Patent Description:
Existing bulk reagent reservoirs for use in robotic liquid handlers that are used to process and analyze biological samples, such as in the preparation of libraries of nucleic acid fragments (e.g., libraries of fragments derived from cellular DNA or RNA molecules) including next-generation sequencing (NGS) libraries, suffer from various drawbacks. One drawback emerges when the amount of a reagent in a kit does not provide enough overage of each reagent to use on automation. The dead volume (e.g., the amount in the reservoir enough to be uniform across the bottom of the well and be pipetted) is far too high in standard reagent reservoirs, which results in a decrease in the number of actual samples the kit can process. Stated differently, entropy and surface tension can create an unequal distribution of liquid across the bottom of traditional/existing bulk reservoirs as the volume decreases. This unequal distribution can cause local depletions of liquid as it is aspirated, resulting in unequal aspiration or completely missed aspiration by probes of a multi-channel pipettor.

<CIT> discloses assay trays for use with solid phase assays, and a machine for adding and removing liquids from wells of assay trays using automated pipettes. <CIT> describes a system and a method for handling and analysing liquids with a reaction well having tapered side sections and side walls, where liquids are aspirated from the deepest portion of the reaction well.

The bulk reagent reservoirs described herein address the drawbacks of existing reagent reservoirs. Briefly, the bulk reagent reservoirs described herein contain a slope from front to back on the bottom of the reservoir. This allows for at least eight pipettor tips (e.g., a <NUM> tip pipettor head at <NUM> spacing/tip that could still access the reservoir) with all tips above the slope from a multi-channel pipettor to simultaneously access a volume greater than, e.g., about <NUM>, or as little as about <NUM>µL. The slope will also contain a taper towards the single tip access point to gather the volume into a concise location for the single tip. After this volume is removed, a single tip from a single-channel pipettor or a single channel of a multi-channel pipettor will be able to access the remaining volume in order to reduce dead volume. The bulk reservoir design will allow increased sample quantity by minimizing dead volume. The sloped area will contain less than <NUM> of volume and can be left in the reservoir for non-volume critical reagents or user-supplied bulks such as water and ethanol.

The disclosure therefore relates to a method of transferring liquid using a robotic liquid handler as according to the features of the enclosed claim <NUM>.

Further, the disclosure relates to a method of transferring liquid using a robotic liquid handler, the method comprising:.

The disclosure also relates to a method of transferring liquid using a robotic liquid handler, the method comprising:.

And the disclosure relates to a method of transferring liquid from a bulk storage vessel using a robotic liquid handler, the method comprising:.

A further embodiment relates to the method above, wherein:
the multi-channel pipettor extends between the first end-wall and the second end-wall above the first volume; and.

the single-channel pipettor or a single channel of a multi-channel pipettor extends across the deep end.

Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings.

Making reference to <FIG>, reagent reservoir <NUM> has longitudinal axis <NUM> and a sloped bottom <NUM> along a width <NUM> of the reagent reservoir <NUM>. The sloped bottom <NUM> defines a shallow end <NUM> and a deep end <NUM> of the reagent reservoir <NUM>, where the shallow end <NUM> is proximal to a first side-wall <NUM> of the reagent reservoir <NUM> and the deep end <NUM> is proximal to a second side-wall <NUM> of the reagent reservoir <NUM> opposite the first side-wall <NUM>. The reagent reservoir <NUM> shown in <FIG> can have a lid (not shown) that can span from an outer edge <NUM> of the first side-wall <NUM> to an outer edge <NUM> of the second side-wall <NUM>. The lid functions to prevent contamination and to at least reduce evaporation of reagents located inside reagent reservoir <NUM>.

The reagent reservoir <NUM> also has protuberance <NUM>, which functions to elevate first end <NUM> relative to second end <NUM>. Protuberance <NUM> functions to keep the top of the reservoir level/horizontal when resting on a countertop or flat surface <NUM>. Protuberance <NUM> can take any suitable form, such as fin, tab or a notch, so long as it elevates first end <NUM> relative to second end <NUM>. Protuberance <NUM> creates an angle (θ) <NUM> between flat surface <NUM> and first end <NUM>. The angle θ can be any suitable angle, sch as an angle from about <NUM>° to about <NUM>°.

Reagent reservoir <NUM> optionally comprises volume markings <NUM> as shown in <FIG>.

<FIG> shows an end-view of reagent reservoir <NUM> from first end <NUM>. The end-view shown in <FIG> shows an example of a reagent reservoir <NUM> having a plurality of protuberances (in this case two tabs or fins) <NUM>A and <NUM>B, which elevate first end <NUM> relative to second end <NUM>. Further, the end view of this reagent reservoir shows that the reservoir can have a first channel <NUM> and a second channel <NUM> spanning width <NUM> of reagent reservoir <NUM> and divided by a dividing wall (numeral <NUM> in <FIG>), also spanning width <NUM>, and which serves to separate first channel <NUM> from second channel <NUM>. In short, the reagent reservoir can have multiple chambers, each with sloped bottom.

A top view of reagent reservoir <NUM>, without a lid, is shown in <FIG> shows reagent reservoir <NUM>, first channel <NUM> and second channel <NUM>, spanning width <NUM>. The channels are separated by dividing wall <NUM>, which also spans width <NUM>. Also shown in <FIG> is lip <NUM>, which goes around a top portion of reagent reservoir <NUM> and on which a lid, when present, can sit. First channel <NUM> and second channel <NUM> can slope at a constant slope from first end <NUM> to second end <NUM>. <FIG> is an example of a reagent reservoir <NUM> having channels <NUM> and <NUM> (not shown in <FIG>) having a constant slope from first end <NUM> to second end <NUM>. Or the slope of first channel <NUM> and second channel <NUM> can vary from first end <NUM> to second end <NUM>. The specific slope can be chosen based on what may be required for aqueous liquids to effectively pool in the shallow end (e.g., second end <NUM>), while not being so steep that suspended particles (e.g. the magnetic beads described herein) would concentrate at that same end (e.g., second end). This slope can also affect the volume accessible to all tips versus a single tip of a pipettor.

Further, first channel <NUM> can have a floor <NUM> having any suitable shape. Likewise, second channel <NUM> can have a floor <NUM> having any suitable shape, with floor <NUM> having the same shape or a different shape relative to floor <NUM>. In the example presented in <FIG>, floors <NUM> and <NUM> have substantially the same triangular shape from first end <NUM> to second end <NUM>.

<FIG> is a bottom view of reagent reservoir <NUM>. <FIG> shows reagent reservoir <NUM>, first channel <NUM> and second channel <NUM>, spanning width <NUM>. The channels are separated by dividing wall <NUM>, which also spans width <NUM>. Also shown in <FIG> is lip <NUM>, which goes around a top portion of reagent reservoir <NUM> and on which lid <NUM> sits. <FIG> also shows first around a top portion of reagent reservoir <NUM> and on which lid <NUM> sits. <FIG> also shows first and second protuberances (e.g., tabs or fins) <NUM>A and <NUM>B, which elevate first end <NUM> relative to second end <NUM>.

<FIG> is a cross section of <FIG> along an axis <NUM>A perpendicular to longitudinal axis <NUM> in <FIG>, at the shallow end <NUM>, looking down the longitudinal axis toward the shallow end <NUM> (not shown). <FIG> shows first and second protuberances (e.g., tabs or fins) <NUM>A and <NUM>B, which elevate first end <NUM> relative to second end <NUM> (not shown in <FIG>). The reagent reservoir <NUM> in <FIG> also has lip <NUM> which goes around a top portion <NUM> of reagent reservoir <NUM>. First channel <NUM> has a floor <NUM> and second channel <NUM> has a floor <NUM> having any suitable shape, such as a shape with decreasing cross-section along a width of the reagent reservoir from a first side-wall to a second side-wall, such as a V-shape, with floor <NUM> having the same shape or a different shape relative to floor <NUM>. In the example presented in <FIG>, floors <NUM> and <NUM> have substantially the same flat shape along width <NUM>. But floors <NUM> and <NUM> could also be, independently rounded from first end <NUM> to second end <NUM>.

Finally, <FIG> is a cross section of <FIG> along an axis <NUM>B perpendicular to longitudinal axis <NUM> in <FIG>, at the shallow end <NUM>, looking down the longitudinal axis toward the deep end <NUM> (not shown). The reagent reservoir <NUM> in <FIG> also has lip <NUM> which goes around a top portion <NUM> of reagent reservoir <NUM>. First channel <NUM> has a floor <NUM> and second channel <NUM> has a floor <NUM> having any suitable shape, such as a V-shape, with floor <NUM> having the same shape or a different shape relative to floor <NUM>. In the example presented in <FIG>, floors <NUM> and <NUM> have substantially the same flat shape along width <NUM>. Floors <NUM> and <NUM> could also be, independently rounded from first end <NUM> to second end <NUM>.

Reagent reservoirs contemplated herein, such as reagent reservoir <NUM>, can be made of any suitable material including, but not limited to, polymers such as polycarbonate, polyethylene, polypropylene, polyethylene terephthalate (PET), and the like. It should be understood that one portion of the reagent reservoirs contemplated herein can be made of a first material, while other portions can be made of a second material, so long as the first material is compatible with the second material.

The reagent reservoirs contemplated herein can be used in the context of robotic liquid handler <NUM> (see <FIG>). For explanatory purposes, robotic liquid handler <NUM> will mainly be described herein as a system for processing and analyzing biological samples, such as the preparation of libraries of nucleic acid fragments (e.g., libraries of fragments derived from DNA or RNA molecules) including but not limited to next-generation sequencing (NGS) libraries.

The reagent reservoirs described herein can be used in a method of transferring liquid using a robotic liquid handler, the method comprising: aspirating a first volume of liquid out of a reagent reservoir <NUM> using a multi-channel pipettor <NUM> (e.g., any pipettor with the ability to aspirate/dispense liquid into more than one channel simultaneously, which encompasses Span-<NUM> style multichannel pipettors with independent motion and aspirate/dispense functions between the different channels and other multichannel pipettors that do not have independent motion and aspirate/dispense functions. It should be immaterial if the multi-channel pipettor has independent probes versus fixed probes, so long as a fixed system allows for loading a single tip for accessing the sloped section and act as a single-channel pipettor or the independent system allows for enough vertical difference between probes to accommodate the slope. ) of the robotic liquid handler, the reagent reservoir <NUM> having a sloped bottom <NUM> along a width <NUM> of the reagent reservoir <NUM>, the sloped bottom <NUM> defining a shallow end <NUM> and a deep end <NUM> of the reagent reservoir <NUM>, wherein the shallow end <NUM> is proximal to a first side-wall <NUM> of the reagent reservoir <NUM>, wherein the deep end <NUM> is proximal to a second side-wall <NUM> of the reagent reservoir <NUM> opposite the first side-wall <NUM>; and aspirating a second volume of liquid out of the deep end <NUM> of the reagent reservoir <NUM> using a single-channel pipettor <NUM> (see <FIG>; or a single channel of a multi-channel pipettor) of the robotic liquid handler, wherein aspiration of the second volume out of the deep end <NUM> results in depletion of liquid in the shallow end <NUM> of the reagent reservoir <NUM>.

The multi-channel pipettor can be any suitable pipettor, including a four-channel pipettor such as those available from manufactures such as Genex Laboratory Products, Eppendorf, Raning, and Gilson. Further, the multi-channel pipettor can be positioned in any suitable position of the deep end <NUM> of the reagent reservoir <NUM>. For example, making reference to <FIG>, the multi-channel pipettor can be positioned such that a plurality of pipettor tips <NUM> of the multi-channel pipettor are arranged longitudinally, along longitudinal axis <NUM>, as shown. Accordingly, aspirating the first volume <NUM> of liquid out of the reagent reservoir using the multi-channel pipettor <NUM> of the robotic liquid handler <NUM> can include aspirating the first volume using the multi-channel pipettor along the width (e.g., along longitudinal axis <NUM>) of the reagent reservoir <NUM> from the first side-wall <NUM> to the second side-wall <NUM>.

The single-channel pipettor (or a single channel of a multi-channel pipettor) can be any suitable pipettor and can be positioned in any suitable position of the shallow end <NUM>. Making reference to <FIG>, the single-channel pipettor or a single channel of a multi-channel pipettor can be positioned at a deepest portion <NUM>A of reagent reservoir <NUM>, wherein the deepest portion <NUM>A is proximal to the second side-wall <NUM> of the reagent reservoir <NUM>.

The methods described herein can further comprise depositing particles (e.g., beads, such as magnetic beads) in the reagent reservoir <NUM> first, followed by depositing reagents/liquid therein or by depositing beads reagents/liquid (already in the reagent reservoir); and allowing the beads to become suspended in the liquid; wherein the beads settle (e.g., evenly) along the sloped bottom, where the slope can be from about <NUM>° to about <NUM>°, as described in <FIG> in terms of the angle θ. The beads can be, for example, magnetic particles. Suitable magnetic particles for use in the methods described herein include, but are not limited to AMPure XP beads available from Beckman Coulter, Inc. , Brea, CA. Suitable magnetic particles also include those described in <CIT>; <CIT>; and <CIT>, and in <CIT>.

When beads are deposited in reagent reservoir <NUM>, maintaining a uniform suspension in the reservoir for liquids or reagents that contain particles is desired. Over time, the particles will settle to the bottom of the reservoir. The slope of the reservoir is such that the particles will settle uniformly across the length of the reservoir without accumulating in the shallow end (or the deep end, for that matter). In this way, a multichannel pipettor can be used to efficiently resuspend the particles to re-create a uniform suspension just prior to use.

The magnetic particles of the disclosure can comprise a magnetic or a paramagnetic core, surrounded by a coating. In an example, the magnetic or a paramagnetic particles are coated with one or more layers of a non-magnetic material. The use of coated magnetic particles, having no exposed iron, on their surfaces, can eliminate the possibility of iron interfering with certain downstream manipulations of a sample. The coating can be, for example, a polymer layer, or a silica layer.

Example polymer layers can include polyethylene, polystyrene, poly methyl methacrylate, polyvinyl alcohol, or any other suitable polymer. Example silica layers can include silicon dioxide, borosilicate, soda lime, barium titanate, and other types of glass. The polymer or silica layer can be for adjusting the density of the magnetic particles. For example, the polymer or silica layer can adjust the density of the magnetic particles to be close to the density of the sample, for example, an aqueous sample (e.g., approximately <NUM>/cm<NUM>).

The coating can also comprise a ligand such as capture reagent or a functional group, including those mentioned herein, for selectively or non-selectively binding target analytes. The functional group can be for adsorbing biomolecules, such as nucleic acids, which can non-sequence-specifically and reversibly bind to the functional group coating the magnetic particles. The polynucleotides can be DNA, RNA, or polyamide nucleic acids (PNAs). In an example, the functional group is a carboxyl group. Various coatings comprising functional groups suitable for these purposes are described in <CIT>, <CIT>, and <CIT>. Any of the coatings described herein can be functionalized with surface chemicals as described herein, for example, with carbolic acid, streptavidin, amine, hydrazide, silanol, azide. And those can be further functionalized with biological molecules such as antibodies, enzymes, DNA or RNA fragments, catalysts, etc..

In some examples, the coating can comprise a capture reagent. The capture reagent can be for capturing an analyte in a sample. The surface of the magnetic particles can be coated with a capture reagent that is a suitable ligand or receptor (e.g., antibodies, lectins, oligonucleotides, other affinity groups, or any of the other capture reagents mentioned herein), which can selectively bind a target analyte or a group of analytes in a mixture. In some examples, the capture reagent can be an antibody.

Those of skill will recognize that any number of capture reagents can be used for this purpose, e.g. aptamers, nanoparticles, binding proteins, and the like. The capture reagent can be designed to capture a specific analyte or a specific panel of analytes, e.g., drug panel or endocrine panel, etc..

Alternatively, the ligand can include an enzyme. In some embodiments the enzyme can be linked to the coating in order to selectively interact with a substrate of that enzyme. Upon interacting with the substrate, the enzyme can function to modify, degrade or digest the substrate. This can lead to generation of a substance of interest through enzyme's action or to remove a substrate from a sample. According to various embodiments, the enzyme can be trypsin.

As discussed herein, the reagent reservoir <NUM> can be used in the context of a robotic liquid handler. The reagent reservoir <NUM> can have protuberances, such as tabs or fins 120A and <NUM> that can be aligned with slots on the liquid handler to position the reagent reservoir on a deck of the robotic liquid handler, as described in greater detail herein. Tabs or fins 120A and <NUM> can be engaged with a deck of the robotic liquid handler <NUM> to elevate the shallow end <NUM> above the deep end <NUM> and the first side-wall <NUM> and the second side-wall <NUM> stand vertical on the deck.

<FIG> is a high-level block diagram of robotic liquid handler <NUM>. Robotic liquid handler <NUM> can comprise control computer <NUM> operatively coupled to structure <NUM>, transport device <NUM>, processing apparatus <NUM> and thermalcycler system <NUM>. Input/output interfaces may be present in each of these devices to allow for data transmission between the illustrated devices and external devices. Robotic liquid handler <NUM> can comprise a fluid handling system as described herein. Fluids can include various liquids such as reagents and the like. An example of a processing system in which the present disclosure can be implemented is the Biomek i7 Automated Workstation marketed by the Beckman Coulter, Inc. of Brea, California.

For explanatory purposes, robotic liquid handler <NUM> will mainly be described as a system for processing and analyzing biological samples, such as the preparation of libraries of nucleic acid fragments (e.g., libraries of fragments derived from DNA or RNA molecules) including, but not limited to, next-generation sequencing (NGS) libraries.

Structure <NUM> can include a housing (e.g., housing <NUM> of <FIG>), legs or casters to support the housing, a power source, deck <NUM> loadable within the housing, and any other suitable feature. Deck <NUM> can include a physical surface (e.g., platform <NUM> of <FIG>) such as a planar physical surface upon which components can be placed and accessed for experiments, analyses, and processes. In some instances, deck <NUM> can be a floor or a tabletop surface. Deck <NUM> can be subdivided into a plurality of discrete deck locations (e.g., locations L1 - L16 of <FIG>) for placing different components. The locations can be directly adjacent or can be spaced apart from each other. Each deck location can include dividers, inserts, and/or any other support structure for separating the different deck locations and containing components. For example, <FIG> shows first location 905A, second location 905B, and third location 905C on deck <NUM>, though additional locations can be included. One or more of locations 905A -905C can be loaded with a carousel (e.g., carousel <NUM> of <FIG>) or one or more reagent reservoirs or liquid vessels (e.g. reaction vessel <NUM> of <FIG>) that can include spaces for holding one or more components. Structure <NUM> can additionally include a motor or another device for rotating the carousel relative to deck <NUM> to facilitate, among other things, interaction with transport device <NUM>, a reagent reservoir and thermalcycler system <NUM>. Furthermore, motor of structure <NUM>, or an additional motor of structure <NUM>, can be used to rotate individual vials loaded onto deck <NUM>, a tray or reagent reservoir loaded on deck <NUM> or a carousel located on deck <NUM>.

Transport device <NUM>, which can comprise a trolley, bridge or carriage system having moving capabilities in x and y directions and hoisting capabilities in a z direction, which can represent multiple transport devices, can prepare and/or transport components between deck <NUM> and processing apparatus <NUM>, as well as between different locations on deck <NUM>. Examples of transport devices may include conveyors, cranes, sample tracks, pick and place grippers, laboratory transport elements that can move independently (e.g., pucks, hubs or pedestals), robotic arms, and other tube or component conveying mechanisms. In some embodiments, transport device <NUM> includes a pipetting head configured to transfer liquids. Such a pipetting head may transfer liquids within removable pipette/pipettor tips and may include grippers suitable for grasping or releasing other labware, such as microwell plates or lids for reagent reservoir <NUM>.

Processing apparatus <NUM> can include any number of machines or instruments for executing any suitable process. For example, processing apparatus <NUM> can include an analyzer, which may include any suitable instrument that is capable of analyzing a sample such as a biological sample. Examples of analyzers include spectrophotometers, luminometers, mass spectrometers, immunoanalyzers, hematology analyzers, microbiology analyzers, and/or molecular biology analyzers. In some embodiments, processing apparatus <NUM> can include a sample staging apparatus. A sample staging apparatus can include a sample presentment unit for receiving sample tubes with biological samples, a sample storage unit for temporarily storing sample tubes or sample retention vessels, a means or device for aliquotting a sample, such as an aliquottor, a means for holding at least one reagent pack comprising the reagents needed for an analyzer, and any other suitable features.

Thermalcycler system <NUM> can be positioned relative to deck <NUM> and can be configured to receive a liquid vessel. Reaction vessels (e.g., <NUM> in <FIG>) can be loaded manually into thermalcycler system <NUM> or via transport device <NUM>. Thermalcycler system <NUM> can be configured to provide a plurality of different heating zones, as will be discussed below in greater detail with reference to <FIG>, that can heat different portions of liquid vessels to different temperatures. For example, thermalcycler system <NUM> can comprise three stacked or vertical levels of heating to provide top, middle and bottom heating zones to liquid vesses. Thus, for example, depending on the amount and type of liquid disposed in a liquid vessel, different amounts of heating can be applied, such as to perform thermalcycling and incubating processes.

Robotic liquid handler <NUM> can be provided with an imaging system, e.g., a camera, to read labels of reagent vials loaded onto deck <NUM>. The imaging system can ensure that all portions of any single reagent vial label loaded into robotic liquid handler <NUM> is in view of at least one camera. Thus, for a reagent vial label that is wrapped around the circumference of a reagent vial, one or more imaging devices, with or without the use of mirrors or turntables, can have complete three-hundred-sixty-degree view of each reagent vial. The imaging device can be any suitable device for capturing an image of deck <NUM> and any components on deck <NUM> or the entirety of structure <NUM>. The imaging device can comprise one of a plurality of imaging devices mounted to or nearby structure <NUM>. In additional examples, multiple imaging devices can be mounted to obtain multiple views of reagent vials disposed on deck <NUM>. For example, the imaging device can be any suitable type of camera, such as a photo camera, a video camera, a three-dimensional image camera, an infrared camera, etc. Some embodiments can also include three-dimensional laser scanners, infrared light depth-sensing technology, or other tools for creating a three-dimensional surface map of objects and/or a room. In examples, the imaging device can utilize slit-scan technology to produce panoramic images. Images taken by the imaging system can be analyzed for recognition of visual indicators, e.g., numbers, text or symbols, by the fluid handling system.

Control computer <NUM> can control the processes run on processing system <NUM>, initially configure the processes, and check whether a component setup has been correctly prepared for a process. Control computer <NUM> can control and/or transmit messages to processing apparatus <NUM>, transport device <NUM>, and/or thermalcycler system <NUM>. Control computer <NUM> can comprise data processor 908A, non-transitory computer readable medium 908B and data storage 908C coupled to data processor 908A, one or more input devices 908D and one or more output devices 908E. Although control computer <NUM> is depicted as a single entity in <FIG>, it is understood that control computer <NUM> may be present in a distributed system or in a cloud-based environment. Additionally, embodiments allow some or all of control computer <NUM>, processing apparatus <NUM>, transport device <NUM>, and/or thermalcycler system <NUM> to be combined as constituent parts in a single device.

Output device 908E can comprise any suitable devices that can output data. Examples of output device 908E can include display screens, video monitors, speakers, audio and visual alarms and data transmission devices. Input device 908D can include any suitable device capable of inputting data into control computer <NUM>. Examples of input devices can include buttons, a keyboard, a mouse, touchscreens, touch pads, microphones, video cameras and sensors (e.g., light sensor, position sensors, speed sensor, proximity sensors).

Data processor 908A can include any suitable data computation device or combination of such devices. An example of a data processor may comprise one or more microprocessors working together to accomplish a desired function. Data processor 908A can include a CPU that comprises at least one high-speed data processor adequate to execute program components for executing user and/or system-generated requests. The CPU may be a microprocessor such as AMD's Athlon, Duron and/or Opteron; IBM and/or Motorola's PowerPC; IBM's and Sony's Cell processor; Intel's Celeron, Itanium, Pentium, Xeon, and/or XScale; ARM-based/family processors and/or the like processor(s).

Computer readable medium 908B and data storage 908C can be any suitable device or devices that can store electronic data.

Computer readable medium 908B can comprise code, executable by data processor 908A to perform any suitable method. For example, computer readable medium 908B can comprise code, executable by processor 908A, to cause processing system <NUM> to perform automated reagent processing and heating methods including mixing of various reagents within labware to different levels, heating the labware to different levels, adding additional reagents and performing additional heating using thermalcycler system <NUM>.

Computer readable medium 908B can comprise code, executable by data processor 908A, to receive and store process steps for one or more protocols (e.g., a protocol for processing a biological sample or a protocol for a library construction process), as well as to control thermalcycler system <NUM>, structure <NUM>, transport device <NUM>, and/or processing apparatus <NUM> to execute the process steps for the one or more protocols, such as those described with reference to the Examples section below. Computer readable medium 908B can also include code, executable by data processor 908A, for receiving results from processing apparatus <NUM> (e.g., results from analyzing a biological sample) and for forwarding the results or using the results for additional analysis (e.g., diagnosing a patient). Additionally, computer readable medium 908B can comprise code, executable by data processor 908A, for obtaining an image of deck <NUM>, identifying information in the images of deck <NUM>, deciphering information in the images using information stored in data storage 908C or computer readable medium 908B by comparing the deciphered information to information contained in protocol 908F, and loading thermalcycler system <NUM> accordingly.

Data storage component 908C can be internal or external to control computer <NUM>. Data storage component 908C can include one or more memories including one or more memory chips, disk drives, etc. Data storage component 908C can also include a conventional, fault tolerant, relational, scalable, secure database such as those commercially available from Oracle™ or Sybase™. Data storage 908C can store protocols 908F and images <NUM>. Data storage component 908C can additionally include instructions for data processor 908A, including protocols. Computer readable medium 908B and data storage component 908C can comprise any suitable storage device, such as non-volatile memory, magnetic memory, flash memory, volatile memory, programmable read-only memory and the like.

Protocols 908F in data storage component 908C can include information about one or more protocols. A protocol can include information about one or more processing steps to complete, components used during the process, a component location layout, loading of reagent reservoir <NUM> and/or any other suitable information for completing a process. For example, a protocol can include one or more ordered steps for processing a biological sample or processing a DNA library. A protocol can also include steps for preparing a list of components before starting the process. The components can be mapped to specific locations in the reagent reservoir (e.g., reagent reservoir <NUM>), in the carousel (e.g., carousel <NUM>) or in a microplate mounted to ambient storage location containing index adapters or other reagents in microplates where transport device <NUM> can obtain the components in order to transport them or the container they are loaded into to processing apparatus <NUM> or thermalcycler system <NUM>. This mapping can be encoded as instructions for operating transport device <NUM>, such as instructions directing a pipettor to aspirate a volume of liquid from a labware in the carousel and to dispense the volume at a predetermined destination, and the mapping can also be represented by a virtual image shown to a user such that the user can place the components on deck <NUM>, the reagent reservoir and the carousel. Robotic liquid handler <NUM> can be used for multiple processes (e.g., multiple different sample processes or preparation procedures). Accordingly, information about multiple protocols 908F can be stored and retrieved when needed. Components on deck <NUM>, the reagent reservoirs and the carousel can be rearranged, changed, and/or replenished as necessary when changing from a first process to a second process, or when re-starting a first process.

Images <NUM> in data storage 908C can include a real-world or simulated visual representation of deck <NUM>, the reagent reservoirs and the carousel, as well as of components disposed on or in deck <NUM>, the reagent reservoirs and the carousel and labels disposed on those components. In each image, deck <NUM>, the reagent reservoirs and the carousel can be shown in a ready state for beginning a certain process, with components for executing a protocol placed in locations accessible to transport device <NUM>. Each of images <NUM> can be associated with a specific protocol from the stored protocols 908F. There can be a single image for certain protocol or there can be multiple images (e.g., from different angles, with different lighting levels, or containing acceptable labware substitutions in some locations) for a certain protocol. Images <NUM> can be stored as various types or formats of image files including JPEG, TIFF, GIF, BMP, PNG, and/or RAW image files, as well as AVI, WMV, MOV, MP4, and/or FL V video files.

Deck <NUM> can be subdivided into a plurality of discrete deck locations for staging different components. The discrete locations may be of any suitable size. An example of deck <NUM> with a plurality of locations is shown in <FIG>. Deck <NUM> in <FIG> shows separate areas numbered L1 through L16, as well as thermal cycler <NUM>, which can operate as a separate location for separate types of components or packages of components. Deck <NUM> can have additional locations or fewer locations as desired. While these locations can be numbered or named, they may or may not be physically labeled or marked on deck <NUM> in physical embodiments of the system.

Images, such as images <NUM>, can be used to verify if the proper components are loaded into deck <NUM>, the reagent vessels and lids, if needed, the carousel and thermalcycler system <NUM> for completing protocol 908F programmed into processing system <NUM> by an operator, and if those components are located in correct positions for executing the programmed protocol, if required by the protocol. As discussed herein, processing system <NUM> can thereafter execute mixing procedures for liquids loaded into reagent reservoirs, e.g., reagent reservoir <NUM>, and controllably heat the reagent reservoir using thermalcycler system <NUM> in a variety of different manners depending on the liquids loaded into the reagent reservoir, thereby eliminating the need for having different types and sizes of reagent reservoirs and different capacities and configurations of thermalcycler systems included in processing system <NUM>.

<FIG> is perspective view of liquid handling system <NUM> that can comprise an example of robotic liquid handler <NUM> of <FIG>. Liquid handling system <NUM> can comprise housing <NUM>, carousel <NUM>, reaction vessel <NUM>, imaging device <NUM> and thermalcycler system <NUM>. Note, components of <FIG> are not necessarily drawn to scale for illustrative purposes. Housing <NUM> can comprise a plurality of walls or panels that form an enclosure into which carousel <NUM> can be positioned. The enclosure can have an opening over which door or other access point for a user <NUM> can be positioned to encapsulate carousel <NUM>, imaging device <NUM> and thermalcycler system <NUM> within the enclosure. Housing <NUM> can additionally include platform <NUM> on which a deck, such as deck <NUM> (<FIG>) or deck <NUM> (<FIG>) can be positioned. The deck can include a slot or socket for receiving carousel <NUM> and one or more of reagent reservoirs <NUM>. In examples, the slots or sockets can be configured to hold carousel <NUM> and reagent reservoir <NUM> in a predetermined or know position relative to imaging device <NUM>. Platform <NUM> can hold the deck in a predetermined or known position relative to imaging device <NUM>. Housing <NUM> can additionally comprise space for holding controller <NUM>, such as those of control computer <NUM> (<FIG>). Controller <NUM> can be configured to communicate with network <NUM>, such as via a wireless or wired communication link.

Imaging device <NUM>, which may comprise imaging device described with reference to of <FIG>, can be located within housing <NUM> in a stationary location. One or more imaging devices <NUM> can be configured to point at a single location or multiple locations in housing <NUM>. Simultaneously, a pipettor of transport device <NUM> or processing apparatus <NUM> (<FIG>) can be located within housing <NUM> to access a location of carousel <NUM>. Transport device <NUM> can additionally be configured to move reagent reservoir <NUM> into thermalcycler system <NUM>. Carousel <NUM> can spin or rotate to present different locations to the pipettor and imaging device <NUM>. In other examples, imaging device <NUM> can be mounted within housing <NUM> to move a viewing area over different portions of the interior of housing <NUM>.

Controller <NUM> can be configured to execute a protocol for components loaded into carousel <NUM> and reagent reservoir <NUM> and loaded onto the deck within housing <NUM>. In order for controller <NUM> to perform one or more sequences of steps on a set of vials loaded into carousel <NUM> and reagent reservoir <NUM> per the protocol, controller <NUM> should know the location of each vial within carousel <NUM> and reagent reservoir <NUM>, e.g., the contents of each vial at each location within carousel <NUM> and reagent reservoir <NUM>. As discussed herein, controller <NUM> can be configured to operate imaging device <NUM> to obtain images of carousel <NUM> and reagent reservoir <NUM> and components loaded therein. In particular, carousel <NUM> can be loaded with vials of material, wherein each vial can have a label that provides identifying information as to the contents of each vial, a set of vials to which each vial belongs, a manufacturer of the set of vials, one or more protocols for liquid handling system <NUM> to execute with the set of vials, etc. Images of the vial labels can be read by controller <NUM> to recognize information presented in the labels. The information read from the labels can be compared to information, such as information obtained from network <NUM>, stored in a computer readable medium, such as medium 908B of <FIG>. The information stored in the computer readable medium can include a protocol for the set of vials that includes one or more sequences of steps for interacting with the set of vials, such as an order for which transport device <NUM> can interact with each vial, such as for moving reagents into carousel <NUM> and reagent reservoir <NUM> and therebetween.

Reaction vessels <NUM> can be moved into thermalcycler system <NUM>, either manually or automatically by transport device <NUM>. Controller <NUM> can operate thermalcycler system <NUM> to execute or partially execute various protocols and protocol steps. Controller <NUM> can operate thermalcycler system <NUM> and transport device <NUM> to heat liquid vessels loaded into thermalcycler system <NUM>. Thermalcycler system <NUM> can comprise a plurality of heating zones and reaction vessel can have a geometry forming a plurality of different shaped storage volumes, that each can have a different wall thickness for interacting with the heating zones. As such, a single thermalcycler system <NUM> and a single reaction vessel can be used to perform a large quantity of procedures using the different combinations of heating zones and storage volumes without the need for additional equipment or reaction vessels, such as those described in the Examples section below.

<FIG> is plan view of deck <NUM> for loading onto platform <NUM> of housing <NUM> of <FIG>. Deck <NUM> can include spaces or locations for various components, including carousel <NUM>. Imaging device <NUM> can be mounted within housing <NUM> relative to platform <NUM> such that imaging device can produce a field of view that covers all of platform <NUM>. However, in various examples, the field of view can be configured to cover only portions of platform <NUM> and multiple imaging device can be used or an articulating imaging device can be used that can move the field of view across platform <NUM> to different locations to achieve total coverage. Likewise, a transport system, such as transport device <NUM> of <FIG>, can be configured to reach the entirety of platform <NUM>.

<FIG> shows deck <NUM> including locations numbered L1 - L16, as well as other components such as thermalcycler system <NUM>, which can operate as a separate location for separate types of components or packages of components. Examples of deck <NUM> can have additional locations or fewer locations, as desired. While these locations can be numbered or named, the locations may or may not be physically labeled or marked on deck <NUM> in physical embodiments of liquid handling system <NUM>. In examples of liquid handling system <NUM>, some or all of the locations can be occupied by a pre-defined type of component according to a certain protocol. For example, locations L1 - L10 can comprise storage locations for pipette tip racks <NUM> and <NUM> (e.g., pipette tips of various volumes), and location L11 can be loaded with carousel <NUM>. Location L12 can comprise a cold reagent storage area for reaction vessels, lids, and plugs. Location L13 can comprise a warm reagent storage area for reaction vessels. Location L15 can comprise a storage area for reagent reservoirs <NUM>. Location L14 can comprise an reaction vessel stack storage area. Location L16 can comprise a waste storage area for bin <NUM>. Some of locations L1 - L16 can include the same type of component. The components can comprise test tubes, microwell or microtiter plates, pipette tips, plate-lids, reagent reservoirs <NUM> or any other suitable labware component. The components can also comprise an item of laboratory equipment, such as a shaker, stirrer, mixer, temperature-incubator, vacuum manifold, magnetic plate, thermalcycler, centrifuge or the like. In examples, one or more locations can be physically part of structure <NUM> (<FIG>), housing <NUM> (<FIG>) or deck <NUM> (<FIG>), or can be a separate component disposed on platform <NUM>. Each of locations L1 - L16 can be accessed by transport device <NUM> (<FIG>). For example, locations L1 - L16, and thermal cycler <NUM> can be physically separate from structure <NUM> or deck <NUM>.

Imaging device <NUM> can be configured to recognize the presence of one or more components at each of locations L1 - L16, the presence of carousel <NUM> at location L11, and the presence of reagent reservoirs <NUM> at location L15, for example. Furthermore, imaging device <NUM> can be configured to read information from the one or more components located at each of locations L1 - L16. Components, e.g., vials of liquid, can be loaded into carousel <NUM> in a desired manner, e.g., according to a protocol and liquid therefrom, or from another location, can be loaded into one of reaction vessels <NUM> for loading into thermalcycler system <NUM> according to the protocol. Images of carousel <NUM> taken by imaging device <NUM> can be used to read information from labels of vials loaded into carousel <NUM>. Thereafter, thermalcycler system <NUM> can execute a heating method, such as those discussed with reference to the Examples section below, to heat the liquid loaded into the thermalcycler system <NUM> according to the protocol.

Other methods contemplated herein include a method of transferring liquid using a robotic liquid handler, the method comprising:
filling a reagent reservoir with a liquid such that a sloped bottom of the reagent reservoir, wherein the sloped bottom forms a deep end and a shallow end; removing a first portion of the liquid from the reagent reservoir using a multi-channel pipettor having a first pipettor tip extending into a first position near the shallow end and a second pipettor tip extending into a second position near the deep end; and removing a second portion of the liquid from the reagent reservoir using a multi-channel pipettor having a single pipettor tip extending into a second position near the deep end or a pipettor tip of a single-channel pipettor or a single channel of a multi-channel pipettor extending into the deep end. Removing the first portion of the liquid from the reagent reservoir using the multi-channel pipettor can result in the liquid emptying from the shallow end of the reagent reservoir. The removing the first portion of the liquid from the reagent reservoir can further comprise repeating aspiration of liquid from the second volume with the second pipettor. The removing a second portion of the liquid from the reagent reservoir using a multi-channel pipettor having a single pipettor tip extending into a second position near the deep end or a pipettor tip of a single-channel pipettor extending into the deep end can result in removing all of the liquid in the first volume.

Still other methods contemplated herein include a method of transferring liquid using a robotic liquid handler, the method comprising:.

Additional methods contemplated herein include a method of transferring liquid from a bulk storage reservoir using a robotic liquid handler, the method comprising:
adding liquid to the bulk storage reservoir such that:.

In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading can occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and can include modification thereto and permutations thereof.

The present invention can be better understood by reference to the following examples which are offered by way of illustration. The present invention is not limited to the examples given herein.

The bulk reagent reservoirs described herein decrease the dead volume/waste while allowing the systems described herein to process samples in a way that increases speed and throughput. The bulk reagent reservoirs can have a dead volume of <NUM>µL if accessed by <NUM> tip, and approximately <NUM>µL when using <NUM> tips. A <NUM>% grade from horizontal at the bottom portion of the reservoir can allow the decreasing volume in the reservoir over the application run to pool at the lowest point at the back of the reservoir. At the beginning of a method, reservoirs can be filled with the estimated volume of a reagent needed to complete the application. As reagent is aliquoted from the bulk reservoir and volume decreases, the application can track the remaining amount of liquid in the reservoir. When the automation reaches a point where the next pipetting action will take the calculated remaining volume below <NUM>µL, the systems described herein can switch from using <NUM> tips, to using <NUM> tip. This reduces the dead volume required by the application to use the reservoir and allows the automation to process samples efficiently.

If the user wishes to run <NUM> samples and plans to use <NUM>µL sample of AmpureXP beads, the required volume for the run would be <NUM>µL total assuming no dead volume. Dead volume is estimated at <NUM>µL in the bulk reservoir when we program the pipettor to run as follows:.

By utilizing the reagent reservoirs described herein, the dead volume needed for a bulk reagent can be reduced by approximately <NUM>µL. This is especially important with reagents that come in application kits that are not packaged for automated systems. Low volume overages are a problem when automating applications. The reagent reservoirs described herein will enable the system to reduce the cost of a run. Using standard costs, the reagent described above would cost the user $<NUM>/mL. The reagent reservoirs described wherein will reduce cost from $<NUM> to $<NUM> per <NUM> sample run.

To wash the beads aliquoted from above, the user will utilize <NUM>µL of <NUM>% Ethanol. The total volume needed would be <NUM>*<NUM>. The total volume for the run would be <NUM> total assuming no dead volume. Dead volume is estimated at <NUM>µL in the bulk reservoir. When programmed as follows:.

The system could be programmed for a larger dead input in this case if the choice was to optimize for speed. This would depend on the desire for a speed over dead volume as well as the cost of the reagent being pipetted. The bulk reagent reservoir design allows for the reduction in the volume needed. In the case above, it is not the cost that is a concern. However, ethanol is flammable. Using the bulk reagent reservoir will decrease the volume of harmful chemicals that are deployed on our pipetting systems. This will also reduce the amount of hazardous waste created by an automation platform.

This example uses Illumina TruSight Oncology <NUM> (Document number <NUM> v02) Utilizing the <NUM> DNA ONLY workflow:
SPB (Sample Prep Beads) will be used in a bulk reagent reservoir, one time per <NUM> day in the <NUM> day protocol for library creation and enrichment.

Day <NUM> Library Generation: The standard protocol calls for the addition of <NUM>µL beads to each well to clean up the ligation reaction. The systems described herein can use millitips to pipette mix the bead solution by aspiration and dispensation of the bead mixture until the beads are fully suspended in the solution. The speed of the aspiration and dispensation will push beads off the bottom of the bulk reservoir. When tested in the lab, the beads do not "slide" down the slope of the reservoir if we maintain the <NUM>% grade. The Ampure XL beads do not fall out of solution very quickly. Only <NUM> mix per transfer group will be needed.

Total volume needed for the bulk reservoir: <NUM> ul/sample plus 50ul dead volume; total volume in reservoir is: <NUM>µL.

Day <NUM> (lllumina manual page <NUM>): The standard protocol calls for the addition of 110ul beads to each well to clean up the amplified enriched library. The NGeniuS system will use millitips to pipette mix the bead solution by aspiration and dispensation of the bead mixture until the beads are fully suspended in the solution. The Ampure XL beads do not fall out of solution very quickly. Only <NUM> mix per transfer group will be needed.

Total volume needed for the bulk reservoir: <NUM> ul/sample plus dead volume Total volume in reservoir is: 2690ul.

The described procedure described in this Example can also be used for nonmagnetic reagents in the Illumina kit (RSB-Resuspension Buffer,<NUM>% EtOH-Ethanol, EEW-Enhanced Enrichment Wash, LNA1-Library Normalization Additives (contains formamide) and other magnetic bead solutions (LNB1-Library Normalization Beads and SMB-Streptavidin Magnetic Beads).

LNA1 reagent listed above does emit gas and is dangerous if inhaled. It is wise to keep volumes as low as possible for use on the system.

An experiment was conducted to determine whether beads (e.g., AMPureXP and Stretavidin beads) accumulate at the deeper end of the reagent reservoir when they are added to the reagent reservoir and allowed to settle. To that end, two separate reagent reservoirs were prepared: <NUM> of AMPureXP was added to one section and <NUM> of AMPureXP was added to a the second section. Photos were taken of the sides of the trough before settling. The reagent reservoir was covered with an adhesive seal and left to settle overnight. The next morning, the seal was removed. Photos (not included herein) showed that there does not appear to be any noticeable settling towards the deep end of the reagent reservoir when allowed to settle overnight.

This experiment was meant to investigate how difficult is it to resuspend beads (e.g., AMPureXP and Stretavidin beads) in the reagent reservoir. To that end, <NUM> of AmpureXP were added to one side of a reagent reservoir and the beads were allowed to settle overnight. The next morning a multi-channel pipettor, specifically a i5 Span-<NUM> available from Beckman Coulter, Brea, CA, was used to resuspend the beads that had settled. After three to four mixes the beads appeared to be completely resuspended.

The AmpureXP suspension from Example <NUM> was completely removed from the reagent reservoir and <NUM>µL were returned to the reservoir. Utilizing a modified pipetting template, eight <NUM>µL of AmpureXP from the <NUM>µL suspension were added to eight wells of a PCR plate, removing <NUM>µL of the <NUM>µL from the reagent reservoir. Pipetting was successful for all eight samples.

Claim 1:
A method of transferring liquid using a robotic liquid handler (<NUM>), the method comprising:
aspirating a first volume of liquid (<NUM>) out of a reagent reservoir (<NUM>) using a multi-channel pipettor (<NUM>) of the robotic liquid handler,
the reagent reservoir having a sloped bottom (<NUM>) along a length of the reagent reservoir (<NUM>), the sloped bottom (<NUM>) defining a shallow end (<NUM>) and a deep end (<NUM>) of the reagent reservoir (<NUM>),
wherein
the shallow end (<NUM>) is proximal to a first side-wall (<NUM>) of the reagent reservoir, wherein the deep end (<NUM>) is proximal to a second side-wall (<NUM>) of the reagent reservoir (<NUM>) opposite the first side-wall (<NUM>);
and
then aspirating a second volume of liquid out of the deep end (<NUM>) of the reagent reservoir (<NUM>) using a single-channel pipettor (<NUM>) or a single channel of a multi-channel pipettor of the robotic liquid handler,
wherein
aspiration of the second volume out of the deep end (<NUM>) results in depletion of liquid in the shallow end (<NUM>) of the reagent reservoir (<NUM>); and
aspirating the first volume of liquid out of the reagent reservoir using the multi-channel pipettor (<NUM>) of the robotic liquid handler (<NUM>) comprises aspirating the first volume (<NUM>) using the multi-channel pipettor (<NUM>) along the width (<NUM>) of the reagent reservoir (<NUM>) from the first side-wall (<NUM>) to the second side-wall (<NUM>).