Fluidics system for reagent delivery to the flow cell

Abstract

The present application relates to a fluid system for delivering reagents to a flow cell. A fluid system includes: a reagent manifold including a plurality of channels configured for fluid communication between a reagent cartridge and an inlet of a flow cell; a plurality of reagent pipettes extending downwardly from a port in the manifold, each reagent pipette configured to be placed into a reagent reservoir of the reagent cartridge so that a liquid reagent can be drawn into the pipette from the reagent reservoir; and at least one valve configured to regulate fluid communication between the reservoir and the inlet of the flow cell. The reagent manifold can also include a reservoir for reagent reuse.

Description

[0001] This application is a divisional application of an application filed on August 7, 2014, with application number 201810252278.6 and invention name “Fluid system for delivering reagents to a flow cell”.

[0002] The application with the application date of August 7, 2014, the application number of 201810252278.6, and the invention name of “Fluid system for delivering reagents to a flow cell” is a divisional application of the application with the application date of August 7, 2014, the application number of 201480042435.0, and the invention name of “Fluid system for delivering reagents to a flow cell”.

[0003] Priority claim

[0004] This application claims the benefit of currently pending U.S. Provisional Application No. 61 / 863,795, filed August 8, 2013, which is incorporated herein by reference. background

[0005] Generally speaking, embodiments of the present disclosure relate to apparatus and methods for fluid manipulation and optical detection of samples, such as in nucleic acid sequencing processes.

[0006] Our genome provides a blueprint for predicting many inherent tendencies such as our preferences, talents, susceptibility to disease, and responsiveness to therapeutic drugs. The human genome alone contains over 3 billion nucleotide sequences. Differences in just a small fraction of those nucleotides give us many unique characteristics. The research community has made great progress in figuring out the features that make up the blueprints and, with it, a more complete understanding of how the information in each blueprint relates to human health. However, our understanding is far from complete, and this has hindered the transfer of information from the research laboratory to the clinic, where the hope is that, one day, each of us will have a copy of our own personal genome, enabling us to sit down with our doctors to determine the appropriate choices for a healthy lifestyle or an appropriate course of treatment.

[0007] Current bottleneck is the problem of throughput and scale.The basic component of figuring out the blueprint of any given individual is to determine the accurate sequence of 3,000,000,000 nucleotides in its genome.Technology can be used to do this, but those technologies usually spend many days and tens of thousands of dollars to perform.In addition, the clinical relevance of any individual's genome sequence is the problem of comparing the unique characteristics of its genome sequence (that is, its genotype) and the reference genome related to known characteristics (such as phenotype).When considering that the reference genome is created based on the correlation of genotype and phenotype (it usually uses thousands of individuals, so that it is statistically effective), the problem of scale and throughput becomes apparent.Therefore, billions of nucleotides can be sequenced to thousands of individuals, so as to identify any clinically relevant genotype and phenotype correlation.Further connected with the quantity of disease, drug response and other clinically relevant features, the needs of very cheap and fast sequencing technology become more and more obvious.

[0008] What is needed is a reduction in sequencing costs that facilitates large genetic association studies performed by research scientists and makes sequencing accessible in clinical settings for treatment of individual patients to make life-changing decisions. Embodiments of the invention presented herein meet this need and provide other advantages as well. Summary of the invention

[0009] The present disclosure provides a fluid system, which includes: a reagent manifold, including a plurality of channels configured for fluid circulation between a reagent cartridge and an inlet of a flow cell; a plurality of reagent pipettes extending downward from a port in the manifold, each reagent pipette being configured to be placed in a reagent cell of the reagent cartridge so that a liquid reagent can be sucked into the pipette from the reagent cell; and at least one valve configured to regulate fluid circulation between the cell and the inlet of the flow cell.

[0010] One or more of the channels in the reagent manifold include a reservoir.

[0011] The volume of one or more of the reservoirs is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% of the volume of a flow cell channel in fluid communication with the reservoir.

[0012] The reservoir includes sufficient volume to allow a certain amount of liquid reagent in one or more flow cell channels to flow into the reservoir, such that the liquid reagent from the flow cell is not directed back into the reagent reservoir after contacting the flow cell.

[0013] The amount of liquid reagent comprises 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the liquid reagent in one or more flow cell channels.

[0014] One or more of the reservoirs are in fluid communication with a pump, wherein the pump is configured to move liquid reagents from the reservoirs to the flow cell and from the flow cell back to the reservoir, wherein reagents enter the flow cell and reagents leave the flow cell through the same port of the flow cell.

[0015] At least one valve is configured to variously direct liquid reagent from the flow cell back to the reservoir or to waste.

[0016] The reservoir is configured to reduce mixing of fluid in the reservoir, thereby maintaining a gradient of liquid reagent along the length of the reservoir from an end proximal to the flow cell to an end distal to the flow cell.

[0017] The reservoir includes a plurality of mixing elements configured to facilitate mixing of fluid in the reservoir.

[0018] The mixing element comprises a static feature in the reservoir or on an interior surface of the reservoir.

[0019] The mixing element comprises a baffle element.

[0020] The reservoir includes a serpentine channel.

[0021] The reservoir includes a non-cylindrical channel.

[0022] The reagent manifold is configured to deliver a reagent from a first reagent reservoir to a first valve via a first channel and from the first reagent reservoir to a second valve via a second channel.

[0023] The reagent manifold includes multiple layers, and each of the first channel and the second channel resides in a separate layer of the multiple layers.

[0024] The first channel and the second channel converge to form a T-junction at the port.

[0025] Each of the plurality of reagent pipettes includes a tubular body having a proximal end and a distal end, the distal end tapering to a tip configured to pierce a foil layer covering a reagent reservoir in the reagent cartridge.

[0026] The plurality of reagent pipettes are provided with a single lumen extending through the tubular body from the distal end to the proximal end, the single lumen being configured for fluid communication between the reagent cartridge and the reagent manifold.

[0027] A subset of the plurality of reagent pipettes has a shorter length than other reagent pipettes in the plurality of reagent pipettes.

[0028] The length of the subset is at least 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mm shorter than the other reagent pipettes.

[0029] At least one of the plurality of reagent pipettes includes a compliant tip configured to bend when the compliant tip strikes a bottom of a reagent well in a reagent cartridge.

[0030] The fluidic system is operatively associated with the detection device in a configuration for detecting, by the detection device, a characteristic of nucleic acids in the flow cell.

[0031] The detection apparatus includes a plurality of microfluorometers, wherein each of the microfluorometers includes an objective configured for widefield image detection in an image plane along x and y dimensions.

[0032] The reagent manifold includes between 10 and 20 ports, each port configured to couple a reagent pipette to a channel in fluid communication with the at least one valve.

[0033] The fluid system also includes one or more alignment pins extending downward from the reagent manifold along an axis parallel to the plurality of reagent pipettes, the alignment pins being longer than the plurality of reagent pipettes and configured to engage with corresponding interface slots on the reagent cartridge.

[0034] The at least one valve has a plurality of inlet ports and one or more outlet ports, each of the inlet ports being in fluid communication with a reagent pipette and each outlet port being in fluid communication with a flow cell inlet or a waste container.

[0035] The fluidic system includes a first valve and a second valve configured to independently deliver separate reagents across a first channel and a second channel of a flow cell.

[0036] Reagent flow across the first and second channels occurs in opposite directions.

[0037] The fluid system also includes a sensor for detecting bubbles, the sensor being positioned along a fluid pathway between the reagent cell and the flow cell.

[0038] The sensor is positioned along the fluid line between the at least one valve and the flow cell.

[0039] The sensor is positioned along the fluid line between the reagent reservoir and the at least one valve.

[0040] The present disclosure also provides a kit configured to interact in fluid communication with a fluid system as described above.

[0041] The present disclosure also provides a test kit, which includes: a plurality of reagent pools configured to simultaneously engage with a plurality of reagent pipettes of a fluid system along the z dimension, so that liquid reagents can be sucked into the pipettes from the reagent pools, the reagent pools being arranged along the x and y dimensions to the top row, the middle row, and the bottom row, wherein the reagent pools along the top row and the bottom row of the box are deeper along the z dimension than the reagent pools in one or more middle rows; and at least two interface grooves configured to engage with corresponding alignment pins of the fluid system.

[0042] The test kit also includes multiple flushing pools, which are arranged in a configuration corresponding to the multiple reagent pools, whereby the flushing pools are configured to simultaneously engage the multiple reagent pipettes when the multiple reagent pipettes are not engaged with the multiple reagent pools, so that the flushing buffer can be sucked into the multiple reagent pipettes from the flushing pools.

[0043] The reagent kit also includes at least two additional interface slots configured to engage with corresponding alignment pins of the fluid system so that the reagent pipette of the fluid system is positioned aligned with the wash reservoir.

[0044] The reagent kit includes a foil layer covering at least one of the plurality of reagent cells.

[0045] Also provided is a multilayer diffusion-bonded reagent manifold comprising at least 10, 15, or at least 20 ports, each port configured to draw reagent from a separate reagent reservoir via a pipette, wherein the ports are in fluid communication with one or more channels of a flow cell via fluid channels in the manifold.

[0046] The reagent manifold includes a first valve and a second valve, wherein a channel connected to the first valve resides on a first layer and a channel connected to the second valve resides on a second layer.

[0047] One or more of the channels in the reagent manifold include a reservoir.

[0048] The volume of one or more of the reservoirs is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% of the volume of a flow cell channel in fluid communication with the reservoir.

[0049] The reservoir includes sufficient volume to allow a certain amount of liquid reagent in one or more flow cell channels to flow into the reservoir, such that the liquid reagent from the flow cell is not directed back into the reagent reservoir after contacting the flow cell.

[0050] The amount of liquid reagent comprises 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the liquid reagent in one or more flow cell channels.

[0051] One or more of the reservoirs are in fluid communication with a pump configured to move liquid reagents from the reservoirs to the flow cell and from the flow cell back to the reservoirs, wherein reagents enter the flow cell and reagents leave the flow cell through the same port of the flow cell.

[0052] At least one valve is configured to variously direct liquid reagent from the flow cell back to the reservoir or to waste.

[0053] The reservoir is configured to reduce mixing of fluid in the reservoir, thereby maintaining a gradient of liquid reagent along the length of the reservoir from an end proximal to the flow cell to an end distal to the flow cell.

[0054] The reservoir includes a plurality of mixing elements configured to facilitate mixing of fluid in the reservoir.

[0055] The mixing element comprises a baffle element.

[0056] The reservoir includes a serpentine channel.

[0057] The reservoir includes a non-cylindrical channel.

[0058] The reagent manifold is configured to deliver a reagent from a first reagent reservoir to a first valve via a first channel and from the first reagent reservoir to a second valve via a second channel.

[0059] The reagent manifold includes a plurality of reagent pipettes extending downwardly from the port, each of the plurality of reagent pipettes being configured to be placed into a reagent reservoir of a reagent cartridge such that a liquid reagent can be drawn from the reagent reservoir into each of the plurality of reagent pipettes.

[0060] Each of the plurality of reagent pipettes includes a tubular body having a proximal end and a distal end, the distal end tapering to a tip configured to pierce a foil layer covering a reagent reservoir in the reagent cartridge.

[0061] The plurality of reagent pipettes are provided with a single lumen extending through the tubular body from the distal end to the proximal end, the single lumen being configured for fluid communication between the reagent cartridge and the reagent manifold.

[0062] A subset of the plurality of reagent pipettes has a shorter length than other reagent pipettes in the plurality of reagent pipettes.

[0063] The length of the subset is at least 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mm shorter than the other reagent pipettes.

[0064] At least one of the plurality of reagent pipettes includes a compliant tip configured to bend when the compliant tip strikes a bottom of a reagent well of a reagent reagent cartridge.

[0065] The present disclosure also provides a method for reusing a reagent, comprising: a) sucking a liquid reagent from a reagent cell into a reservoir, the reservoir being in fluid communication with the reagent cell and at least one channel of a flow cell; b) transferring the reagent from the reservoir to at least one channel of the flow cell; c) transferring at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the reagent on the flow cell channel to the reservoir, so that the liquid reagent from the flow cell is not directed back into the reagent cell after contacting the flow cell; and d) repeating steps b) and c) to achieve reuse of the liquid reagent on the flow cell.

[0066] One or more of the reservoirs are in fluid communication with a pump configured to move liquid reagents from the reservoirs to the flow cells and from the flow cells back to the reservoirs, wherein liquid reagents enter the flow cells and liquid reagents leave the flow cells through the same port of the flow cells.

[0067] Liquid reagent from the flow cell that is not transferred to the reservoir in step c) is transferred to a waste reservoir.

[0068] The transferring in one or both of steps b) and c) is performed via a valve fluidly connecting the reservoir and the flow cell.

[0069] The transport in one or both of steps b) and c) is performed with fluid flow in a single direction.

[0070] The transport in one or both of steps b) and c) is performed using a reciprocating flow.

[0071] The liquid reagents include reagents for performing nucleic acid sequencing.

[0072] The liquid reagent includes a polymerase.

[0073] The liquid reagent comprises nucleotides or a mixture of different nucleotides.

[0074] One or more different reagents are flowed onto the flow cell prior to performing step d).

[0075] The liquid reagent that flows back into the reservoir in step c) mixes with a similar liquid reagent in the reservoir, thereby forming a mixed reagent comprising multiple repetitions of similar liquid reagents from steps b) and c).

[0076] Mixing of liquid reagent flowing back into the reservoir with fresh reagent is minimized within the reservoir.

[0077] The method is performed on a fluidic system or reagent manifold as described above.

[0078] The present disclosure also provides a sequencing system, comprising a detection device and the fluid system as described above.

[0079] The present disclosure also provides a sequencing method, which includes the following steps: (a) providing a fluid system, which includes: (i) a flow cell including an optically transparent surface, (ii) a nucleic acid sample, (iii) a plurality of reagents for a sequencing reaction, and (iv) a fluid system for transporting the reagents to the flow cell; (b) providing a detection device, which includes (i) a plurality of microfluorometers, wherein each microfluorometer includes an objective lens, the objective lens is configured for wide-field image detection along x and y dimensions in an image plane, and (ii) a sample stage; and (c) performing fluid operations of a nucleic acid sequencing process in a box and detection operations of a nucleic acid sequencing process in the detection device, wherein (i) the reagents are transported to the flow cell by the fluid system, (ii) the wide-field image of nucleic acid features is detected by a plurality of microfluorometers, and (iii) at least part of the reagents are moved from the flow cell to a storage tank.

[0080] The fluidic system further includes a waste reservoir, and fluidic operations of the nucleic acid sequencing process further include moving at least some of the plurality of reagents from the flow cell to the waste reservoir.

[0081] The reservoir is in fluid communication with a pump configured to move liquid reagent from the reservoir to the flow cell and from the flow cell back to the reservoir, wherein reagent enters the flow cell and reagent exits the flow cell through the same port of the flow cell.

[0082] Reagent from the flow cell that is not transferred to the reservoir in step (c)(iii) is transferred to a waste reservoir.

[0083] The reagent transfer in step (c)(i) or step (c)(iii) is performed via a valve fluidly connecting the reservoir and the flow cell.

[0084] The reagent transfer in step (c)(i) or step (c)(iii) is performed using fluid flow in a single direction.

[0085] The reagent transfer in step (c)(i) or step (c)(iii) is performed using a reciprocating flow.

[0086] The liquid reagent is a reagent for performing nucleic acid sequencing.

[0087] The reagent is a polymerase.

[0088] The reagent is a nucleotide or a mixture of different nucleotides.

[0089] The details of one or more implementations are set forth in the accompanying drawings and the description that follows. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1A A fluidic system is shown with a reagent pipette interacting with a reagent cartridge.

[0091] Figure 1B An isometric view of a manifold assembly is shown, and an example of a layout of fluid passages in the manifold is shown.

[0092] Figure 2 A front view of the manifold assembly with reagent pipettes, valves and alignment pins is shown. It also shows pipettes of different lengths.

[0093] Figure 3 is a top view of a manifold assembly showing one possible layout of fluid channels in the manifold.

[0094] Figure 4 A cross-sectional view of the passages in the manifold is shown, including a cross-sectional view of the storage lines and non-storage fluid passages.

[0095] Figure 5 Various connectors are shown for connecting the reagent ports to the two valves.

[0096] Figure 6 A cross-sectional view of a reagent cartridge having wells of variable depth is shown.

[0097] Fig. 7A A simplified top view of storage lines in a manifold assembly is shown according to one embodiment.

[0098] Figure 7B Various stages of reagent reuse in a method utilizing reciprocating flow of reagent from a reservoir line to a flow cell, followed by partial refilling of the reservoir line from the flow cell are shown.

[0099] Figure 8 Shown is a top view of a reagent tray interface having reagent wells and interface slots for alignment pins.

[0100] Fig. 9 A fluid diagram for a fluid system is shown.

[0101] Fig.10 Shown is a detailed view of a reagent pipette including a compliant pipette and a piercing pipette. DETAILED DESCRIPTION

[0102] The present disclosure provides a fluid system and method for providing reagents to a chamber, such as a flow cell. A particularly useful application is the detection of fixed biological samples. For example, the method and system proposed herein can be used in nucleic acid sequencing applications. A variety of nucleic acid sequencing techniques can be used, which utilize optical and non-optical detectable samples and / or reagents. These technologies are particularly suitable for the method and apparatus of the present disclosure, and therefore highlight the various advantages of a specific embodiment of the present invention. A part of those advantages is proposed below for ease of explanation, although the nucleic acid sequencing application is demonstrated, the advantages can also be extended to other applications.

[0103] The fluidic systems presented herein are particularly useful utilizing any of the detection device configurations and sequencing methods presented in U.S. Patent Application Serial No. 13 / 766,413 (filed on February 13, 2013, entitled “INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGE USEFUL FORNUCLEIC ACID SEQUENCING”), the contents of which are incorporated herein by reference in their entirety.

[0104] In a specific embodiment, the sample to be tested can be provided to the detection chamber using a fluid system as provided herein. To give a more specific example of a nucleic acid sequencing application, the fluid system can include: a manifold assembly that can be placed in fluid communication with one or more of a reservoir for holding sequencing reagents, a reservoir for holding sample preparation reagents, a reservoir for holding waste generated during sequencing, and / or pumps, valves, and other components capable of moving fluids through a flow cell.

[0105] In a specific embodiment, the fluid system can be configured to allow the reuse of one or more reagents. For example, the fluid system can be configured to transport the reagent to the flow cell, then remove the reagent from the flow cell, and then reintroduce the reagent into the flow cell. The advantage of reusing reagents is to reduce the volume of waste and reduce the cost of processes that utilize expensive reagents and / or reagents that are transported in high concentrations (or in large quantities). Reagent reuse benefits from the following understanding: the exhaustion of reagents occurs only or primarily on the surface of the flow cell, so the majority of the reagents become unused and can be reused.

[0106] Figure 1AAn exemplary fluid system 100 is shown with reagent pipettes 103, 104 and valve 102, which utilizes the advantages of the fluid system provided by several embodiments presented herein. The fluid system 100 includes a manifold assembly 101, which includes various fixed components, including, for example, reagent pipettes, valves, channels, pools, etc. There is a reagent cartridge 400 with reagent pools 401 and 402, wherein the reagent pools 401 and 402 are configured to simultaneously engage a set of reagent pipettes 103 and 104 along dimension z so that liquid reagents can be drawn from the reagent pools into the pipettes.

[0107] Figure 1B Shown is a demonstration manifold assembly 101 that can be used to provide liquid reagents from a reagent cell to a flow cell. The manifold includes reagent pipettes 103 and 104 extending downwardly from a port in the manifold along dimension z. Reagent pipettes 103 and 104 can be placed in one or more reagent cells (not shown) of a test kit. The manifold also includes a channel 107 that fluidically connects reagent pipette 103 to valve 102 and valve 109. Reagent pipettes 103 and 104, channel 107, and valve 102 regulate fluid communication between the reagent cell and the flow cell (not shown). Valves 102 and 109 can select reagent pipettes 103 or 104 individually or in combination, and regulate fluid communication between the reagent cell and the flow cell (not shown) through channels, such as 107.

[0108] Figure 1A and Figure 1B The device shown is exemplary. Figure 1A and Figure 1B Other exemplary embodiments of the methods and apparatus of the present disclosure may be used instead of or in addition to the examples.

[0109] Figure 2 Another exemplary manifold assembly with a reagent pipette and a valve is shown. The manifold has an alignment pin 105 that protrudes downward from the manifold along an axis parallel to the reagent pipette. The alignment pin 105 is longer than the test tube pipette along the z dimension, but in an alternative embodiment, they can also have equal length or shorter. The alignment pin 105 is configured to engage with one or more corresponding interface grooves on a reagent box (not shown). Reagent pipettes 103 and 104 are coupled to the manifold via a port 106 included in the manifold body. Reagent pipette 104 is longer than reagent pipette 103 so as to draw liquid from a reagent reservoir of variable depth corresponding to the depth of reagent pipette 103 or 104. In an alternative embodiment, reagent pipettes 103 and 104 can have equal length, or a switchable main length.

[0110] Figure 2Also shown are channels 107A and 107B, which reside on separate xy planes. Separate channels 107A and 107B can originate from a single channel, which then bifurcates at T-junction 119, thereby generating multiple channels that reside on separate planes. The manifold directs liquid reagents from one pipette to one or more valves by having the channels connected to a particular valve 102 reside entirely on the same plane A or on a combination of planes A and B, while the channels connected to any other valve can share this property of a coplanar or inter-plane starting point.

[0111] Figure 3 1 is a top view of manifold assembly 101 showing one possible layout of fluid channels in the manifold. Fluid channels 107A and 107B originate from a single port 106 and connect port 106 to valve 102A or 102B. Certain channels include reservoirs 108 having sufficient volume to allow a certain amount of liquid reagent to flow from a flow cell (not shown) to the reservoir 108, so that the liquid reagent from the flow cell is not redirected back to the reagent reservoir (not shown) after contacting the flow cell. Figure 3 Also shown are exemplary locations of one or more alignment pins 105 . Figure 3 The manifold assembly shown also includes an inlet port 111 for shared buffer. Each of valves 102A and 102B is configured with an inlet port corresponding to each reagent port 106, and a common output port 112, 110 fluidly connected to a flow cell and a waste port 113, 129 fluidly connected to a waste container.

[0112] As shown in the above-described exemplary embodiments, the fluid system for transporting reagents from the test kit to the flow cell can include a reagent manifold, including a plurality of channels configured for fluid circulation between the test kit and the inlet of the flow cell. The use of a manifold in the fluid system provides several advantages over the use of a separate pipeline. For example, a manifold with fixed channels reduces the possibility of errors during assembly, such as misplacing of pipe attachments and over-tightening or under-tightening of connections. In addition, a manifold provides the ease of maintenance, thereby allowing the rapid replacement of the entire unit rather than the time-intensive testing and replacement of separate pipelines.

[0113] One or more of the channels of the manifold can include a fluid track through the solid material. The track can have any diameter to allow the expected level of fluid transfer through the track. The track can have an inner diameter of, for example, less than 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm or less than 10mm. The track configuration can be, for example, straight or curved. Alternatively or in addition, the track can have a combination of curved and straight portions. The cross-section of the track can be, for example, square, circular, "D" shaped, or any other shape that achieves the expected level of fluid transfer through the track. Figure 4 The fluid path through the manifold body is demonstrated, and a cross-sectional view of one channel 302 is shown. Figure 4 The exemplary channel 302 shown has a "D" shaped cross-section formed by a 0.65 mm diameter semicircle combined with an additional 0.65 mm x 0.325 mm rectangle.

[0114] The passage between the straw and the valve can be completely contained within the manifold body. Alternatively or in addition, the passage can include one or more portions external to the manifold. For example, a conduit, such as a flexible conduit, can connect a portion of a fluid track to another portion of a track on the manifold. Alternatively or in addition, a flexible conduit can connect a flow cell to fixed fluid components of the system, including, for example, pumps, valves, sensors, and meters. As an example, a flexible conduit can be used to connect a flow cell or passage of the system to a pump, such as a syringe pump or a peristaltic pump.

[0115] The manifold body can be composed, for example, of any suitable solid material capable of supporting one or more channels therein. Thus, the manifold body can be a resin, such as polycarbonate, polyvinyl chloride, (polyoxymethylene), PCTFE (polychlorotrifluoroethylene); PEEK TM (Polyetheretherketone); PK (Polyketone); Polyethylene; PPS (polyphenylene sulfide); polypropylene; polysulfone; FEP; PFA; high-purity PFA; R; 316 stainless steel; ETFE (ethylene tetrafluoroethylene); (polymethylpentene); titanium; UHMWPE (ultra-high molecular weight polyethylene); (Polyetherimide); Or any other suitable solid material compatible with the solvent and fluid transmitted by the passage of the manifold in the embodiments provided herein. The manifold body can be formed by a single piece of material. As an alternative or in addition, the manifold body can be formed by multiple layers joined together. The bonding method includes the use of, for example, adhesives, gaskets, and diffusion bonding. The passage can be formed in the solid material by any appropriate method. For example, the passage can be drilled, etched, or ground into the solid material. The passage can be formed in the solid material before multiple layers are joined together. As an alternative or in addition, the passage can be formed after the layers are joined together.

[0116] Figure 5 A variety of connectors 300 are shown for connecting a reagent port 301 to two valves. Figure 5 In each example shown, port 301 is fluidly connected to channel 302, which bifurcates into two channels 302A and 302B, each of which supplies a different valve. In a first configuration, the connector separates the fluid flow from port 301 into channels 302A and 302B on separate layers of the manifold. Figure 5 In the second and third configurations shown, the joint 300 includes rounded squares 303 separated in one layer or full circles 304 separated in one layer of the manifold.

[0117] The manifold assembly provided herein is configured to transport liquid reagents from the test kit to the flow cell. Therefore, the manifold can have any number of ports coupled to a reagent pipette. More specifically, the number of ports can correspond to the number and configuration of the reagent cells in the test kit. In certain embodiments, the manifold comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or at least 30 ports, each port being configured to couple a reagent pipette to a channel for fluid communication with at least one valve.

[0118] The fluid system provided herein can also include an array of pipettes extending downwardly along the z dimension from a port in the manifold, each of the reagent pipettes being configured to be inserted into a reagent reservoir of the test kit so that a liquid reagent can be drawn into the pipette from the reagent reservoir. The reagent pipette can include, for example, a tubular body having a proximal end and a distal end. The distal end can taper to a tip that is configured to pierce a film or foil layer that is used as a seal on the reagent reservoir in the test kit. Various exemplary pipette tips are provided in Fig.10 The reagent pipette can be provided with, for example, a single lumen extending from the distal end to the proximal end through the tubular body. The lumen can be configured to provide fluid communication between the reagent cartridge at one end of the pipette and the reagent manifold at the other end of the pipette. Figure 2 As shown, reagent pipettes 103 and 104 are coupled to the manifold via ports 106 contained in the manifold body.

[0119] In some embodiments, Figure 2 As shown, the subset of reagent pipettes has a shorter length than other reagent pipettes. For example, the length of the subset can be shorter than other reagent pipettes by at least 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or at least 2.0 mm. Manifold and reagent pipette can be used in a device with a lifting mechanism, wherein the lifting mechanism is configured to move the test kit bidirectionally along the z dimension so that the reagent pipette is inserted into the corresponding hole or the pool of the test kit. In certain embodiments, the reagent well can be covered with a protective foil. Therefore, the advantage of providing a variable length pipette is that when the test kit is contacted with the piercing pipette, the lifting mechanism adapts to the reduction of the required force of the foil piercing force. The difference in pipette length can advantageously correspond to the depth of the reagent well in the test kit so that each pipette reaches the expected depth in its corresponding reagent well when the pipette and the box are in a fully engaged position.

[0120] In the embodiments provided herein, the straw can be formed of any suitable material that allows fluid transport through the lumen and is compatible with the solvents and fluids transported through the passages of the manifold. The straw can be formed of a single tube. Alternatively or in addition, one or more straws can be made of multiple sections of straws that together form a desired length and diameter.

[0121] In some embodiments, at least one of the reagent pipettes includes a compliant tip that is configured to bend when the tip collides with the bottom of the reagent well in the test kit. By bending or deforming, the compliant tip allows the inner cavity of the pipette to more completely approach or even contact the bottom of the reagent well, thereby reducing or even eliminating the emptying volume in the reagent well. The compliant tip is particularly advantageous for the uptake of samples or reagents in the case of using small volumes or in the case of most or all of the uptake of the liquid in the desired reagent cell. The body of the pipette with the compliant tip can be made entirely of the same flexible material as the tip. As an alternative or in addition, the body of the pipette can be made of a material different from the tip. The compliant tip can be made of any suitable material so that the compliant tip can be deformed or recessed when it is pushed into contact with the bottom of the reagent cell. Some suitable materials include polymeric and / or synthetic foams, rubbers, silicones and / or elastomers, including thermoplastic polymers, such as polyurethanes.

[0122] The fluidic systems provided herein may also include, for example, pumps and valves that are selectively operable to control fluid communication between the reservoir and the inlet of the flow cell. Figure 2 and Figure 3As shown in the manifold assembly, the channel output on the manifold can be configured to be connected to the corresponding inlet port on one or more valves so that each reagent channel is in fluid communication with the inlet port on the valve. Therefore, via the reagent channel of the manifold, one or more or each of the inlet ports can be in fluid communication with the reagent pipette. Each of the one or more valves can be configured with a common output port (110, 112) that is fluidically connected to the inlet of one or more lanes on the flow cell. As an alternative or in addition, each of the one or more valves can be configured with a waste port (129, 113) that is fluidically connected to one or more waste containers.

[0123] In embodiments where the fluid system includes at least a first valve and a second valve, each valve can be configured to simultaneously deliver separate reagents across a first channel and a second channel of a flow cell, respectively. Thus, one valve can deliver one reagent to a first flow cell channel, while a second valve can simultaneously deliver a different reagent to a second flow cell channel. Fig. 9 As shown in the exemplary embodiment of FIG. 1 , valve A (VA) is fluidly connected to inlet V1 of the flow cell, which is a manifold that delivers reagents to channel 1 and channel 3. Similarly, valve B (VB) is fluidly connected to inlet V2 located at the opposite end of the flow cell, and it delivers reagents to channel 2 and channel 4. Inlets V1 and V2 are located at opposite ends of the flow cell, and the reagent flow direction is in the opposite direction for channels 1 and 3 compared to channels 2 and 4.

[0124] Fluid system as herein described can be advantageously used for the fluid manipulation of the flow cell during nucleic acid sequencing.More specifically, fluid system as herein described can be associated in operation with a detection device according to a configuration for detecting nucleic acid features in the flow cell by a detection device.In certain embodiments, the detection device can include a plurality of microfluorometers, wherein each microfluorometer includes an objective configured for wide-field image detection in the image plane along x and y dimensions.The fluid system proposed herein utilizes any of the detection device configurations proposed in U.S. Patent Application Serial No. 13 / 766,413 (filed on February 13, 2013, entitled "INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGE USEFUL FOR NUCLEICACID SEQUENCING"), which is fully incorporated herein by reference.

[0125] As an example, specifically a nucleic acid sequencing embodiment, a flow cell comprising multiple channels can be fluidically manipulated and optically detected in an interleaved manner. More specifically, fluid manipulation can be performed on a first subset of the channels of the flow cell, while optical detection is performed on a second subset of the channels. For example, in one configuration, at least four linear channels can be arranged in parallel with each other in the flow cell (e.g., channels 1 to 4 can be arranged in sequential rows). Fluid manipulation can be performed on each other channel (e.g., channels 1 and 3), while detection is performed on other channels (e.g., channels 2 and 4). This particular configuration can be accommodated by using a read head, wherein the read head has a detector positioned in a spaced-apart configuration so that the objective lens is directed to each other channel of the flow cell. In this case, the valve can be activated to direct the flow of reagents for the sequencing cycle to the alternating channels, while the detected channels remain in the detection state. In this example, a first set of alternating channels can pass through the fluid step of the first sequencing cycle, and a second set of alternating channels pass through the detection step of the second sequencing cycle. Once the fluidic steps of the first cycle are completed and the detection steps of the second cycle are completed, the read head can step to the first set of alternating channels (e.g., along the x-dimension) and the valves can be actuated to deliver sequencing reagents to the second set of channels. Then, the detection steps of the first cycle can be completed (in the first set of channels) and the fluidic steps of the third cycle can be performed (in the second set of channels). The steps can be repeated several times until the desired number of cycles have been performed or until the sequencing process is complete.

[0126] An advantage of staggering fluidics and detection steps as described above is that it provides a faster overall sequencing run. In the above example, if the time required for fluidics manipulation is approximately the same as the time required for detection, a faster sequencing run will result from the staggered configuration (compared to fluidics manipulation of all channels in parallel followed by parallel detection of all channels). Of course, in embodiments where the timing of the detection step is different from the timing of the fluidics step, the staggered configuration can be changed from each other channel to a more appropriate pattern to accommodate parallel scanning of a subset of the channels while another subset of the channels is passing through the fluidics step.

[0127] An additional advantage of having fluid flow in opposite directions is that it provides a means of comparing the performance of individual microfluorometers. For example, where multiple microfluorometers are used per flow cell channel, it can be difficult to distinguish whether reduced microfluorometer performance is caused by the detector or reduced chemical efficiency from one end of the channel to the other. By having opposite directions of liquid flow, microfluorometer performance can be compared across the channels, effectively distinguishing whether reduced performance is due to a microfluorometer.

[0128] The flow diagram of the exemplary fluid system is Fig. 92020 has four passages, each fluidically connected to one of two separate fluid lines FV and RV independently actuated by inlet valves VA and VB. Inlet valves VA and VB control the flow of fluids from the sample reservoir, SBS reagent reservoir, and amplification reagent reservoir in the reagent cartridge or tray 2035 fluidically connected to various ports in the reagent manifold 2030.

[0129] Fluid passing Fig. 9 The flow of the system is driven by two independent syringe pumps 2041 and 2042. The syringe pumps are positioned to pull fluid through the fluid system, and each pump can be activated individually by valves 2051 and 2052. Therefore, the flow of the flow cell through each channel can be controlled individually by a dedicated pressure source. Valves 2051 and 2052 are also configured to control the flow of fluid to a waste tank 2060.

[0130] Fig. 9 A fluid system is demonstrated in which fluid is pulled by the action of a downstream syringe pump. It will be appreciated that useful fluid systems can use other types of devices instead of syringe pumps to drive fluids, including, for example, positive or negative pressure, peristaltic pumps, diaphragm pumps, piston pumps, gear pumps, or Archimedean screws. In addition, these and other devices can be configured to pull fluid from a downstream position relative to the flow cell or to push fluid from an upstream position.

[0131] Fig. 9 Also demonstrate that two syringe pumps are used for four channels of flow cell.Therefore, fluid system comprises a plurality of pumps less than the number of channels in use.It will be appreciated that in fluid box of the present disclosure, useful fluid system can have any number of tips, such as pumps (or other pressure sources) that are suitable with the number of channels in use or less.For example, some channels can be connected to a shared pump fluidically, and valve can be used to start the fluid flow through a separate channel.

[0132] Fig. 9 The fluid system shown also includes a sensor BUB-4 for detecting bubbles, which is positioned along the fluid path RV between the valve VA and the flow cell inlet V1. An additional bubble sensor BUB-3 is positioned along the fluid path RV between the valve VB and the flow cell inlet V2. It will be understood that the fluid lines useful in the fluid system of the present disclosure can include any number of bubble sensors, pressure gauges, etc. The sensors and / or meters can be located at any position along any part of the fluid path in the fluid system. For example, the sensor or meter can be positioned along the fluid line between one of the valves and the flow cell. As an alternative or in addition, the sensor or meter can be positioned along the fluid line between the reagent cell and one of the valves, between the valve and the pump, or between the pump and the outlet or pool, such as a waste pool.

[0133] A cross section of the demonstration kit is shown in Figure 6Compared with hole 402, Figure 6 The reagent kit 400 shown includes a well 401 of variable depth along the z-dimension. More specifically, Figure 6 The illustrated reagent cartridge has holes designed to accommodate the length of corresponding reagent pipettes (not shown) so that each pipette reaches the desired depth in its corresponding reagent well when the pipette and cartridge are in the fully engaged position. Figure 6 In the test kit shown, the hole is arranged along the row or column of y dimension, and those holes 401 of the row or column outside further extend downward along the z dimension than those holes 402 of the row or column inside. Part or all of the hole can have variable depth. As an alternative or supplement, part or all of the hole can have the same depth. When the pipette and the box are in the fully engaged position, the penetration depth of any pipette tip (that is, the distance from the bottom surface of the hole to the tip of the pipette) can be equivalent to the penetration depth of any other pipette tip in any other given hole of the test kit. The penetration depth of any pipette tip does not need to be the same as the penetration depth of any other given hole in the test kit. In the case where at least part of the reagent holes have different hole depths, the hole depth can be, for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or at least 2.0mm shorter than other reagent pipettes. Similarly, when the pipette and box are in a fully engaged position, the penetration depth of any pipette tip can differ from the penetration depth of any other pipette tip in the test kit by at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or at least 2.0 mm.

[0134] Top view of a demonstration reagent tray with reagent wells and interface slots for alignment pins in Figure 8 As shown in Figure 8 As shown in the exemplary reagent kit 400, the box includes a plurality of reagent cells 401A, 401B, 402A and 402B. Figure 8 The reagent cells in are arranged in rows along the x and y dimensions. Figure 8 Also shown, the box includes slots 403 and 404, which are configured to engage with corresponding alignment pins of a manifold assembly (not shown). The box may also include a protective foil covering any number of reagent wells or reservoirs that can be pierced by a piercing pipette when the box is contacted to the piercing pipette.

[0135] The test kit provided herein can include any number of reagent pools or holes. Reagent pools or holes can be arranged to promote the transmission and storage of the reagent in the box along the x and y dimensions according to any format. As an alternative or supplement, reagent pools or holes can be arranged along the x and y dimensions according to any format that is suitable for interacting with the pipette array extending downward along the z dimension from the port in the manifold. More specifically, reagent pools or holes can be arranged according to any format that is suitable for the mixing of the reagent pipettes simultaneously, so that liquid reagent can be sucked in the pipette from the reagent pool.

[0136] Not all reagent wells need not interact with all pipettes of the manifold assembly at the same time. For example, a test kit can include a subset of one or more reagent pools or wells that are configured to remain in a non-interactive state while other pools or wells are engaged by a pipette array. As an example, a box provided herein can include multiple flushing pools that are arranged in a configuration corresponding to multiple reagent pools, whereby the flushing pools are configured to simultaneously engage the reagent pipettes when the reagent pipettes are not engaged with the reagent pools, so that flushing buffer can be drawn from the flushing pools into the pipettes. An exemplary embodiment is Figure 8 , which shows a row of reagent wells 401A. The box also includes a row of corresponding wells 401B, which retain the same orientation along the x dimension relative to each other, but are offset from the wells 401A in the y dimension. The offset wells 401B can include, for example, a flushing buffer provided for cleaning pipettes and fluid lines after using one box and before using another box.

[0137] As an alternative or supplement, other pools that are empty or hold buffers, samples or other reagents can be present on the box. Additional pools can but not necessarily interact with the pipette. For example, the pool can be configured to be filled with flushing or overflow reagents or buffers during the use of the box. Such pools can be accessed, for example, via a port that does not interact with the pipette.

[0138] To facilitate proper alignment of the wells of the cartridge with the corresponding pipettes, an alignment slot can be positioned in the cartridge. For example, in a specific embodiment where the pipette array is moved from one set of wells and relocated to another set of reagent or rinse wells, an alignment slot can be positioned in the cartridge to ensure that the reagent pipette array is properly aligned with one or both sets of wells. Figure 8 As shown, the demonstration box includes groove 404, which retains the same orientation along the x dimension, but it is offset along the y dimension relative corresponding groove 403. The box of the embodiment provided herein can have any number of alignment grooves, which provide suitable alignment with the features of the fluid assembly. For example, the box can include 0,1,2,3,4,5,6,7,8,9,10 or more alignment grooves, which are configured to engage with the corresponding alignment pins of the fluid system so that the reagent pipette of the fluid system is positioned to align with the reagent and / or the flushing pool.

[0139] In certain embodiments, the fluid system can be configured to allow for the reuse of one or more reagents. For example, the fluid system can be configured to deliver a reagent to a flow cell, then remove the reagent from the flow cell, and then reintroduce the reagent into the flow cell. Fig. 7A , which shows a top view of the storage lines in the manifold assembly. Fig. 7A As shown in the schematic diagram at the top, the reagent reservoir can be used to maintain a concentration gradient from the most used to the least used (fresh) reagent. In some embodiments, the reservoir can be configured to reduce mixing of the fluid in the reservoir, thereby maintaining a gradient of liquid reagent along the length of the pool from the end close to the flow cell to the end away from the flow cell. When the reagent is transported from the reservoir to the flow cell again, the gradient is maintained so that the reagent flowing across the flow cell forms a gradient from the most used to the least used (fresh) reagent.

[0140] like Fig. 7A As shown in the bottom diagram, manifold fluid can be configured to make reagent pool carry out fluid communication with the input port of flow cell (not shown) via valve inlet 1804. Valve controls the flow of fluid between each of flow cell (not shown) and CLM pool, SRE pool, IMF pool and LAM1 and LPM1 pool. Channel 1802 connects CLM pool with valve inlet 1804 fluidly via port 1801. A part of channel 1802 includes reagent reservoir 1803, which is configured to keep the reagent volume of the volume of one or more passages equivalent to flow cell (not shown). The volume increased by reagent reservoir 1803 compared with channel 1802 allows the used reagent to be stored for reuse, while the unused reagent reserve is kept in the reagent pool, thus avoiding the unused reagent reserve in the reagent pool to be contaminated with the used reagent.

[0141] Fig. 7AThe configuration shown is exemplary. Other configurations are also likely to achieve reuse. For example, one or more of the reservoirs can have 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more of the volume of the flow cell channel that fluid circulates with the reservoir. Alternatively or additionally, the reservoir can include sufficient volume to allow a certain amount of liquid reagent in one or more flow cell channels to flow to the reservoir such that liquid reagent from the flow cell is not directed back into the reagent reservoir after contacting the flow cell. For example, the amount of liquid reagent can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more of the liquid reagent in one or more flow cell channels.

[0142] As provided in this paper, the storage tank can be configured to reduce the mixing of the fluid in the storage tank. In some such embodiments, the mixing of reduction can keep the gradient of the liquid reagent along the end of the flow cell to the pond length away from the end of the flow cell. As an alternative or supplement, as provided in this paper, the storage tank can include one or more mixing elements, which are configured to promote the mixing of the fluid in the storage tank. Any suitable active or passive mixing element can be used in this embodiment. For example, mixing element can include baffle element, curved structure or any other passive or active or fluid feature, which is configured to promote mixing when fluid is transmitted across the storage tank. As an alternative or supplement, any suitable pump, rotor, blade, inlet, etc. can be used for the active mixing in the storage tank.

[0143] As the storage tank provided by this paper can have any shape, volume and length that are suitable for the storage tank purpose.In a specific embodiment, the storage tank of any shape, volume and / or length can be used in the fluid system provided by this paper, and it allows the liquid reagent of a certain amount in one or more flow cell passages to flow to the storage tank, so that the liquid reagent from the flow cell can not be guided back to the reagent cell again after contacting the flow cell.For example, the storage tank can include serpentine passage.As another example, the storage tank can include the passage of cylinder or non-cylindrical shape.In addition, any number of fluid channels in the fluid system provided by this paper can include one or more independent storage tanks.

[0144] Storage tank as provided herein can be carried out fluid circulation with pump, wherein pump is configured to move liquid reagent from storage tank to flow cell and from flow cell to storage tank again, wherein reagent enters flow cell and reagent leaves flow cell and is carried out through the same port of flow cell.As an alternative or supplement, reagent enters flow cell and reagent leaves flow cell and can be carried out through the different ports of flow cell, but still realizes reagent reuse.For example, fluid system provided herein can utilize any of the described reuse pool and configuration of U.S. Patent Application Serial No. 13 / 766413 (submitted on February 13, 2013, entitled " INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGE USEFUL FOR NUCLEIC ACID SEQUENCING "), and its content is fully incorporated herein by reference.

[0145] Figure 7B The schematic diagram of presents an exemplary illustration of the reuse method provided herein, which utilizes a reciprocating flow of reagents from a reservoir line to a flow cell, followed by a partial refill of the reservoir line from the flow cell. Figure 7B, a reservoir 1903 containing 100 μL of reagent 1906 is in fluid communication with a flow cell passage 1905 via a separator 1904 and a valve 1911. The valve is activated to allow the reagent 1906 to flow into the flow cell passage 1905. At the same time, fresh reagent 1907 is pulled from the reagent reservoir to fill the gap left in the reservoir 1903. After the reagent is used on the flow cell, a portion (75 μL) of the used reagent 1906 is directed back into the reservoir 1903 by valve 1911. Another portion (25 μL) of the used reagent 1906 is diverted by valve 1911 to a waste container. At the end of cycle 1, the reservoir 1903 has a gradient of 25 μL of fresh reagent 1907 and 75 μL of used reagent 1906 across the length of the reservoir. The cycle of reciprocating flow of reagent from the reservoir to the flow cell and back to the reservoir is repeated, with a portion (25 μL) of the used reagent 1906 being diverted to the waste container by valve 1911 at each cycle, and the remainder of the used reagent 1906 flowing back to the reservoir 1903. At the end of four such repeated cycles, the reservoir 1903 contains 25 μL of fresh reagent 1910, 25 μL of reagent 1909 that has been used once, 25 μL of reagent 1908 that has been used twice, and 25 μL of reagent 1907 that has been used three times.

[0146] Fig. 7A and Figure 7B The configuration shown is exemplary.Other configurations are also likely to realize one or more reuses of the reagent used in the ad hoc process.It will be appreciated that in some reagent reuse configurations, the fluid configuration of reagent reuse will only be used for the subset of the reagent used in the ad hoc process.For example, the first subset of reagent can be strong enough to be reused, and the second subset may be easy to pollute, degrade or other undesirable effects after single use.Correspondingly, the fluid system can be configured for the reuse of the first subset of reagent, and the fluid of the second set of reagent will be configured for single use.

[0147] Specific reagent can reuse any number of times that expection is suitable for specific process.For example, one or more of the reagents demonstrated herein, described in the reference cited herein or otherwise known for the process described herein can reuse at least 2,3,4,5,10,25,50 or more times.In fact, any one of multiple expected reagents can reuse at least as much as possible number of times.Any part of specific reagent can turn to storage tank again for reuse. For example, one or more of the reagents shown herein, described in references cited herein, or otherwise known for use in the processes described herein can have 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the reagent volume on one or more flow cell pathways directed back into the reservoir for subsequent reuse. Alternatively or additionally, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the reagent volume on one or more flow cell pathways can be diverted to a waste container or otherwise not used subsequently on the flow cell.

[0148] Although demonstrated for nucleic acid sequencing processes, the liquid configurations and methods of reagent reuse can be applied to other processes, particularly processes involving repeated cycles of reagent delivery. Exemplary processes include sequencing of polymers such as polypeptides, polysaccharides, or synthetic polymers, and also include synthesis of such polymers.

[0149] As demonstrated in the above exemplary embodiments, the method for reagent reuse includes the following steps: a) sucking a liquid reagent from a reagent cell into a reservoir, the reservoir being in fluid communication with at least one channel of the reagent cell and the flow cell; b) transferring the reagent from the reservoir to at least one channel of the flow cell; c) transferring at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the reagent on the flow cell channel to the reservoir, so that the liquid reagent from the flow cell is not directed back to the reagent cell after contacting the flow cell; d) repeating steps b) and c) to achieve the reuse of the liquid reagent on the flow cell. One or more of the reservoirs can be in fluid communication with a pump, wherein the pump is configured to move the liquid reagent from the reservoir to the flow cell and from the flow cell to the reservoir, so that the reagent enters the flow cell and the reagent leaves the flow cell through the same port of the flow cell. As an alternative or supplement, the reagent enters the flow cell and the reagent leaves the flow cell can be performed through different ports of the flow cell, but still achieves reagent reuse. In some embodiments, reagents from the flow cell that are not transferred to the storage tank in step c) can be diverted. As an example, reagents from the flow cell that are not transferred to the storage tank can be transferred to the waste tank. The transfer of reagents in one or both of steps b) and c) can be performed via a valve that fluidically connects the storage tank and the flow cell. The transfer of reagents in one or both of steps b) and c) can be performed, for example, using fluid flow in a single direction, or can be performed using reciprocating flow.

[0150] Embodiments of the current fluid system and method obtain particular use for nucleic acid sequencing technology. For example, synthesis test (SBS) protocol is particularly applicable. In SBS, the extension of nucleic acid primers along nucleic acid templates is monitored to determine the nucleotide sequence in the template. The basic chemical process can be polymerization (e.g., as catalyzed by a polymerase) or connection (e.g., catalyzed by a ligase). In a specific polymerase-based SBS embodiment, fluorescently labeled nucleotides are added to the primers (thereby extending the primers) in a template-dependent manner, so that the detection of the order and type of nucleotides added to the primers can be used to determine the template sequence. A plurality of different templates can be subjected to SBS technology on the surface under conditions that can distinguish events occurring to different templates. For example, the template can be present on the surface of the array so that different templates are spatially distinguishable from each other. Typically, the template appears as features (sometimes referred to as "clusters" or "clones") each having multiple copies of the same template. However, it is also possible to perform SBS on an array, wherein each feature has a single template molecule present, so that the single template molecules are resolvable from each other (sometimes referred to as "single molecule arrays").

[0151] The flow cell provides a convenient substrate for containing a nucleotide array. The flow cell is convenient for sequencing technology because the technology generally involves repeated delivery of reagents in the cycle. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerases, etc. can flow into / flow through the flow cell containing the nucleic acid template array. Wherein the primer extension enables those features of the labeled nucleotide to be combined to be detected, for example, using the method or device described herein. Optionally, the nucleotide can also include a reversible termination property, which terminates other primer extensions once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator half family can be added to the primer so that subsequent extension cannot occur until a blocking agent is delivered to remove the half family. Therefore, for an embodiment using reversible termination, the blocking agent can be delivered to the flow cell (before or after detection occurs). Flushing can be performed between various delivery steps. The cycle can be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary sequencing techniques are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, US 7057026, WO 91 / 06678, WO 07 / 123744, US 7329492, US 7211414, US 7315019, US 7405281, and US2008 / 0108082, each of which is incorporated herein by reference.

[0152] For the nucleotide delivery step of the SBS cycle, a single type of nucleotide can be delivered each time, or a plurality of different nucleotide types (e.g., A, C, T, and G together) can be delivered. For the nucleotide delivery configuration in which only a single type of nucleotide exists each time, different nucleotides do not need to have different labels, because they can be distinguished based on the time separation inherent in the individualized delivery. Accordingly, the sequencing method or device can use monochrome detection. For example, a microfluorometer or a reader only needs to provide excitation at a single wavelength or at a single range of wavelengths. Therefore, a microfluorometer or a reader only needs to have a single excitation source, and multi-band filtering of the excitation is not necessarily required. For the nucleotide delivery configuration in which the delivery causes a plurality of different nucleotides to be present in the flow cell at the same time, the characteristics of the different nucleotide types can be distinguished based on the different fluorescent labels of the corresponding nucleotide types attached to the mixture. For example, four different nucleotides can be used, each with one of four different fluorophores. In one embodiment, four different fluorophores can be distinguished using the excitation of four different regions of the spectrum. For example, a microfluorometer or a reader can include four different excitation radiation sources. Alternatively, the readhead can include fewer than four different excitation radiation sources, but can use optical filtering of the excitation radiation from a single source to produce different ranges of excitation radiation in the flow cell.

[0153] In some embodiments, four different nucleotides can be detected in a sample (or nucleic acid feature array) using less than four different colors. As a first example, a pair of nucleotide types can be detected at the same wavelength, but based on the difference in the intensity of one member of the pair compared to another member or based on the change of one member of the pair (e.g., via chemical modification, photochemical modification, or physical modification) (which makes the apparent signal appear or disappear compared to the signal detected by the other member of the pair) to distinguish. As a second example, three of the four different nucleotide types can be detectable under specific conditions, and the fourth nucleotide type does not have a detectable label under those conditions. In the SBS embodiment of the second example, the first three nucleotide types are incorporated into nucleic acid and can be determined based on the presence of their corresponding signals, and the fourth nucleotide type is incorporated into nucleic acid and can be determined based on the absence of any signal. As a third example, a nucleotide type can be detected in two different images or in two different channels (e.g., a mixture of two species with the same base but different labels can be used, a single species with two labels can be used, or a single species with labels detected in two channels can be used), and other nucleotide types are detected in no more than one image or channel. In this third example, comparison of two images or two channels is used to distinguish between different nucleotide types.

[0154] The three exemplary configurations in the above section are not mutually exclusive, but can be used in various combinations. An exemplary embodiment is an SBS method that uses reversibly blocked nucleotides (rbNTPs) with fluorescent labels. In this format, four different nucleotide types can be delivered to the nucleic acid feature array to be sequenced, and due to the reversible blocking group, a unique binding event will occur in each feature. The nucleotides delivered to the array in this example can include a first nucleotide type detected in the first channel (e.g., rbATP, with a label detected in the first channel when stimulated by the first excitation wavelength), a second nucleotide type detected in the second channel (e.g., rbCTP, with a label detected in the second channel when stimulated by the second excitation wavelength), a third nucleotide type detected in the first and second channels (e.g., rbTTP, with at least one label detected in two channels when stimulated by the first and / or second excitation wavelengths) and a fourth nucleotide type of label not detected in any channel (e.g., rbGTP, without non-intrinsic label).

[0155] Once the four nucleotide types are in contact with the array in the above example, a detection process can be performed, such as to capture two images of the array. Images can be obtained in separate channels, and can be obtained simultaneously or sequentially. The first image obtained using the first excitation wavelength and emission in the first channel will show the features of the first and / or third nucleotide types (e.g., A and / or T). The second image obtained using the second excitation wavelength and emission in the second channel will show the features of the second and / or third nucleotide types (e.g., C and / or T). The unclear identification of the nucleotide types combined with each feature can be determined by comparing the two images to derive as follows: features that appear only in the first channel in combination with the first nucleotide type (e.g., A), features that appear only in the second channel in combination with the second nucleotide type (e.g., C), features that appear in the two channels in combination with the third nucleotide type (e.g., T), and features that do not appear in any channel in combination with the fourth nucleotide type (e.g., G). Note that the position of the feature in combination with G in this example can be determined from other circles (where at least one of the other three nucleotide types is combined). Exemplary apparatus and methods for distinguishing four different nucleotides using detection in fewer than four colors are described, for example, in U.S. Patent Application Serial No. 61 / 538,294, which is incorporated herein by reference.

[0156] In certain embodiments, nucleic acid can be attached to the surface and amplified before or during sequencing. For example, amplification can be performed using bridge amplification to form nucleic acid clusters on the surface. Useful bridging amplification is described, for example, in US 5641658, US2002 / 0055100, US 7115400, US2004 / 0096853, US 2004 / 0002090, US2007 / 0128624 or US2008 / 0009420, each of which is incorporated herein by reference. Another useful method for amplifying nucleic acids on surfaces is rolling circle amplification (RCA), for example, as described in Nat.Genet.19:225-232 (1998) and US2007 / 0099208 A1 of Lizardi et al., each of which is incorporated herein by reference. Emulsion PCR on microspheres can also be used, such as described in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145, US2005 / 0130173, or US2005 / 0064460, each of which is incorporated herein by reference.

[0157] As mentioned above, the sequencing example is an example of an iterative process. The method disclosed herein is fully suitable for iterative processes. Some embodiments are proposed below and in other parts of this article.

[0158] Accordingly, provided herein is a sequencing method comprising: (a) providing a fluid system comprising (i) a flow cell comprising an optically transparent surface, (ii) a nucleic acid sample, (iii) a plurality of reagents for a sequencing reaction, and (iv) a fluid system for delivering the reagents to the flow cell; (b) providing a detection device comprising (i) a plurality of microfluorometers, wherein each microfluorometer comprises an objective lens configured for wide-field image detection along x and y dimensions in an image plane, and (ii) a sample stage; and (c) performing fluid operations of a nucleic acid sequencing process in a cartridge and detection operations of a nucleic acid sequencing process in a detection device, wherein (i) the reagents are delivered to the flow cell by the fluid system, (ii) the wide-field image of nucleic acid features is detected by the plurality of microfluorometers, and (iii) at least a portion of the reagents are moved from the flow cell to a storage tank.

[0159] Throughout this application, various publications, patents and / or patent applications are cited. The entire disclosures of these publications are incorporated into this application by reference.

[0160] The term “comprising” is intended to be unrestricted herein and includes not only the recited elements but also any additional elements.

[0161] Several embodiments have been described. However, it will be appreciated that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A multilayer diffusion bonded reagent manifold comprising at least 10 ports, each port being configured to draw liquid reagent from a separate reagent cell via a reagent pipette, wherein the ports are in fluid communication with one or more channels of a flow cell via a fluid channel in the reagent manifold, and wherein one or more of the fluid channels in the reagent manifold comprises a reservoir, the reservoir comprising sufficient volume to allow a certain amount of liquid reagent in one or more flow cell channels to flow to the reservoir, so that the liquid reagent from the flow cell is not directed back into the reagent cell after contacting the flow cell.

2. A reagent manifold as described in claim 1, comprising at least 15 ports.

3. The reagent manifold of claim 1 comprising at least 20 ports.

4. A reagent manifold as described in any of claims 1-3, comprising a first valve and a second valve, wherein the channel connected to the first valve resides on the first layer and the channel connected to the second valve resides on the second layer.

5. The reagent manifold of any one of claims 1-3, wherein the volume of one or more of the reservoirs is at least 30% of the volume of a flow cell channel in fluid communication with the reservoir.

6. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 40% of the volume of a flow cell channel in fluid communication with the reservoir.

7. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 50% of the volume of a flow cell channel in fluid communication with the reservoir.

8. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 60% of the volume of a flow cell channel in fluid communication with the reservoir.

9. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 70% of the volume of a flow cell channel in fluid communication with the reservoir.

10. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 80% of the volume of a flow cell channel in fluid communication with the reservoir.

11. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 90% of the volume of a flow cell channel in fluid communication with the reservoir.

12. The reagent manifold of any one of claims 1-3, the volume of one or more of the reservoirs being at least 100% of the volume of a flow cell channel in fluid communication with the reservoir.

13. The reagent manifold of any one of claims 1 to 3, wherein: The certain amount of liquid reagent comprises 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the liquid reagent in the one or more flow cell channels.

14. The reagent manifold of any one of claims 1 to 3, wherein: One or more of the reservoirs are in fluid communication with a pump configured to move the liquid reagent from the reservoir to the flow cell and from the flow cell back to the reservoir, wherein reagent enters the flow cell and reagent leaves the flow cell through the same port of the flow cell.

15. The reagent manifold of any one of claims 1 to 3, wherein: At least one valve is configured to variously direct the liquid reagent from the flow cell back to the reservoir or to waste.

16. The reagent manifold of any one of claims 1-3, wherein: The reservoir is configured to reduce mixing of fluid in the reservoir, thereby maintaining a gradient of the liquid reagent along the length of the reservoir from an end proximal to the flow cell to an end distal to the flow cell.

17. The reagent manifold of any one of claims 1 to 3, wherein: The reservoir includes a plurality of mixing elements configured to facilitate mixing of fluid in the reservoir.

18. The reagent manifold of claim 17, wherein: The mixing element comprises a baffle element.

19. The reagent manifold of any one of claims 1 to 3, wherein: The reservoir includes a serpentine channel.

20. The reagent manifold of any one of claims 1-3, wherein: The reservoir includes a non-cylindrical channel.

21. The reagent manifold of any one of claims 1-3, wherein: The reagent manifold is configured to deliver the liquid reagent from a first reagent reservoir to a first valve via a first channel and from the first reagent reservoir to a second valve via a second channel.

22. The reagent manifold as described in any one of claims 1-3, comprising a plurality of reagent pipettes extending downwardly from the port, each of the reagent pipettes being configured to be placed in a reagent reservoir of a reagent cartridge so that the liquid reagent can be sucked into the reagent pipette from the reagent reservoir.

23. The reagent manifold of any one of claims 1-3, wherein: Each of the reagent pipettes includes a tubular body having a proximal end and a distal end, the distal end tapering to a pointed tip configured to pierce a foil layer covering a reagent reservoir in a reagent cartridge.

24. The reagent manifold of claim 23, wherein: The reagent pipette is provided with a single lumen extending through the tubular body from the distal end to the proximal end, the lumen being configured for fluid communication between the reagent cartridge and the reagent manifold.

25. The reagent manifold of claim 23, wherein: A subset of the reagent pipettes has a shorter length than other of the reagent pipettes.

26. The reagent manifold of claim 25, wherein: The length of the subset is at least 1 mm shorter than the other reagent pipettes.

27. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.2 mm shorter than the other reagent pipettes.

28. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.3 mm shorter than the other reagent pipettes.

29. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.4 mm shorter than the other reagent pipettes.

30. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.5 mm shorter than the other reagent pipettes.

31. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.6 mm shorter than the other reagent pipettes.

32. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.7 mm shorter than the other reagent pipettes.

33. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.8 mm shorter than the other reagent pipettes.

34. The reagent manifold of claim 25, wherein: The length of the subset is at least 1.9 mm shorter than the other reagent pipettes.

35. The reagent manifold of claim 25, wherein: The length of the subset is at least 2.0 mm shorter than the other reagent pipettes.

36. The reagent manifold of any one of claims 1-3, wherein: At least one of the reagent pipettes includes a compliant tip configured to bend when the tip strikes the bottom of a reagent well of the reagent cartridge.

37. A method for fluid manipulation and optical detection of a sample in a nucleic acid sequencing process, comprising: (a) A fluid system is provided, the fluid system comprising: (i) a flow cell comprising an optically transparent surface, (ii) Nucleic acid samples, (iii) various liquid reagents for sequencing reactions, and (iv) a reagent manifold for delivering the liquid reagent to the flow cell, one or more of the fluidic channels in the reagent manifold comprising a reservoir; (b) providing a detection device, the detection device comprising: (i) a plurality of microfluorometers, wherein each of the microfluorometers comprises an objective lens, The objective lens is configured for widefield image detection along the x-dimension and the y-dimension in the image plane, and (ii) a sample stage; and (c) performing fluid operations of the nucleic acid sequencing process in the cartridge and detection operations of the nucleic acid sequencing process in the detection device, wherein (i) the liquid reagent is transported to the flow cell by the fluid system, (ii) a widefield image of nucleic acid features is detected by the microfluorometer, and (iii) moving a portion of the reagent from the flow cell to the reservoir that is in fluid communication with one or more flow cell channels of the flow cell, the reservoir comprising a sufficient volume to allow a certain amount of liquid reagent in the one or more flow cell channels to flow into the reservoir, such that the liquid reagent from the flow cell is not directed back into the reagent reservoir of the liquid reagent after contacting the flow cell.

38. The method of claim 37, wherein: The fluidic system further includes a waste reservoir, and fluidic operations of the nucleic acid sequencing process further include moving at least some of the liquid reagents from the flow cell to the waste reservoir.

39. The method of claim 37, wherein: The reservoir is in fluid communication with a pump configured to move the liquid reagent from the reservoir to the flow cell and from the flow cell back to the reservoir, wherein reagent enters the flow cell and reagent exits the flow cell through the same port of the flow cell.

40. The method of claim 37, wherein: The liquid reagent from the flow cell that is not transferred to the reservoir in step (c)(iii) is transferred to a waste reservoir.

41. The method of claim 37, wherein: The reagent transfer in step (c)(i) or step (c)(iii) is performed via a valve fluidly connecting the reservoir and the flow cell.

42. The method of claim 37, wherein: The reagent transfer in step (c)(i) or step (c)(iii) is performed using fluid flow in a single direction.

43. The method of claim 37, wherein: The reagent transfer in step (c)(i) or step (c)(iii) is performed using a reciprocating flow.

44. The method of claim 37, wherein: The liquid reagent is a reagent for performing nucleic acid sequencing.

45. The method of claim 44, wherein: The liquid reagent is a polymerase.

46. ​​The method of claim 44, wherein: The liquid reagent is a nucleotide or a mixture of different nucleotides.

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