Patent Application
20170259262
Field of the Invention
The present invention relates generally to instruments for performing sequencing-by-syntheses or other sequencing processes, and more particularly to flowcells used in such instruments.
Description of the Related Art
Deoxyribonucleic acid (“DNA”) sequencing instruments are used to determine DNA molecular sequences. Such instruments are useful for clinical studies, diagnostics, so-called “personalized medicine” (medical treatment tailored to an individual's genetic content or the like), and so on. Current instruments for performing DNA sequencing use a variety of technologies to analyze the base pairs that form the DNA sequence. For example, some instruments perform sequencing on single-stranded DNA molecule fragments (DNA templates) that are fixed in place inside a flowcell. The flowcell is essentially a small chamber in which the DNA templates are subjected to a series of nucleobase extension processes. Each successive extension is detected to determine the base pair sequence of each DNA template. The flowcell provides an environment to hold the DNA templates during the extension process, and also during the imaging process to read each extended base pair.
Many sequencing-by-synthesis instruments use an optical system such as a microscope to detect the nucleobase extensions, although non-optical systems are also known. A typical optical instrument uses visible chemical labels to determine the identity of each extended base pair. For example, each nucleobase (i.e., adenine, guanine, cytosine and thymine) that makes up the DNA molecule may be labeled with a unique fluorescent probe that is visible through the microscope. The label is read each time the DNA template is extended, and then the label is removed to make way for the next base pair extension.
In modern “next-generation” instruments, millions of DNA templates may be processed simultaneously in a single flowcell. The DNA templates may be randomly ordered within the flowcell, or ordered at specific predetermined locations. A variety of flowcell designs have been developed to hold the immobilized DNA templates, but they usually include certain common features. A typical flowcell includes a flow channel, an optically transparent cover that encloses the channel, and fluid inlets and outlets through which the appropriate reagents are passed to control the growth and extension of the DNA templates. Example of such flowcells are found in U.S. Pat. Nos. 8,481,259, 8,940,481 and 9,146,248 and U.S. Patent Application Publication Nos. 2009/0298131 and 2014/0267669, all of which are incorporated herein by reference.
The flowcell channel may be a so-called “microfluidic” channel, which is loosely defined as one that is less than about one millimeter in height. (As used herein, the “height” is the dimension perpendicular to a plane in which the DNA templates are immobilized.) Such flowcells typically are dimensioned to minimize the cross-sectional area and maximize the surface area-to-volume ratio. (As used herein, the “cross-sectional area” refers to the area in a plane perpendicular to the flow axis of the channel.) Increasing the surface area-to-volume ratio has the beneficial effect of increasing the exposure of the DNA templates to reagents passing through the flowcell. However, decreasing the cross-sectional area increases the amount of pressure differential required to flow reagents through the channel, and increases the likelihood that the channel will clog, and these factors may limit the ability to reduce the cross-sectional area. Typical flowcells are optimized, with respect to the height dimension, to balance these considerations and the maximum surface area-to-volume ratio without unduly decreasing the cross-sectional area.
The inventors have determined that there continues to be a need to advance the state of the art of flowcells for sequencing instruments and similar devices.
Non-limiting examples of embodiments are provided in the following summary.
In one exemplary aspect, there is provided a flowcell for a processing instrument. The flowcell has a flowcell channel having an upstream channel end, a downstream channel end, a longitudinal axis extending from the upstream channel end to the downstream channel end, and a first operative surface extending between the upstream channel end and the downstream channel end and configured to receive a first plurality of DNA templates. A first reagent inlet is fluidically connected to the upstream channel end at a location adjacent the first operative surface. A buffer inlet is fluidically connected to the upstream channel end at a location spaced from the first operative surface. An outlet is fluidically connected to the downstream channel end. The upstream channel end may have a predetermined height in a direction perpendicular to the longitudinal axis, and the first reagent inlet may have an exit portion, adjacent to the upstream channel end, that extends from a first point adjacent to the first operative surface to a second point that is spaced a predetermined distance from the first operative surface in the direction perpendicular the longitudinal axis. Such predetermined distance may be 0.4% to 50% of the predetermined height, 1.4% to 18% of the predetermined height, or 3.6% to 10.7% of the predetermined height. The predetermined height also may be 140 micrometers, and the exit portion may extend from a first point adjacent to the first operative surface to a second point that is spaced from the first operative surface in the direction perpendicular the longitudinal axis by about 0.5 micrometers to about 50 micrometers, about 2 micrometers to about 25 micrometers, or about 5 micrometers to about 15 micrometers. The first reagent inlet may include an exit portion, located immediately upstream of the upstream channel end, that is parallel to and coincident with the first operative surface. The buffer inlet may include an exit portion, located immediately upstream of the upstream channel end, that is parallel to and spaced from the first operative surface. The first operative surface may be a transparent material.
The flowcell also may have a second operative surface extending between the upstream channel end and the downstream channel end and configured to receive a second plurality of DNA templates, and a second reagent inlet fluidically connected to the upstream channel end at a location adjacent the second operative surface, wherein the buffer inlet is at a location spaced from the second operative surface. The first operative surface may be parallel to and facing the second operative surface. The first reagent inlet and the second reagent inlet may be fluidically connected or not fluidically connected upstream of the flowcell channel. The upstream channel end may have a predetermined height in a direction perpendicular the longitudinal axis, the first reagent inlet may have an exit portion adjacent to the upstream channel end that extends from a first point adjacent to the first operative surface to a second point that is spaced a first predetermined distance from the first operative surface in the direction perpendicular to the longitudinal axis, and the second reagent inlet may have an exit portion adjacent to the upstream channel end that extends from a third point adjacent to the second operative surface to a fourth point that is spaced a second predetermined distance from the second operative surface in the direction perpendicular to the longitudinal axis. The first predetermined distance may not equal or it may equal the second predetermined distance. The first predetermined distance and the second predetermined distance may equal to 0.4% to 50%, 1.4% to 18%, or 3.6% to 10.7% of the predetermined height. The predetermine height may be 140 micrometers, and the exit portions of the first and second reagent inlets may extend to points from their respective operative surfaces by distances of about 0.5 micrometers to about 50 micrometers about 2 micrometers to about 25 micrometers, or about 5 micrometers to about 15 micrometers.
In another exemplary aspect, there is provided a sequencing instrument having a flowcell as described above.
In another exemplary aspect, there is provided a method of operating a processing instrument, the method including providing a flowcell channel having an upstream channel end, a downstream channel end, a longitudinal axis extending from the upstream channel end to the downstream channel end, and a first operative surface extending between the upstream channel end and the downstream channel end and comprising a first plurality of DNA templates, providing a first reagent fluid to the upstream channel end channel at a first location adjacent the first operative surface, providing a barrier fluid that is different from the first reagent fluid to the upstream channel end at a second location spaced from the first operative surface, and passing the first reagent fluid and the barrier fluid through the flowcell channel under laminar flow conditions such that the first reagent fluid remains adjacent the first operative surface and the barrier fluid remains spaced from the first operative surface from the upstream channel end to the downstream channel end. The barrier fluid may remain spaced from the first operative surface from the upstream channel end to the downstream channel by a distance equal to 0.4% to 50%, 1.4% to 18%, or 3.6% to 10.7% of a total height of the flowcell channel at the upstream channel end as measured between the first operative surface and an opposite interior wall of the flowcell channel. The total height of the flowcell channel at the upstream channel end as measured between the first operative surface and an opposite interior wall of the flowcell channel may be about 140 micrometers, and the barrier fluid may remain spaced from the first operative surface from the upstream channel end to the downstream channel by a distance equal to about 0.5 micrometers to about 50 micrometers, about 2 micrometers to about 25 micrometers, or about 5 micrometers to about 15 micrometers. The first reagent fluid comprises may be provided in a direction parallel to the first operative surface. The barrier fluid may be provided in a direction parallel to the first operative surface.
The method also may include providing a second reagent fluid to the upstream channel end at a third location adjacent a second operative surface comprising a second plurality of DNA templates, wherein the second operative surface is spaced from the second location, and passing the second reagent fluid through the flowcell channel with the first reagent fluid and the barrier fluid under laminar flow conditions such that the second reagent fluid remains adjacent the second operative surface and the barrier fluid remains spaced from the second operative surface from the upstream channel end to the downstream channel end. The first operative surface may be parallel to and facing the second operative surface. The first reagent fluid and the second reagent fluid may be the same, or different. The second location may be spaced from the first operative surface by a first distance and the second location may be spaced from the second operative surface by a second distance, and the first distance may not equal or may equal the second distance. The first and second distance may be 0.4% to 50%, 1.4% to 18%, or 3.6% to 10.7% of a total height of the flowcell channel at the upstream channel end as measured between the first operative surface and an opposite interior wall of the flowcell channel. The total height of the flowcell channel may be about 140 micrometers, and the barrier fluid may remain spaced from each of the first and second operative surfaces by a distance equal to about 0.5 micrometers to about 50 micrometers, about 2 micrometers to about 25 micrometers, or about 5 micrometers to about 15 micrometers.
In another exemplary aspect, there is provided a method of operating a processing instrument, the method including providing a flowcell channel having an upstream channel end, a downstream channel end, a longitudinal axis extending from the upstream channel end to the downstream channel end, a first operative surface extending between the upstream channel end and the downstream channel end and comprising a first plurality of DNA templates, and a second operative surface extending between the upstream channel end and the downstream channel end and comprising a second plurality of DNA templates, providing a first reagent fluid comprising a first reactive chemistry to the upstream channel end channel at a first location adjacent the first operative surface, providing a second reagent fluid comprising a second reactive chemistry that is different from the first reactive chemistry to the upstream channel end at a second location adjacent the second operative surface, providing a barrier fluid that is different from the first reagent fluid and the second reagent fluid to the upstream channel end at a third location spaced from the first operative surface and the second operative surface, and between the first location and the second location, and passing the first reagent fluid and the barrier fluid through the flowcell channel under laminar flow conditions such that the first reagent fluid remains adjacent the first operative surface, the second reagent fluid remains adjacent to the second operative surface, and the barrier fluid remains spaced from the first operative surface and the second operative surface from the upstream channel end to the downstream channel end. The method may also include periodically passing the second reagent fluid through flowcell channel at the first location and the first reagent fluid through the flowcell at the second location, simultaneously with passing the barrier fluid through the flowcell channel at the third location, under laminar flow conditions such that the second reagent fluid remains adjacent the first operative surface, the first reagent fluid remains adjacent to the second operative surface, and the barrier fluid remains spaced from the first operative surface and the second operative surface from the upstream channel end to the downstream channel end. The method also may include imaging the first plurality of DNA templates when the first reagent fluid it passed through the flowcell channel at the first location, and imaging the second plurality of DNA templates when the first reagent fluid is passed through the flowcell at the second location.
In another exemplary aspect, there is provided a processing instrument programmed to perform the methods described above.
Other alternatives will be apparent to persons of ordinary skill in the art in view of the present disclosure.
The recitation of this summary of the invention is not intended to limit the claims of this or any related or unrelated application. Other aspects, embodiments, modifications to and features of the claimed invention will be apparent to persons of ordinary skill in view of the disclosures herein.
A better understanding of the exemplary embodiments may be had by reference to the attached drawings, in which like reference numbers designate like parts. The drawings are exemplary and not intended to limit the claims.
The inventors have determined that typical processing instrument flowcells can operate under conditions that waste a significant proportion of the expensive reactive chemicals that are necessary to perform the sequencing and imaging processes. In sequencing instruments, for example, these reactive chemicals may include an imaging chemistry comprising a buffer used during the imaging process, extension chemistry comprising fluorescently-labeled nucleotides that are used to extend the DNA templates, and a cleaving chemistry that is used to remove the fluorescent labels from the added nucleotides and remove a termination group to allow subsequent addition of nucleotides. Such reagents are expensive, and account for a large part of the operating costs of sequencing instruments. The exemplary embodiments described herein are provided in the context of DNA sequencing instruments, it will be readily appreciated that embodiments may be used in other kinds of processing instruments that use flowcells.
A typical microfluidic flowcell has very small dimensions (e.g., less than 1 mm in height), and has a relatively high surface area-to-volume ratio. As noted above, the height of the flowcell often is optimized between minimal reagent consumption, which urges the design towards reducing the flowcell height, and providing a reasonable pressure drop across the flowcell, which urges against reducing the flowcell height.
It has been determined that the fluid behavior in typical microfluidic flowcells is defined mainly by the fluid's interactions with the channel surfaces where viscous forces dominate over inertial forces. This leads to laminar flow conditions inside the channel, in which the reagent fluid (and the reactive chemicals located in the reagent fluid) moves in layers that maintain their relationship with one another throughout their passage through the channel (i.e., fluid at the top of the channel generally remains at the top, and fluid at the bottom generally remains at the bottom). For example, in a system having a flowcell channel with a height of 0.14 mm and a width of 6 mm, and a reagent fluid having approximately the same kinematic viscosity of water at 20° C. (˜1 mm2/s) flowing at 67 μL/s, the Reynolds number for the flow is approximately 22, which indicates that the flow is laminar.
Furthermore, it has been determined that the reactive chemicals in the reagent fluid can take considerable time to diffuse between the layers, even when the flow of reagent fluid has stopped. For example, fluorescently-labeled nucleotides having a diffusion coefficient of ˜200 μm2/s can take an average of about one minute to diffuse a 140 μm distance from the bottom of a flow cell to the top of a flow cell (and vice-versa).
As a result of these laminar flow and slow diffusion conditions, it is expected that a large proportion of the reactive chemicals may never be used. For example, in the exemplary flowcell system described above (i.e., a height of 0.14 mm, etc.), each sequencing cycle may include sequentially flooding the flowcell channel with a series of reactive chemicals, following each with a respective incubation time. The reactive chemicals located in layers furthest from the immobilized DNA templates are likely to never be used because they remain in the distant layer during the flooding process, and do not have sufficient time to diffuse to react with the DNA templates before the next sequencing step begins (the sequencing time potentially could be increased to increase the likelihood of reaction, but this sacrifices processing speed). In this diffusion-limited environment, a large proportion of reactive chemicals may be discarded in each reagent flooding cycle.
In view of the foregoing, it has been determined that the fluidic layers closest to the surfaces holding the DNA templates are the most relevant layers of the flow, because these layers are the first ones to bring reagents necessary for the biochemical reactions to the DNA templates, and because reactive chemicals beyond the closest layers are unlikely to diffuse into range to react with the DNA templates.
To address the problem of waste caused by laminar flow and the slow reaction kinetics of slowly-diffusing reactive chemicals, the inventors have provided a new flowcell design in which a supplemental barrier fluid flow is introduced adjacent to the reagent fluid to restrict the reagent fluid to a region proximal to the operative surface upon which the DNA templates are immobilized. Descriptions of exemplary embodiments follow, but it will be appreciated that the scope of the invention is not limited to any particular example, and the examples may be combined and modified in various ways, as will be understood by one of ordinary skill in the art in view of the present disclosure.
A first exemplary embodiment is illustrated in
The instrument 100 further includes a barrier fluid supply 122 that is fluidically connected to a barrier fluid inlet 204 to the flowcell 102. A barrier fluid pump 124 also may be provided in the fluid path between the barrier fluid supply 122 and the secondary flowcell inlet 204, and configured to convey the barrier fluid to the flowcell 102 under positive pressure. Alternatively, the barrier fluid may be conveyed under negative pressure by a pump downstream of the flowcell 102.
The channel 206 includes at least one operative surface upon which a plurality of DNA templates 212 are immobilized. Suitable structures for the operative surface and the manners in which DNA templates can be immobilized thereto are known in the art, and need not be described herein. Typically, the operative surfaces will be flat, so as to present the DNA templates in a single plane to facilitate imaging. In
The reagent inlet 200 is configured to direct its flow of reagent fluid into contact with the operative surface (i.e., the upper surface in
The proportional sizes of the exit portions 218, 220 may be selected as desired. For example, the exit portion 218 of the reagent inlet 202 may extend from a proximal location 226 that is located at the level of the operative surface, to a distal location 228 that is located at a distance d from the operative surface. For example, in a flowcell 102 having a channel height H of 140 μm at the upstream end, the distance d may be from 0.5 μm to 50 μm, from 2 μm to 25 μm, or most preferably from 5 μm to 15 μm. As another example, this distance d may be equal to about 0.4% to 36%, 1.4% to 18%, or 3.6% to 10.7% of the total channel height H as measured at the upstream end.
In use, reagent fluid including the reactive chemistry is directed through the reagent inlet 200 at the same time that barrier fluid is directed through the barrier fluid inlet 204. Operating under laminar flow conditions such as those described above, the barrier fluid abuts the reagent fluid at a boundary layer 222. A small amount of mixing may occur at the boundary layer 222, but for the most part the two fluid flows are expected to remain separate as they pass through the chamber 206 from the chamber inlet 208 to the chamber outlet 210. Thus, the barrier fluid effectively restricts the reagent fluid to a boundary region 224 adjacent the operative surface.
The position of the boundary layer 222 may be modified to adjust the size of the boundary region 224. It is expected that the boundary layer 222 will begin at or near the same distance d as the distal region 228 of the exit portion 218 of the reagent inlet 202. Assuming similar flow velocities for the reagent fluid and the barrier fluid, it is also expected that the boundary layer 222 will be generally parallel to the operative surface. For example, in a flowcell 102 having a channel height H of 140 μm, the boundary layer 222 may be positioned a distance from the operative surface of 0.5 μm to 50 μm, from 2 μm to 25 μm, or most preferably from 5 μm to 15 μm (or as another example, this boundary layer may be at a distance equal to about 0.4% to 50%, 1.4% to 18%, or 3.6% to 10.7% of the total channel height H). Some variation and degradation of the boundary layer 222 may occur, particularly at greater distances from the upstream end 208 of the channel 206. Where the boundary layer 222 is not entirely parallel to the operative surface, the measurement of the distance from the surface will be understood to be the distance at the upstream flow channel end 208. Degradation of the boundary layer 222 may be accounted for and the amount of or effects of such degradation reduced by modifying the starting position of the boundary layer 222, the length of the channel 206 in the flow direction F, the flow rates and pressures of the two fluids, and so on. Such modifications will be within the ability of a person of ordinary skill in the art, in view if this disclosure, without undue experimentation. Preferably, the boundary layer 222 remains intact for the full length of the operative surface that includes DNA templates, but this is not strictly required.
It is expected that a flowcell that uses a barrier fluid to restrict the reagent fluid to a boundary region 224 adjacent the operative surface will provide several benefits. For example, the barrier fluid effectively replaces a portion of the reagent fluid that would normally be used in the flowcell 102, which reduces reagent fluid consumption. This can provide a significant cost benefit by replacing a large portion of the reagent fluid with relatively inexpensive barrier fluid. Furthermore, the concentration of reactive chemicals in the reagent fluid can be increased in order to provide faster kinetics of diffusion-limited biological reactions, while still obtaining a cost reduction as compared to conventional systems. For example, the barrier fluid may replace 75% of the reagent fluid, and the concentration of reactive chemicals in the reagent fluid can be doubled, to obtain a 50% reduction in reactive chemical costs and nearly a 50% reduction in fluid chemistry costs (assuming the barrier fluid has a negligible cost as compared to the reactive chemicals). While similar reductions in costs might be obtained by simply reducing the size of the channel 206, doing so is expected to result in a significantly higher pressure drop across the flowcell 102, which may not be practical and may result in excessive clogging. The use of the barrier fluid allows the flowcell 102 to operate at conventional pressure differentials (and even reduced pressure differentials) while still obtaining the benefit of a concentrated flow of reagent fluid.
Any suitable barrier fluid may be used. For example, the barrier fluid may comprise an inexpensive and inert chemical, such as saccharide or a derivative thereof. The barrier fluid also may comprise a viscous fluid, such as polyethylene glycol, to decrease the diffusion of the reactive chemicals across the boundary layer 222 and out of the reagent flow. The barrier fluid also may comprise an immiscible fluid, such as mineral oil or silicone oil, to help prevent any exchange of reactive chemicals across the boundary layer 222. The buffer fluid also may comprise a non-reactive composition—i.e., a composition that does not chemically react as part of the sequencing process. Other alternatives will be apparent to persons of ordinary skill in the art in view of the present disclosure.
The first channel layer 302 includes a first channel 310 having a first end 312, a middle region 314, and a second end 316. The second channel layer 306 includes a second channel 318 having a first end 320, a middle region 322, and a second end 324. A series of first reagent openings 326 are provided in the layers above the first channel layer 302, and aligned with one another to provide a fluid passage through the cover 308 and to the first end 312 of the first channel 310. Similarly, one or more barrier fluid openings 328 are provided in the layers above the second channel layer 306, and aligned with one another to provide a fluid passage through the cover 308 and to the first end 320 of the second channel 318. A series of outlet openings 330 are provided in the cover 308 and divider layer 304, and aligned with one another to form a fluid passage through the cover 308 and to the second end 316 of the first channel 310, and the second end 324 of the second channel 318. Finally, the divider layer 304 includes a channel opening 332 that is aligned with the middle regions 314, 322 of the channels 310, 318, to combine and form a flowcell channel 400 that passes through the flowcell 102.
When assembled, the various parts and openings form the passages through the flowcell 102. A flow of reagent fluid can enter the reagent opening 326 through the cover 308, flow along the flowcell channel 400, and exit through the outlet openings 330. Similarly, a flow of barrier fluid can enter the barrier fluid opening 328 through the cover 308, flow along flowcell channel 400, and exit through the outlet openings 330. It will be appreciated that the arrangement of the openings and channels can be modified in any suitable way. For example, one or more of the reagent openings 326, the barrier fluid openings 328, or the outlet openings 330 may be configured to pass through the base 300, rather than the cover 308. The various openings 326, 328, 330 also may pass through the sides of the flowcell. Any configuration in which one or more openings are provided to convey fluid into and out of the first channel 310 and the second channel 318 may be used in other embodiments. Other alternatives will be apparent to persons of ordinary skill in the art in view of the present disclosure.
While not strictly necessary, the divider 304 provides a gasket to seal the first channel layer 302 to the second channel layer 306. Additional gasket layers (not shown) may be provided between the base layer 300 and the first channel layer 302, and the second channel layer 306 and the cover 308. The divider 304 also may be configured to divide the first channel 310 from the second channel 318 until they reach the flowcell channel 400, as shown in
The foregoing construction is expected to provide suitable results and allow relatively simple manufacturing techniques. For example, the base layer 300, first channel layer 302 and second channel layer 314 may be made from sheet metal using conventional machining processes or from plastic using conventional molding and machining processes. The divider 306 may be integral with one of the other layers, constructed as a separate part in the same way as the other layers, or made of a pliable material (e.g., plastic film or sheet) that is formed using a stamping process. All embodiments are not intended to be limited to these construction techniques and materials, however, and other embodiments may use different constructions. For example, the flowcell 102 may be made of metal or plastic using machining, injection molding or three-dimensional printing to form the passages instead of stacking layers to form the passages. Other alternatives will be apparent to persons of ordinary skill in the art in view of the present disclosure.
It is also expected that the foregoing benefits of using a barrier fluid may be obtained in flowcells having multiple operative surfaces. For example,
A first reagent inlet 516, a second reagent inlet 518 and a barrier fluid inlet 520 are fluidically connected to the upstream end 512 of the channel 510. The downstream end 514 of the channel 510 is fluidically connected to a flowcell outlet 526. As in the embodiment of
As with the earlier embodiments, the first and second reagent inlets 516, 518 are fluidically connected to one or more reagent supplies, and may be have their own independent supply pumps or operate through a common pump. The barrier fluid inlet 520 is fluidically connected to a barrier fluid supply. Pumps are provided to convey reagents and barrier fluid into the flowcell under positive or negative pressure (or a combination of both).
This embodiment is expected to operate in the same manner as the earlier-described embodiments. In particular, reagent fluids may be directed through the first and second reagent inlets 516, 518 simultaneously with a flow of barrier fluid through the barrier fluid inlet 520. The fluids establish a laminar flow through the channel 510, and the barrier fluid creates first boundary layer 522 at the border of the first reagent flow, and a second boundary layer 524 at the border of the second reagent flow, with little or no mixing or diffusion across either boundary layer 522, 524.
This arrangement provides the benefits described above, such as reducing the consumption of expensive reactive chemical. This arrangement also allows the first and second operative surfaces 502, 504 to be subjected to two different reagent fluids. For example, the first operative surface 502 may be subjected to a first reagent flow A, and the second operative surface 504 may be subjected to a second reagent flow B that is different from the first reagent flow A. The first and second reagent flows A, B may comprise any chemistry used during the sequencing and imaging process. For example, one reagent flow may comprise chemistry used to cleave blockers from existing DNA templates to prepare the DNA templates to assimilate additional nucleotides during the extension process, and the other reagent flow may comprise fluorescently-labeled nucleotides that join to the DNA templates to perform the extension process. Other alternatives will be apparent to persons of ordinary skill in the art in view of the present disclosure. In use, the reagent flows can be alternatively applied to each operative surface 502, 504 in any desired sequence of operation, such as simultaneous, alternating, staggered, etc.
This ability to simultaneously expose the first and second operative surfaces 502, 504 to different chemistries can be used to simultaneously perform imaging on the DNA templates on one operative surface, while performing nucleobase extension on the DNA templates on the other operative surface. This may significantly increase the throughput rate of the instrument by reducing the lag time between subsequent imaging operations. To facilitate this, both the cover 506 and the base 508 may comprise a transparent surface, so that imaging can be performed from both sides of the flowcell 500. Furthermore, the barrier fluid may be opaque to provide a dark background during each imaging session, or have other properties helpful to the sequencing process. Other alternatives and advantages will be apparent to persons of ordinary skill in the art in view of the present disclosure.
The first channel layer 602 includes a first channel 614 having a first end 616, a middle region 618, and a second end 620. The second channel layer 606 includes a second channel 622 having a first end 624, a middle region 626, and a second end 628. The third channel layer 610 includes a third channel 630 having a first end 632, a middle region 634, and a second end 636.
A series of first reagent openings 638 are provided in the layers above the first channel layer 602, and aligned with one another to provide a fluid passage through the cover 612 and to the first end 616 of the first channel 614. Similarly, a series of barrier fluid openings 640 are provided in the layers above the second channel layer 606, and aligned with one another to provide a fluid passage through the cover 612 and to the first end 624 of the second channel 622. A second reagent opening 642 is provided in the layer or layers above the third channel layer 610, and aligned to provide a fluid passage through the cover 612 and to the first end 632 of the third channel 630. A series of outlet openings 644 are provided in the divider layers 604, 608 and the cover 612, and aligned with one another to form a fluid passage through the cover 612 and to the second end 620 of the first channel 614, the second end 628 of the second channel 622, and the second end 636 of the third channel 630. Finally, the first and second divider layers 604, 608 include channel openings 646 that are aligned with the middle regions 618, 626, 634 of the channels 614, 622, 630, and combine to form a flowcell channel that passes through the flowcell 500. As with the embodiment of
When assembled, the various parts and openings form the necessary passages through the flowcell 500. This construction is expected to provide suitable results and allow relatively simple manufacturing techniques, as discussed in relation to the embodiment of
A further exemplary embodiment and its operation are illustrated in
The reagent inlet 702 and barrier fluid inlet 704 are provided as openings through the flowcell base 710 and/or cover 712. The outlet 706 may be an opening through the base 710 or cover 712. One or more of the reagent inlet 702, barrier fluid inlet 704 and outlet 706 alternatively may be formed as a passage through the perimeter spacer 714.
The reagent inlet 702 and barrier fluid inlet 704 may be spaced along a longitudinal axis 716 of the channel 708 by a distance z, with the reagent inlet 702 preferably (but not necessarily) being downstream of the barrier fluid inlet 704. The use of spaced inlets 702, 704 is expected to provide relatively straightforward manufacturing using existing production technologies for conventional flowcells. The distance z may be selected to optimize the development of suitable laminar flow conditions. The distance z also may be selected to accommodate constraints of the instrument, such as the requirement to provide fittings to the inlets 702, 704 and the like. In exemplary embodiments, the distance z may be 1-2 millimeters, but other distances may be used.
An inlet spacer 718 may be provided around the barrier fluid inlet 704 to space the barrier fluid inlet 704 relative to the reagent inlet 702 in a direction perpendicular to the longitudinal axis 716 by a predetermined distance d. The inlet spacer 718 may be integral to or separate from the perimeter spacer 714. The inlet spacer 718 also may be movable to alter the distance d. For example, the inlet spacer 718 may comprise a tube that is movable through the opening that defines the barrier fluid inlet 704 to place the end of the barrier fluid inlet 704 at alternative distances d from the reagent inlet 702. Alternatively, the inlet spacer 718 may be removable and replaceable with an inlet spacer 718 of a different thickness to define a new distance d.
The inlet spacer 718 is expected to guide the inert barrier fluid over the reagent fluid in a way that reduces or eliminates uncontrolled effects at the meeting zone between the fluids that might impair the development of the desired laminar flow conditions. For example, a Venturi effect may occur when the barrier fluid passes over the reagent inlet 702, in which the moving reagent fluid exerts additional low pressure to the reagent inlet fluid and pulls portions of the reagent fluid into the barrier fluid. This can lead to microfluidic droplets of reagent fluid forming within the barrier fluid. Localized mixing at the junction of the fluids also may occur due to other fluid mechanics. The inlet spacer 718 may reduce or eliminate this and other phenomena, and embodiments are not intended to be bound to any particular theory of operation.
As shown in an exemplary manner in the plan view of
The first width x preferably is equal to the width of the reagent inlet 702. This is expected to ensure that the incoming reagent distributes across the full width of the intermediate zone 722 of the channel 708. In contrast, if the first width x is greater than the reagent inlet 702 width, it is expected that laminar flow effects may prevent the reagent from expanding laterally to the full width y of the intermediate zone 722. Nevertheless, in embodiments in which it is not necessary for the reagent to extend across the full width y of the intermediate zone 722, the first width x may be greater than the width of the inlet channel 702. The second width y may be any width (e.g. 6 mm), but preferably is selected such that the fluids maintain a sufficient degree of laminar flow and separation throughout the channel 708.
It is expected that, when the reagent and barrier fluid are pumped through their respective inlets 702, 704, the fluids will meet at the reagent inlet 702 and create an overlapping multiphase laminar flow over the whole channel width x within the inlet zone 720. When the channel 708 expands to the intermediate zone 722, overlapping laminar flow will expand accordingly due to laminar behavior of the fluids. In some embodiments, it may be necessary or desirable to provide a certain distance between the reagent inlet 702 and the end of the inlet zone 720 (i.e., before lateral expansion) to stabilize the laminar behavior and reduce or eliminate fluidic effects that might disturb the balanced expansion of the two fluids when they reach the intermediate zone 722. For example a gap distance g in the range of 0.2 to 5.0 mm or even longer may be provided between the reagent inlet 702 and the end of the inlet zone 720.
Examples of how the foregoing flowcell 700 (or similar flowcells) may be used are described and illustrated with reference to
One or more of the reagent inlet 702, barrier fluid inlet 704 and outlet 706 may include a flow valve to control the passage of fluid. Such valves may be located anywhere along the flow passage leading to the respective opening, but it may be preferable for the valve to be as close as possible to the opening to minimize residual flow when the valve is closed. For example, the reagent inlet 702 may have a reagent inlet valve 904 that, when opened, allows reagent to pass into the channel 708 when a downstream pump is activated to pull the barrier fluid into the channel 708. The reagent valve 904 preferably may be gradually adjusted to vary the flow resistance and thus flow rate of the reagent passing through the reagent inlet 702. This feature may be used, for example, to gradually adjust and selectively choose the volume proportions of the reagent and the barrier fluid.
A second operation mode is illustrated in
A third operation mode is illustrated in
It will be appreciated that the foregoing embodiments may be modified in various ways. For example, the barrier fluid inlet 704 may be downstream of the reagent inlet 702, or the flowcell channel 708 may have a third inlet to receive a second flow of reagent fluid to provide reactive chemistry to the top and bottom of the channel 708. Also, the feature of an expanding-width flowcell channel also may be provided in the earlier embodiments describe herein in relation to
The present disclosure describes a number of new, useful and nonobvious features and/or combinations of features that may be used alone or together. It is expected that embodiments may be particularly helpful to reduce the cost of goods associated with high-throughput nucleic acid sequencing systems, but other benefits may be provided, and it will be appreciated that reduced cost is not necessarily required in all embodiments. While the embodiments described herein have generally been explained in the context of sequencing by syntheses processes, it will be appreciated that embodiments may be configured for use in other sequencing processes that use visual observation of chemical labels. The embodiments described herein are all exemplary, and are not intended to limit the scope of the inventions. It will be appreciated that the inventions described herein can be modified and adapted in various and equivalent ways, and all such modifications and adaptations are intended to be included in the scope of this disclosure and the appended claims.
This application claims the benefit of U.S. Provisional Application No. 62/305,636, entitled LAMINAR FLUIDIC SEPARATION IN FLOWCELLS FOR MINIMAL REAGENT USAGE filed Mar. 9, 2016, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62305636 | Mar 2016 | US |
