Hydration and homogenization of freeze-dried reagents

Abstract

A system and method are provided that include the steps of hydrating a lyophilized reagent and homogenizing the hydrated reagent under the control of a control circuit that implements a hydration and homogenization protocol. A lyophilized reagent nozzle sipper that includes a distal tip extends into a lyophilized reagent well such that the distal tip does not contact the associated lyophilized reagent, and a specified amount of hydration fluid is automatically aspirated from a corresponding hydration reservoir by a corresponding sipper and dispensed into the lyophilized reagent well based on a hydration and homogenization protocol implemented by the control circuit. The method may also include extending the lyophilized reagent nozzle sipper into the lyophilized reagent well such that the distal tip contacts the hydrated reagent and automatically aspirating and discharging the hydrated reagent based on a hydration and homogenization protocol implemented by the control circuit.

Description

Technical Field

[0001] The present disclosure generally relates to systems and methods for hydrating and homogenizing one or more lyophilized reagents. More specifically, the present disclosure includes a method for performing a hydration operation and a mixing operation, wherein a lyophilized reagent nozzle sipper extends into a well containing a lyophilized reagent to a first position during hydration and to a second position during mixing. The present disclosure also generally relates to a system for performing a method that includes a fluid manifold with one or more lyophilized reagent nozzle sippers, a pump, a bypass valve, and a control circuit for implementing the method.

Background Art

[0002] Devices for sequencing molecules of interest, particularly DNA, RNA, and other biological samples, have been developed and continue to evolve. Prior to the sequencing operation, a sample of the molecule of interest is prepared to form a library or template, which is then mixed with reagents and ultimately introduced into a flow cell where individual molecules attach to sites and are amplified to enhance detectability. The sequencing operation then includes repeating a cycle of steps of binding molecules at each site, tagging the bound components, imaging the components at each site, and processing the resulting image data. In such a sequencing system, a fluid system (or subsystem) provides the flow of substances (e.g., reagents) under the control of a control system such as a programmed computer and a suitable interface.

[0003] The stability of reagents involved in sample preparation varies depending on various factors. Historically, reagents have often been wet, i.e., in liquid form at room temperature, and thus current systems and methods are designed for the use of wet reagents, which often involve freezing for transport and storage. The move to dry reagents could enable ambient transport and storage. However, dry reagents can be more sensitive than wet reagents to undesirable environmental conditions during manufacturing, transport, storage, and sample preparation. Lyophilized reagents can involve different systems and methods for adaptation prior to use (e.g., rehydration) and serve as an alternative to wet reagents. Current systems and methods can benefit from the rationalized use of lyophilized reagents.

[0004] Accordingly, improved systems and methods for sample preparation are needed. In particular, there is a need for systems and methods that utilize lyophilized sequencing reagents.

[0005] This disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY OF THE INVENTION

[0006] Details of one or more embodiments of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims.

[0007] One aspect is a fluid manifold that includes a plurality of lyophilized reagent nozzle sippers and a bypass valve. Each of the lyophilized reagent nozzle sippers includes a distal tip that extends into a corresponding lyophilized reagent well containing the lyophilized reagent such that the distal tip does not contact the lyophilized reagent before rehydration and contacts the rehydrated reagent after rehydration. The bypass valve is fluidly connected to the lyophilized reagent nozzle sippers, a fluid manifold, a pump fluidly connected to the bypass valve, and a control circuit operably connected to the lyophilized reagent nozzle sippers, the bypass valve, and the pump. The control circuit controls the lyophilized reagent nozzle sippers, the bypass valve, and the pump to automatically rehydrate the lyophilized reagent and homogenize the rehydrated reagent, related to a system including.

[0008] In one embodiment, the system includes a bypass line between the bypass valve and the pump, and the bypass line is fluidly connected to the pump. In another embodiment, the system further includes a bypass cache between the pump and the bypass valve, the bypass cache includes a heating chamber, and the bypass cache is fluidly connected to the bypass valve. In yet another embodiment, the fluid manifold includes one or more rehydration sippers, and each of the one or more moisture sippers includes a distal tip that extends into a corresponding moisture replenishment reagent reservoir containing the rehydration fluid.

[0009] In yet another embodiment, the control circuit controls the pump to rehydrate the lyophilized reagent by aspirating a volume of rehydration fluid and dispensing the volume of rehydration fluid onto the lyophilized reagent in the lyophilized reagent well. In a further embodiment, the control circuit controls the pump to dilute the rehydrated reagent by aspirating a second volume of rehydration fluid and dispensing the second volume of rehydration fluid into the lyophilized reagent well. In yet a further embodiment, the flow rate when aspirating the second volume of rehydrated reagent is less than or equal to the flow rate when dispensing the second volume of rehydrated reagent. In yet a further embodiment, the flow rate when aspirating the second volume of rehydrated reagent is less than the flow rate when dispensing the second volume of rehydrated reagent.

[0010] In another embodiment, the control circuit positions the lyophilized reagent nozzle sipper so that the distal tip contacts the rehydration reagent, aspirates the rehydration reagent, dispenses it back into the lyophilized reagent well, and repeats the steps of aspiration and dispensing until the rehydration reagent is homogeneous, thereby controlling the pump and the lyophilized reagent nozzle sipper to homogenize the rehydration reagent.

[0011] In one embodiment, the control circuit controls the pump and the bypass cache to polish the homogeneous rehydration reagent. The homogeneous rehydration reagent is aspirated into the bypass cache, heated, dispensed back into the lyophilized reagent well, aspirated into the bypass cache a second time, heated a second time, cooled, and dispensed into a buffer well containing a buffer fluid. In another embodiment, the control circuit controls the pump to add a third component to the buffer well by aspirating an amount of the third component and dispensing the amount of the third component into the buffer well.

[0012] Another aspect is a method of using the system, comprising: (a) performing a rehydration operation, the substeps of which include operating a pump to aspirate a rehydration fluid, instructing one of a plurality of lyophilized reagent nozzle sippers to extend to a first position within a corresponding lyophilized reagent well, and operating the pump to dispense the rehydration fluid into the corresponding lyophilized reagent well, thereby forming a rehydration reagent; and (b) performing a mixing operation, the substeps of which include instructing one of a plurality of lyophilized reagent nozzle sippers to extend to a second position within a corresponding lyophilized reagent well, and operating the pump to mix the rehydration reagent.

[0013] In one embodiment, the method further includes (c) performing a dilution operation before the mixing operation, the substeps of which include operating a pump to aspirate a dilution fluid and operating the pump to dispense the dilution fluid into a corresponding lyophilized reagent well.

[0014] In another embodiment, the method further includes the step of performing a polishing operation after the mixing operation, the step including: a sub-step of operating a pump to draw a hydration reagent into a bypass cache including a heating chamber; a sub-step of instructing the heating chamber to heat the hydration reagent; a sub-step of dispensing and returning the hydration reagent into a corresponding lyophilized reagent well; a sub-step of operating the pump to draw the hydration reagent into the heating chamber of the bypass cache; a sub-step of instructing the heating chamber to heat the hydration reagent for a second time; and a sub-step of cooling the hydration reagent, thereby forming a polished reagent.

[0015] In yet another embodiment, the method further includes the step of performing a second mixing operation, the step including: a sub-step of operating a pump to dispense a polished reagent into a buffer well containing a buffer fluid; a sub-step of operating the pump to draw a third component; a sub-step of operating the pump to dispense the third component into the buffer well; and a sub-step of operating the pump to draw a solution in the buffer well and dispense and return it into the buffer well, where the solution is mixed.

[0016] Yet another aspect includes the step of implementing a hydration protocol under the control of a control circuit, the step including: a sub-step of extending a lyophilized reagent nozzle sipper into a first position above a lyophilized reagent in a lyophilized reagent well; a sub-step of drawing a certain volume of hydration fluid from a hydration reservoir; and a sub-step of dispensing the certain volume of hydration fluid into the lyophilized reagent well to form a hydration reagent, and the step of implementing a homogenization protocol under the control of the control circuit, the step including: a sub-step of extending the lyophilized reagent nozzle sipper to a second position where the lyophilized reagent nozzle sipper contacts the hydration reagent; a sub-step of drawing a certain amount of hydration reagent; and a sub-step of dispensing the certain amount of hydration reagent into the same well.

[0017] In one embodiment, the method comprises performing a dilution protocol under the control of a control circuit after the hydration protocol has been performed and before the homogenization protocol is performed, the dilution control including sucking a dilution fluid from a dilution reservoir into a bypass cache and dispensing the dilution fluid into a lyophilized reagent well.

[0018] In another embodiment, the method comprises performing a polishing protocol under the control of a control circuit after the homogenization protocol has been performed, the polishing protocol including sucking a homogenized reagent into a bypass cache, a first sub-step of heating the homogenized reagent in the bypass cache, a sub-step of dispensing and returning the homogenized reagent into a lyophilized reagent well, sucking the homogenized reagent into the bypass cache, a second sub-step of heating the homogenized reagent in the bypass cache, a sub-step of cooling the homogenized reagent, and a sub-step of dispensing the resulting polished reagent into a buffer well.

[0019] In yet another embodiment, the method comprises performing a mixing protocol under the control of a control circuit after the polishing protocol has been performed, the mixing protocol including sucking a third component and dispensing the third component into a buffer well, and sucking a mixture of the polished reagent and the third component and dispensing and returning the mixture into the buffer well.

[0020] In yet another embodiment, during the homogenization protocol, the flow rate during dispensing is greater than or equal to the flow rate during suction. In yet another embodiment, during the homogenization protocol, the flow rate during dispensing is greater than the flow rate during suction.

[0021] In a further embodiment, during the mixing protocol, the flow rate during dispensing is greater than or equal to the flow rate during suction. In yet a further embodiment, during the mixing protocol, the flow rate during dispensing is greater than the flow rate during suction.

[0022] Yet another aspect is a system including a flow path fluidly connected to a flow cell, a plurality of hydration sippers fluidly connected to the flow path, a plurality of lyophilized reagent nozzle sippers fluidly connected to a bypass cache, a selector valve fluidly connected to the plurality of hydration sippers and the plurality of lyophilized reagent nozzle sippers, a bypass valve fluidly connected to the selector valve and the bypass cache, a pump fluidly connected to the bypass cache, and a control circuit operably coupled to the plurality of lyophilized reagent nozzle sippers, the selector valve, the bypass valve, and the pump, the control circuit including one or more processors and a memory storing machine-executable instructions that, when executed by the one or more processors, cause the control circuit to: (a) cause the selector valve to select a hydration sipper associated with a hydration fluid from among the plurality of hydration sippers; (b) cause the pump to draw the hydration fluid from the selected hydration sipper and deliver the hydration fluid to the bypass cache; (c) cause the selector valve to select a lyophilized reagent nozzle sipper associated with a lyophilized reagent to be rehydrated; (d) position the selected lyophilized reagent nozzle sipper at a first position above the associated lyophilized reagent; (e) cause the pump to dispense the drawn hydration fluid from the bypass cache into a well containing the lyophilized reagent, thereby forming a hydrated reagent; (f) cause the selector valve to reselect a hydration sipper associated with the hydration fluid; (g) cause the pump to draw the hydration fluid from the selected hydration sipper and deliver the hydration fluid to the bypass cache, and dispense the drawn hydration fluid into a first well containing the hydrated reagent; (h) position the selected lyophilized reagent nozzle sipper at a second position where the distal tip contacts the hydrated reagent; (i) cause the pump to draw the hydrated reagent and deliver the hydrated reagent to the bypass cache and dispense the hydrated reagent into the first well, thereby homogenizing the hydrated reagent.

[0023] In one embodiment, the bypass cache includes a heating chamber, and the instructions further cause (j) the pump to aspirate the homogenized hydrated reagent, deliver the homogenized hydrated reagent to the bypass cache, heat the homogenized hydrated reagent for the first time, and dispense it into the first well; (k) the pump to aspirate the homogenized hydrated reagent, deliver the homogenized hydrated reagent to the bypass cache, heat the homogenized hydrated reagent for the second time, thereby polishing the hydrated reagent; and (l) cool the polished reagent in the bypass cache.

[0024] In another embodiment, the instructions further cause (m) the pump to dispense the polished hydrated reagent into a second well containing a second component; and (n) the pump to aspirate the mixture, deliver the mixture to the bypass cache, and dispense the mixture into the second well, thereby mixing the polished hydrated reagent and the second component.

[0025] In yet another embodiment, the instructions further cause (o) the pump to aspirate a third component into the bypass cache and dispense the third component into a second well containing the polished hydrated reagent and the second component; and (p) the pump to aspirate the mixture of the polished hydrated reagent, the second component, and the third component into the bypass cache and dispense the mixture into the second well, thereby mixing the polished hydrated reagent, the second component, and the third component.

[0026] A further aspect is a method of using a system, comprising: (a) performing a hydration operation, the sub-steps of which include: commanding a selector valve to select a hydration sipper that extends into a reservoir associated with a hydration fluid among a plurality of hydration sippers; operating a pump to draw the hydration fluid into a bypass cache; commanding the selector valve to select a lyophilized reagent nozzle sipper that extends into a first well associated with a lyophilized reagent; commanding the selected lyophilized reagent nozzle sipper to extend to a first position within the first well; operating the pump to dispense the hydration fluid from the bypass cache into the first well associated with the lyophilized reagent, thereby forming a hydrated reagent; commanding the selector valve to select a hydration sipper that extends into a reservoir associated with the hydration fluid; operating the pump to draw the hydration fluid into the bypass cache; commanding the selector valve to select a lyophilized reagent nozzle sipper that extends into the first well containing the hydrated reagent; operating the pump to dispense the hydration fluid from the bypass cache into the well associated with the lyophilized reagent, thereby diluting the hydrated reagent; (b) performing a homogenization operation, the sub-steps of which include: commanding the selected lyophilized reagent nozzle sipper to extend to a second position within the well; operating the pump to homogenize the hydrated reagent; and (c) performing a polishing operation, the sub-steps of which include: operating the pump to draw the homogenized hydrated reagent into the bypass cache; commanding a heating chamber within the bypass cache to heat the homogenized hydrated reagent for the first time.

[0027] In one embodiment, the method further comprises: (c) performing a polishing operation, the sub-steps of which include: operating the pump to draw the homogenized hydrated reagent into the bypass cache; commanding a heating chamber within the bypass cache to heat the homogenized hydrated reagent for the first time. A step further includes sub-steps of operating a pump to dispense the homogenized hydration reagent into a first well, operating the pump to aspirate the homogenized hydration reagent into a bypass cache, commanding a heating chamber in the bypass cache to heat the homogenized hydration reagent for the second time, and operating the pump to dispense the polished hydration reagent into a second well.

[0028] In another embodiment, the method further includes a step of performing a mixing operation, which includes sub-steps of commanding a selector valve to select a reagent, operating a pump to aspirate the reagent into a bypass cache, operating the pump to dispense the reagent into a second well containing the polished hydration reagent to form a mixture, operating the pump to aspirate the mixture, and operating the pump to dispense the mixture to mix the mixture.

[0029] In yet another embodiment, the second well contains a buffer solution. In yet another embodiment, the flow rate during aspiration of the mixture is less than or equal to the flow rate during dispensing of the mixture. In a further embodiment, the flow rate during aspiration of the mixture is less than the flow rate during dispensing of the mixture.

[0030] Yet a further aspect includes a fluid system, a plurality of hydration sippers, and a plurality of lyophilized reagent nozzle sippers, the fluid system including a plurality of hydration channels, a plurality of lyophilized reagent channels, a selector valve, and a bypass cache, each of the plurality of hydration channels having a first end configured to be in fluid connection with a different hydration receptacle of a plurality of hydration receptacles and a second end in fluid connection with the selector valve, each of the plurality of lyophilized reagent channels having a first end configured to be in fluid connection with a different lyophilized reagent receptacle of a plurality of lyophilized reagent receptacles and a second end in fluid connection with the selector valve, the selector valve being in fluid connection with the bypass cache, the plurality of hydration sippers being in fluid connection with the plurality of hydration channels, the plurality of lyophilized reagent nozzle sippers being in fluid connection with the plurality of lyophilized reagent channels, and the system extending to a first position when the fluid system hydrates a lyophilized reagent with a hydration fluid to form a hydrated reagent and extending to a second position where the lyophilized reagent nozzle sipper contacts the hydrated reagent when the fluid system homogenizes the hydrated reagent.

[0031] Yet a further aspect includes a housing and a fluid manifold disposed within the housing, the fluid manifold including a plurality of channels fluidly connected to lyophilized reagent nozzle sippers that extend to a first position or a second position into different corresponding wells, each well being associated with a lyophilized reagent, the lyophilized reagent nozzle sipper contacting the hydrated reagent when in the second position, a reagent selector valve disposed within the housing and operably connected to at least two of the channels of the manifold, a bypass valve disposed within the housing and operably connected to the reagent selector valve, a bypass cache disposed within the housing and operably connected to the bypass valve, and a pump disposed within the housing, operably connected to the channels of the fluid manifold, and fluidly connected to the bypass cache.

[0032] Another aspect relates to an apparatus including a lyophilized reagent sipper that is extendable from a first position to a second position and a third position, wherein the distance between the first position and the third position is greater than the distance between the first position and the second position. In one embodiment, the apparatus includes a removable cartridge including wells, wherein the lyophilized reagent sipper extends into the wells of the cartridge when the lyophilized reagent sipper is in the second and third positions, and the lyophilized reagent sipper does not extend into the wells of the cartridge when the lyophilized reagent sipper is in the first position. In another embodiment, the lyophilized reagent sipper extends into the well above the lyophilized cake contained in the well when the lyophilized reagent sipper is in the second position, and the lyophilized reagent sipper extends into the well and into the rehydrated lyophilized reagent contained in the well when the lyophilized reagent sipper is in the third position.

[0033] In yet another embodiment, the lyophilized reagent sipper further includes a centerline and a distal end, the distal end includes facets and a nozzle, the facets intersect at a vertex offset or eccentric with respect to the centerline, and the centerline extends through the nozzle. In yet another embodiment, the distal end includes four facets. In a further embodiment, the lyophilized reagent sipper further includes a nozzle insert, the lyophilized reagent sipper has an inner diameter, the nozzle insert has an inner diameter, and the inner diameter of the nozzle insert is about half of the inner diameter of the lyophilized reagent sipper.

Brief Description of the Drawings

[0034] These features, aspects, and advantages of the present disclosure, as well as other features, aspects, and advantages, will be more fully understood upon reading the following detailed description with reference to the accompanying drawings, in which like features represent like parts throughout the drawings.

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[0035] All embodiments and combinations thereof of the foregoing concepts and additional concepts, to be discussed in more detail below (so long as such concepts are not mutually inconsistent), are intended to be part of the subject matter of the invention disclosed herein and are understood to be usable to achieve the benefits and advantages described herein.

DETAILED DESCRIPTION OF THE INVENTION

[0036] FIG. 1 shows an embodiment of a sequencing system 10 configured to process a molecular sample that can be sequenced to determine its components, component order, and generally the structure of the sample. The system includes equipment 12 for receiving and processing a biological sample. A sample source 14 often provides a sample 16 that includes a tissue sample. The sample source can include, for example, an individual or subject such as a human, animal, microorganism, plant, or other donor (including environmental samples), or any other subject that includes organic molecules of the object whose sequence is to be determined. The system can be used with samples other than those taken from organisms, including samples that include synthesized molecules. Often, the molecules include DNA, RNA, or other molecules having base pairs, and their sequences can define genes and variants that have a specific function of the ultimate object.

[0037] Sample 16 is introduced into a sample / library preparation system 18. This system can isolate, break down, and otherwise prepare the sample for analysis. The resulting library contains target molecules in lengths that facilitate the sequencing operation. The resulting library is then provided to an instrument 12 on which the sequencing operation is performed. In practice, the library, which may also be referred to as a template, is combined with reagents in an automated or semi-automated process and then introduced into a flow cell prior to sequencing.

[0038] In the embodiment shown in FIG. 1, the instrument includes a flow cell or array 20 that receives the sample library. The flow cell may include one or more fluid channels that enable sequencing chemistry to occur, including attachment of library molecules and amplification at positions or sites that can be detected during the sequencing operation. For example, the flow cell / array 20 may include a sequencing template immobilized on one or more surfaces at positions or sites. A “flow cell” may include a patterned array such as a microarray, nanoarray, etc. In practice, the positions or sites may be arranged in a regular repeating pattern, a complex non-repeating pattern, or a random array on one or more surfaces of a support. To enable sequencing chemistry to occur, the flow cell may also allow introduction of various reagents, buffers, and other reaction media such as substances that can be used for reactions, flushing, etc. The substances can flow through the flow cell and contact the molecules of interest at individual sites. In some embodiments, the substances may first bypass the flow cell during preparation via a bypass cache 300. For example, lyophilized reagents may be hydrated, mixed, and / or milled via a bypass circuit that includes the bypass cache 300 as described herein. In some embodiments, the lyophilized reagent is in the form of a cake and is referred to as a lyophilized reagent cake.

[0039] In one embodiment, the flow cell 20 can be mounted on a movable stage 22 that can be moved in one or more directions, as indicated by reference numeral 24. The flow cell 20 can be provided in the form of a removable and replaceable cartridge that can interface with ports on the movable stage 22 or other components of the system, for example, to enable reagents and other fluids to be delivered to or from the flow cell 20. The stage can be associated with an optical detection system 26 that can direct radiation or light 28 at the flow cell during sequencing. The optical detection system can employ various methods such as fluorescence microscopy for the detection of analytes disposed at sites of the flow cell. By way of non-limiting example, the optical detection system 26 can employ confocal line scanning to generate progressive pixelated image data that can be analyzed to locate individual sites in the flow cell and to determine the type of nucleotide that was most recently attached or bound to each site. Other suitable imaging techniques can also be employed, such as techniques that scan one or more emission points along the sample, or techniques that employ “step and shoot” imaging techniques. The optical detection system 26 and the stage 22 can cooperate to maintain a static relationship between the flow cell and the detection system while acquiring area images, or, as described above, the flow cell can be scanned in any suitable mode (e.g., point scanning, line scanning, “step and shoot” scanning).

[0040] Many different techniques can be used for imaging or, more generally, for detecting the molecules at each site, but one embodiment can utilize confocal optical imaging at wavelengths that cause excitation of the fluorescent tags. The tags can be excited by their absorption spectra and can return a fluorescent signal by their emission spectra. The optical detection system 26 can be configured to capture such signals in order to process pixelated image data at a resolution that enables analysis of the signal emission sites and to process and store the resulting image data (or data derived therefrom).

[0041] In the array determination operation, the cycle operation or process may be implemented in an automatic or semi-automatic manner. The reaction may be facilitated, for example, using a single nucleotide or oligonucleotide, followed by flushing, imaging, and deblocking in the preparation for subsequent cycles. A sample library prepared for array determination and immobilized on a flow cell can undergo a number of such cycles before all useful information is extracted from the library. The optical detection system can generate image data from the scanning of the flow cell (and its sites) during each cycle of the array determination operation by using an electronic detection circuit (e.g., a camera or imaging electronics circuit or chip). The resulting image data is then analyzed to identify the positions of the individual sites in the image data, and to analyze and characterize the molecules present at those sites, for example, by reference to the specific color or wavelength of light detected at a particular position (the characteristic emission spectrum of a particular fluorescent tag), as indicated by a group or cluster of pixels in the image data at that position. In DNA or RNA sequencing applications, for example, the four common nucleotides can be represented by distinguishable fluorescent emission spectra (wavelength or wavelength range of light). Each emission spectrum can then be assigned a value corresponding to that nucleotide. Based on this analysis and tracking the cycle values determined for each site, the individual nucleotides and their order can be determined for each site. These sequences can then be further processed to assemble longer segments, including genes, chromosomes, etc. As used in this disclosure, the terms "automated" and "semi-automated" mean that the operation is carried out by system programming or configuration with little or no human interaction once the operation is initiated or the process including the operation is initiated.

[0042] In the illustrated embodiment, the reagent 30 is drawn or aspirated into the flow cell through the valve regulating section 32. The valve regulating section can access the reagent from a receptacle or container in which the reagent is stored, such as through a pipette or sipper (not shown in FIG. 1). The valve regulating section 32 can enable the selection of the reagent based on a defined sequence of operations to be performed. The valve regulating section can further receive a command to direct the reagent through the flow path 34 toward the flow cell 20. The outlet or effluent flow path 36 directs the used reagent from the flow cell. In the illustrated embodiment, the pump 38 serves to move the reagent through the system. The pump can also perform other useful functions, such as measuring the reagent or other fluid through the system, aspirating air or other fluid, and the like. An additional valve regulating section 40 downstream of the pump 38 enables the used reagent to be properly directed to a disposal container or receptacle 42.

[0043] The machine further includes various circuits that assist in commanding the operation of various system components, monitoring their operation via feedback from sensors, collecting image data, and at least partially processing the image data. In the embodiment illustrated in FIG. 1, the control / monitoring system 44 includes a control system 46 and a data acquisition and analysis system 48. Both systems include one or more processors (e.g., digital processing circuits such as microprocessors, multi-core processors, FPGAs, or any other suitable processing circuits) and an associated memory circuit 50 (e.g., solid-state memory devices, dynamic memory devices, on and / or off-board memory devices, etc.) that may store machine-executable instructions for controlling, for example, one or more computers, processors, or other similar logic devices to provide specific functionality. Special-purpose or general-purpose computers may at least partially constitute the control system as well as the data acquisition and analysis system. The control system may include, for example, circuits configured (e.g., programmed) to process commands for fluid systems, optical systems, stage control, and any other useful functions of the instrument. The data acquisition and analysis system 48 may interface with an optical detection system to command, for example, the movement of the optical detection system and / or the stage, the emission of light for periodic detection, the reception and processing of return signals, etc. The instrument may also include various interfaces such as an operator interface, indicated by reference numeral 52, that permits control and monitoring of the instrument, sample transfer, initiation of automated or semi-automated sequencing operations, generation of reports, etc. Finally, in the embodiment of FIG. 1, an external network or system 54 is coupled to and may cooperate with the instrument, for example, for analysis, control, monitoring, maintenance, and other operations.

[0044] In some embodiments, reagent 30 includes other substances used in the system, such as wash buffer, hydration fluid, etc. Such substances may be in liquid form or lyophilized. In some embodiments, the liquid substance is selected via valve regulator 32 and drawn into or aspirated into bypass cache 300 and dispensed into a well (e.g., a container or receptacle) containing the lyophilized reagent 30 or other lyophilized substance, thereby hydrating the lyophilized reagent or substance. In some embodiments, valve regulator 32 can receive a command to direct reagent 30 or the substance toward bypass cache 300, and bypass cache 300 can receive a command to heat the reagent 30 or the substance to mill them. For example, a lyophilized fully functionalized nucleotide (ffN) can be hydrated, homogenized, and milled by repeating aspiration and heating into bypass cache 300. In one example, the lyophilized substance or reagent, or the hydrated solution, can include a polishing polymerase, such that the solution containing the rehydrated lyophilized ffN also includes the polishing polymerase. In another example, the polishing polymerase may be included in the bypass cache into which the rehydrated lyophilized ffN is aspirated, or alternatively, the polishing polymerase may be aspirated or added to the rehydrated lyophilized ffN within the bypass cache.

[0045] A single flow cell and fluid path, and a single optical detection system are illustrated in FIG. 1, but it should be noted that in some devices, more than two flow cells and fluid paths can be accommodated. For example, in currently contemplated embodiments, two such arrays are provided to improve sequencing and throughput. In fact, any number of flow cells and flow paths can be provided. These can utilize the same or different reagent receptacles, disposal receptacles, control systems, image analysis systems, etc. When provided, the multiple fluid systems can be controlled individually or in a coordinated manner. The phrase "fluidly connected" can be used in this specification to represent a connection between two or more components that fluidly communicate with each other, much as the phrase "electrically connected" can be used to represent an electrical connection between two or more components. The phrase "fluidly interposed" can be used, for example, to describe a particular order of components. For example, if component B is fluidly interposed between component A and component C, the fluid flowing from component A to component C flows through component B before reaching component C.

[0046] FIG. 2 shows an exemplary fluid system of the array determination system of FIG. 1. In the illustrated embodiment, the flow cell 20 includes a series of paths or lanes 56A and 56B that can be grouped in pairs to receive flowing substances (e.g., reagents, buffers, reaction media) during the array determination operation. Lane 56A is coupled to a common line 58 (the first common line), and lane 56B is coupled to a second common line 60. A bypass line 62 is also provided to enable the fluid to bypass the flow cell without entering the flow cell. As described above, a series of containers or receptacles 64 enable the storage of reagents and other fluids that can be utilized during the array determination operation. The reagent selector valve 66 is mechanically coupled to a motor or actuator (not shown) and enables the selection of one or more of the reagents introduced into the flow cell. The selected reagent is then advanced to a common line selector valve 68 that also includes a motor (not shown). The common line selector valve is instructed to select one or more, or both, of the common lines 58 and 60 and cause the reagent 64 to flow into lanes 56A and / or 56B in a controlled manner, or cause one or more of the reagents to flow through the bypass line 62. Note that the bypass line enables other useful operations such as the ability to prime all reagents (and liquids) to the reagent selector valve (and common line selector valve) without drawing air through the flow cell, the ability to perform cleaning of the reagent channels and sippers independently of the flow cell (e.g., automatic or semi-automatic cleaning), and the ability to perform diagnostic functions (e.g., pressure and volume delivery tests) on the system.

[0047] In some embodiments, bypass line 62 may further include a bypass cache 302, and a selected reagent may be advanced to the bypass cache 302 to bypass the flow cell 20. For example, a substance may be selected by the reagent selector valve 66, advanced to the bypass cache 302, and then dispensed into a lyophilized reagent well or container. Further, by using a bypass circuit that includes the bypass line 62 and the bypass cache 302, two or more substances can be mixed. For example, substances may be aspirated into the bypass cache 302 and dispensed into different wells or containers containing different substances. Further, the bypass circuit may be used to bypass the flow cell 20 in order to rehydrate a lyophilized reagent or substance. For example, a rehydration fluid may be selected by the reagent selector valve 66, aspirated into the bypass cache 302, and dispensed or released into a lyophilized reagent well or container. Additionally, the bypass cache 302 may include a heating chamber for abrasive reagents / substances such as ffN.

[0048] "Polishing" means purifying 3'-blocked nucleotides by removing unblocked (3'-OH) nucleotides from solution prior to initiating a synthesis-based sequencing (SBS) or genotyping operation. For example, a 3'-blocked nucleotide can include a blocking group coupled to the nucleotide at the 3'-position, such as an azidomethyl group. The nucleotide can also be coupled to a detectable moiety, such as a fluorophore. Depending on the complementary polynucleotide (e.g., the template to be sequenced), when an SBS polymerase polymerizes a 3'-blocked nucleotide by adding a given one of the nucleotides to a growing polynucleotide, that one of the nucleotides can be detected and identified by detection of the detectable moiety, thus enabling identification of the nucleotide complementary to the nucleotide of the template. However, the polymerase may not be able to add another nucleotide to the growing polynucleotide until the 3'-blocking group is removed using a suitable reagent. After the 3'-blocking group is removed, the detectable moiety is cleaved from the nucleotide and another 3'-blocked nucleotide can be added to the growing polynucleotide. Such a process can be repeated any suitable number of times, for example, to identify one or more bases in the sequence of a complementary polynucleotide. The detectable moieties of various 3'-blocked nucleotides can be detected via a suitable detection circuit. In some examples, the detectable moiety can include a fluorophore that can be detected via a suitable optical detection circuit. However, it will be understood that the detectable moiety can be detected in any suitable manner and is not limited to detection via fluorescence.

[0049] The presence of 3'-unblocked nucleotides (nucleotides with an unblocked 3') can interfere with sequencing. For example, storage or transport can cause deblocking of 3'-blocked nucleotides by hydrolysis of the bond coupling the blocking group to the nucleotide, such that the 3'-blocked nucleotide can be converted to a 3'-OH nucleotide. Such hydrolysis can be reduced by lyophilizing the 3'-blocked nucleotide prior to storage or transport, but nevertheless, some 3'-OH nucleotides can be mixed with the 3'-blocked nucleotides by the time the nucleotides are used. In addition or alternatively, if the 3'-blocking group is first added during the synthesis of the 3'-blocked nucleotide, the reaction yield may not necessarily be 100%, and thus, some residual 3'-OH nucleotides can be mixed with the 3'-blocked nucleotides. If 3'-OH nucleotides are mixed with 3'-blocked nucleotides, for example, during polymerization using an SBS polymerase and a complementary polynucleotide, the 3'-OH nucleotides can cause errors in the sequencing of the complementary polynucleotide. For example, an SBS polymerase may sometimes add a 3'-OH nucleotide to a growing polynucleotide, but since such a 3'-OH nucleotide lacks a 3'-blocking group, without waiting for the addition of a reagent to remove the blocking group, the SBS polymerase may quickly add another nucleotide to the growing polynucleotide. Thus, the 3'-OH nucleotides can accelerate polymerization (such acceleration is also called "prephasing"), and the increased rate can inhibit the ability of the detection circuit to accurately detect and identify a detectable moiety coupled to the 3'-OH nucleotide. Accordingly, the sequence of the complementary polynucleotide may not be determined completely or accurately.

[0050] Polishing can be performed via a polishing reagent. In one embodiment, the polishing reagent can include a polishing polymerase. Non-limiting examples of polishing polymerases are heat-stable polymerases that polymerize 3'-OH nucleotides at a rate significantly higher than that of 3'-blocked nucleotides, or many other examples of polymerases that cannot substantially polymerize 3'-blocked nucleotides, such as those not specifically engineered for use in SBS. The polishing polymerase can polymerize the 3'-OH nucleotides in the mixture to remove these nucleotides from the solution, while the 3'-blocked nucleotides can remain in the solution. Then, for example, in an SBS or genotyping process, an SBS polymerase can be used to polymerize the 3'-blocked nucleotides while reducing interference from the 3'-OH nucleotides. "Polishing polymerase" is intended to mean an enzyme that polymerizes 3'-OH nucleotides, for example, by adding 3'-OH nucleotides to a growing polynucleotide using a complementary polynucleotide, can polymerize 3'-blocked nucleotides at a rate significantly reduced compared to 3'-OH nucleotides, and cannot actually substantially polymerize 3'-blocked nucleotides. Thus, the polishing polymerase can be considered to "selectively" polymerize 3'-OH nucleotides.

[0051] Non-limiting examples of polishing polymerases are "thermostable" polymerases, which refer to polymerases that can function well at relatively high temperatures, such as from about 30°C to about 100°C, or from about 40°C to about 80°C, or from about 50°C to about 70°C. Examples of thermostable polymerases include Pyrococcus sp. (strain GB-D) DNA polymerase with the trade name DEEP VENT® DNA polymerase (exemplary working temperature 75°C), Thermus aquaticus DNA polymerase I (Taq polymerase) (exemplary working temperature 75°C), Bst (exemplary working temperature 65°C), Sulfolobus DNA polymerase IV (exemplary working temperature 55°C), and Pfu (Phusion) (exemplary working temperature 75°C), all of which are commercially available from New England Biolabs, Inc. (Ipswich, MA). Other non-limiting examples of polishing polymerases include Escherichia coli DNA polymerase I proteolytic (Klenow fragment) (exemplary working temperature 37°C) and Bsu (exemplary working temperature 37°C), which are commercially available from New England Biolabs, Inc.

[0052] The concentration of 3'-OH nucleotides can be reduced compared to 3'-blocked nucleotides by selectively polymerizing the 3'-OH nucleotides. For example, a polishing polymerase and a polynucleotide (template) can be mixed in an aqueous solution with a mixture of 3'-blocked nucleotides and 3'-OH nucleotides. Unlike SBS polymerases that can polymerize both 3'-blocked nucleotides and 3'-OH nucleotides relatively well, polishing polymerases can polymerize 3'-OH nucleotides relatively well, but can polymerize 3'-blocked nucleotides at a significantly lower rate than 3'-OH nucleotides or, in some instances, not polymerize them at all. Non-limiting examples of polishing polymerases are thermostable polymerases, but there are many other examples of polymerases that polymerize 3'-OH nucleotides at a significantly higher rate than 3'-blocked nucleotides or that cannot substantially polymerize 3'-blocked nucleotides, e.g., polymerases that have not been specifically engineered for use in SBS. The polishing polymerase can polymerize the 3'-OH nucleotides in the mixture to remove these nucleotides from the solution, while the 3'-blocked nucleotides can remain in the solution. Then, for example, in an SBS or genotyping process, an SBS polymerase can be used to polymerize the 3'-blocked nucleotides while reducing interference from the 3'-OH nucleotides.

[0053] In some embodiments, the 3'-blocked nucleotides can be purified on the same instrument that performs subsequent polymerization operations. For example, both purification and polymerization of the 3'-blocked nucleotides can be performed on the same SBS instrument. As described in more detail below, the instrument can include a device such as a "cache manifold" that is used to heat or cool the solution for purification so that, for example, a polishing polymerase can be used at a suitable temperature and, for example, to heat or cool the solution for polymerization so that an SBS polymerase can be used at a suitable temperature. The cache manifold can include a heat exchanger having an inner sleeve and an outer sleeve, one or both of which can be heated or cooled, and a coiled fluid path located between the sleeves through which the solution to be heated or cooled can flow. In some embodiments, the cache manifold is a bypass cache that includes a heating chamber.

[0054] Used reagents can exit the flow cell through a line connected between the flow cell and pump 38. In the illustrated embodiment, the pump includes a syringe pump having a pair of syringes 70 that are controlled and moved by an actuator 72 to aspirate reagents and other fluids and to discharge reagents and fluids during different operations of a test cycle, a verification cycle, and a sequencing cycle. The pump assembly can include various other components and elements, including valve regulators, instrumentation, actuators, etc. (not shown). In the illustrated embodiment, pressure sensors 74A and 74B sense the pressure on the inlet line of the pump, and pressure sensor 74C is provided to sense the pressure output by the syringe pump.

[0055] The fluid used by the system may enter from the pump into the used reagent selector valve 76. This valve enables the selection of one from a plurality of flow paths for the reagent and other fluids used. In the illustrated embodiment, the first flow path leads to the first used reagent receptacle 78, while the second flow path leads through the flow meter 80 to the second used reagent receptacle 82. Depending on the reagent used, it may be advantageous to collect the reagent or a particular reagent in a separate container for disposal, and the used reagent selector valve 76 enables such control.

[0056] It should be noted that the valve adjustment section in the pump assembly may enable various fluids including reagents, solvents, cleaning agents, air, etc. to be sucked by the pump and injected or circulated through one or more of the common line, bypass line, and flow cell. Further, as described above, in the presently contemplated embodiments, two parallel embodiments of the fluid system shown in FIG. 2 are provided under common control. Each of the fluid systems may be part of a single sequencing device and may perform functions including sequencing operations for different flow cells and sample libraries in parallel.

[0057] The fluid system operates under the instructions of a control system 46 that implements a defined protocol for testing, verification, sequencing, etc. The defined protocol may be pre-established and may include a series of events or operations for activities such as sucking reagents, sucking air, sucking other fluids, discharging such reagents, air, and fluids, etc. The protocol may enable the coordination of such fluid operations with other operations of the device such as reactions occurring in the flow cell, imaging of the flow cell and its parts, etc. In the illustrated embodiment, the control system 46 uses one or more valve interfaces 84 configured to provide command signals for the valves, and a pump interface 86 configured to command the operation of the pump actuator. It is also possible to provide various input / output circuits 88 to receive feedback from the pressure sensors 74A - C and the flow meter 80, etc., and process such feedback.

[0058] Figure 3 shows the specific functional components of the control / monitoring system 44. As shown, the memory circuit 50 stores predetermined routines that are executed during testing, commissioning, troubleshooting, servicing, and alignment operations. Many such protocols and routines may be implemented and stored in the memory circuit, and these may be updated or changed from time to time. As shown in Figure 3, these include not only fluid control protocols 90 for controlling various valves, pumps, and any other fluid actuators, but also for receiving and processing feedback from fluid sensors such as valves and flow rate and pressure sensors. The stage control protocol 92 enables the flow cell to be moved as desired during imaging and the like. The optical control protocol 94 enables commands to be issued to the imaging components for irradiating each part of the flow cell and for receiving the signals returned for processing. The image acquisition and processing protocol 96 enables the image data to be at least partially processed for extraction of useful data for alignment determination. Other protocols and routines may be provided in the same or different memory circuits, as indicated by reference numeral 98. In practice, the memory circuit may be provided as one or more memory devices, such as both volatile and non-volatile memory. This memory may be within the device, and some may be off-board.

[0059] One or more processors 100 access the stored protocols and implement them on the device. As described above, the processing circuitry may be part of an application-specific computer, a general-purpose computer, or any suitable hardware, firmware, and software platform. The operation of the processor and the device may be commanded by a human operator via an operator interface 101. The operator interface may not only enable testing, commissioning, troubleshooting, and servicing, but also may enable reporting of any problems that may occur in the device. The operator interface may also enable starting and monitoring of the array determination operation.

[0060] FIG. 4 shows a non-limiting embodiment of an array determination system having a bypass flow path adapted for hydration and homogenization of lyophilized reagents. The bypass flow path corresponds to aspirating and dispensing the reagent through a bypass valve into a bypass cache for hydration and mixing of the lyophilized reagent such that the preparation of the lyophilized reagent bypasses the flow cell. The sipper manifold assembly includes a reagent selection valve, a bypass valve, and a plurality of sippers. The lyophilized reagent nozzle sipper may be mounted, for example, on a movable stage that can move in one or more directions.

[0061] FIGS. 5A and 5B show two different views of a non-limiting example of a sipper manifold assembly including a reagent selector valve and a bypass valve.

[0062] FIG. 6 shows a sipper manifold assembly including a reagent selector valve, a bypass valve, wells for the hydration fluid, buffer, sample, and lyophilized reagent, and sippers and nozzle sippers for each. The lyophilized reagent nozzle sipper may move in one or more directions including a direction along the z-axis, for example, vertically within the lyophilized reagent well or container, to vary the distance between the nozzle sipper tip and the bottom of the well bottom depending on which step, for example, hydration, dilution, mixing, etc. is being performed.

[0063] As disclosed herein, the use of a mixing channel with a nozzle sipper can promote vorticity in the destination receptacle and provide excellent mixing of the reagent and template despite significant differences in the fluid properties of the reagents. Further, these structures and techniques enable automated mixing with little or no human interaction. Exemplary nozzle sippers used in these techniques are shown in FIGS. 7 and 8A-8C. As shown in FIG. 7, the nozzle sipper has an elongated body with a central lumen (hollow) extending along its length and a tip at its distal end. A nozzle is provided at the tip to decrease the inner diameter of the sipper at the location of the tip and increase the velocity of the fluid being aspirated and discharged through the sipper. In the illustrated embodiment, the nozzle is formed as an insert 158 that is inserted into the distal end or tip of the sipper. Other structures such as caps, machining, molding, mounting regions, etc. can form the nozzle.

[0064] In the illustrated embodiment, the sipper has a nominal outer diameter 160 of about 0.125 inches (3.175 mm) and a nominal inner diameter 162 of 0.020 inches ± 0.001 inches (0.508 mm). In some examples, the lyophilized reagent sipper has a nominal inner diameter 162 of from about 0.0200 inches ± 0.002 inches to about 0.030 inches ± 0.002 inches, including all values, ranges, and sub-ranges therein (e.g., 0.0215 inches ± 0.002 inches). On the other hand, the nozzle has a nominal inner diameter 164 of 0.010 inches ± 0.001 inches (0.254 mm), although some embodiments may feature a nozzle inner diameter in the range of up to 0.20 to 0.28 mm. Of course, other sizes and dimensions may be utilized to provide the desired mixing. Further, in the illustrated embodiment, the nozzle sipper 116 is positioned at a height 166 of from about 2 mm to about 10 mm above the bottom of the receptacle 138, including all values, ranges, and sub-ranges therein. When the reagent is injected into the receptacle, as indicated by reference numeral 168, the vorticity within the receptacle is enhanced by the increased velocity of the reagent moving through the nozzle, thereby enhancing the mixing in the receptacle as indicated by the arrow 170 in FIG. 7. The mixed reagent is raised within the receptacle as indicated by reference numeral 172.

[0065] Figure 8A shows the distal end of the nozzle sipper in somewhat more detail. As can be seen in the figure, the nominal inner diameter 162 of the sipper is reduced by the nozzle insert 158, in this case to approximately half the inner diameter of the sipper (the nozzle insert is tubular in shape in this example). Embodiments of the distal end are shown in FIGS. 8B, 8C, and 8D. As shown here, the nozzle sipper has a faceted lower end that includes four facets 174, giving the nozzle sipper tip a wedge-like appearance. The sipper has a centerline 176, and the facets intersect at vertices 178 that are offset or eccentric with respect to the centerline 176. This geometry of the distal end reduces or avoids dragging or scraping of the receptacle when the sipper is lowered into the receptacle or when the receptacle is lifted around the sipper. However, note that in the illustrated embodiment, the insert has a lower profile that matches the profile of the tip (e.g., one or more of the angled facets). Stated another way, the insert may be shaped to conform to the faceted or wedge shape of the distal end of the nozzle sipper. Further, note that in presently contemplated embodiments, the sipper and nozzle are made of an engineering plastic such as polyetheretherketone (PEEK). Such materials can provide chemical resistance to the reagents and any solvents used in the process.

[0066] Figure 9 shows a non-limiting embodiment of the sipper within the well, where the sipper nozzle tip is positioned within 0° to 10° horizontal. The position of the ± sipper nozzle tip affects mixing performance, and uncontrolled rotation of the sipper can result in significant variations in mixing performance.

[0067] FIG. 10 is a flowchart showing exemplary logic for aspirating and mixing a reagent and a sample template. In accordance with the flowchart of FIG. 10, control logic 204 may begin by aspirating air at 206 to remove existing liquid from the flow path through which a previous mixture of the reagent was sent. For example, any remaining liquid in flow path 142 that connects reagent selector valve 66 to destination receptacle 136 may be aspirated with air (i.e., such that the liquid is replaced with air) so that any new mixture of the reagent subsequently delivered through flow path 142 does not contact the remaining liquid. The transfer sequence may then begin with a priming sequence as indicated by reference numeral 208 in FIG. 10. Generally, these events first enable drawing the reagent into the system. Somewhat more specifically, returning to FIG. 10, a buffer solution may be aspirated as shown at 210. This buffer solution can include a liquid selected to be non-reactive or relatively inert with respect to the reagent and can be used as an incompressible working fluid that at least partially extends between the pump and the reagent to enable, if desired, more accurate metering of the reagent into the mixing volume in subsequent steps. The first reagent may then be aspirated in a priming event as shown at 212 in FIG. 10, followed by aspiration of any number of other reagents through aspiration of the final reagent at 214. In presently contemplated embodiments, for example, three such reagents are aspirated in the priming sequence.

[0068] In the logic shown in FIG. 10, the reagents to be mixed are then aspirated in transfer sequence 218. The transfer sequence continues to aspirate the first reagent as shown at 220 and then continues to aspirate one by one each additional reagent until the final reagent is aspirated as shown at 222. As previously described, in the presently contemplated embodiments, three reagents are aspirated in this order. As noted above, in the presently contemplated embodiments, some sets of reagents are aspirated in relatively small amounts to generate a sequence of reagents, thereby facilitating premixing. Thus, at 224, the logic may determine whether all sets of reagents have been aspirated and, if not, return to 220 to continue aspirating additional sets. It should also be noted that in the presently contemplated embodiments, all sets include all the reagents selected for mixing, although this need not be the case. Further, different volumes or amounts of reagents may be aspirated in the various sets. When all the reagents have been aspirated, control can proceed beyond the transfer sequence.

[0069] As shown in FIG. 10, each successive aspiration (or discharge) of a reagent or pre-mixed reagent may involve controlling one or more of the valves described above as well as a pump. That is, to aspirate an individual reagent, the reagent selector valve may be shifted to direct a negative pressure to the sipper for the corresponding receptacle of the selected reagent. The pump may likewise be instructed to draw in the reagent (or air or buffer or template) and squeeze out the fluid aspirated according to a predetermined protocol. The mixing protocol may be pre-determined and stored in the memory circuit described above and may be executed in an automatic or semi-automatic manner based on an array determination operation defined within the memory circuit. The protocol may be executed by a processing and control circuit that instructs the operation of the valves and pump through a suitable interface circuit.

[0070] When the reagent is aspirated, the aspirated fluid can be discharged into the destination receptacle as shown at 226 in FIG. 10. As described above, in one embodiment, this can be done through a nozzle sipper where mixing begins due to an increase in the velocity of the reagent through the nozzle and the resulting vorticity within the destination receptacle. In some embodiments, aspiration can be further performed as indicated by reference numeral 228 in FIG. 10. Thereafter, the aspirated reagent can be discharged into the destination receptacle. Following this sequence, aspiration of air can be performed as indicated by reference number 230 in FIG. 10 (e.g., to remove as much liquid as possible from the bypass line, mixing channel, template channel, and sipper). It should also be noted that in some embodiments, the nozzle sipper, or the receptacle, or both can be moved relative to the other (e.g., vertically) during aspiration and discharge to further aid in the mixing of the striped sample and reagent.

[0071] Following the mixing volume by the above operation or suction and partial premixing in the channel, the mixing may be performed by repeatedly moving the reagent through the nozzle sipper within the channel and between the channel and the destination receptacle. For this purpose, a series of mixing cycles may be performed in mixing sequence 234. In this sequence, the combined reagent and template may be aspirated at 236 and discharged back to the destination receptacle at 238. The logic may repeatedly determine at 240 whether all of these desired mixing cycles have been executed and continue until all such cycles are completed. As can be seen, each may involve a relatively short negative pressure event followed by a relatively short positive pressure event. These events can effectively aspirate the combined reagent and template into the mixing volume or channel through the nozzle sipper and return the gradually mixed reagent and template to the destination receptacle through the nozzle. Any desired volume may be replaced in this process, but in currently contemplated embodiments, about 2000 μL to about 4000 μL (including all values, ranges, and sub-ranges therein) is aspirated from and discharged into the destination receptacle in each mixing cycle, while in other embodiments, depending on the size of the flow cell used, about 500 μL or 1500 μL may be dispensed. At the end of the mixing process, the mixed reagent and template may be returned to the destination receptacle to proceed with the sequencing operation.

[0072] Note that in one embodiment, the nozzle sipper can effectively increase the velocity of the reagent when the reagent (and mixed reagent) is being mixed during aspiration and discharge. The increased velocity can increase the kinetic energy to assist in mixing. For example, in one embodiment, the nozzle can accelerate the mixture to at least about 1600 mm / sec at a flow rate of at least about 5000 μL / min. In a non-limiting embodiment, the lyophilized nozzle sipper accelerates the mixture such that the flow rate is about 2800 μl / min to about 6000 μl / min.

[0073] FIG. 11 is a schematic diagram of an exemplary protocol for rehydration and homogenization of a lyophilized reagent. In the preparation step, the sipper is anti-primed with air, the bypass cache and fluid path are primed with wash buffer, and the rehydration fluid is primed. In the rehydration step, the lyophilized reagent nozzle sipper is extended into the lyophilized reagent well or container to a first position (position 1), the lyophilized reagent is rehydrated, and then diluted with the rehydration fluid. In the homogenization step, the lyophilized reagent nozzle sipper is further extended into the lyophilized reagent well or container to a second position (position 2), and the rehydrated reagent is mixed to the desired degree of homogenization.

[0074] FIG. 12 is a flowchart showing exemplary logic for rehydrating and homogenizing a lyophilized reagent. During preparation, air is aspirated into the anti-priming sipper, wash buffer is aspirated to prime the bypass cache and fluid path, and rehydration fluid is aspirated for priming. During rehydration, the lyophilized reagent nozzle sipper is controlled to move to a first position within the lyophilized reagent well or container, and the rehydration fluid is aspirated into the bypass cache and discharged into the lyophilized reagent well, thereby rehydrating the lyophilized reagent. The rehydration fluid may be aspirated into the bypass cache and discharged into the rehydrated reagent well to dilute the rehydrated reagent. In some embodiments, the rehydration step includes over-aspiration from the rehydration fluid well and under-dispensing into the lyophilized reagent well. In any dilution step, a second volume of rehydration fluid is aspirated into the bypass cache and then dispensed into the lyophilized reagent well, thereby diluting the rehydrated reagent. During homogenization, the lyophilized reagent nozzle sipper may be controlled to move to a second position within the lyophilized reagent well or container, and the rehydrated reagent may be aspirated and discharged back into the same well. In some embodiments, the flow rate during aspiration and discharge varies to increase the efficiency of the homogenization step.

[0075] In the mixing step, the lyophilized reagent nozzle sipper extends into the lyophilized reagent well to a second position, which is closer to the bottom of the well than the first position. A mixing volume is aspirated and then dispensed back into the well, and the aspiration and dispensing of the mixing volume are repeated one or more times to homogenize the hydrated reagent. In some embodiments, the lyophilized reagent is in the form of a cake.

[0076] FIG. 13 is a schematic diagram of a non-limiting exemplary workflow for the hydration and homogenization of a lyophilized reagent (e.g., an exclusion amplification reagent or ExAmp). In the hydration preparation step, a lyophilized reagent nozzle sipper (e.g., an ExAmp nozzle sipper) extends into the lyophilized reagent well to a first position and is anti-primed with air. The hydration fluid sipper is also anti-primed with air. A bypass circuit (e.g., including a bypass cache and bypass flow path) is primed with wash buffer and the hydration fluid is primed. In some embodiments, the hydration fluid sipper may be primed and an air slug of about 100 μl to about 200 μl (including all values, ranges, and sub-ranges therein) may be generated between the wash buffer and the hydration fluid. In the hydration step, a first volume of hydration fluid is aspirated into the bypass cache and then dispensed into the lyophilized reagent well, thereby forming a hydrated reagent. In some embodiments, the hydration step includes over-aspiration from the hydration fluid well and under-dispensing into the lyophilized reagent well. In any dilution step, a second volume of hydration fluid is aspirated into the bypass cache and then dispensed into the lyophilized reagent well, thereby diluting the hydrated reagent. In the mixing step, the lyophilized reagent nozzle sipper extends into the lyophilized reagent well to a second position, which is closer to the bottom of the well than the first position. A mixing volume is aspirated and then dispensed back into the well, and the aspiration and dispensing of the mixing volume are repeated one or more times to homogenize the hydrated reagent. In some embodiments, the lyophilized reagent is in the form of a cake. In some embodiments, the lyophilized reagent is ExAmp. In some embodiments, the lyophilized reagent is an incorporated lyophilized reagent such as a fully functionalized nucleotide or ffN.

[0077] Figure 14 is a schematic diagram of an exemplary protocol for rehydration, homogenization, and milling of a lyophilized reagent. The preparation, rehydration, and homogenization steps are described above in FIG. 11. In the milling step, the rehydrated and homogenized reagent is aspirated into a bypass cache, heated, dispensed back into the same well or container. The once-heated reagent is then aspirated into the bypass cache, a second heating is performed, and then after being cooled within the bypass cache, it is discharged back into the same well.

[0078] Figure 15 is a flowchart showing exemplary logic for rehydrating, homogenizing, and milling a lyophilized reagent. In some embodiments, the milled reagent may be discharged into different wells containing different substances, and a mixing protocol is implemented to mix the milled reagent with the different substances. In some embodiments, the milled reagent may be further mixed with one, two, or more additional substances. By way of non-limiting example, the lyophilized reagent can include ffN, which can be rehydrated, homogenized, milled, and mixed with additional substances such as polymerase, taq, and buffer, as described in more detail below.

[0079] FIG. 16 is a flowchart showing an exemplary workflow for hydration and homogenization of an incorporated lyophilized reagent, followed by milling and further mixing. In the preparation step, the lyophilized reagent nozzle sipper is extended into the lyophilized reagent well to a first position above the lyophilized reagent. In some embodiments, the lyophilized reagent is in the form of a cake. In some embodiments, the lyophilized reagent may contain fully functional nucleotides (ffN). The lyophilized reagent nozzle sipper, buffer sipper, and additional reagent sipper are anti-primed with air. In some embodiments, the additional reagent may be polymerase. The bypass circuit (e.g., bypass cache and bypass flow path) may be primed with wash buffer and the incorporated hydration fluid is primed. In the hydration step, a first volume of the incorporated hydration fluid is aspirated into the bypass cache and then dispensed into the lyophilized reagent well, thereby forming a hydrated reagent. In some embodiments, the hydration step may include over-aspiration from the hydration fluid well and under-dispensing into the lyophilized reagent well. In any dilution step, a second volume of the hydration fluid is aspirated into the bypass cache and then dispensed into the lyophilized reagent well, thereby diluting the hydrated reagent. In the two-step milling step, the hydrated reagent is aspirated into the bypass cache, heated and milled the first time, dispensed and returned into the hydrated reagent well, aspirated into the bypass cache, heated and milled the second time, and cooled in the bypass cache. In the transfer and mixing step, the milled reagent is dispensed into a buffer well containing buffer fluid. In the lyophilized reagent well flushing step, the buffer fluid and the milled reagent mixture are aspirated into the bypass cache, dispensed into the lyophilized reagent well, aspirated into the bypass cache, and dispensed into the buffer well. In a further mixing step, an additional reagent is aspirated into the bypass cache and then dispensed into the buffer well. The lyophilized reagent nozzle sipper is extended into the buffer well to a second position that is between the first position and the bottom of the well. Subsequently, the mixture of the milled reagent, buffer fluid, and added reagent is aspirated and dispensed one or more times to mix the reagents.

[0080] In another aspect, a device includes a housing, a fluid manifold disposed within the housing, the fluid manifold including a plurality of channels fluidly connected to a lyophilized reagent nozzle sipper extending to a first position or a second position within different corresponding wells, each well being associated with a lyophilized reagent, the lyophilized reagent nozzle sipper contacting a hydrated reagent when in the second position, a reagent selector valve disposed within the housing and operably connected to at least two of the channels of the manifold, a bypass valve disposed within the housing and operably connected to the reagent selector valve, a bypass cache disposed within the housing and operably connected to the bypass valve, and a pump disposed within the housing, operably connected to a channel of the fluid manifold and fluidly connected to the bypass cache.

[0081] It should be understood that any features of this device can be combined in any desirable manner. Further, it should be understood that any combination of features of this device and / or features of this exemplary system and / or features of this method can be used together, and / or that any features from any of these aspects or any can be combined with any of the examples disclosed herein.

[0082] In this disclosure and the claims, when present, the use of indicators of order, such as (a), (b), (c), etc., should be understood not to convey any particular order or sequence (except to the extent that such order or sequence is explicitly indicated). For example, if there are three steps labeled (i), (ii), and (iii), it should be understood that, unless otherwise stated, these steps may be performed in any order (or, if not otherwise prohibited, even simultaneously). For example, if step (ii) involves the handling of an element generated in step (i), step (ii) can be seen as being performed at some point after step (i). Similarly, if step (i) involves the handling of an element generated in step (ii), the reverse should be understood.

[0083] It should be understood that specific aspects, modes, embodiments, variations, and features of the present disclosure are described below at various levels of detail to provide a substantial understanding of the technology. Unless otherwise described, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" and other forms is not limiting. The use of the term "having" and other forms is not limiting. When used in this disclosure, whether in a transitional phrase or in the body of a claim, the terms "include(s)" and "including" should be interpreted as having an open-ended meaning. That is, these terms should be interpreted as synonymous with the phrase "having at least" or "including at least".

[0084] Also, it should be understood that the use of "for", such as "a valve for switching between two flow paths", may be replaceable with phrases such as "configured to", such as "a valve configured to switch between two flow paths".

[0085] Terms such as "about," "approximately," "substantially," and "nominal," when used in reference to a quantity or similar quantifiable characteristic, are to be understood as including values within ±10% of the specified value, unless otherwise indicated.

[0086] In this disclosure, reference is made to the accompanying drawings, which form a part hereof and illustrate specific embodiments that can be implemented. These embodiments are described in detail to enable those skilled in the art to practice the disclosure, and other embodiments can be utilized and structural, logical, and electrical changes can be made without departing from the scope of the disclosure.

[0087] It is to be understood that all combinations of the foregoing concepts and additional concepts, as discussed in more detail herein, are intended to be part of the subject matter of the invention disclosed herein (provided such concepts are not mutually inconsistent). Specifically, all combinations of the claimed subject matter that appear at the end of this disclosure are intended to be part of the subject matter of the invention disclosed herein.

[0088] Although the preferred embodiments have been illustrated and described in detail herein, various changes, additions, substitutions, etc. can be made without departing from the spirit of the subject matter disclosed herein, and thus it will be apparent to those skilled in the art that these are considered to be within the scope of the invention as defined by the following claims.

Claims

1. It is a system, A fluid manifold comprising a plurality of lyophilized reagent nozzle shippers and bypass valves, wherein each lyophilized reagent nozzle shipper includes a distal tip and extends into a corresponding lyophilized reagent well containing the lyophilized reagent such that the distal tip does not contact the lyophilized reagent before hydration, but contacts the hydration reagent after hydration, and the bypass valves are fluidly connected to the lyophilized reagent nozzle shippers. A pump connected to the bypass valve, The present invention relates to a system comprising a control circuit operably connected to a lyophilized reagent nozzle shipper, a bypass valve, and a pump, the control circuit controlling the lyophilized reagent nozzle shipper, the bypass valve, and the pump to automatically hydrate the lyophilized reagent and homogenize the hydrated reagent.

2. The system according to claim 1, further comprising a bypass line between the bypass valve and the pump, wherein the bypass line is fluidly connected to the pump.

3. The system according to claim 1, further comprising a bypass cache between the pump and the bypass valve, wherein the bypass cache includes a heating chamber and the bypass cache is fluidly connected to the bypass valve.

4. The system according to claim 1, wherein the fluid manifold further comprises one or more hydration shippers, each of which comprises a distal tip and extends into a corresponding hydration reagent reservoir containing a hydration fluid.

5. The system according to claim 1, wherein the control circuit controls the pump to hydrate the freeze-dried reagent by aspirating a certain volume of the hydration fluid and dispensing the certain volume of the hydration fluid onto the freeze-dried reagent in the freeze-dried reagent well, thereby obtaining a hydrated reagent.

6. The system according to claim 1, wherein the control circuit controls the pump to dilute the hydrating reagent by aspirating a second volume of the hydrating fluid and dispensing the second volume of the hydrating fluid into the freeze-dried reagent well.

7. The system according to claim 1, wherein the flow rate when aspirating the second volume of hydration reagent is less than or equal to the flow rate when dispensing the second volume of hydration reagent.

8. The system according to claim 1, wherein the flow rate when aspirating the second volume of hydration reagent is less than the flow rate when dispensing the second volume of hydration reagent.

9. The system according to claim 1, wherein the control circuit controls the pump and the freeze-dried reagent nozzle shipper so aspirate the freeze-dried reagent nozzle shipper so aspirate the freeze-dried reagent nozzle shipper so aspirates

10. The system according to claim 1, wherein the control circuit controls the pump and the bypass cache to polish the homogeneous hydration reagent, the homogeneous hydration reagent is drawn into the bypass cache, heated, dispensed back into the freeze-dried reagent well, drawn into the bypass cache a second time, heated a second time, cooled, and dispensed into a buffer well containing buffer fluid.

11. The system according to claim 10, wherein the control circuit controls the pump to add the third component to the buffer well by aspirating a certain amount of the third component and dispensing the certain amount of the third component into the buffer well.

12. A method for using the system described in any one of claims 1 to 11, (a) A step of performing a hydration operation, A substep of operating the pump to draw in the hydrated fluid, A substep of instructing one of the plurality of lyophilized reagent nozzle shippers to extend to a first position in the corresponding lyophilized reagent well, A step comprising: operating the pump to dispense the hydrated fluid into the corresponding freeze-dried reagent well, thereby forming the hydrated reagent; (b) A step of performing a mixing operation, A substep of commanding one of the plurality of freeze-drying reagent nozzle shippers to extend to a second position in the corresponding freeze-drying reagent well, A method comprising the steps of: a substep of operating the pump to mix the hydration reagent.

13. (c) A step of performing a dilution operation before the mixing operation, A substep of operating the pump to draw in the dilution fluid, The method according to claim 12, further comprising the step of operating the pump to dispense the diluted fluid into the corresponding freeze-dried reagent well.

14. (d) A step of performing a polishing operation after the mixing operation, A substep of operating the pump to draw the hydration reagent into a bypass cache including a heating chamber, A substep of instructing the heating chamber to heat the hydration reagent, A substep of dispensing the hydration reagent back into the corresponding freeze-dried reagent well, A substep of operating the pump to draw the hydration reagent into the heating chamber of the bypass cache, A substep of instructing the heating chamber to heat the hydration reagent a second time, The method according to claim 12, further comprising the steps of: a substep of cooling the hydrated reagent, thereby forming a polished reagent.

15. (e) A step of performing a second mixing operation, A substep of operating the pump to dispense the polished reagent into a buffer well containing buffer fluid, A substep of operating the pump to aspirate a third component, A substep of operating the pump to dispense the third component into the buffer well, The method according to claim 12, further comprising the step of operating the pump to draw the solution into the buffer well and dispense it back into the buffer well, thereby mixing the solution.

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