Apparatus, systems and methods for sequencing using capillary electrophoresis

Abstract

In one aspect, a biosequencing device is disclosed that includes a cartridge configured to be removed from an instrument. In various embodiments, the cartridge may include one or more capillaries suitable for capillary electrophoresis, a reservoir, and a pump. In various embodiments, the reservoir may contain a separation matrix. In various embodiments, the pump may load the capillary with the separation matrix. In another aspect, the biosequencing device may include one or more capillaries and an integrated valve assembly. In various embodiments, the integrated valve assembly may provide a polymer to the one or more capillaries.

Description

[0001] This application is a divisional application of the invention patent application with application number 201580019797.2, application date March 6, 2015, and invention name “Device, system and method for sequencing using capillary electrophoresis”. Technical Field

[0002] The present invention generally relates to devices, systems and methods for simplifying the user experience during sequencing of biological samples using capillary electrophoresis. Background Art

[0003] The present invention relates to a multi-capillary electrophoresis apparatus and components thereof. The present invention further relates to structures and mechanisms designed to accommodate multiple components of a multi-capillary electrophoresis apparatus.

[0004] Capillary electrophoresis devices typically provide certain major components, including, for example, a capillary array, a separation medium source for providing the medium to the capillaries, a sample injection mechanism, an optical detector assembly, electrodes, and an anode buffer source on one end of the capillaries and a cathode buffer source on the other end of the capillaries. Capillary electrophoresis devices also typically provide various heating components and areas to regulate the temperature of many of the aforementioned components. Regulating the temperature of many of these components can improve the quality of the results.

[0005] In order to provide the main components of the capillary electrophoresis device when adjusting the temperature of many of these components, the current capillary electrophoresis device uses multiple structures to accommodate these components, and these structures are connected or coupled together to provide a functional capillary electrophoresis device. The use of multiple structures has disadvantages. For example, each of the interconnected structures may require its own temperature adjustment mechanism, thus creating independent temperature control areas. Each of these areas will thus require an associated individual control mechanism. This multi-structure design will increase the total number of components in the device, complicate the temperature control scheme, and increase the probability of component failure due to the absolute number of components involved. Figure 1 One such design illustrating multiple temperature control zones TCM0 through TCM5 is shown.

[0006] The use of multiple interconnect structures is also not user friendly. For example, attaching a polymer source to a capillary array can be complicated, and there can be a risk of introducing air bubbles or other artifacts each time the array is detached and attached to a polymer source. Furthermore, the user, rather than the manufacturer, typically must attach a buffer source to the array, and must do so many times during the life of the capillary array.

[0007]

[0006] Therefore, there is a need to provide a capillary electrophoresis device having a reduced number of interconnected structures to reduce the number of necessary heating zones, reduce user handling of the structures, reduce the likelihood of component failure, and reduce the introduction of bubbles and other artifacts into the device. Summary of the invention

[0008] Disclosed are devices and methods for simplifying the user experience during sequencing of biological samples using capillary electrophoresis

[0009] Figure 2 A basic schematic representation of a portion of a conventional capillary electrophoresis apparatus is provided. Specifically, Figure 2 Capillary array, electrode assembly (anode and cathode), polymer source, buffer source, and polymer introduction mechanism (illustrated as a syringe pump) are described. As described, a coupling is provided to connect the capillary array assembly to a polymer / buffer structure, which includes a polymer source, a buffer source, an anode, and a syringe pump. As provided, the temperature controlled region controls only the enclosed capillary array assembly and the cathode. Thus, additional temperature control is required for the polymer source and the delivery path. In addition, the user must couple the capillary array assembly to the polymer / buffer structure.

[0010] In one aspect, a biological sequencing apparatus may include a sequencing instrument and a sequencing apparatus configured to Figure 3 The cartridge can include one or more capillaries suitable for capillary electrophoresis, a reservoir containing a polymer, and a pump for introducing the polymer into the capillary. It is known in the art that a sequencing instrument can include a container containing a polymer and a second container containing a buffer. The polymer contained in the reservoir of the cartridge can also serve as a buffer, thereby reducing the number of vessels in the instrument. Configuring the cartridge as a removable element can further enable a user to quickly replace the capillary and the polymer to speed up the evaluation of another sequencing analysis.

[0011] There are many advantages in providing the capillary array, cathode, polymer / buffer source and polymer introduction mechanism in a single cartridge. For example, such a single cartridge allows a buffer to be combined with a polymer as an anode buffer, and provides this polymer / buffer package in a small volume to fit in the cartridge and thus make it a low-use item. The polymer / buffer reservoir can advantageously be attached to the array when the cartridge is manufactured so that the customer only needs to install the entire cartridge in the capillary electrophoresis device. Alternatively, the cartridge can be designed so that the customer only needs to attach the polymer / buffer reservoir to the array before closing and installing the cartridge, so that for the life of the array and the polymer, the polymer / buffer reservoir 208 and the array 202 are combined.

[0012] In another aspect, an integrated valve assembly may be used. The integrated valve assembly may include an interface to couple the integrated valve to a capillary cartridge, an onboard manifold, containment for the polymer, printed electrodes, and two linear actuators. In various embodiments, the integrated valve assembly may be disposable. In other embodiments, the integrated valve assembly may be sufficient to run multiple sequencing experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For a more complete understanding of the principles disclosed herein and its advantages, reference is now made to the following description taken in conjunction with the accompanying drawings, in which:

[0014] Figure 1 A block diagram illustrating the current capillary electrophoresis apparatus.

[0015] Figure 2 A block diagram illustrating another capillary electrophoresis device.

[0016] Figure 3 A block diagram illustrating a sequencing device according to various embodiments.

[0017] Figures 4A to 4D A block diagram illustrating a disposable integrated valve assembly.

[0018] Figure 5 is a representation of a blister package according to various embodiments.

[0019] Figure 6 For similar Figure 3 Another embodiment of a polymer reservoir of the element depicted in .

[0020] Figure 7 is a block diagram of one embodiment that may be used to heat a polymer in a capillary.

[0021] Figure 8 Another embodiment is shown to illustrate an alternative for heating the capillary. DETAILED DESCRIPTION

[0022] Embodiments of devices, systems, and methods for providing a simplified workflow for nucleic acid sequencing are described in this specification.The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0023] Reference will now be made in detail to various aspects of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts.

[0024] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced with or without these specific details. In other cases, structures and devices are shown in block diagram form. Furthermore, those skilled in the art may readily appreciate that the particular order in which the methods are presented and performed is illustrative, and it is contemplated that the order may be changed and still remain within the spirit and scope of the various embodiments disclosed herein.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. When the definition of a term in an incorporated reference appears to be different from the definition provided in the present teachings, the definition provided in the present teachings shall prevail.

[0026] It will be understood that the use of the singular encompasses the plural unless expressly stated otherwise. Furthermore, the use of “comprise / comprises / comprising,” “contain / contains / containing,” and “include / includes / including” are not intended to be limiting. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative and do not restrict the present teachings.

[0027] Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, as those skilled in the art will appreciate, the present teachings encompass various alternatives, modifications, and equivalents.

[0028] In addition, when describing various embodiments, this specification may present methods and / or processes as steps in a specific order. However, to the extent that methods or processes do not rely on the specific order of the steps set forth herein, the methods or processes should not be limited to the specific order of the steps described. As will be appreciated by those skilled in the art, other orders of steps may be possible. Therefore, the specific order of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for methods and / or processes should not be limited to carrying out their steps in the order in which they are written, and what those skilled in the art can easily understand is that the order can be changed and still remain within the spirit and scope of the various embodiments.

[0029] In general, where instruments are provided to biological laboratories for biological sequencing, uncomplicated workflows can be beneficial for at least the following reasons. First, laboratories often have concerns about the economics of performing experiments that may include the use of instruments by less trained individuals. Second, less time required for user interaction with the instrument can increase the number of experiments that can be run in a day.

[0030] Figure 2 An embodiment of a common sequencer known in the art is depicted. Instrument 200 includes a fluidics section 210 and a capillary section 220. These sections are connected via a coupling 205 and can be thermally controlled independently of each other, as indicated by dashed outlines 201 and 202. Capillary section 220 can include a capillary 222, an optics access 224, and a cathode 226. Fluidics section 210 can further include a buffer container 212, a polymer container 214, a pump 216, an anode 211, and a valve 218. In various embodiments, capillary 222 can be a capillary array. Although instrument 200 may have advantages over Figure 1 , the fluidic section 210 may still represent a complex fluidic design, especially when a capillary array is coupled to the fluidic section.

[0031] refer to Figure 2 , valve 218 can be used to connect the outlet of the capillary or capillary array, polymer pump 216, buffer container 214 and polymer container 212. In essence, valve 218 can be configured to fill capillary 222 with fresh polymer, or to prepare the cartridge for electrophoresis. In various embodiments, the valve can be a custom-made, finely machined, light-transmitting plexiglass component. In various embodiments, the processed light-transmitting plexiglass can be referred to as a "gel block". Such a gel block can be found in, for example, a Model 310 sequencer purchased from Life Technologies in South San Francisco, California. In various embodiments, valve 218 can be constructed from two pieces. Each piece can be a custom-made, finely machined, light-transmitting plexiglass component. Such a two-piece embodiment can be found in, for example, a Model 3130 sequencer purchased from Life Technologies in South San Francisco, California.

[0032] In various embodiments, a lower cost solution may be utilized to provide the functionality just described. The inventors have discovered that the fluidic functionality of both the Model 310 and Model 3130 clear glass assemblies can be largely emulated by a medium pressure 4-way 2-position switch valve. In various embodiments, the switch valve may be similar to the switch valve used in liquid chromatography (and specifically, for example, in biological HPLC). Such a 4-way 2-position switch valve may be similar to the valve manufactured by Upchurch Scientific of Oak Harbor, Washington and sold by IDEX Health and Science.

[0033] In addition to providing a lower cost solution, the 4-way 2-position on-off valve may also provide additional advantages. Advantages may include, but are not limited to, reduced polymer sludge volume, improved availability from multiple suppliers with off-the-shelf components. The 4-way 2-position valve may also eliminate the need for a custom fabricated clear glass assembly and the non-standard needle valve and high pressure cannula associated with the clear glass assembly.

[0034] In addition, there are complexities surrounding the anode 211 and cathode 226. The anode 211 and cathode 226 can be electrically connected to a high voltage source to provide a high potential across the capillary 222. Because the nucleic acids carry a charge, this potential tends to cause sample fragments to migrate through the capillary for separation and detection. Because the cathode is in the capillary section 220 and the anode is in the fluidic section 210, it can become challenging to isolate the high voltage from the rest of the instrument to prevent any risk of electric shock to the user.

[0035] Figure 3 The embodiment shown in FIG. provides advantages over Figure 2 The cartridge assembly 300 can combine many elements of the instrument 200 into an integrated unit. The integrated unit can include a capillary 322, a pump 324, a container 326a, an optical channel 328, a cathode 325, and a channel to an anode 323. In various embodiments, the container 326a can contain a separation matrix 326b. In various embodiments, the pump 324 can be a syringe pump. In yet another embodiment, the pump 324 can include an electrokinetic pump.

[0036] As shown, the capillary 322, pump 324, container 326a, optical channel 328, cathode 325 and the passage to anode 323 can all be enclosed in a single temperature controlled area 301, thereby further simplifying the requirements of the instrument. In various embodiments, the separation matrix 326b can include a polymer and a buffer, thereby making it unnecessary to include a separate container for the buffer and further simplifying the system. In various embodiments, the container 326a can provide enough separation matrix to perform multiple sequencing rounds. In various embodiments, the container 326a can provide enough separation matrix to perform 100 sequencing rounds.

[0037] In various embodiments, the cartridge 300 can be an easily removable cartridge that can enable a user to easily replace the capillary 322, the container 326a, and the pump 324 as a single unit. The cartridge 300 can further enable a user to replace the capillary 322, the container 326a, and the pump 324 by removing the cartridge 300. Figure 2 The sequencing workflow is simplified by showing valves, fluid lines, and multiple containers in the figure.

[0038] refer to Figures 4A to 4D , shows integrated valve assembly 400 connected to capillary tube 460. Integrated valve assembly 400 can include manifold 410, polymer container 420, pinch point 430, pinch valve 440, and dispenser 450. In various embodiments, integrated valve assembly 400 can be disposable.

[0039] Figure 4A Depicts conditions suitable for performing capillary electrophoresis with a pinch valve 440 and dispenser 450. For these conditions, the pinch valve 440 does not contact the pinch point 430, and the dispenser 450 does not contact the polymer container 420. It is known in the art that prior to sequencing a biological sample, the capillary needs to be pre-loaded with fresh polymer.

[0040] Figure 4B and 4C Depicts the loading process using integrated valve assembly 400. Figure 4B In the embodiment of the present invention, the pinch valve 440 is in contact with the pinch point 430. The pinch valve 440 is in contact with the pinch point 430 to compress the pinch point 430 and isolate the manifold 410 from the capillary 460. Figure 4C Depicted is a dispenser 450 in contact with the polymer container 420 to push the polymer from the polymer container 420 into the capillary 460. Figure 4D This is accomplished in which both the pinch valve 440 and the dispenser 450 are retracted to place the integrated valve assembly in suitable conditions for performing electrophoresis.

[0041] In various embodiments, manifold 410 can hold enough polymer to perform multiple sequencing runs. In some embodiments, the manifold can hold enough polymer to run up to four capillary arrays ten times.

[0042] In various embodiments, the pinch valve 440 and the dispenser 450 can be manually operated. Manual operation can be achieved through the use of a mechanical lever, a handle, a button, or any other suitable mechanism known in the art.

[0043] In various embodiments, the operation of the pinch valve 440 and the dispenser 450 can be automated. Automating the action of the pinch valve 440 and the dispenser 450 can eliminate the need for expensive instrument valves known in the art. Automation can be achieved through the use of solenoids, motors, linear actuators, or any other suitable mechanism known in the art.

[0044] Figure 5 4. The blister package 500 is a representation of the integrated valve assembly depicted in FIG. 4. Where possible, the same reference numerals are used to refer to the same or similar parts. As shown, the blister package 500 can be a molded fluidic device that can guide fresh polymer to the capillary to refill or preload the capillary 460 before sequencing as discussed above. In various embodiments, the capillary 460 can be a capillary array.

[0045] As shown, bubble shell package 500 can include manifold 410, pinch point 430 and polymer container 420. In various embodiments, manifold 410 can contain 1 ml of polymer. In various embodiments, 1 ml of polymer can be sufficient to run multiple sequencing experiments. In various embodiments, 1 ml of polymer can support up to four capillary arrays for ten rounds.

[0046] In addition, the bubble shell package 500 may further include a printed electrode 510. The printed electrode 510 may be used as Figure 3 Anode 323 is depicted in FIG. Anode 323 can be used as an electrical terminal to apply a high voltage to the capillary to promote migration of nucleic acid fragments through the capillary.

[0047] In various embodiments, the blister package 500 can be loaded with polymer and shipped to the customer ready for use. In such embodiments, the user can attach the blister package 500 to the capillary 460 and does not need to manually load the polymer in the system. In various embodiments, the blister package 500 can be a disposable component.

[0048] refer to Figure 3 , pump 324 is depicted as a separate element from 326a. Figure 6 For similar Figure 3640 to provide the newly formed polymer 640 to the capillary 620. In various embodiments, the electrode 630 may be located in the polymer 640. In various embodiments, the electrode 630 may be connected to a high voltage source (not shown). In various embodiments, the high voltage source may form gas and ions at the interface 650 between the electrode and the polymer. In various embodiments, a gas permeable membrane or vent (not shown) may be included as part of the polymer reservoir to allow any formed gas or ions to escape.

[0049] It is well known in the art that separation of nucleic acid fragments during capillary electrophoresis can be improved when the polymer in the capillary is heated to a temperature above ambient temperature. In various embodiments, the temperature of the polymer can be between 40°C and 60°C. Figure 7 700 may include a temperature controlled region 710. The temperature controlled region 710 may enclose a capillary 740. Prior to sequencing, the capillary 740 may be refilled or pre-loaded with freshly made polymer delivered from a polymer reservoir 750, as previously discussed.

[0050] The temperature controlled area 710 may be connected to an outlet duct 760 at one end of the area 710 and to an inlet duct 770 at the opposite end. The outlet duct 760 may be further connected to the inlet of the blower 720, and the outlet of the blower 720 may be connected to the inlet of the heat source 730. The outlet of the heat source 730 may be connected to the inlet duct 770 to form a complete air path with the temperature controlled area 710. In operation, the heat source 730 may be controlled at a temperature between 40° C. and 60° C. as desired, and the blower 720 may disperse the hot air through the temperature controlled area 710. The temperature controlled area 710 may then be filled with hot air to raise the temperature of the capillary 740 to an optimal temperature.

[0051] In another embodiment, the Figure 8800 heats the capillary. The heating diagram 800 may include a temperature control region 810 surrounding the capillary 840. In this embodiment, one end of the temperature control region may be connected to an outlet duct 860, and the opposite end of the temperature control region may be connected to an inlet duct 870. The outlet duct 860 may be further connected to the inlet of a blower 820, and the outlet of the blower 820 may be connected to the inlet of an air bag 880. The outlet of the air bag 880 may be connected to the inlet duct 870 to form a complete air path with the temperature control region 810. In this embodiment, a heat source 830 is thermally coupled to the underside of the temperature control region 810. In operation, the heat source 830 may be controlled at a temperature between 40°C and 60°C as desired, and the blower 820 may disperse the hot air through the temperature control region 810 to provide uniform heating for the polymer in the capillary 840.

[0052] Although the foregoing embodiments have been described in some detail for the purpose of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the embodiments disclosed herein. For example, all of the above-described techniques, devices, systems, and methods may be used in various combinations.

Claims

1. A biological sequencing system, comprising: one or more capillaries; an integrated valve assembly configured to dispense a polymer to the one or more capillaries; Optical detection system; one or more heat cyst chambers; as well as a processor capable of executing machine-readable code to control the integrated valve assembly, the optical detection system, and the one or more thermal cell chambers, wherein the integrated valve assembly is a disposable blister package configured to direct polymer to the capillary to refill or preload the capillary prior to sequencing, the blister package comprising a manifold, a pinch point, and a polymer container, and The manifold is connected to a printed electrode disposed on one end of the bubble package, and the capillary is connected to the other end of the bubble package.

2. The biological sequencing system according to claim 1, wherein the pinch valve and the dispenser are automatically controlled.

3. The biological sequencing system of claim 1, wherein the manifold is capable of containing an amount of polymer sufficient for 10 runs. 4 . The biological sequencing system according to claim 1 , wherein the bubble shell package further comprises the printed electrode, and the printed electrode is configured to apply a predetermined voltage to the one or more capillaries.

5. The biological sequencing system according to claim 1, wherein the one or more thermal capsule chambers are configured to form a complete air path with a temperature control area, and the temperature control area encloses the one or more capillaries.

6. The biological sequencing system according to claim 5, further comprising a heat source and a blower, wherein the heat source is configured to be heated to a predetermined temperature, and the blower is configured to disperse hot air through the one or more heat cell chambers to the temperature control area, thereby heating the polymer in the one or more capillaries.

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