may store or will store instructions executable by another machine, which, when executed by the one or more processors, further control the one or more processors to repeat (b) one or more times before performing (c) or (d) for the analysis operation.

[0015] In some of these embodiments of the system, the memory may store or will store additional machine-executable instructions that, when executed by the one or more processors, further control the one or more processors to: (e) cause the selector valve to select an additional reagent pipette associated with the additional reagent, (f) cause the pump to draw fluid from the additional reagent pipette, and (g) cause the pump to deliver the drawn additional fluid from the additional reagent pipette into the mixing volume, wherein the additional reagent pipette is not in the set of reagent pipettes from (a) and wherein (e) to (g) are performed between (b) and (c).

[0016] In some embodiments of the system, the system may further include a reagent receiver containing at least three reagents of different specific gravities to be premixed.

[0017] In some embodiments, a method may be provided comprising: (a) performing a reagent premixing operation for two or more reagents; (b) actuating the pump to cyclically move the aspirated reagent into and out of the mixing volume to mix the reagents; and (c) actuating the pump to spray the mixed reagents into a destination receiver. The reagent premixing operation in (a) may include: commanding a selector valve to select a first reagent; actuating the pump to aspirate a portion of the first reagent from a first receiver into the mixing volume; commanding the selector valve to select a second reagent; and actuating the pump to aspirate a portion of the second reagent from a second receiver into the mixing volume.

[0018] In some embodiments of the method, the mixing volume may include or may be a serpentine channel.

[0019] In some implementations of this method, the destination receiver may contain the DNA to be sequenced.

[0020] In some embodiments of the method, the reagent premixing operation of (a) may further include commanding the selector valve to select a third reagent and actuating the pump to draw a portion of the third reagent from the third receiver into the mixing volume.

[0021] In some embodiments of the method, the reagent premixing operation in (a) may further include selecting and aspirating at least one of the reagents more than once before performing (b). In some embodiments of the method, the premixing operation in (a) may be repeated once or more before performing (b).

[0022] In some embodiments of the method, the method may further include drawing air into the mixing volume before drawing the first reagent.

[0023] In some embodiments, a method may be provided comprising: actuating a pump to draw gas into a mixing volume; controlling a selector valve to select a plurality of liquid reagents for an analytical operation; for each selected reagent, actuating the pump to draw the selected reagent from a corresponding receiver containing the selected reagent into a serpentine mixing volume; controlling the selector valve to fluidly connect the serpentine mixing volume to a destination receiver; circulating the pump to move the reagent back and forth between the serpentine mixing volume and the destination receiver to mix the reagent, wherein the destination receiver contains an analyte of interest to be analyzed in the analytical operation; and actuating the pump to spray the mixed reagent into the destination receiver.

[0024] In some embodiments of this method, the method may further include selecting and aspirating each reagent more than once before circulating the pump to move the reagent.

[0025] In some embodiments of this method, the pump can be used to circulate the reagent while a volume of air exists between the pump and the reagent.

[0026] In some embodiments of this method, the reagent may include at least three reagents with different specific gravities.

[0027] Details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims. It should be noted that the relative dimensions in the following figures are not drawn to scale. Brief description of the attached diagram

[0028] These and other features, aspects, and advantages of the present invention will become more readily understood when the following embodiments are read with reference to the accompanying drawings, wherein like characters throughout the drawings denote like parts, in which:

[0029] Figure 1 A graphical overview of an example sequencing system in which the revealed technology can be used;

[0030] Figure 2 for Figure 1A graphical overview of a sequencing system and a fluid system;

[0031] Figure 3 for Figure 1 A graphical overview of the processing and control system of an example sequencing system;

[0032] Figure 4 A perspective view of an example of a reagent manifold with a selector valve;

[0033] Figure 5 for Figure 4 A top view of an example manifold and valve configuration;

[0034] Figure 6A A graphical view configured for an instance of aspirating and mixing reagents and sample templates, while Figure 6B One example demonstrates how to add stripes to the reagents and sample template before mixing;

[0035] Figure 7 A graphical view of an example of how reagents to be mixed can be individually aspirated into a mixing volume;

[0036] Figure 8 A graphic segment illustrating an example destination receiver vessel for a mixed reagent and sample template, using a nozzle pipette to spray the mixed reagent into the receiver;

[0037] Figures 9A to 9D Examples of nozzle pipettes that can be used to mix reagents;

[0038] Figure 10 A graphical representation of an example cycle of aspirating and mixing reagents and sample templates; and

[0039] Figure 11 A flowchart illustrating the example logic used for aspirating and mixing reagents and sample templates. Detailed Implementation

[0040] Figure 1 This describes an embodiment of a sequencing system 10 configured to process molecular samples, which can be sequenced to determine their components, component ordering, and the general structure of the sample. The system includes an instrument 12 that receives and processes biological samples. A sample source 14 provides a sample 16 that, in many cases, will include tissue samples. For example, the sample source may include a person or individual, such as a human, animal, microorganism, plant, or other donor (including environmental samples), or any other individual containing the organic molecule of interest whose sequence will be determined. The system can be used with samples other than those obtained from organisms, including synthetic molecules. In many cases, the molecule will include DNA, RNA, or other molecules with base pairs whose sequence may define genes and variants with a specific function of ultimate interest.

[0041] Sample 16 is introduced into sample / library preparation system 18. This system can isolate, split, and otherwise prepare the sample for analysis. The resulting library includes molecules of interest of length that facilitate the sequencing operation. The resulting library is then provided to instrument 12, where the sequencing operation is performed. In practice, libraries, sometimes referred to as templates, are combined with reagents in an automated or semi-automated process and then introduced into a flow cell prior to sequencing. In some such embodiments, the library may be premixed with reagents before being transferred to the flow cell; for example, the library may be transferred via a selector valve system such as described below and mixed with reagents in the destination receiver before being transferred to the flow cell.

[0042] exist Figure 1 In the embodiments described herein, the instrument includes a flow cell or array 20 for receiving a sample library. The flow cell includes one or more fluid channels that allow sequencing chemistry reactions to occur, including the attachment of molecules from the library and amplification at sites or locations detectable during sequencing operations. For example, the flow cell / array 20 may include sequencing templates fixed at sites or locations on one or more surfaces. A “flow cell” may include patterned arrays, such as microarrays, nanoarrays, etc. In practice, sites or locations may be arranged in regular repeating patterns, complex non-repetitive patterns, or randomly configured on one or more surfaces of a support. To enable sequencing chemistry reactions to occur, the flow cell also allows the introduction of substances for reactions, rinsing, etc., such as various reagents, buffers, and other reaction media. Substances flow through the flow cell and may come into contact with molecules of interest at individual sites.

[0043] In the instrument, flow cell 20 is mounted on a movable stage 22, which in this embodiment is movable in one or more directions as indicated by reference numeral 24. For example, flow cell 20 may be provided as a removable and replaceable cartridge that can dock with ports on the movable stage 22 or other components of the system to allow delivery of reagents and other fluids to or from flow cell 20. The stage is associated with an optical detection system 26 that directs radiation or light 28 to the flow cell during sequencing. The optical detection system may use various methods (such as fluorescence microscopy) to detect analytes positioned at sites in the flow cell. As a non-limiting example, optical detection system 26 may use confocal column scanning to generate progressively pixelated image data, which can be analyzed to locate individual sites in the flow cell and determine the type of nucleotides most recently attached or bound to each site. Other suitable imaging techniques may also be used (such as techniques that scan one or more radiation points along the sample), or techniques using a "step and shoot" imaging method. The optical inspection system 26 and the stage 22 can cooperate to maintain a static relationship between the flow cell and the inspection system while acquiring a regional image (or, as mentioned, scanning the flow cell in any suitable mode (e.g., point scan, column scan, "step and shoot" scan)).

[0044] While many different techniques can be used for imaging or, more generally, for detecting molecules at sites, the embodiments currently covered utilize confocal optical imaging at the wavelength that induces excited fluorescent labeling. The label, excited by its absorption spectrum, transmits a fluorescent signal by its emission spectrum. An optical detection system 26 is configured to capture these signals in order to process pixelated image data at a resolution that allows for analysis of the signal emission sites, and to process and store the resulting image data (or data derived therefrom).

[0045] In sequencing operations, cycling or processes are performed automatically or semi-automatically, promoting reactions (e.g., by using mononucleotides or oligonucleotides), followed by rinsing, imaging, and unpacking in preparation for subsequent cycles. The sample library, prepared for sequencing and immobilized on the flow cell, can undergo several such cycles before all useful information is extracted from the library. An optical detection system generates image data by scanning the flow cell (and its sites) during each cycle of the sequencing operation using electronic detection circuitry (e.g., a camera or imaging electronics or chip). The resulting image data can then be analyzed to locate individual sites within the image data and to analyze and characterize the molecules present at the sites, such as by referring to the specific color or wavelength of light detected at a particular site (characteristic emission spectra of specific fluorescent labels), as indicated by groups or clusters of pixels in the image data at that site. For example, in DNA or RNA sequencing applications, four common nucleotides can be represented by distinguishable fluorescence emission spectra (wavelengths or wavelength ranges of light). Each emission spectrum can then be assigned a value corresponding to that nucleotide. Based on this analysis, and by tracking the cycle value determined for each point, individual nucleotides and their order can be determined for each point. These sequences can then be further processed to assemble longer fragments, including genes, chromosomes, etc. As used in this invention, the terms "automatic" and "semi-automatic" mean that once the operation begins, or once the process including the operation begins, the operation is performed by a system program or configuration with little or no human interaction.

[0046] In the illustrated embodiment, reagent 30 is drawn or aspirated into the flow cell via valve 32. The valve may be located via, for example, a pipette or tube. Figure 1 (Not shown) Reagents are obtained from the receiver or vessel where they are stored. Valve 32 allows selection of reagents based on a specified sequence of operations performed. The valve may further receive a command to direct the reagents into the flow cell 20 via flow path 34. The used reagents are directed out of or through flow path 36 from the flow cell. In the illustrated embodiment, pump 38 is used to move reagents through the system. The pump may also provide other useful functions, such as measuring reagents or other fluids passing through the system, drawing air or other fluids, etc. An additional valve 40 downstream of pump 38 allows the used reagents to be properly directed to the disposal vessel or receiver 42.

[0047] The instrument further includes a series of circuits that help command the operation of various system components, monitor the operation of components via feedback from sensors, collect image data, and at least partially process the image data. Figure 1In the implementation described herein, the control / monitoring system 44 includes a control system 46 and a data acquisition and analysis system 48. Both systems will include one or more processors (e.g., digital processing circuitry, such as microprocessors, multi-core processors, FPGAs, or any other suitable processing circuitry) and associated memory circuitry 50 (e.g., solid-state memory devices, dynamic memory devices, onboard and / or offboard memory devices, etc.), which can store machine-executable instructions for controlling, for example, one or more computers, processors, or other similar logic devices to provide a certain functionality. A dedicated or general-purpose computer may at least partially constitute the control system and the data acquisition and analysis system. The control system may include circuitry configured (e.g., programmed) to process commands for any other useful functions related to fluid dynamics, optics, stage control, and instrumentation. The data acquisition and analysis system 48 interfaces with the optical inspection system to control the movement of the optical inspection system or stage, or both, the emission of light for cyclic inspection, the reception and processing of returned signals, etc. The instrument may also include various interfaces as indicated by reference numeral 52, such as operator interfaces that allow control and monitoring of the instrument, transfer of samples, initiation of automated or semi-automated sequencing operations, generation of reports, etc. Finally, Figure 1 In one implementation, an external network or system 54 may be coupled to and cooperate with the instrument, for example, for analysis, control, monitoring, servicing and other operations.

[0048] It should be noted that, although in Figure 1 The document describes a single flow cell and fluidic pathway, as well as a single optical detection system; however, some instruments may accommodate more than one flow cell and fluidic pathway. For example, in the currently covered embodiments, two such configurations are provided to enhance sequencing and throughput. In practice, any number of flow cells and pathways can be provided. These flow cells and pathways may utilize the same or different reagent containers, disposal containers, control systems, image analysis systems, etc. In the case of multiple fluidic systems, these systems may be controlled individually or in a coordinated manner. It should be understood that the term "fluidly connected" may be used herein to describe a connection between two or more components that fluidly communicates with each other, and "electrically connected" may be used to describe electrical connections between two or more components that are substantially identical. The term "fluidly inserted" may be used, for example, to describe a particular ordering of components. For example, if component B is fluidly inserted between component A and component C, the fluid flowing from component A to component C will flow through component B before reaching component C.

[0049] Figure 2 illustrate Figure 1An example fluid system for a sequencing system. In the illustrated embodiment, flow cell 20 includes a series of paths or pathways 56A and 56B, which can be grouped in pairs for receiving fluid substances (e.g., reagents, buffers, reaction media) during sequencing operations. Pathway 56A is coupled to a common line 58 (first common line), while path 56B is coupled to a second common line 60. A bypass line 62 is also provided to allow fluid to bypass the flow cell without entering it. As mentioned above, a series of vessels or receivers 64 allows for the storage of reagents and other fluids that can be used during sequencing operations. A reagent selector valve 66 is mechanically coupled to a motor or actuator (not shown) to allow selection of one or more reagents to be introduced into the flow cell. The selected reagent then proceeds to a common line selector valve 68, which similarly includes a motor (not shown). The common line selector valve can be commanded to select one or more of the common lines 58 and 60, or two of the common lines, to allow reagent 64 to flow in a controlled manner to passages 56A and / or 56B, or to select bypass line 62 to allow one or more of the reagents to flow through the bypass line. It should be noted that other useful operations can be achieved by the bypass line, such as the ability to fill all reagents (and liquids) into the reagent selector valve (and the common line selector valve) without drawing air through the flow cell, the ability to perform cleaning of reagent channels and pipettes independently of the flow cell (e.g., automatic or semi-automatic cleaning), and the ability to perform diagnostic functions on the system (e.g., pressure and volume delivery tests).

[0050] Used reagents exit the flow cell via a line coupled between the flow cell and pump 38. In the illustrated embodiment, the pump includes a syringe pump with a pair of syringes 70, which are controlled and moved by actuator 72 to aspirate and eject reagents and other fluids during different operations of the testing, validation, and sequencing cycles. The pump assembly may include various other parts and components (including valves, gauges, actuators, etc. (not shown)). In the illustrated embodiment, pressure sensors 74A and 74B sense the pressure on the pump inlet line, while pressure sensor 74C senses the pressure output from the syringe pump.

[0051] Fluid used by the system is pumped into the used reagent selector valve 76. This valve allows selection of one of several flow paths for used reagents and other fluids. In the illustrated embodiment, a first flow path leads to a first used reagent container 78, while a second flow path leads to a second used reagent container 82 via a flow meter 80. Depending on the reagents used, it may be advantageous to collect the reagents, or some of the reagents, in separate containers for disposal, and the used reagent selector valve 76 allows for this control.

[0052] It should be noted that the valves within the pump assembly allow various fluids (including reagents, solvents, cleaning agents, air, etc.) to be drawn by the pump and injected or circulated via one or more of a common line, bypass line, and flow tank. Furthermore, as mentioned above, in the currently covered embodiments, under common control... Figure 2 The diagram illustrates two parallel implementations of a fluid dynamics system. Each component of the fluid dynamics system can be part of a single sequencing instrument and can perform sequencing operations in parallel on different flow cells and sample libraries.

[0053] The fluid dynamics system operates under commands from a control system 46 that implements specified protocols for testing, validation, sequencing, etc. These specified protocols are pre-established and include a series of events or operations for the activity, such as aspirating reagents, aspirating air, aspirating other fluids, spraying such reagents, air, and fluids, etc. The protocols will allow coordination of such fluid operations with other instrument operations, such as reactions occurring in the flow cell, imaging of the flow cell and its sites, etc. In the illustrated embodiment, the control system 46 uses one or more valve interfaces 84 and a pump interface 86, the valve interfaces 84 being configured to provide command signals to valves, and the pump interface 86 being configured to command the operation of pump actuators. Various input / output circuits 88 may also be provided for receiving and processing feedback, such as from pressure sensors 74A to 74C and flow meters 80.

[0054] Figure 3 This describes certain functional components of the control / monitoring system 44. As described, the memory circuitry 50 stores specified routines executed during testing, debugging, fault handling, servicing, and sequencing operations. Many such protocols and routines can be implemented and stored in the memory circuitry, and these protocols and routines can be updated or changed from time to time. Figure 3 As described herein, these protocols and conventions may include fluid control protocol 90, which controls various valves, pumps, and any other fluid actuators, and receives and processes feedback from fluid sensors and flow and pressure sensors, such as valves. Stage control protocol 92 allows the flow cell to be moved as needed, such as during imaging. Optical component control protocol 94 allows commands to be issued to the imaging assembly to illuminate portions of the flow cell and to receive return signals for processing. Image acquisition and processing protocol 96 allows image data to be processed at least partially for the extraction of useful data for sequencing. Other protocols and conventions may be provided in the same or different memory circuitry as indicated by reference numeral 98. In practice, the memory circuitry may be provided as one or more memory devices, such as both volatile and non-volatile memory. This memory may be located within the instrument, and some memory may be off-board.

[0055] One or more processors 100 access the stored protocol and implement the protocol on the instrument. As mentioned above, the processing circuitry can be part of a dedicated computer, a general-purpose computer, or any suitable hardware, firmware, and software platform. The processors and the operation of the instrument can be controlled by a human operator via operator interface 101. The operator interface allows for testing, debugging, troubleshooting, and servicing, as well as reporting any problems that may occur in the instrument. The operator interface also allows for initiating and monitoring sequencing operations.

[0056] Figure 4 This describes a valve assembly for drawing reagents and other fluids from a receiver and delivering them to a flow cell. The valve assembly 102 includes a manifold structure 104, in which channels are formed to define flow paths for the reagents and other fluids. Figure 4 As can be seen, valves 66 and 68 are driven and controlled by motors 106 and 108. One or more motor interfaces or connectors 110 supply power and (if necessary) signals to and from the motors. As mentioned above, the motors (and thus the valves) are controlled by control circuitry during testing, commissioning, and servicing, as well as during sequencing operations.

[0057] The reagent and fluid pathway within the manifold is coupled to pipette 112, which, during operation, draws reagents and other fluids from the corresponding receiver (not shown). Typically, this is achieved by... Figure 4 The flow path of the reagents and fluids specified by reference numeral 114 may be formed by molding, etching, or any other suitable process to allow the reagents and fluids to move from the pipette to the valve when the pump described above is commanded to aspirate the reagents and fluids. At least one of the pipettes is configured as a nozzle pipette 116 to assist in mixing reagents during sequencing operations (e.g., prior to reaction and imaging). Figure 4 The diagram also describes a mixing volume configured as channel 118, in which reagents and fluids can be drawn and moved for mixing. In some embodiments, the mixing volume may be a portion or all of bypass line 62. For example, reagents may be drawn into bypass line 62 in the desired sequence, such that the reagents do not traverse the entire length of the bypass line (which could result in the reagents being delivered for disposal). Once the bypass line (or which serves as part of the mixing volume) has been loaded with the reagents of the desired sequence, a valve can be used to switch the end of the bypass line through which the reagents are introduced to connect fluidly to a flow path leading to, for example, a destination receiver, so that the entire set of reagents loaded into the bypass line can then be discharged from the bypass line and into the destination receiver. In other embodiments, the mixing volume may be, for example, a destination receiver (e.g., to which the premixed fluid will be delivered) or a separate destination receiver (e.g., a destination receiver completely emptied before delivery of the selected reagents).

[0058] Figure 5 This is a top view of valve assembly 102. Also here, valves 66 and 68 are visible in the manifold and coupled to the flow paths of reagents and fluids. Reagent selector valve 66 receives reagents via a self-priming tube and directs aspirated fluid to common line selector valve 68. Mixing passage 118 is coupled to the common line selector valve to allow mixing of reagents, as described below. Figure 5 The diagram also shows a port 120 disposed in the manifold to allow coupling of the manifold to a pipette. One of the ports 120 (indicated by reference numeral 122) is coupled to a nozzle pipette to allow injection of reagent into a destination receiver and to allow aspiration of reagent from the destination receiver for mixing. For example, the destination receiver may be a container, tube, or other vessel designed to contain reagents. For example, the destination receiver may serve as a temporary processing space to which reagents and / or other materials can be transferred, for example by mixing, to prepare reagents and / or other materials for delivery to a flow cell. Thus, reagents and other fluids can be prepared in the destination receiver and then transferred from the destination receiver to the flow cell.

[0059] Figure 6A The present embodiment describes the mixing channel 118 used for mixing and the reagent flow path. As mentioned above, the mixing channel 118 is coupled to a common line selector valve 68, which in turn is coupled to the outlet of a reagent selector valve 66. The mixing channel 118 is also coupled to a pump 38 to allow for the suction and ejection of reagents and fluids, as described below. Figure 6A In the embodiments described herein, reagent receivers or containers 124, 126, and 128 store reagents 130, 132, and 134, respectively. In this example, another destination receiver 136 stores a pre-prepared sample template or library 138. For mixing operations, reagents 130, 132, and 134 are pre-mixed and then combined with template 138. To allow such pre-mixing, via... Figure 6A The reagent is drawn into the mixing channel 118 one by one through the corresponding flow path indicated by reference numeral 140. Another flow path 142 allows the reagent, along with the template, to be deposited in the destination receiver 136. In the illustrated embodiment, the mixing channel 118 forms a serpentine internal volume with a loop 144, which allows the desired volume of reagent to be drawn and mixed in a relatively compact region of the manifold.

[0060] In the currently covered embodiments, reagents 130, 132, and 134 have different fluid properties that pose a challenge to mixing. For example, the reagents have different densities, and there can be substantial differences between their viscosity and oil interfacial tension. For instance, in the currently covered embodiments, the viscosity varies between approximately 1.5 cP and 50 cP (e.g., 2.4 cP at 25°C), while the oil interfacial tension varies between approximately 5.0 and approximately 19.2 dynes / cm. In contrast, the template can still have different densities and lower viscosity (e.g., approximately 1 cP at 25°C) and different oil interfacial tensions (e.g., approximately 9.8 dynes / cm). Figure 6B The diagram illustrates stripes of the reagents and template in destination receiver 136 when unmixed. In the illustrated embodiment, the template comprises approximately 30% of the total volume, while reagent 130 comprises approximately 22%, reagent 132 comprises approximately 42%, and reagent 134 comprises approximately 6%. In this context, the term "approximately" means that the indicated value is not precise, and the actual value may vary within the range of values ​​indicated in a manner that does not substantially alter the operation.

[0061] To enable automated mixing of reagents and templates, the fluid dynamics system and its controls allow reagents to be selectively drawn into mixing channels, injected into destination receivers, and cyclically drawn and reinjected for mixing. Figure 7 This describes the currently covered technique for reagent aspiration. As shown, reagents 130, 132, and 134 are aspirated one by one via the control of valve 66. With the reagents directed to the mixing channel via a common line selector valve, several volume groups of each reagent are aspirated (as indicated by reference numerals 146, 148, 150, 152, and 154). To reduce pressure spikes during mixing, the pump can also aspirate a certain volume of air before aspirating the reagents. The air volume provides a buffer to limit positive and negative pressure spikes during mixing. Figure 7 In the implementation scheme described herein, air is aspirated to the upper left of the reagent assembly. Additionally, a liquid buffer can be aspirated to aid in filling, rinsing, and displacing the reagents. Once... Figure 7 As described above, if aspiration is performed, the valve can then be controlled to allow the pump to inject the reagent into the template 138 that will be preloaded into the template receiver 136 described above. That is, one or both selector valves can be controlled to fluidly connect the mixing volume or mixing channel (e.g., a serpentine mixing channel) to the destination receiver.

[0062] In another technique, where three or more reagents can be selected for mixing in a destination receiver, at least two of the selected reagents for mixing are introduced sequentially and repeatedly into a mixing channel, while at least one other selected reagent is held in reserve until the reagents introduced sequentially into the mixing channel have been completely delivered to the mixing channel. The reserved reagent can then be added to the mixing channel all at once. For example, with ABCABCABCABCABC (which can be derived, for example, from similar to...) Figure 7 In contrast to the techniques described, if reagents A and B are repeatedly introduced into a mixing channel one after another, followed by the introduction of the retained reagent C, the reagents in the mixing channel will typically separate into ABABABABABC. This technique is considered advantageous in preventing or reducing the occurrence of undesirable reaction byproducts of some reagents. For example, the retained reagent may react with one of other reagents isolated in one particular manner, but may react with two or more of other reagents combined in another manner. The latter may be the desired reaction that may occur after the reagents have been thoroughly mixed, while the former may occur during premixing when the reagents can still be relatively separated and can only be mixed with directly adjacent reagents. In another example, the retained reagent may react with the material forming the structure of the mixing channel and produce undesirable byproducts. Since introducing reagents one by one into the mixing channel can take several minutes (e.g., 5 minutes, 10 minutes, 15 minutes, or longer depending on the number and quantity of each reagent required), reserving the introduction of potentially problematic reagents until other reagents have been delivered into the mixing channel significantly reduces the amount of time spent on the reserved reagents coming into contact with other reagents and the structure of the mixing channel, thereby reducing the likelihood of unwanted reaction byproducts. Of course, in these embodiments, the reserved reagents may not benefit from the premixing that other reagents benefit from, but the reduction in unwanted reaction byproducts may be more important than the lack of premixing of the reserved reagents. Specifically, if the reserved reagent is a low-viscosity liquid, the lack of premixing of the reserved reagents ultimately has almost no effect.

[0063] Using channel-like mixing volumes (e.g., volumes much longer than their width (e.g., at least 10x, 100x, 150x to 170x, 160x, 200x, or 500x the width)) allows sequentially delivered reagents to maintain a relatively stratified configuration relative to each other within the channel by reducing the interfacial contact interface area between each layer of reagent (the reagent is a liquid and will therefore likely diffuse across this boundary to some extent over time; therefore, the boundary / contact interface area mentioned herein should actually be understood theoretically; however, reducing such theoretical areas slows down the rate of diffusion). Additionally, for reagents that may be slightly immiscible with each other, mixing volumes shaped, for example, spheres or having a large width-to-length ratio, allow various reagent doses delivered to the mixing volume to float within the mixing volume and potentially recombine with earlier doses of the same reagent, thus losing the stratification achievable with channel-like mixing volumes. For example, a mixing channel with a diameter or width of approximately 2.25 mm and a length of approximately 360 mm can provide advantageous stratification of the delivered reagents during the premixing process. Once the mixing volume has been loaded with the required amounts of multiple reagents, the contents of the mixing volume can be delivered to the destination receiver (a portion of the fluid in the mixing volume may be lost to the dead volume of the fluid system; the total volume of reagent delivered to the mixing volume can be calibrated to account for this loss). After delivery to the destination receiver, the delivered premixed reagent can be repeatedly aspirated and ejected back into the destination receiver to facilitate further mixing. In some embodiments, the premixed (or pre-mixed) reagent can be aspirated from the destination receiver and returned to the mixing volume before being ejected back into the destination receiver. Thus, in such embodiments, the premixed reagent can repeatedly move in and out of the mixing volume during aspiration / ejection mixing operations.

[0064] It has been found that using a mixing channel with a nozzle pipette promotes vortexing in the destination receiver and provides excellent mixing of reagents and templates, even when the fluid properties of the reagents vary considerably. Furthermore, such structures and technologies enable automated mixing with little or no human interaction. Figure 8 and 9A to Figure 9C This describes an example nozzle suction pipe used in such techniques. For example... Figure 8 As shown, the nozzle suction tube has an elongated body and a tip 156 at its distal end, the elongated body having a central inner cavity (cavity) extending along its length. The nozzle is positioned at the tip to reduce the inner diameter of the suction tube at this location, thereby increasing the velocity of the fluid drawn in and ejected through the suction tube. In the illustrated embodiment, the nozzle is formed as an insert 158 ​​received in the distal end or tip of the suction tube. Other structures, such as caps, machined, formed, forged regions, etc., can form the nozzle.

[0065] In the illustrated embodiment, the nominal outer diameter 160 of the pipette is approximately 0.125 inches (3.175 mm), and the nominal inner diameter 162 is 0.020 inches ± 0.001 inches (0.508 mm). On the other hand, the nominal inner diameter 164 of the nozzle is 0.010 inches ± 0.001 inches (0.254 mm, although some embodiments may feature a nozzle inner diameter ranging from at most 0.20 to 0.28 mm). Of course, other sizes and dimensions can be used to provide the desired mixing. Furthermore, in the illustrated embodiment, the nozzle pipette 116 is positioned at a height 166 approximately 2 mm above the bottom of the receiver 138. When the reagent is injected into the receiver, as indicated by reference numeral 168, the mixing in the receiver is enhanced by increasing the velocity of the reagent moving through the nozzle, thereby increasing the vortex within the receiver. Figure 8 Arrow 170 indicates that the mixed reagent is allowed to rise in the receiver, as indicated by reference numeral 172.

[0066] Figure 9A The distal end of the nozzle straw is described in slightly more detail. As can be seen in the figure, the nominal inner diameter 162 of the straw is reduced by the nozzle insert 158, in this case to approximately half the inner diameter of the straw (in this example, the nozzle insert is tubular in shape). Figure 9B , Figure 9C and Figure 9D The current covered form of the distal end is best illustrated herein. As shown here, the nozzle straw has a faceted lower end comprising four facets 174, thereby giving the nozzle straw tip a wedge-shaped appearance. The straw has a centerline 176, and the facets meet at vertices 178 offset or eccentric relative to the centerline 176. This geometry of the distal end reduces or avoids dragging or scraping the receiver when the straw is lowered into the receiver or when the receiver is raised around the straw. However, it should be noted that in the illustrated embodiment, the insert has a lower profile (e.g., one or more angled facets) that matches the profile of the tip. In other words, the shape of the insert can conform to the faceted or wedge shape of the distal end of the nozzle straw. Furthermore, it should be noted that in the currently covered embodiment, the straw and nozzle are made of an engineering plastic such as polyetheretherketone (PEEK). Such materials provide chemical resistance to reagents and any solvents used in the process.

[0067] Figure 10 This is a graphical representation of an instance cycle involving aspiration, mixing, and spraying of reagents and sample templates. Figure 11 This is a flowchart illustrating an example of the logic used for aspirating and mixing reagents and sample templates. Figure 10In this process, reference numeral 180 specifies the suction, mixing, and jetting cycles, wherein the pressure applied by the pump is indicated by shaft 182, and the cycle time is indicated by shaft 184. Negative pressure indicates the suction of one or more reagents, while positive pressure indicates jetting. The process can be considered to include a “transfer” sequence 186, followed by a “mixing” sequence 196, as discussed below.

[0068] follow Figure 11 The flowchart shows that control logic 204 can begin by evacuating air at 206 to remove existing liquid from the flow path through which the previous mixture of reagents can be delivered. For example, any residual liquid remaining in the flow path 142 connecting reagent selector valve 66 and destination receiver 136 can be evacuated with air (that is, the liquid is replaced with air), so that any new mixture of reagents subsequently delivered to the destination receiver via flow path 142 will not be mixed with the residual liquid. The transfer sequence can then be... Figure 11 The filling sequence begins, indicated by reference number 208. This filling sequence is initiated by... Figure 10 Reference number 188 generally indicates a series of negative pressure or aspiration events. Generally, these events allow for the initial aspiration of reagents into the system. Return to Figure 11 Slightly more specifically, a pumpable buffer, as indicated at 210. This buffer may comprise a liquid selected to be non-reactive or relatively inert relative to the reagent and may serve as an incompressible working fluid extending at least partially between the pump and the reagent to allow for more precise metering of the reagent to be added to the mixing volume as needed in the following steps. The first reagent may then be pumped during the filling event, as in Figure 11 As indicated at position 212, any number of other reagents are then aspirated via aspirating the final reagent at position 214. For example, in the currently covered embodiment, three such reagents are aspirated in sequence for filling.

[0069] exist Figure 11In the logic described above, the reagents to be mixed are then aspirated using transfer sequence 218. The transfer sequence continues by aspirating the first reagent as indicated at 220, and then each of the additional reagents is aspirated one by one until the final reagent is aspirated as indicated at 222. As previously mentioned, in the currently covered embodiment, three reagents are aspirated in this sequence. As mentioned above, in the currently covered embodiment, a certain number of reagent groups are aspirated in relatively small amounts to generate a series of reagents and thereby promote premixing. Therefore, at 224, the logic can determine whether all groups of reagents have been aspirated, and if not, it returns to 220 to continue aspirating additional groups. It should also be noted that in the currently covered embodiment, all groups contain all the reagents selected for mixing, but this is not necessary. Furthermore, different volumes or quantities of reagents can be aspirated in each group. Once all reagents have been aspirated, the control can proceed beyond the transfer sequence. The transfer sequence is... Figure 10 The reference number 190 indicates a negative pressure event.

[0070] like Figure 11 As shown in the document, and if it is from Figure 10 Each individual negative (and positive) pressure event is clearly defined; each successive aspiration (or jet) of reagent or premixed reagent involves controlling one or more of the valves described above, as well as the pump. That is, to aspirate an individual reagent, the reagent selector valve will be displaced to direct negative pressure to the pipette of the corresponding receiver for the selected reagent. The pump will similarly be commanded to draw reagent (or air, buffer, or template) and expel the aspirated fluid according to a specified protocol. This mixing protocol will be predetermined and stored in the memory circuitry described above, and will be performed automatically or semi-automatically based on sequencing operations also defined in the memory circuitry. These protocols are executed by processing and control circuitry that commands the operation of the valves and pump via appropriate interface circuitry.

[0071] Once all reagents have been aspirated, the aspirated fluid can be sprayed into the destination receiver, such as... Figure 11 Location 226 is indicated. As mentioned above, in the currently covered embodiment, this is done via a nozzle suction tube, wherein mixing is initiated by increasing the velocity of the reagent through the nozzle and the resulting vortex in the destination receiver. The jetting to the destination receiver is... Figure 10 Positive pressure event 192 is indicated. In some implementations, aspiration can be as follows: Figure 11 Further execution is performed as indicated by reference number 228. Subsequently, the aspirated reagent can be sprayed into the destination receiver. This sequence can be performed as indicated by... Figure 11 Reference number 230 indicates the suction air and Figure 10Prior to the negative pressure event 194 (e.g., to remove as much liquid as possible from the bypass line, mixing channel, template channel, and pipette). It should also be noted that in some embodiments, during aspiration and jetting, the nozzle pipette or receiver, or both, may be moved relative to the other (e.g., vertically) to further aid in mixing the striped sample and reagents.

[0072] After aspiration and partial premixing in the mixing volume or channel by the operations described above, mixing is performed by repeatedly moving the reagent through a nozzle pipette in the channel and between the channel and the destination receiver. For this purpose, a series of mixing cycles are performed with mixing sequence 234. In this sequence, the combined reagent and template are aspirated at 236 and sprayed back into the destination receiver at 238. The logic can repeatedly determine whether all such desired mixing cycles have been performed at 240 and continue execution until all such cycles are completed. Figure 10 In the graphical illustration, the overall cycle is indicated by reference numeral 198. As can be seen, each cycle involves a relatively short negative pressure event followed by a relatively short positive pressure event. These events effectively aspirate the combined reagents and template into the mixing volume or channel via a nozzle pipette, and gradually transfer the mixed reagents and template back to the destination receiver via the nozzle. While any desired volume can be moved during this process, in the currently covered embodiment, approximately 2,000 μL is aspirated from the destination receiver and sprayed into it in each mixing cycle; however, other embodiments may dispense approximately 500 μL or 1,500 μL, depending on the size of the flow cell used. At the end of the mixing process, the mixed reagents and template can be transferred back to the destination receiver to continue sequencing operations.

[0073] If present, the use of ordinal indicators such as (a), (b), (c), etc., in this invention and claims, unless explicitly indicated in a particular order or sequence, should be understood as not conveying any such order or sequence. For example, if there are three steps labeled (i), (ii), and (iii), it should be understood that, unless otherwise specified, these steps may be performed in any order (or even simultaneously if not otherwise prohibited). For example, if step (ii) involves disposing of an element produced in step (i), then step (ii) can be considered to occur at some point after step (i). Similarly, if step (i) involves disposing of an element produced in step (ii), the reverse should be understood.

[0074] It should also be understood that the use of “for” (e.g., “a valve for switching between two flow paths”) can be replaced by language such as “configured to” (e.g., “a valve configured to switch between two flow paths”) or similar terms.

[0075] When used for reference quantities or similar quantifiable properties, unless otherwise indicated, terms such as “about,” “approximately,” “substantially,” “nominal,” etc., should be understood to include values ​​within ±10% of the specified value.

[0076] In addition to the claims listed herein, the following additional embodiments should also be understood to be within the scope of this invention:

[0077] Implementation Scheme 1: A system includes: a flow cell for supporting an analyte of interest; a selector valve coupled to the flow cell to select a variety of reagents for analytical operations; a pump coupled to the flow cell and to a bypass line to displace fluid via the flow cell during analytical operations and via the bypass line during reagent mixing operations; a mixing volume in fluid communication with the bypass line; and control circuitry coupled to the selector valve and the pump to command the selector valve to select a reagent to be mixed and to command the pump to draw the selected reagent from a corresponding receiver into the mixing volume one by one, to circulate the drawn-in reagent in the mixing volume to mix the reagent, and to spray the mixed reagent into a destination receiver.

[0078] Implementation Scheme 2: The system as described in Implementation Scheme 1, wherein the mixing volume includes a serpentine channel.

[0079] Implementation Scheme 3: The system as described in Implementation Scheme 1, wherein the destination receiver includes the DNA to be sequenced.

[0080] Implementation Scheme 4: The system as described in Implementation Scheme 1, wherein the pump includes an injection pump.

[0081] Implementation Scheme 5: The system of Implementation Scheme 1 includes a valve to allow the pump to draw in air before drawing in the reagent.

[0082] Implementation Scheme 6: The system of Implementation Scheme 5, wherein the pump circulates the aspirated reagent while a certain volume of air exists between the pump and the aspirated reagent.

[0083] Implementation Scheme 7: The system of Implementation Scheme 1, wherein the reagent comprises at least three reagents with different specific gravities.

[0084] Implementation Scheme 8: A method includes: commanding a selector valve to select a first reagent; actuating a pump to draw the first reagent from a first receiver into a mixing volume; commanding the selector valve to select a second reagent; actuating the pump to draw the second reagent from a second receiver into the mixing volume; actuating the pump to circulate the drawn-in reagent in the mixing volume to mix the reagent; and actuating the pump to spray the mixed reagent into a destination receiver.

[0085] Implementation Scheme 9: The method of Implementation Scheme 8, wherein the mixing volume includes a serpentine channel.

[0086] Implementation Scheme 10: The method of Implementation Scheme 8, wherein the destination receiver comprises DNA to be sequenced.

[0087] Implementation Scheme 11: The method of Implementation Scheme 8, which includes commanding the selector valve to select a third reagent before actuating the pump to cyclically move the aspirated reagent, and actuating the pump to aspirate the third reagent from the third receiver into the mixing volume.

[0088] Implementation Scheme 12: The method of Implementation Scheme 11, which includes selecting and aspirating at least one of the reagents more than once before actuating the pump to cyclically move the aspirated reagent.

[0089] Implementation Scheme 13: The method of Implementation Scheme 12, which includes selecting and aspirating all of the reagent more than once before actuating the pump to cyclically move the aspirated reagent.

[0090] Implementation Scheme 14: The method of Implementation Scheme 8, which includes drawing air into the mixing volume before drawing the first reagent.

[0091] Implementation Scheme 15: The method of Implementation Scheme 14, wherein the first reagent is selected to provide a meniscus that prevents air from entering the reagent during suction or movement within the mixing volume.

[0092] Implementation Scheme 16: A method includes: actuating a pump to draw gas into a mixing volume; commanding a selector valve to select multiple reagents one by one for an analytical operation; for each selected reagent, actuating the pump to draw the selected reagent from a corresponding receiver into a serpentine mixing volume; circulating the pump to move the reagent in the mixing volume to mix the reagent; and actuating the pump to spray the mixed reagent into a destination receiver, the destination receiver including the analyte of interest to be analyzed in the analytical operation.

[0093] Implementation Scheme 17: The method of Implementation Scheme 16, which includes selecting and aspirating each reagent more than once before circulating the pump to move the reagent.

[0094] Implementation Scheme 18: The method of Implementation Scheme 17, wherein the first reagent selected and aspirated is selected to provide a meniscus that prevents air from entering the reagent during aspiration or movement within the mixing volume.

[0095] Implementation Scheme 19: The method of Implementation Scheme 16, wherein the pump circulates the aspirated reagent while a certain volume of air exists between the pump and the aspirated reagent.

[0096] Implementation Scheme 20: The method of Implementation Scheme 16, wherein the reagent comprises at least three reagents with different proportions. It should be understood that all combinations of the foregoing concepts (assuming these concepts are not inconsistent with each other) are contemplated as part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this invention are contemplated as part of the inventive subject matter disclosed herein. It should also be understood that terms expressly used herein that may also appear in any disclosure incorporated by reference should have a meaning substantially consistent with the specific concepts disclosed herein.

Claims

1. A reagent mixing method, comprising: (a) Performing reagent premixing operations for two or more reagents, including: The selector valve, which is fluidly coupled to multiple reagent pipettes, selects the first reagent. An actuated pump is used to draw a portion of the first reagent from a first reagent receiver through a corresponding reagent nozzle of a plurality of reagent nozzles into a mixing volume in a bypass line, wherein the pump is fluidly connected to the bypass line and displaces fluid via the bypass line during reagent premixing operations, and wherein the bypass line is decoupled from the flow path and fluidly coupled to the selector valve, wherein a portion of the bypass line serves as the mixing volume; The selector valve is commanded to select the second reagent; and Actuate the pump to draw a portion of the second reagent from the second reagent receiver through the respective reagent nozzles of the plurality of reagent nozzles into the mixing volume of the bypass line; (b) Actuating the pump to circulate aspirated reagent through the selector valve into and out of the mixing volume, and through a nozzle pipette into the destination receiver to mix the reagent, the nozzle pipette being fluidly coupled to the selector valve, wherein the destination receiver contains the molecule to be sequenced; and (c) Actuate the pump to draw the mixed reagent from the destination receiver.

2. The method of claim 1, wherein the mixing volume comprises a serpentine channel.

3. The method of claim 1, wherein the destination receiver contains DNA to be sequenced.

4. The method of claim 1, wherein (a) further comprises commanding the selector valve to select a third reagent and actuating the pump to draw a portion of the third reagent from the third receiver into the mixing volume.

5. The method of claim 4, wherein (a) further comprises selecting and aspirating at least one of the first reagent, the second reagent, and the third reagent more than once prior to performing (b).

6. The method of claim 5, further comprising repeating (a) one or more times before performing (b).

7. The method of claim 1, wherein air is aspirated into the mixing volume prior to aspirating the first reagent.

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