Low-Volume Sequencing System and Method of Use
Various embodiments of a low-volume sequencing system are provided herein. The system can include a low-volume flowcell having at least one reaction chamber of a defined volume (e.g., less than about 100 μl). The system can also include an automated reagent delivery mechanism configured to reversibly couple with the inlet port corresponding to a target reaction chamber thereby placing allowing for reagent to be accurately moved from a storage container to the reaction chamber with minimal reagent waste. The flowcells can include a plurality of reaction chambers (e.g., 6) thereby allowing for parallel analysis of multiple samples. Various methods of analyzing a biomolecule are also provided herein.
This application claims priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61/238,633, filed on Aug. 31, 2009, entitled “Enhanced System and Methods For Sequence Detection,” U.S. Provisional Patent Application Ser. No. 61/238,667, filed on Aug. 31, 2009, entitled “Enhanced Flow Cell and Reagent Delivery For Sequence Detection,” U.S. Provisional Patent Application Ser. No. 61/307,623, filed on Feb. 24, 2010, entitled “Methods of Bead Manipulation and Forming Bead Arrays,” U.S. Provisional Patent Application Ser. No. 61/307,492, filed on Feb. 24, 2010, entitled “Flowcells and Methods of Filling and Using Same,” U.S. Provisional Patent Application Ser. No. 61/307,641, filed on Feb. 24, 2010, entitled “Flowcells and Methods of Filling and Using Same,” and U.S. Provisional Patent Application Ser. No. 61/307,486, filed on Feb. 24, 2010, entitled “Flowcell, Flowcell Delivery System, Reagent Delivery System, and Method For Sequence Detection,” the entirety of each of these applications being incorporated herein by reference thereto.FIELD
The present disclosure is directed towards molecular sequencing, in particular towards low-volume flowcell design and reagent delivery optimization.BACKGROUND
Nucleic acid sequencing techniques are of major importance in a wide variety of fields ranging from basic research to clinical diagnosis. The results available from such technologies can include information of varying degrees of specificity. For example, useful information can consist of determining whether a particular polynucleotide differs in sequence from a reference polynucleotide, confirming the presence of a particular polynucleotide sequence in a sample, determining partial sequence information such as the identity of one or more nucleotides within a polynucleotide, determining the identity and order of nucleotides within a polynucleotide, etc.
Next generation sequencing techniques commonly utilize fluidic technologies for performing aspects of sample analysis. For example, Assignee's PCT Application Publication No. WO 2006/084132, entitled “Reagents, Methods, And Libraries for Bead-Based Sequencing,” the entirety of which is incorporated herein by reference thereto, provides various techniques, systems, and methods for sequencing a sample coupled to a solid-support (e.g., a bead, particle, surfaces and surface features, etc.) wherein a plurality of supports are disposed over the surface of a flowcell. Flowcells allow for a large number of samples, or samples coupled to other solid-supports, to be immobilized in random and/or ordered fashion across reaction chamber(s) while reagents are added to, removed from, or pumped through the chamber(s) to produce the desired effect (e.g., reaction, wash, etc.). Typical systems can also include imaging, optics, or other detection components in communication with the reaction chambers thereby allowing sample images or other properties to be rapidly captured and analyzed.
In view of the ever-increasing benefits of genomic analysis, demand continues for, among other things, faster sample analysis, higher throughput, enhanced sequence accuracy, and reduced cost (e.g., on a per-run or per-genome basis).SUMMARY
Various embodiments of a sample analysis and sequencing system are provided herein. In some embodiments, the system includes a low-volume flowcell having at least one reaction chamber of a defined volume with at least one fluid transfer port (e.g., inlet/outlet port). The systems can also include an automated reagent delivery mechanism having a fluidic dispenser with an internal reservoir such as a compartment configured to retain an amount of reagent. In some exemplary embodiments, the dispenser can include a distal portion configured to reversibly couple with the inlet port thereby placing an internal compartment of the dispenser into fluid communication with at least one reaction chamber, and optionally further configured to dispense to the reaction chamber a volume substantially equal to the defined volume of the reaction chamber.
The systems can also include a controller (e.g., a computer, processor, etc.) in communication with the delivery mechanism, and configured to couple/decouple the delivery mechanism with the inlet port, and further configured to dispense reagent to the reaction chamber. In some embodiments, instructions for coupling and/or decoupling the delivery mechanism are embodied in hardware, firmware, software, or combinations thereof. In some aspects, the instructions can be contained in computer-readable media and optionally transferable between different systems.
The delivery mechanism can be configured in some embodiments to withdraw a volume of reagent substantially equal to the volume of the reaction chamber from a storage container and transfer this volume of reagent to the reaction chamber. Further, volume of the reaction chamber can be in a range from as low as flowcell manufacturing technology will allow and about 100 between about 10 μl and about 40 μl, between about 20 μl and about about 25 μl, etc. These features can, among other things, assist in reducing reagent use and in reducing or eliminating reagent waste.
Flowcells of the systems can include a single reaction chamber of a discrete volume or can include a plurality of reaction chambers each having a discrete volume. The volume of each reaction chamber can be the same for each chamber or different. The reaction chamber(s) can extend between distinct inlet and outlet ports, or can have a common inlet and outlet ports. In those embodiments having a plurality of reaction chambers, the flowcells can have 2, 3, 4, 5, 6, 7, 8, etc. chambers. Additionally, when delivering reagent to multiple chambers, one or more internal compartments of the delivery mechanism can be configured to transfer/deliver a volume of a reagent substantially equal to a total volume of the plurality of reaction chambers.
Various flowcell configurations are also disclosed herein. For example, at least one fluid transfer port can be incorporated into a top portion of the flowcell, or can be incorporated into a bottom portion of the flowcell. Also, at least a portion of the flowcell's surface can be configured to bind, immobilize, or trap a sample or other reagents or components (e.g., polynucleotides, solid-supports configured to bind a polynucleotide). In some embodiments, the flowcells can include an interior portion configured to bind, immobilize, or trap sample, reagents, or other components.
In some embodiments, the reagent delivery mechanism includes a robotic assembly having x-, y-, and z- functionality. Additionally, the system can include a reagent storage container housing a plurality of reagents (which can be in the form of a kit). The delivery mechanism can be configured to retrieve sample from a storage container and dispense reagent into a reaction chamber without plumbing or tubing requirements therebetween. In some exemplary embodiments, dead volume associated with plumbing, tubing, or other fluid connection means can be reduced or eliminated, which can assist in reducing reagent use, reducing or eliminating reagent waste, and enhancing reagent delivery and system accuracy and efficiency.
Various embodiments of a low-volume flowcell are also provided herein. In some embodiments, the flowcell includes a substrate having at least one reaction chamber of a defined volume extending between an inlet port and an outlet port, the inlet port being configured to reversibly couple with an automated reagent delivery mechanism which is configured to deliver a pre-determined amount of reagent to the reaction chamber. To reduce reagent requirements and waste, the reaction chamber can be sized and configured to minimize the pre-determined amount of reagent required to affect a desired result. For example, the defined volume of the reaction chamber can be a volume within a range of between about a minimum amount (e.g., to limits of flowcell manufacturing technology) and about 100 μl between about 10 μl and about 40 μl or about 25 μl.
Methods of analyzing biomolecules are also provided herein, including methods employing various embodiments of flowcells and reagent delivery mechanisms disclosed herein. In some embodiments, a method can include providing an automated reagent delivery mechanism configured to withdraw a pre-determined volume of a reagent from a reagent storage container, withdrawing a pre-determined amount of the reagent from the storage container by the automated reagent delivery mechanism, and coupling a portion of the automated reagent delivery mechanism to an inlet port of a flowcell, the inlet port being in fluid communication with a reaction chamber having a defined volume. Methods can also include dispensing into the reagent chamber a reagent volume substantially equal to the defined volume of the reagent chamber, and decoupling the automated reagent delivery mechanism from the inlet port.
In those embodiments relating to flowcells with multiple chambers (e.g., 2, 3, 4, 5, 6, 7, 8, etc.), methods of analyzing biomolecules can include repeating the coupling and decoupling steps for each of the corresponding inlet ports in fluid communication with distinct reaction chambers, each reaction chambers having discrete volumes which can be the same or different than other reaction chambers. In one embodiment, the method can include dispensing the same reagent to each (or at least two) of the plurality of chambers, and other embodiments can include delivering different reagents to each (or at least two) of the chambers.
Instructions for performing or implementing the various functions and features described above and in the remainder of this disclosure can be embodied in hardware, firmware, software, or combinations thereof. Instructions can also be contained in computer-readable media, and optionally be transferable between different systems and reconfigurable and updatable via on-line services, by the user, or by other processes and means.
These and other embodiments are described in greater detail below.
The present disclosure will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Low-volume sample analysis or sequencing systems and methods of use are provided herein. The presently disclosed system utilizes various embodiments of a low-volume analysis apparatus and a precision reagent delivery mechanism. The system can include an embodiment of a low-volume flowcell having discrete reaction chambers sized and configured to minimize reagent volume necessary to achieve the desired result (e.g., reaction, wash, etc.). For example, various embodiments reduce reagent volume per chamber to within a range of between about a minimal amount possible in view of flowcell manufacturing technology and about 100 μl. The precision reagent delivery mechanism can include an x-y-z functional robot capable of receiving a pre-determined amount of reagent from a reagent storage container, reversibly coupling to a dedicated fluid transfer port (e.g., an inlet port), and dispensing this minimal volume to the reaction chamber. In one embodiment, the pre-determined amount of reagent is substantially equal to the amount of reagent to be dispensed into the reaction chamber thereby substantially eliminating any amount of reagent waste. As detailed below, such a system significantly reduces reagent requirements thereby providing cost savings and simplifying maintenance over typical systems utilizing extensive plumbing/tubing requirements.
The analysis systems, flowcells, fluid and reagent delivery systems, software, and other systems, apparatuses, and methods disclosed herein can be used in connection with various sequencing techniques and processes, such as chain termination or dideoxynucleotide sequencing, chemical degradation sequencing, sequencing by synthesis, pyrosequencing, sequencing by hybridization, oligonucleotide-based sequencing, and single-molecule sequencing. The analysis systems, flowcells, fluid and reagent delivery systems, software, and other systems, apparatuses, and methods disclosed herein can also be used in connection with automated, partially automated, and manual sequencing instruments and processes. For example, low-volume flowcells disclosed herein can be used for performing next-generation sequencing reactions such as oligonucleotide-based reactions and sequencing-by-synthesis reactions. An advantage of some embodiments of the disclosed flowcells is a reduction in the amount of sample or reagent that is used; where such samples or reagents are available only in small quantities or are expensive or otherwise difficult to make or acquire, substantial reduction in cost can be realized (sequencing of a whole genome for $1,000 or less) and the sequencing of samples that were previously difficult or impossible to sequence.
Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the systems and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the systems and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure.
While the apparatus 12 is shown to include a variety of components, those skilled in the art will appreciate that any or all of these components may be removed, substituted, new components added, etc. Additionally, any or all of these components may be considered to be independent of the apparatus 12. All such variations are within the spirit and scope of the present disclosure.
In the preferred embodiment, the apparatus 12 includes a low-volume flowcell 14 in communication with a temperature block 16 and processing stage 18. As detailed below, the low-volume flowcell 14 can be configured to retain sample in any number of discrete reactions chambers. The reaction chambers can likewise be sized and configured to minimize reagent volume required to affect the desired result within each chamber. That is, the width, length, and/or height of the chamber can be selected to maintain desired flow characteristics while ensuring the desired reaction takes place. Additionally, the discrete chambers allow for multiple, distinct reactions or distinct samples to be analyzed simultaneously, or the same reaction may be performed multiple times in parallel as a quality control check. Thus, the entire surface area of the flowcell 14 does not need to be utilized when a single chamber (e.g., 1 of 6 available chambers) can provide the desired result thereby limiting the required reagent volume. Additionally, the system 10 can be configured to include multiple independently operated flowcells (e.g., 2 flowcells located on the processing stage 18). In various embodiments, the low volume characteristics of the flowcell may be determined on the basis of the amount of reagents needed to effectuate an efficient reaction between the sample and the reagents with little or no excess. For example, if the principle reactivity between the sample and the reagents takes place only in close proximity with the flowcell surface with little or no reaction taking place in the volume contained above the flowcell surface then the cross-sectional area or overall volume of the flowcell may be decreased such that reagents contained within the flowcell are principally positioned where reactivity is substantial or optimal.
In maintaining an accurate temperature profile within each reaction chamber, a temperature block 16 can be in placed into thermal communication with the flowcell(s). Various such temperature blocks are within the spirit and scope of the present disclosure. For example, the temperature block 16 can be a device capable of both heating and cooling, such as a peltier device. Other temperature control devices can also be used, such as heating/cooling units or blocks, thermal controllers, heat transfer devices, reaction processors. In some embodiments, there is a water-based variant referred to as a hydrocycler which uses as temperature controlled water bath. In some embodiments, thermocycling of the reaction components (e.g., cycling between higher and lower temperatures) is not necessarily required and the reaction may also proceed isothermally which may or may not require a heat block (e.g., reaction proceeds at ambient temperature). As detailed below, flowcell temperature control is facilitated by the reduced reagent volume requirements of the presently disclosed system where lower fluidic volumes may be thermally regulated more efficiently and quickly (e.g., lower heat capacity in the fluidic volume allowing faster ramping in heating and cooling) because the system no longer must account for large fluctuations in reagent volume.
The flowcell 14 and temperature block 16 can be coupled to a movable or motorized processing stage 18 configured to move between, for example, a flowcell loading stage position and an analysis position. Those skilled in the art will appreciate that various types of motors can be utilized to drive the processing stage 18 between such positions.
In the analysis position, the low-volume flowcell and associated sample disposed therein can be placed into communication with an optics/imaging mechanism 20. The optics/imaging mechanism 20 may be configured to transmit and/or filter excitation energy from an excitation source (e.g., an arc lamp, a laser, etc.) through one or more lenses, filters, etc. such that a sample (or selected area or panel of the flowcell) is imaged at one or more selected wavelengths prior to moving onto the next sample (or selected area or panel of the flowcell). The optics/imaging mechanism 20 can further be configured to include one or more filters (not shown) such that the emission signals pass through a corresponding filter prior to impinging upon the detector 24 (e.g., a CCD). One embodiment of the optics/imaging mechanism 20 is described in greater detail in co-pending U.S. patent application Ser. No. ______, filed Aug. 31, 2010, entitled “Fast-Indexing Filter Wheel and Method of Use,” the entirety of which is incorporated herein by reference thereto.
The fluid delivery mechanism 26 can include an x-y-z robot configured to accurately receive a predetermined amount of reagent from a reagent storage container 28, reversibly couple to a fluid transfer port (e.g., an inlet port) in fluid communication with a target reaction chamber, and dispense the selected volume of reagent to the chamber. In one embodiment, the fluid delivery mechanism is configured to eliminate or at least substantially reduce the amount of reagent utilized in typical systems by eliminating commonly found extensive plumbing/tubing requirements. Additionally, the mechanism 26 can be configured to retain and deliver a volume of reagent that is substantially identical to the volume of the reaction chamber thus essentially eliminating any wasted reagent. The mechanism 26 can also include various sensors capable of determining when a reagent reservoir needs to be filled, capable of accurately determining volume within the reservoir, etc. In various embodiments, the method of fluidic dispensation described herein desirably reduces potential wasted reagents that would be contained in the instrument plumbing and fluidic transfer lines. Furthermore, the system desirably improves thermal control over the sample and/or reagents which may be held at desired temperatures and dispensed as desired without substantial temperature deviations which may accompany movement through complex instrument plumbing and fluid transfer lines.
In use, sample can be incorporated/bound to either or both the top and bottom surfaces of the flowcell. The target sample can be bound directly on the substrate layer or can be coupled to solid-supports (e.g., particles or beads) which can then bind to the desired substrate. In various embodiments, the target sample can be bound to both the top and bottom layers to achieve a higher target sample density and furthermore to achieve more efficient utilization of reagents in the flowcell. In a preferred embodiment, sample is bound to solid-supports which are then bound to the top layer of the flowcell 14.
The inlet port 44 of the flowcell 14 allows for, as detailed below, the precision reagent delivery mechanism 26, to deliver a substantially exact pre-determined amount of reagent to the target reaction chamber(s) 42. Additionally, the outlet port 46 allows for waste to be removed from the chamber(s) 42. While each chamber 42 is shown to be in communication with a distinct outlet port 46, other embodiments can include a single outlet port in communication with all chambers. In such an embodiment, waste streams from each chamber 42 can pool together prior to exiting the flowcell from the one outlet port. In one embodiment, the fluidics interface (i.e., the inlet and outlet ports 44, 46) is incorporated into the top layer 36 of the low-volume flowcell. In other embodiments, the fluidics interface can be incorporated into the bottom of the flowcell 14. In another embodiment, the inlet/outlet ports can be incorporated into a side portion of the flowcell. In various embodiments, fluidic dispensation and withdrawal of fluid from the sample chamber can be achieved by positive or negative pressure or fluidic displacement methods such as purging air through the sample dispenser and/or flowcell.
The presently disclosed low-volume flowcell 14 can be configured to include any number of discrete channels defining reaction chambers 42. Thus, the flowcell 14 can simultaneously analyze a plurality of samples, can analysis the same sample multiple times, and/or can analyze the same sample under distinct reaction conditions. Additionally, each chamber 42 can be sized and configured to minimize reagent volume. That is, the height, width, and/or length can be modified in order to reduce reaction volume while also maintaining fluid flow characteristics (e.g., avoiding bubble formation) and also allowing for the desired reaction to occur.
As shown, the low-volume flowcell 14 can include 4 reaction chambers 42. Various other embodiments of the low-volume flowcell 14 can include any number of reaction chambers 42. For example, the flowcell 14 can include 1, 2, 3, 4 (as shown in
The length and width of an embodiment of reaction chambers of the 4-chamber flowcell is represented as “C” and “D” in
As indicated above, to reduce reagent volume and associated waste, the chambers 42 can be sized and configured to utilize a minimum volume of reagent while maintaining desired flow characteristics and also allowing for the desired reaction to occur. In one embodiment, the height of the reaction chamber 42 can be configured to provide for such reagent optimization. For example, referring again to the embodiment of the 4-chamber flowcell of
The length and width of an embodiment of reaction chambers of the 6-chamber flowcell is represented as “C′” and “D′” in
During imaging and analysis, defined portions of each reaction chamber 42 can be imaged individually. In one embodiment, a single area is imaged at multiple wavelengths (e.g., 4 images taken of each area at 4 distinct wavelengths) prior to imaging another area.
In one embodiment, the flowcell 14 is a presealed, low volume device, capable of being placed directly onto a precision monitoring or retaining surface on the stage 18, which is pre-aligned with the optical axis. As the flowcell 14 is pre-sealed, clamping forces, and thus deformation forces, are very minimal. Such an arrangement can be used with the optics either above or below the flowcell 14. Reagent consumption can be limited to the absolute minimum required to fill the specially designed low-volume flowcell, thus achieving minimal operational costs. In view of the minimal volume requirements, optical alignment and distortion reduction is also greatly improved.
As shown in
The various components/substrates of the flowcell can be constructed of various materials and/or mixtures of materials. For example, the components can include glass, sapphire, plastic, ceramic, metal, etc. Additionally, any or all of the components/substrates can be layered components. For example, one layer can include a composition capable of facilitating sample binding via a covalent coupling between the particle and substrate or between the sample and substrate. The layer may also comprise a surface treatment such as a plasma-oxygen treatment which facilitates binding of the desired sample and/or particle. Depending on the nature of the sample (e.g., protein, lipid, nucleic acid, etc.) the layer can be selected to provide a desired selective binding property. Similarly, particles or beads can be secured to the surface via a functional group present on either the particle/bead or the surface, or both.
In one embodiment, various components of the flowcell 14 can be constructed of a moldable polymer, copolymer, and/or polymer blend that have relatively low background fluorescence when exposed to excitation light. Furthermore, it may be desirable for such material to exhibit little or no reactivity with the reagents (e.g., diminishing the activity of any enzymes present in the reagents). Various thermoplastics are thus suitable for use in flowcell construction including, by way of example cycloolefin polymers, polypropylene, polystyrene, etc. This results in high quality components at relatively low cost, with low fluorescence optical properties that further enable the incorporation of the molded microfeatures (e.g., positioning element 48). In various embodiments, small particles or beads can be immobilized to the top/bottom of the flowcell 14 by means of attachment to a functionalized surface. To facilitate this approach, the surface can be functionalized prior to assembly, and the opposing surface can remain unfunctionalized. In some embodiments, functionalizing can be done after assembly, to functionalize both surfaces. According to some embodiments, the surface functionalization can comprise the deposition of a thin layer of a binding agent such as a metal oxide that is only a few atoms thick, such as ten or fewer or five or fewer atoms thick.
In various embodiments the flowcell can be assembled in different manners. Two such methods are described for the final assembly. In the first method, the cover surface (unfunctionalized) is bonded to the bead deposition surface (functionalized), prior to bead deposition. The beads are then introduced through a port in the closed assembly for deposition. In the second method, the beads are deposited on the functionalized surface prior to the assembly of the opposing surfaces. For this method, a very thin (for example a few microns thick) layer of pressure sensitive adhesive, patterned to match the channel ribs, is provided on the mating surface.
Other flowcell configurations capable of improving system performance are also provided herein. For example, as shown in
In another embodiment, as shown in
Such a system can make use of a flexible bead substrate consumable 64 (e.g., a polymer or plastic slide). The reaction chamber 42 can be pressurized facilitating the flattening of the flexible bead substrate 64 against the thermal block 16 both for thermal transfer aspects as well as for flatness of the flexible substrate 64, which is desirable for narrow depth of field systems. The substrate/optical window 62 can be desirably configured to be robust enough to tolerate working loads, and as described above and shown in
Another benefit of a sapphire substrate is depicted in
In yet another embodiment, as represented in
In summary, these low-volume flowcell designs provide numerous potential advantages, including the ability to use materials with higher stiffness and strength that enables thinner substrates, high thermal conductivity that provides better thermal uniformity properties, optical grade qualities such as flatness and smoothness, novel fluidic filling possibilities to minimize bubbles, and pressurization capable of flattening the consumable for reducing focal variation.
As shown in
Prior to securing the low-volume flowcell to the processing stage 18 for analysis, sample can be introduced to the reaction chambers 42. In one embodiment, the sample is coupled to a solid-support (e.g., beads). Beads can be introduced to the chambers 42 in many manners, for example, through the inlet and/or outlet ports 44, 46. As the dimensions of the chambers 42 are relatively small, there may be some difficulties adding the beads into the chambers 42 while avoiding sample loss (e.g., bead spilling over the top of the flowcell 14).
Those skilled in the art will appreciate that various other embodiments of a deposition tool are within the spirit and scope of the present disclosure. For example, in one embodiment, not shown, the tool can be built onto a clamp to hold the flowcell in place with the beads are introduced into the inlet/outlet couplings. All such embodiments are within the spirit and scope of the present disclosure.
In addition to facilitating sample introduction, in one embodiment, the deposition tool 70 can be utilized during shipment and/or processing of the flowcells. That is, flowcells may be shipped as part of the deposition assembly in order to provide added stability/security to the flowcell thereby preventing or reducing damage. Also, the flowcell can remain part of the deposition assembly during processing steps (e.g., rotation and/or centrifugation of the flowcells during bead incorporation/binding to the flowcell substrate) prior to analysis.
In addition to the various embodiment of the low-volume sequencing apparatus 12 provided above, the presently disclosed system 10 further includes various embodiments of a precision liquid delivery system 26 in communication with the apparatus 12. That is, prior sequencing systems waste large amounts of reagents due to extensive plumbing requirements wherein reagents must be transported through extensive tubing to the reaction chambers. Additionally, many reagent delivery systems involve delivery lines, valves, etc., which suffer from a residual ‘dead volume’ of reagents required for the actual transport process, but not actually utilized in the destination reaction chamber.
Minimizing reagent consumption in sequencing analysis is important for cost effectiveness. The reagent consumption can essentially be broken into two components including the actual reagent volume contained in the sequencing/reaction chamber and any reagent ‘overhead’ required in the process of transporting the reagent from its storage location to the actual point of use in the reaction chamber. As disclosed herein, the precisions reagent delivery mechanism can reduce the system's ‘dead volume’ to zero or near zero.
The currently disclosed system 10 includes a precision liquid reagent delivery mechanism 26 configured to receive a pre-determined amount of reagent from a reagent storage container, and further configured to deliver the entire pre-determined amount to the reaction chamber via the inlet port thus eliminating essentially all plumbing/tubing requirements. Additionally, in one embodiment, this pre-determined volume can be substantially equal to the volume of the target reaction chamber. Thus, the delivery mechanisms can be configured to withdraw an exact amount of reagent from a storage container and then dispense this entire or substantially this entire amount into the reaction chamber to achieve the desired effect (e.g., reaction). In other embodiment, the reagent delivery mechanism 26 can be configured to withdraw and retain an amount of reagent substantially equal to that amount required to introduce reagent into each of the reaction chambers (or at least some subset thereof). These improvements decrease analysis time, increase accuracy as a precise amount of reagent is being delivered to a desired location, and substantially reduce waste thus lowering total cost.
When it is time to empty the reaction chamber for the next reagent/reaction, the syringe pump 78, charged with air or a wash reagent, forces the reagent from the reaction chamber 42 thereby leaving the chamber 42 in a state where it is occupied only by air or wash liquid. Additional washes can then be performed as required, again leaving the chamber 42 in a state occupied only by air. Subsequent reagent deliveries and washes are repeated as outlined above. Because each delivery is bounded by air, there is no liquid diffusion border effect that results in dilution.
Also disclosed herein is a mechanism for converting a relatively high-volume sequencing system to a low-volume system. That is, low-volume applications of instruments for next generation sequencing such as for use with a sample volume of approximately 25 μl can be desirable. Certain conventional systems have a significantly larger sample volume of approximatly 400 μl or more and may not be able to efficently handle small volumes. As shown in
The port 95 can be configured to act like a valve that allows the existing robot tip or other pipetting tip to be plugged into it to shorten the fluidic path and avoid moving the sample through the syringe and through the existing long tubing. The port can also be configured with a lid or top 96 that has at least one tubing 98 connected to it. This tubing 98 can be attached to a valve block so it can configured for self washing of the port 95 and the sample slide, and can also pump bulk reagents through the slide. The port 95 can further include a plurlaity of sealing mechansims 100, 102, 104 (e.g., O-rings) configured to effectively seal the connection between the input tubing 96 and the output tubing 106 running to the flowcell. Desirable features of such designs allow the system to handle smaller volumes with little or no dead volumes and fluid losses. Additionally, the port 95 can be used on existing products that are required to pipette small volumes and/or load a sample.
In various embodiments, the low volume flowcell and robotically assisted reagent delivery system can be configured for use in next generation sequencing systems such as that provided by Life Technologies (e.g., SOLiD). For example, Assignee's PCT Application Publication No. WO 2006/084132, entitled “Reagents, Methods, And Libraries for Bead-Based Sequencing,” the entirety of each of these applications being incorporated herein by reference thereto, describe a system for nucleic acid sequence anaysis using a flowcell imaging apparatus which may benefit from the presently disclosed low-volume flowcell designs disclosed herein, as well as from the automation capabilities provided by the robotically assisted reagent delivery apparatus.
Referring again to
The controller can include various embodiments of a computer system configured to control the flowcell(s), processing stage, temperature profile, precision reagent delivery mechanism, etc. For example,
Computer system 200 also includes a memory 206, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus 202 for issuing instructions to be executed by processor 204. Memory 206 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 204. Computer system 200 further includes a read only memory (ROM) 208 or other static storage device coupled to bus 202 for storing static information and instructions for processor 204. A storage device 210, such as a magnetic disk, optical disk, EPROM or the like is provided and coupled to bus 202 for storing information and instructions.
Computer system 200 may be coupled via bus 202 to a display 212 (e.g., the user interface 32), such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 214, including alphanumeric and other keys, touchscreen, etc. may be coupled to bus 202 for communicating information and command selections to processor 204. Another type of user input device is cursor control 216, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 204 and for controlling cursor movement on display 212. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.
A computer system 200 can be implemented in connection with the present teachings for purposes of executed predefined instructions, scripts, or real-time operator issued commands. Consistent with certain implementations of the present teachings, the computer system may perform various operations associated with control, monitoring, and data acquisition to thereby permit automated or semi-automated functionalities. For example, the computer system 200 may be used to invoke and execute desired workflows on the instrument, perform and evaluate instrument diagnostics and operational assessments for the various instrument components, obtain signals and information from the instrument or components thereof, acquire sample data and process results, and output information and data to the user. As will be appreciated by one of skill in the art, information and commands issued and received by the computer system 200 may be in response to processor 204 executing one or more sequences of one or more instructions contained in memory 206. Such instructions may be read into memory 206 from another computer-readable medium, such as storage device 210 or transmitted across a network by another remote computer.
In one exemplary embodiment, execution of the sequences of instructions contained in memory 206 may cause processor 204 to perform the desired operational functionalities including for example, control, monitoring, data acquisition, and data analysis processes. Alternatively hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus implementations of the present teachings are not limited to any specific combination of hardware circuitry and software. Furthermore, computer system 200 may be a remotely located computer or part of a network of computers such as a distributed or cloud computing environment. Furthermore, other electronic devices such as PDAs, cellular phones, laptops or other portable or detached devices may be interconnected with computer system 200 as well as directly or indirectly to the instrument to provide selected functionalities as described above. In various embodiments, these other electronic devices may desirably be used to provide selected functionalities such as monitoring the runtime operation of the instrument, obtaining/downloading sample data, transmitting operations instructions, etc.
The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor 204 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 210. Volatile media includes dynamic memory, such as memory 206. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 202.
Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, papertape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.
Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 204 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a network or telephone line. A network or modem interface local to computer system 200 can receive the data and convert the data to a signal or format of instructions recognized by the instrument. The instructions may optionally be stored on storage device 210 either before or after execution by processor 204.
In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.
Processor 420 is in communication with nucleic acid sequencer 410. Processor 420 can be, but is not limited to, a computer, microprocessor, or any device capable of sending and receiving control signals and data from nucleic acid sequencer 410 and processing data. Processor 420 may be configured to perform a number of steps. Processor 420 may obtain the raw data or reads from sequencer 410. Processor 420 may further obtain a reference sequence or genomic information used in further analysis and assembly of the data obtained from the sequencer. In various embodiments, the reference sequence may be retrieved from a database, for example. The database can be a physical storage device with its own processor (not shown) that is connected to processor 420 across a network, or it can be a physical storage device connected directly to processor 420, for example. Processor 420 may be configured to perform selected analysis in addition to the operations/functionalities described above.
In addition to the various devices, systems, and computer systems provided above, various methods for optimizing sample analysis are also provided herein. For example, the methods include automating fluid delivery to a low-volume flowcell thereby eliminating (or substantially reducing) reagent waste and allowing for system optimization. In one embodiment, the method includes providing an automatic reagent delivery mechanism capable of moving between one or a plurality of reagent sample containers and the low-volume flowcell.
That is, in one embodiment, the method allows for the automated reagent delivery mechanism to withdraw a precise amount of a specific reagent from a reagent sample container and deliver this pre-determined, specific amount of reagent to a desired channel of the low-volume flowcell. The mechanism can be an x-, y-, z-configured robotic instrument having, for example, a syringe pump associated therewith. Once the syringe pump of the mechanism withdraws the pre-determined amount of reagent, the mechanism can reversibly couple with an inlet port of the low-volume flowcell in order to place an internal compartment of the delivery mechanism into fluid communication with the reaction chamber associated with the inlet port. As detailed above, the inlet port can be sized in configured in various manners in order to allow for an effecting coupling (e.g., formation of a seal) between the mechanism and the inlet port.
In other embodiments, the delivery mechanism can be configured and/or programmed to perform many functions and/or to move between various inlet ports. For example, the delivery mechanism can be configured to couple/decouple a plurality of distinct inlet ports in order to dispense a pre-determined amount of a reagent to a plurality of distinct channels. Thus, the mechanism can allow a pre-determined amount of the same reagent to be delivered to a plurality of channels, or can allow for distinct reagents to be delivered to different channels thereby allowing for distinct protocols to be performed in each (or at least two) of the reaction channels.
One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.
1. A low-volume sequencing system, comprising:
- a low-volume flowcell having at least one reaction chamber of a defined volume and at least one fluid transfer port formed therein; and
- an automated reagent delivery mechanism having a fluid dispenser with an internal compartment configured to retain a selected amount of reagent, the fluid dispenser having a distal portion configured to reversibly couple with the inlet port thereby placing the internal compartment into fluid communication with the at least one reaction chamber, and further configured to dispense to the reaction chamber a volume substantially equal to the defined volume of the reaction chamber.
2. The system of claim 1, wherein the delivery mechanism is configured to retain a volume of reagent substantially equal to the volume of the reaction chamber.
3. The system of claim 1, further comprising a controller in communication with the delivery mechanism, and configured to couple or decouple the delivery mechanism with the fluid transfer port, and further configured to dispense reagent to the reaction chamber.
4. The system of claim 1, wherein the defined volume of the reaction chamber is less than about 100 μl.
5. The system of claim 1, wherein the flowcell includes a plurality of reaction chambers, each reaction chamber having a discrete volume.
6. The system of claim 5, wherein each reaction chamber extends between at least two discrete fluid transfer ports.
7. The system of claim 5, wherein the internal compartment of delivery mechanism is configured to retain a volume of a reagent substantially equal to a total volume of the plurality of reaction chambers.
8. The system of claim 1, wherein at least one fluid transfer port is incorporated into a top portion of the low-volume flowcell.
9. The system of claim 1, wherein at least one fluid transfer port is incorporated into a bottom portion of the low-volume flowcell.
10. The system of claim 1, wherein at least a portion of the flowcell surface is configured to bind a sample.
11. The system of claim 10, wherein the sample is a polynucleotide or the sample is a solid support configured to bind a polynucleotide or a polynucleotide.
12. The system of claim 1, wherein an interior surface of the flowcell is configured to bind sample.
13. The system of claim 1, wherein the reagent delivery mechanism includes a robotic assembly having x-, y-, and z-functionality.
14. The system of claim 1, wherein the low-volume flowcell includes a plurality of discrete reaction chambers.
15. The system of claim 1, further comprising a reagent storage container housing a plurality of reagents.
16. A low-volume flowcell, comprising:
- a substrate having at least one reaction chamber of a defined volume extending between an inlet port and an outlet port, the inlet port being configured to reversibly couple with an automated reagent delivery mechanism which is configured to deliver a pre-determined amount of reagent to the reaction chamber, the reaction chamber being sized and configured to minimize the pre-determined amount of reagent required to effect a desired result.
17. The low-volume flowcell of claim 16, wherein the defined volume is between about 15 μl and about 35 μl.
18. The low-volume flowcell of claim 16, wherein the defined volume is between about 10 μl and about 40 μl.
19. The low-volume flowcell of claim 16, wherein the defined volume is about 25 μl.
20. A method of analyzing a biomolecule, comprising:
- providing an automated reagent delivery mechanism configured to withdraw a pre-determined volume of a reagent from a reagent storage container;
- withdrawing a pre-determined amount of the reagent from the storage container by the automated reagent delivery mechanism;
- coupling a portion of the automated reagent delivery mechanism to an inlet port of a flowcell, the inlet port being in fluid communication with a reaction chamber having a defined volume;
- dispensing into the reagent chamber a reagent volume substantially equal to the defined volume of the reagent chamber; and
- decoupling the automated reagent delivery mechanism from the inlet port.
Filed: Sep 26, 2014
Publication Date: Mar 26, 2015
Inventors: Jon A. HOSHIZAKI (Cupertino, CA), Joon Mo YANG (Redwood City, CA), Maryam SHARIATI (Sunnyvale, CA), David M. COX (Foster City, CA), Kirk M. HIRANO (Foster City, CA), John BRIDGHAM (Hillsborough, CA), George Stefan GOLDA (El Granada, CA), Sam Lee WOO (Redwood City, CA)
Application Number: 14/498,979
International Classification: C12Q 1/68 (20060101);