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    • 专利摘要:
    fluidic system for delivering reagent to a flow cell. the present invention provides a fluid system that includes a reagent manifold comprising a plurality of channels configured for fluid communication between a reagent cartridge and an inlet of a flow cell; a plurality of reagent aspirator tubes extending downward from holes in the manifold, each of the reagent aspirator tubes configured to be placed within a reagent reservoir in a reagent cartridge so that liquid reagent can be taken from the reagent container to the aspirator tube; at least one valve configured to mediate fluid communication between the reservoirs and the inlet of the flow cell. the reagent manifold may also include deposit reservoirs for reagent reuse.
    公开号:BR112016000456B1
    申请号:R112016000456-6
    申请日:2014-08-07
    公开日:2021-06-01
    发明作者:Michael Stone；Drew Verkade
    申请人:Illumina, Inc；
    IPC主号:
    专利说明:

    PRIORITY CLAIM
    [001] This application claims the benefit of United States Provisional Application No. 61 / 863,795, filed August 8, 2013, currently pending, which is incorporated herein by reference. BACKGROUND
    [002] Embodiments of the present description generally refer to an apparatus and methods for handling fluids and optically detecting samples, for example, in nucleic acid sequencing procedures.
    [003] Our genome provides a blueprint for predicting many of our inherent predispositions, such as our preferences, talents, susceptibility to disease, and responsiveness to therapeutic drugs. An individual human genome contains a sequence of more than 3 billion nucleotides. Differences in just a fraction of these nucleotides convey many of our unique characteristics. The research community is making impressive progress in unraveling the characteristics that make up the diagram, and with it a more complete understanding of how the information in each diagram relates to human health. However, our understanding is far from over and this is impeding the movement of information from the research labs to the clinic where the hope is that one day each of us will have a copy of our own personal genome for us to discuss with the to determine appropriate choices for a healthy lifestyle or an appropriate course of treatment.
    [004] The current hurdle is a matter of yield and scale. A key component of unlocking the blueprint for any individual is determining the exact sequence of the 3 billion nucleotides in their genome. Techniques are available for this, but these techniques typically take many days and thousands and thousands of dollars to execute. Furthermore, the clinical relevance of any individual genomic sequence is a matter of comparing the original features of its genomic sequence (ie, its genotype) to reference genomes that are correlated with known features (ie, phenotypes). The issue of scale and production becomes evident when considering that reference genomes are created based on the correlation of genotype to phenotype that arise from research studies, which typically use thousands of individuals, in order to be statistically valid. Thus, billions of nucleotides can eventually be sequenced by thousands of individuals to identify any clinically relevant correlation from genotype to phenotype. Multiplied still by the number of diseases, drug responses and other clinically relevant characteristics, the need for very cheap and fast sequencing technologies becomes increasingly evident.
    [005] What is needed is a reduction in the cost of sequencing that drives large genetic correlation studies conducted by research scientists and that makes sequencing accessible in the clinical setting for treating individual patients who make life-changing decisions. Embodiments of the invention presented herein fulfill this need and provide other advantages as well. SUMMARY
    [006] The present invention provides a fluid system that includes a reagent manifold comprising a plurality of channels configured for fluid communication between a reagent cartridge and an inlet of a flow cell; a plurality of reagent aspirator tubes extending downward from holes in the manifold, each of the reagent aspirator tubes configured to be placed within a reagent reservoir in a reagent cartridge so that liquid reagent can be taken from the reagent container to the aspirator tube; at least one valve configured to mediate fluid communication between the reservoirs and the inlet of the flow cell.
    [007] This invention further provides a reagent cartridge that includes a plurality of reagent reservoirs configured to simultaneously engage a plurality of aspirator tubes with reagents from a fluid system along a Z dimension such that the liquid reagent can be removed from the reagent container in the aspirator tubes, the reagent reservoirs arranged in X and Y dimensions at the top, middle and lower lines, in a reagent reservoir along the upper and lower lines of the cartridge are deeper along the z dimension than reagent reservoirs in one or more midlines; and at least two interface slots configured to engage with corresponding fluid system alignment pins.
    [008] A multi-layer diffusion-attached reagent collector is also provided, comprising at least 10, 15, or at least 20 holes, each hole configured to pull reagent from a separate reagent reservoir through a container. aspirator, wherein the orifices are in fluid communication with one or more channels of a flow cell through fluid channels in the manifold.
    [009] This invention further provides a method of reusing reagents that includes a) extracting a liquid reagent from a reagent reservoir to a cache reservoir, the cache reservoir being in fluid communication with the reagent reservoir and at least one channel of a flow cell; b) transporting the reagent from the cache reservoir to the at least one flow channel of the cell; c) transport at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the reagent over the flow cell channel to the cache reservoir such that, the liquid reagent from the flow cell is not directed back to the reagent reservoir after contacting the flow cell; and d) repeating steps b) and c) to achieve reuse of the liquid reagent in the flow cell.
    [010] This invention further provides a sequencing method that includes the steps of (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 apparatus comprising (i) a plurality of microfluorimeters, each of the microfluorimeters comprising an objective configured for wide-field image detection in an image plane in dimensions, X and Y, and (ii) a sample phase; and (c) performing fluid operations of a nucleic acid sequencing procedure in the cartridge operations and detection of the nucleic acid sequencing process in the detection apparatus, wherein (i) reagents are supplied to the flow cell by the fluid system, (ii) wide-field images of nucleic acid characteristics are detected by the plurality of microfluorimeters, and (iii) at least some reagents are removed from the flow cell to a cache reservoir.
    [011] Details of one or more embodiments are presented in the attached drawings and in the description below. Other features, objects and advantages will be evident from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS
    [012] Figure 1A shows a fluid system with reagent aspirator tubes that interact with a reagent cartridge.
    [013] Figure IB shows an isometric view of a manifold assembly and shows an example of a schematic of fluid channels within the manifold.
    [014] Figure 2 shows a front perspective view of a manifold assembly having reagent aspirator tubes, valves and alignment pins. Also shows tubes of different lengths.
    [015] Figure 3 shows a top view of a manifold assembly showing a possible schematic of the fluid channels inside the manifold.
    [016] Figure 4 shows a cross-sectional view of the channels within a manifold, including a cross-sectional view of a deposit line, and a non-deposit fluid channel.
    [017] Figure 5 shows a series of junctions for connecting a reagent orifice with two valves.
    [018] Figure 6 shows a cross-sectional view of a reagent cartridge that has wells of different depths.
    [019] Figure 7A shows a simplified top view of deposit lines in a collector assembly according to an embodiment.
    [020] Figure 7B shows different stages of reagent reuse in a method that uses the reciprocal flow of reagent from a deposit line to a flow cell, followed by partial refilling of the flow cell deposit line.
    [021] Figure 8 shows a top view of a reagent tray interface that has reagent wells and interface slots for pin alignment.
    [022] Figure 9 shows a fluid map for a fluid system.
    [023] Figure 10 shows a detailed view of reagent aspirator tubes including compatible aspirator tubes and a perforated bottle. DETAILED DESCRIPTION
    [024] This invention provides systems and methods for supplying reagent fluids to a chamber, such as a flow cell. A particularly useful application is the detection of an immobilized biological sample. For example, the methods and systems presented here can be used in nucleic acid sequencing applications. A variety of nucleic acid sequencing techniques that utilize optically and non-optically detectable samples and/or reagents can be used. These techniques are particularly well suited to the methods and apparatus of the present disclosure and therefore highlight several advantages for particular embodiments of the invention. Some of these advantages are presented below for illustrative purposes, and although nucleic acid sequencing applications are exemplified, the advantages can be extended to other applications as well.
    [025] The fluidic systems described herein are particularly useful with any of the detection apparatus configurations and sequencing methods set forth in US Patent Application Serial Number 13/766,413 filed February 13, 2013 and entitled "INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGE USEFUL FOR NUCLEIC ACID SEQUENCING" the contents of which are hereby incorporated by reference in their entirety.
    [026] In particular embodiments, a sample that is being detected may be supplied to a detection chamber using a fluid system as provided herein. Taking the 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 the reservoirs for the maintenance of sequencing reagents, reservoirs for holding reagents sample preparation, reservoirs to hold waste products generated during sequencing, and/or pumps, valves and other components capable of displacing fluids through a flow cell.
    [027] In particular embodiments a fluid system can be configured to allow 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 to the flow cell. One advantage of reuse reagents is the reduction in waste volume and cost reduction of processes that use expensive reagents and/or reagents that are released in high concentrations (or in high amounts). The reuse of reagents has the advantage of understanding that the depletion of the reagent occurs only or mainly on the surface of the flow cell, and, therefore, a greater part of the reagent is not used and can be subjected to a reuse.
    [028] Fig. 1A shows an exemplary fluidic system 100 with reagent aspirator tubes 103 and 104 and valves 102, which exploit advantages of fluid systems that are provided by various embodiments presented herein. Fluid system 100 includes a manifold assembly 101, which contains various fixed components, including, for example, reagents, aspirator tubes, valves, channels, reservoirs, and the like. A reagent cartridge 400 is present with reagent reservoirs 401 and 402 configured to simultaneously engage a set of reagent aspirator tubes 103 and 104 along a z dimension so that liquid reagent can be drawn from the reagent reservoirs to the suction tubes.
    [029] In FIG. 1B an exemplary manifold assembly 101 is illustrated, which can be used to deliver liquid reagents from reagent reservoirs to a flow cell. The manifold includes reagent drip containers 103 and 104 that extend down in a z dimension from holes in the manifold. Dropping containers 103 and 104 can be placed in one or more reagent reservoirs (not shown) in a reagent cartridge. The dispenser also includes channels 107 that fluidly connect the drip container 103 to a valve 102 and a valve 109. The drip containers 103 and 104, channels 107 and valve 102 mediate fluid communication between the reagent reservoirs and a flow cell (not shown). Valves 102 and 109 can, individually or together, select suction tubes from containers 103 or 104, and through channels such as 107, mediate fluid communication between the reagent reservoirs and a flow cell (not shown).
    [030] The apparatus shown in Figs. 1A and 1B are exemplary. Other exemplary embodiments of the apparatus and methods of the present disclosure which may be used alternatively or in addition to the example of Figs. 1A and 1B are presented in more detail below.
    [031] Fig. 2 shows another exemplary dispenser assembly having dripping containers with reagents and valves. The manifold has alignment pins 105 that protrude downward from the manifold on an axis parallel to the reagent containers. Alignment pins 105 are longer along the Z dimension compared to reagent containers, although in alternative embodiments they may also be of equal length or shorter. Alignment pins 105 are configured to mate with one or more corresponding interface slots on a reagent cartridge (not shown). Reagent containers 103 and 104 are coupled to the dispensing tube via holes 106 that are housed in the dispenser body. Reagent containers 104 are longer, compared to reagent containers 103, in order to draw liquid from reagent reservoirs of variable depth that corresponds to the depth of reagent containers 103 or 104. In alternative embodiments , containers 103 and 104 may be of equal length, or may alternate dominant lengths.
    [032] Still in Fig. 2 are channels 107A and 107B, which reside in separate x-y planes. Separate channels 107A and 107B can originate from a single channel which then forks at a T trunk 109 generating multiple channels that reside in separate planes. The manifold directs liquid reagent from a vessel to one or more valves, the channels connecting to a special valve 102 residing either entirely in the same plane A, or a combination of plane A and B, while the channels connecting to any other valve may share this characteristic of coplanar or interplanar origin.
    [033] Fig. 3 shows a top view of a manifold assembly 101 showing a possible arrangement of fluid channels within the manifold. Fluid channels 107A and 107B originate from a single port 106 and connect port 106 to either valve 102A or 102B. Some channels include a cache reservoir 108, which has a volume sufficient to allow an amount of liquid reagent to flow from a flow cell (not shown) to the cache reservoir 108 such that the liquid reagent from the flow cell does not. is directed back to the reagent container (not shown) after contacting the flow cell. Also shown in Fig. 3 are exemplary positions of one or more alignment pins 105. The manifold assembly shown in fig. 3 also includes 111 inlet holes for shared dampers. Each of valves 102A and 102B are configured with inlet ports corresponding to each reagent port 106, and with common external ports 112 and 110, which fluidly connect to a flow cell and a flow port 113 and 109, which fluidly attach to a waste container.
    [034] As demonstrated by the examples of embodiments above, a fluid system for dispensing reagents from a reagent cartridge to a flow cell may include a reagent manifold comprising a plurality of configured channels for communication fluid between a reagent cartridge and an inlet of a flow cell. Using a manifold in a fluid system provides several advantages over using piping alone. For example, a manifold with fixed channels reduces the likelihood of errors during assembly, such as poor placement of piping accessories, as well as overtightening or undertightening of connections. In addition, a manifold provides ease of maintenance, allowing, for example, quick replacement of an entire unit instead of time-consuming testing and replacement of individual lines.
    [035] The one or more of the collector channels may include a fluidic band through a solid material. The band can be any diameter to allow the desired level of fluid transfer through the band. The strip may have an inside diameter of, for example, less than 0.1 mm, 0.2 mm, 0.3 mm 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0 .8mm, 0.9mm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm or less than 10mm in diameter. The range setting can be, for example, linear or curved. Alternatively or additionally, the strip may have a combination of curved parts and straight parts. The cross section of the strip can be, for example, square, round, "D" shaped, or any other shape that allows for a desired level of fluid transfer across the strip. Fig. 4 exemplifies a fluidic band through a collector body and shows a cross-sectional view of a band 302. The exemplary channel 302 shown in Fig. 4 has a "D" shaped cross section formed by a half circle of 0 .65mm in cast diameter with an additional 0.65mm x 0.325mm rectangle.
    [036] The channel between the drip container and the valve can be entirely enclosed within the distributor body. Alternatively or additionally, the channel may include one or more parts that are external to the collector. For example, piping, such as, for example, flexible tubes can connect a portion of the fluidic band to another part of the band in the manifold. Alternatively or additionally, flexible tubes can connect a flow cell to fixed fluidic components of the system, including, for example, pumps, valves, sensors and gauges. As an example, flexible tubing can be processed to connect a flow cell or channel of the present system to a pump such as a syringe pump or a peristaltic pump.
    [037] The distributor body can be, for example, made of any suitable solid material that is capable of supporting one or more channels in it. Thus, the dispenser body can be a resin such as polycarbonate, polyvinyl chloride, DELRIN® (Polyoxymethylene); HALAR®; PCTFE (PolyChloroTriFluorEthylene); PEEK™ (Polyether-etherketone); PK (Polyketone); PERLAST®; Polyethylene; PPS (Polyphenylene Sulphide); Polypropylene; Polysulfone; FEP; PFA; high purity PFA; RADEL® R; 316 stainless steel; TEFZEL® ETFE (Ethylene Tetrafluoroethylene); TPX® (Polymethylpentene); Titanium; UHMWPE (Ultra High Molecular Weight Polyethylene); ULTEM® (polyetherimide); VESPEL® or any other suitable solid material that is compatible with the solvents and liquids transported through the collector channels in the embodiments presented herein. The distributor body can be formed from a single piece of material. Alternatively or additionally, the dispenser body can be formed from multiple layers that are bonded together. Bonding methods include, for example, the use of adhesives, sealants, and diffusion bonding. Channels can be formed in the solid material by any suitable method. For example, the channels can be perforated, acid etched or bleached into a solid material. Channels can be formed in the solid material prior to bonding the multiple layers together. Alternatively or additionally, channels can be formed after the layers connecting together.
    [038] Fig. 5 shows a series of junctions 300 for connecting a reagent port 301 with two valves. In each example shown in Fig. 5, an orifice 301 is fluidly connected to a channel 302 that bifurcates into two channels 302A and 302B in each supply channel of a different valve. In the first configuration, the junction divides the fluid flow from port 301 to channels 302A and 302B into separate layers of the manifold. In the second and third configurations shown in Fig. 5 the seam 300 includes a rounded square 303 divided within a layer or a complete round division 304 within a layer of the collector.
    [039] The manifold sets presented here are configured to supply liquid reagents from a reagent cartridge to a flow cell. Thus, the manifold (or manifold) can have any number of holes coupled to the reagent aspirator tubes. More specifically, the number of holes can match the number and configuration of reagent reservoirs in a reagent cartridge. In some embodiments, the dispenser 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 configured to couple a reagent aspirator container to a channel in fluid communication with the at least one valve.
    [040] The fluidic systems presented herein may also include an array of sniffer tubes that extend downward along the Z dimension from holes in the manifold, each of the reagent snip tubes configured to be inserted into a reservoir of reagent in a reagent cartridge so that liquid reagent can be drawn from the reagent reservoir into the aspirator container. Reagent aspirator tubes may comprise, for example, a tubular body with a proximal end and a distal end. The distal end can taper to a sharp point that is configured to pierce a sheet or layer of film used as a seal over a reagent reservoir in a reagent cartridge. Several exemplary sniffer tube tips are shown in Fig. 10. Reagent sip containers can be provided with, for example, a single lumen that traverses the tubular body from the distal to the proximal end. The lumen can be configured to provide fluid communication between the reagent cartridge on one end of the reaction aspirator container and the reagent manifold on the other end of the aspirator container. As shown in Fig. 2 exemplary sipper containers 103 and 104 are coupled to the dispensing tube through holes 106 that are housed in the dispenser body.
    [041] In some embodiments, as exemplified in Fig. 2, a subset of the sink containers is of a length that is shorter than the other sink containers. For example, the subset length can be 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 shorter than other sipper pans. The collector tube and the reagent sip containers can be used in a device that has an elevator mechanism configured to move a reagent cartridge bidirectionally along the Z dimension such that the reagent collectors are inserted into corresponding wells or reservoirs in the reagent cartridge. In certain embodiments, reagent wells can be covered with protective sheets. Thus, an advantage of providing variable length collection containers is a reduction in the force required by the elevator mechanism to accommodate a sheet piercing force when a reagent cartridge is brought into contact with the piercing absorber containers. The difference in aspirator container length can advantageously correspond to the depth of reagent wells in a reagent cartridge, so that each aspirator container reaches a desired depth in its corresponding reagent well when the aspirator tubes and cartridge are in a fully engaged position.
    [042] The suction tubes can be formed of any suitable material that allows the transfer of fluid through a lumen and that is compatible with the solvents and liquids transported through the collector channels in the embodiments presented here. Aspirating tubes can be formed from a single tube. Alternatively or additionally, one or more suction tubes can be made of several segments which together form a suction tube of a desired length and diameter.
    [043] In some embodiments, at least one of the reagent tubes includes a compatible tip configured to flex when the tip collides with the bottom of a reagent well right on a reagent cartridge. By bending or deforming, a compatible tip allows the suction tube lumen to approach more completely, or even make contact with the bottom of the reagent well, reducing or even eliminating the evacuation volume in the reagent well. A compatible tip may be especially advantageous for absorbing sample or reagents where small volumes are used, or in situations where it is desirable to absorb most or all of the liquid in a reagent reservoir. The suction tube body having a compatible tip may be entirely made of the same flexible material as the tip. Alternatively or additionally, the suction tube body can be made of a material other than that of the tip. The compatible tip can be made of any suitable material, such that the compatible tip can deform or yield it when pushed into contact with the bottom of a reagent reservoir. Some suitable materials include polymeric and/or synthetic foams, rubbers, silicone, and/or elastomers including thermoplastic polymers such as polyurethane.
    [044] The fluidic systems presented here may also include, for example, pumps and valves that are selectively operable to control fluid communication between the reservoirs and the inlet of the flow cell. As exemplified by the manifold assembly shown in Figs. 2 and 3, the channel outlets in the manifold may be configured to mate with corresponding inlet ports in one or more valves so that each reagent channel is in fluid communication with an inlet port of the valve. Thus, through the reagent manifold channels, one or more or each of the inlet ports may be in fluid communication with a reagent tube. Each of one or more valves may be configured with a common outlet port (110, 112), which fluidly connects to an inlet of one or more strips in a flow cell. Alternatively or additionally, each of the one or more valves may be configured with a discharge port (109, 113) fluidly connected to one or more waste containers.
    [045] In embodiments where the fluid system comprises at least a first valve and a second valve, each valve may be configured to simultaneously supply separate supply reagents through a first channel and a second channel. a flow cell, respectively. Thus, one valve can deliver a reagent to a first flow cell channel, while the second valve can simultaneously deliver a different reagent to a second flow cell channel. As shown in the exemplary embodiments of FIG. 9, a valve (VA) is hydraulically connected to input V1 of the flow cell, which is a manifold to supply reagents for range 1 and range 3. Similarly, valve B (VB) is hydraulically connected to input V2 located at the opposite end of the flow cell, and which provides reagents for lane 2 and lane 4. Inputs V1 and V2 are located on opposite sides of the flow cell and the reagent flow direction is in opposite directions for lane 1 and 3, compared to lanes 2 and 4.
    [046] The fluidic systems described here can be used advantageously for the manipulation of fluid from flow cell channels during the sequencing of nucleic acids. More specifically, a fluid system described herein can be operatively associated with a detection apparatus of an arrangement for detecting nucleic acid characteristics in the flow cell by the detection apparatus. In some embodiments, the detection apparatus may comprise a plurality of microfluorimeters, each of the microfluorimeters comprising an objective configured for wide-field image detection in an image plane in x and y dimensions. The fluidic systems set forth in this document are particularly useful with any of the detection apparatus configurations set forth in US Patent Application Serial Number 13 / 766,413 filed February 13, 2013 and entitled "INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGE USEFUL FOR NUCLEIC ACID SEQUENCING,” the contents of which are incorporated herein by reference in their entirety.
    [047] As an example, in particular nucleic acid sequencing embodiments, a flow cell containing a plurality of channels can be fluidly manipulated and optically detected in a staggered manner. More specifically, fluid manipulations can be performed on a first subset of channels in the flow cell, while optical detection occurs for a second subset of channels. For example, in one configuration, at least four linear channels can be arranged parallel to each other in the flow cell (for example channels 1 to 4 can be arranged in sequential lines). Fluid manipulations can be performed on all other channels (eg channels 1 and 3), while detection occurs for other channels (eg channels 2 and 4). This particular configuration can be accommodated by the use of a header that has detectors positioned in a spaced-apart configuration such that goals are directed to all other channels of the flow cell. In this case, the valves can be activated to direct the reagent flow for a sequencing cycle to alternate channels, while the channels being detected are kept in a detect state. In this example, a first set of alternate channels may undergo fluidic steps of a first sequencing cycle and a second set of alternate channels undergo detection steps of a second sequencing cycle. Once the fluid steps of the first cycle are completed and the detection steps of the second cycle are completed, the header can be scaled along (eg along dimension X) for the first set of alternating channels and valves can be actuated to deliver reagents in sequence to the second set of channels. Then, detection steps for the first cycle can be completed (in the first set of channels) and fluid steps for a third cycle can occur (in the second set of channels). The steps can be repeated in this way several times, until a desired number of cycles is carried out, or until the sequencing procedure is complete.
    [048] An advantage of the detection and scaled fluid steps set out above is to provide a faster execution of global sequencing. In the example above, a faster sequencing run will result from the staggered setup (compared to fluidic manipulation of all channels in parallel followed by detection of all channels in parallel), if the time required for fluid manipulation is approximately the same as the time required for detection. Of course, in embodiments where the time for the detection steps is not the same as the timing of the fluidic steps, the staggered configuration can be changed from all other channels to a more appropriate model to accommodate scanning in parallel of a subset of channels, while another subset of channels passes through the fluidic steps.
    [049] An additional advantage of having the fluid flow in opposite directions is to provide a means of comparing the performance of the individual microfluorimeter. For example, when multiple microfluorimeters are used per flowcell range, it can be difficult to distinguish whether the microfluorimeter's decreased performance is caused by the detector or by decreasing chemical efficiency from one end of the range to the other. By having opposite liquid flow directions, the performance of the microfluorimeter between the ranges can be compared, effectively distinguishing whether a decrease in performance is due to the microfluorimeter or not.
    [050] A fluid map for an exemplary fluid system is shown in Fig. 9. The Flow Cell 2020 has four bands each of which is fluidly connected to one of two individual fluid lines FV and RV that are individually actuated by valves of admission VA and VB. The VA inlet valve and VB inlet valve control fluid flow from sample reservoirs, SBS reagent reservoirs, and amplifying reagent reservoirs in the reagent cartridge or tray 2035 fluidly connected to various ports within the 2030 reagent manifold.
    [051] Fluid flow through the system of Fig. 9 is driven by two separate syringe pumps 2041 and 2042. The syringe pumps are positioned to pull fluid through the fluid system and each pump can be individually actuated by valves 2051 and 2052. Thus, although each flow channel of the flow cell can be individually controlled by a dedicated pressure source. The 2051 and 2052 valves are also configured to control fluid flow to the 2060 waste reservoir.
    [052] Fig. 9 exemplifies a fluid system in which the fluids are pulled by the action of the syringe pumps downstream. It will be understood that a useful fluidic system may use other types of devices in place of syringe pumps in conducting fluids, including, for example, positive or negative pressure, peristaltic pump, diaphragm pump, piston pump, gear pump or Archimedes screw. In addition, these and other devices can be configured to pull fluid from a position downstream relative to a flow cell or push fluid from an upstream position.
    [053] Fig. 9 also exemplifies the use of two syringe pumps for four channels of a flow cell. Thus, the fluid system includes a number of pumps that is less than the number of channels in use. It should be understood that a fluid system that is useful in a fluid cartridge of the present invention may have any number of pumps, e.g., an equivalent or fewer number of pumps (or other pressure sources) than the number of channels in use. For example, multiple channels can be fluidly connected to a common pump and a valve can be used to drive fluid flow through an individual channel.
    [054] The fluid system exemplified in Fig. 9 also includes a BUB-4 sensor for detecting air bubbles, positioned along the fluid path between valve VA and the inlet of flow cell V1. An additional BUB-3 air bubble sensor is positioned along the PV fluid path between valve VB and flow cell inlet V2. It should be understood that a fluidic line, which is useful in a fluid system of the present invention, can include any number of bubble sensors, pressure gauges, and the like. Sensors and/or gauges can be located in any position along any part of the fluid path in the fluid system. For example, a sensor or gauge can be positioned along a fluid line between one of the valves and the flow cell. Alternatively or additionally, a sensor or gauge may be positioned along a fluid line between a reagent reservoir and one of the valves, between a valve and a pump, or between a pump and an outlet or reservoir such as a waste reservoir. .
    [055] A cross-section of an exemplary reagent cartridge is illustrated in Fig. 6. The reagent cartridge 400 shown in Fig. 6 includes wells 401 of various depths along the Z dimension compared to those of wells 402. More specifically , the reagent cartridge exemplified in Fig. 6 has wells designed to accommodate the length of a corresponding reagent suction tube (not shown) such that each suction tube reaches a desired depth in its corresponding reagent well when the suction tubes and cartridge are in a fully engaged position. In the reagent cartridge exemplified in Fig. 6, the wells are arranged in a row or column along the y dimension, where those wells 401 outside the row or column extend further down along the Z dimension than the wells 402 on the inside of the row or column. Some or all of the wells may be of different depths. Alternatively or additionally, some or all of the wells may be of the same depth. When the sniffer tubes and cartridge are in the fully seated position, the depth of penetration of any end of the suction tube (ie, the distance from the well bottom surface to the end of the suction tube tip) can be equivalent to the penetration depth of any other end of the suction tube into any other well given in the reagent cartridge. The penetration depth of any end of the suction tube need not be the same as the penetration depth of any other well determined in the reagent cartridge. When at least some reagent wells have a different well depth, the well 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 2.0 mm shorter than other reagent aspirator tubes. Likewise, when the sniffer tubes and cartridge are in a fully engaged position, the penetration depth of any suction tube tip can be 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 different from the penetration depth of any other suction tube end in the reagent cartridge.
    [056] The top view of an exemplary reagent tray interface that has reagent wells and interface slots for alignment pins is shown in Fig. 8. As shown on the exemplary reagent cartridge 400 in Fig. 8, the cartridge includes a plurality of reagent reservoirs 401A, 401B, 402A and 402B. The reagent reservoirs in Fig. 8 are arranged in X and Y dimensions in rows. Also shown in Fig. 8, the cartridge includes interface slots 403 and 404 configured to engage with corresponding alignment pins of a dispenser assembly (not shown). The cartridge may also include a protective film covering any number of wells or reservoirs of reagents, which may be pierced by drill pipes when the cartridge is brought into contact with drill suction tubes.
    [057] The reagent cartridges presented here can include any number of reagent reservoirs or wells. Reagent reservoirs or wells can be arranged in any shape along the X and Y dimensions to facilitate transport and storage of reagents in the cartridge. Alternatively or additionally, the reagent reservoirs or wells can be arranged in any shape along the X and Y dimensions suitable for interaction with a series of suction tube extending downward along the Z dimension from holes in the collector. More specifically, the reagent reservoirs or wells may be arranged in any suitable shape to simultaneously engage an array of reagent aspirator tubes such that liquid reagent can be withdrawn from the reagent reservoir into the aspirator tubes.
    [058] Not all reagent wells need to interact simultaneously with all sniffer tubes in a dispenser set. For example, the reagent cartridge can include a subset of one or more reagent reservoirs or wells that are configured to remain in a state of no interaction with each other, while other reservoirs or wells are surrounded by an array of sniffer tubes. As an example, a cartridge shown herein may comprise a plurality of wash reservoirs disposed in a configuration corresponding to the plurality of reagent reservoirs, wherein the wash reservoirs are configured to simultaneously engage the reagent aspirator tubes when the reagent aspirator tubes. reagent are not nested with the reagent reservoirs, so the wash buffer can be drawn from the wash reservoirs into the aspirator tubes. An exemplary embodiment is shown in Fig. 8, which shows a row of reagent wells 401A. The cartridge also includes a row of matching wells 401B that retain the same orientation in dimension X with respect to each other, but which are offset in dimension y from wells 401A. Displaced wells 401B may include a wash buffer, for example, provided for washing aspirator tubes and fluid lines after using one cartridge and before using another cartridge.
    [059] Alternatively or additionally, other reservoirs that are empty, or that contain buffer, sample, or other reagents may be present in the cartridge. Additional reservoirs can, but need not, interact with a suction tube. For example, a reservoir can be configured to be filled with waste or excess reagent or buffer over the course of use of the cartridge. Such a reservoir can be accessed, for example, through an orifice that does not interact with a suction tube.
    [060] To facilitate the correct alignment of the cartridge reservoirs with the corresponding sniffer tubes, alignment grooves can be positioned on the cartridge. For example, in particular embodiments where an array of sniffer tubes is removed from one set of reservoirs and translocated to another set of wash reagent reservoirs, alignment grooves can be positioned on the cartridge to ensure correct alignment array of reagent aspirator tubes with one or both sets of reservoirs. As shown in Fig. 8, the exemplary cartridge includes alignment grooves 404 that retain the same orientation in dimension X, but which are offset in dimension y relative to corresponding alignment groove 403. A cartridge of the embodiments shown herein may have any number of alignment grooves that provide proper alignment with the characteristics of a fluidic assembly. For example, a cartridge may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more alignment slots configured to engage with corresponding fluidic system alignment pins such that tubes Fluidic system reagent aspirators are positioned in alignment with the reagent and/or wash reservoirs.
    [061] In particular embodiments a fluidic system can be configured to allow the reuse of one or more reagents. For example, the fluidic 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 to the flow cell. One configuration is exemplified in Fig. 7A, which shows a top view of the deposit lines in a collector assembly. As shown in the schematic diagram in the upper portion of fig. 7A, a reagent depot can be used to maintain a concentration gradient from more used to less used (fresh) reagent. In some embodiments, the cache reservoir can be configured to reduce fluid mixing within the cache reservoir, thereby maintaining a liquid reagent gradient along the length of the reservoir from the proximal end to the flow cell to the end. distal to the flow cell. As reagent is supplied back to the flow cell from the cache reservoir the gradient is maintained such that the reagent that has flowed through the flow cell forms a reagent gradient from more used to less used (fresh) .
    [062] As exemplified in the diagram in the lower portion of Fig. 7A, the manifold fluids can be configured such that a reagent reservoir is in fluid communication with the inlet port of a flow cell (not shown) through the 1804 inlet valve. The 1804 valve controls fluid flow between the flow cell (not shown) and each of the CLM reservoirs, SRE reservoir, IMF reservoir, and LAM1 and LPM1 reservoirs. Channel 1802 fluidly connects the CLM reservoir through port 1801 with valve inlet 1804. A channel portion 1802 includes a reagent reservoir 1803 configured to contain a volume of reagent equivalent to the volume of one or more flow cell strips (not shown). Increasing the volume of reagent tank 1803 compared to other parts of channel 1802 allows a used reagent to be stored for reuse, keeping an unused reagent stock in the reagent reservoir, thus preventing contamination of the unused reagent stock in the reagent reservoir with the reagent used.
    [063] The configuration shown in Fig. 7A is exemplary. Other configurations are possible as well to achieve reuse. For example, one or more of the deposit reservoirs may have a volume that is 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 a cell channel flow in fluid communication with the cache reservoir. Alternatively or additionally, the cache reservoir may comprise a volume sufficient to allow an amount of liquid reagent in one or more flow cell channels to flow into the cache reservoir such that the liquid reagent from the flow cell is not directed from returns to the reagent reservoir after contact with the flow cell. For example, the amount of liquid reagent may 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.
    [064] A cache reservoir as presented herein can be configured to reduce fluid mixing within the cache reservoir. In some such embodiments, the reduced mixture can thus maintain a gradient of liquid reagent along the length of the reservoir from the proximal end to the flow cell to the distal end to the flow cell. Alternatively or additionally, a cache reservoir as presented herein may comprise one or more mixing elements configured to promote fluid mixing within the cache reservoir. Any suitable active or passive mixing element can be used in such embodiments. For example, the mixing element may comprise deflecting elements, curved structures, or any other passive or active structural or fluidic feature configured to promote mixing when fluid is transported through a cache reservoir. Alternatively or additionally, any suitable pump, impeller, blade, inlet and the like can be used for active mixing within a cache reservoir.
    [065] A cache reservoir as presented here may have any shape, volume and length that is suitable for the purposes of a cache reservoir. In specific embodiments, depot reservoirs of any shape, volume and/or length can be used in the fluidic systems disclosed herein that allow an amount of liquid reagent in one or more flow cell channels to flow into the reservoir. cache such that liquid reagent from the flow cell is not directed back into the reagent reservoir after contacting the flow cell. For example, a cache reservoir can comprise a serpentine channel. By way of another example, a cache reservoir may comprise a cylindrical or non-cylindrical shaped channel. In addition, any number of fluid channels in the fluid system presented herein can include one or more individual sump reservoirs.
    [066] A cache reservoir as presented herein may be in fluid communication with a pump configured to move liquid reagent from the cache reservoir to the flow cell and from the flow cell back to the cache reservoir, where the inlet of the reagent to the flow cell and output of the reagent from the flow cell occurs through the same orifice as the flow cell. Alternatively or additionally, reagent input to the flow cell and reagent output from the flow cell can occur through different orifices of the flow cell and still achieve reagent reuse. For example, the fluidic systems presented herein may make use of any of the reuse reservoirs and configurations described in connection with apparatus configurations in accordance with US Patent Application Serial Number 13/766,413 filed February 13, 2013, and entitled "INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGE USEFUL FOR NUCLEIC ACID SEQUENCING," the contents of which are incorporated herein by reference in their entirety.
    [067] The diagram in fig. 7B presents an exemplary illustration of a reuse method disclosed herein that utilizes reciprocal flow of reagent from a deposit line to a flow cell, followed by partial refilling of the flow cell deposit line. In the state shown in the top panel of Fig. 7B, cache reservoir 1903 containing 100μL of reagent 1906 is in fluid communication with flow cell tracks 1905 via divider 1904 and valve 1911. Valve 1904 is actuated to allow reagent 1906 flow into the 1905 flow cell tracks. At the same time, fresh reagent 1907 is pulled from the reagent reservoir to fill the void left in cache reservoir 1903. After use of the reagent in the flow cell, valve 1911 directs a portion (75 μL) of used reagent 1906 back to cache reservoir 1903. Another portion (25 μL) of used reagent 1906 is diverted by valve 1911 to a waste container. At the end of cycle 1, cache reservoir 1903 has a gradient with 25μl of fresh 1907 reagent and 75μl of 1906 reagent used over the entire length of cache reservoir. The cycle of reverse flow reagent from the cache reservoir to the flow cell and back to the cache reservoir is repeated, with a portion (25μL) of used reagent 1906 diverted in each cycle by valve 1911 to a waste container and the remainder of used reagent 1906 is drained back to cache reservoir 1903. At the end of four equal repeated cycles, cache reservoir 1903 contains 25μL of fresh reagent 1910, 25μL of reagent that has been used only once 1909, 25μL of reagent which was used twice 1908, and 25μL of reagent which was used three times 1907.
    [068] The configurations shown in Fig. 7A and fig. 7B are exemplary. Other configurations are possible as well to achieve reuse of one or more of the reagents used in particular processes. It will be understood that, in some reagent reuse configurations, fluidic configurations for reagent reuse will only be used for a subset of the reagents used in a particular process. For example, a first subset of reagents may be robust enough to be reused while a second subset may be prone to contamination, degradation or other unwanted effects after a single use. Therefore, the fluid system can be configured to reuse the first subset of reagents, while the fluids for the second set of reagents will be configured for a single use.
    [069] A specific reagent can be reused any number of times desired to meet a given process. For example, one or more of the reagents exemplified herein, described in the reference cited herein, or otherwise known for use in a process set forth herein may be reused at least 2, 3, 4, 5, 10, 25, 50 or more. times. In fact, any of a variety of desired regents can be reused at least multiple times. Any portion of a particular reagent can be diverted back to a cache reservoir for reuse. For example, one or more of the reagents exemplified herein, described in the cited reference, or otherwise known to be used in a process set forth herein may be 1%, 2%, 3%, 4%, 5%, 6%, %, 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 in one or more flow cell ranges routed back to the cache 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 in one or more ranges of flow may be diverted to a waste container or otherwise removed from subsequent use in a flow cell.
    [070] Fluid configurations and methods for reagent reuse, although exemplified by a nucleic acid sequencing process, can be applied to other processes, in particular, processes that involve repeated cycles of reagent supply. Exemplary processes include the sequencing of polymers such as polypeptides, polysaccharides or synthetic polymers and also include the synthesis of these polymers.
    [071] As demonstrated by the exemplary embodiments above, a reagent reuse method may include the steps of: a) withdrawing a liquid reagent from a reagent reservoir to a cache reservoir, the cache reservoir in fluid communication with the reservoir of reagent and at least one channel of a flow cell; b) transporting the reagent from the cache reservoir to the at least one flow cell channel; c) the transport of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the reagent in the flow cell channel to the cache reservoir in such a way that the liquid reagent from the flow cell is not directed back to the reagent reservoir after contacting the flow cell; d) repeat steps b) and c) to achieve reuse of the liquid reagent in the flow cell. The one or more of the sump reservoirs may be in fluid communication with a pump configured to move liquid reagent from the cache reservoir to the flow cell and from the flow cell back to the cache reservoir, such that the inlet of reagent to the flow cell and the output of reagents from the flow cell occurs through the same orifice of the flow cell. Alternatively or additionally, reagent input to the flow cell and reagent output from the flow cell can occur through different orifices of the flow cell and still achieve reagent reuse. In some embodiments, the flow cell reagent that is not transported to the cache reservoir in step c) can be bypassed. As an example, flow cell reagent that is not transported to the cache reservoir can be transported to a waste reservoir. Reagent transport in one or both of steps b) and c) can be carried out through a valve, which fluidly connects the cache reservoir and the flow cell. Reagent transport in one or both of steps b) and c) can be carried out, for example with one-way fluid flow, or it can be carried out with alternative flow.
    [072] Embodiments of the present fluidic systems and methods find particular use for nucleic acid sequencing techniques. For example, sequencing by synthesis (SBS) protocols are particularly applicable. In SBS, the extension of a nucleic acid primer along a nucleic acid template is monitored to determine the nucleotide sequence in the template. The underlying chemical process may be polymerization (eg, as catalyzed by a polymerase enzyme) or binding (eg, catalyzed by a ligase enzyme). In a particular polymerase-based SBS embodiment, the fluorescently labeled nucleotides are added to a primer (thus extended the primer) in a template-dependent manner, such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the model. A plurality of different models can be subjected to an SBS technique on a surface under conditions where events occurring in different models can be distinguished. For example, patterns can be present on the surface of a matrix in such a way that the different patterns are spatially distinguishable from one another. Templates typically occur on resources each having multiple copies of the same template (sometimes called "groupings" or "colonies"). However, it is also possible to perform SBS on arrays where each function has a single template molecule present, such that individual template molecules can be resolved to one another (sometimes called "single-molecule arrays").
    [073] Flow cells provide a convenient substrate to house a matrix of nucleic acids. Flow cells are convenient for sequencing techniques because the techniques typically involve repeated cycled release of reagents. For example, to initiate a first cycle of SBS, one or more labeled nucleotides, DNA polymerase etc. can be poured into/through a flow cell that houses an array of nucleic acid templates. These characteristics, where primer extension causes a labeled nucleotide to be incorporated, can be detected, for example, using the methods and apparatus set forth herein. Optionally, nucleotides can further include a reversible termination property that terminates beyond primer extension once a nucleotide has been added to a primer. For example, a nucleotide analogue having a reversible terminator moiety can be added to a primer such that further extension cannot occur until a deblocking agent is released to remove the moiety. Thus, for embodiments using a reversible termination an unlocking reagent can be supplied to the flow cell (before or after detection takes place). Washes can be carried out between the various distribution steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thus detecting a sequence of length n. Examples of 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 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US 2008/0108082, each of which is incorporated herein by reference.
    [074] For the nucleotide delivery step of an SBS cycle, either a single nucleotide type can be delivered at a time, or several different nucleotide types (eg, A, C, T and G together) can be distributed. For a nucleotide delivery configuration where only a single nucleotide type is present at a time, the different nucleotides need not have distinct labels, as they can be distinguished based on the temporal separation inherent in individualized delivery. Therefore, a sequencing method or apparatus may use single color detection. For example, a microfluorimeter or header need only provide excitation at a single wavelength, or over a single wavelength range. Thus, a microfluorimeter or header need only have a single excitation source and filtration in multiple excitation ranges is not necessary. For a nucleotide delivery configuration where delivery results in several different nucleotides being present in the flow cell at the same time, features that incorporate different nucleotide types can be distinguished based on different fluorescent labels that are linked to the respective nucleotide types in the mix. For example, four different nucleotides can be used, each having four different fluorophores. In one embodiment, the four different fluorophores can be distinguished using excitation in four different regions of the spectrum. For example, a microfluorimeter or header can include four different excitation radiation sources. Alternatively, a read head may include less than four different sources of excitation radiation, but may use optical filtration of excitation radiation from a single source to produce different bands of excitation radiation in the flow cell.
    [075] In some embodiments, four different nucleotides can be detected in a sample (for example, nucleic acid characteristic matrix) using less than four different colors. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in the intensity of one member of the pair relative to the other, or based on a shift for one member of the pair. pair (eg via chemical modification, photochemical modification or physical modification) which causes the evident signal to appear or disappear compared to the detected signal for the other member of the pair. As a second example, three of the four different nucleotide types can be detectable under particular conditions, while a fourth nucleotide type lacks a label that is detectable under those conditions. In an SBS embodiment of the second example, incorporation of the first three nucleotide types in a nucleic acid can be determined based on the presence of their respective signals, and incorporation of the fourth nucleotide type in the nucleic acid can be determined based on the absence of any sign. As a third example, a nucleotide type can be detected in two different images or in two different channels (eg a mixture of two species with the same base but different labels can be used, or a single species with two labels can be used or individual species having a label that is detected in both channels can be used equally, whereas other types of nucleotides are detected in no more than one of the images or channels. In this third example, the comparison of the two images or two channels lends itself to distinguishing the different types of nucleotides.
    [076] The three exemplary configurations in the previous paragraph are not mutually exclusive and can be used in various combinations. An exemplary embodiment is an SBS method that uses reversibly blocked nucleotides (rbNTPs) having fluorescent labels. In this format, four different types of nucleotides can be released to a set of nucleic acid functionalities that must be sequenced and, due to reversible blocking groups, one and only one incorporation event will occur in each trait. Nucleotides supplied to the array in this example may include a first type of nucleotide which is detected in a first channel (eg rbATP having a label which is detected in the first channel when excited by a first excitation wavelength), a second type of nucleotide that is detected in a second channel (eg rbCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third type of nucleotide that is detected in both the first and second channel (eg rbTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is detected in either of the channels (eg rbGTP which does not have an extrinsic label).
    [077] Once the four types of nucleotides have been placed in contact with the array in the example above, a detection process can be carried out, for example, to capture two images of the array. Images can be taken in separate channels and can be taken simultaneously or sequentially. A first image obtained using the first excitation and emission wavelength in the first channel will show features that have incorporated the first and/or third nucleotide type (eg, A and/or T). A second image obtained using the second excitation and emission wavelength in the second channel will show features that have incorporated the second and/or third nucleotide type (eg, C and/or T). Unambiguous identification of the type of nucleotide incorporated in each feature can be determined by comparing the two images to arrive at the following features: features that appear only in the first channel incorporated into the first type of nucleotide (eg, A), features that appear only in the second channel incorporated in the second type of nucleotides (eg C), features that appear in both channels incorporated the third type of nucleotide (eg T) and features that did not appear in either channel incorporated the fourth type of nucleotide (eg G ). Note that the location of the features that incorporated G in this example can be determined from other cycles (where at least one of the other three types of nucleotides are incorporated). Apparatus and methods for distinguishing four different nucleotides using less than four color detection are described for example in U.S. Pat. US App. Serial No. 61 / 538,294, which is incorporated herein by reference.
    [078] In some embodiments, nucleic acids can be attached to a surface and amplified before or during sequencing. For example, amplification can be performed using bridging amplification to form nucleic acid groups on a surface. Useful bridge amplification methods are described, for example, in US 5,641,658; US 2002/0055100; US 7,115,400; US 2004/0096853; US 2004/0002090; US 2007/0128624; or US 2008/0009420, each of which is incorporated herein by reference. Another useful method for amplifying nucleic acids on a surface is circular bearing amplification (RCA), for example, as described in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US 2007/0099208 A1, each of which is incorporated herein by reference. Microsphere PCR emulsion can also be used, for example, as described in Dressman et al, Proc. Natl. Academic Sci. USA 100:8817-8822 (2003), WO 05/010145, US 2005/0130173 or US 2005/0064460, each of which is incorporated herein by reference.
    [079] As described above the sequencing embodiments are an example of a repetitive process. The methods of the present invention are well suited for repetitive processes. Some embodiments are shown below and elsewhere.
    [080] Accordingly, provided herein are sequencing methods that include: (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 apparatus comprising (i) a plurality of microfluorimeters, each of the microfluorimeters comprising an objective configured for wide-field image detection in an image plane in X and Y dimensions, and (ii) a sample phase; and (c) the performance of fluidic operations of a nucleic acid sequencing procedure in the cartridge operations and detection of the nucleic acid sequencing process in the detection apparatus, in which (i) the reagents are supplied to the flow cell by the fluid system, (ii) wide-field images of nucleic acid characteristics that are detected by the plurality of microfluorimeters, and (iii) at least some reagents are removed from the flow cell of a cache reservoir.
    [081] Throughout this patent application, various publications, patents and/or patent applications have been referenced. The disclosure of these publications in their entirety is hereby incorporated by reference into this application.
    [082] The term comprising is intended here to be undefined, including not only the elements described, but also encompassing any additional elements.
    [083] Some embodiments have been described here. Nevertheless, it will be understood that several modifications can be made. Therefore, other embodiments are within the scope of the appended claims.
    权利要求:
    Claims (12)
    [0001]
    1. System, comprising: a fluid system (100) for dispensing reagents from a reagent cartridge (400) to a flow cell CHARACTERIZED in that it comprises: a reagent collector assembly (101) comprising a manifold body having a solid material and a plurality of channels (107) configured to fluidly connect a reagent cartridge (400) to an inlet of a flow cell, wherein the plurality of channels (107) of the reagent manifold assembly (101 ) each includes a fluid channel (302) through the solid material of the manifold body; a plurality of reagent aspirator tubes (103, 104) coupled to the reagent manifold assembly (101) and extending downwardly from holes that are housed in the reagent manifold assembly (101), each of the plurality of reagent aspirator tubes (103, 104) configured to be placed within a reagent reservoir (401, 402) in a reagent cartridge (400) so that liquid reagents can be drawn from the reagent reservoirs (401, 402) to the aspirator tube (103, 104); at least one valve (102, 109) configured to mediate fluid communication between the reagent reservoirs (401, 402) and the inlet of the flow cell; at least one of the plurality of channels (107) in the manifold (101) comprising a cache reservoir (108, 1903) in the reagent manifold assembly (101) and in fluid communication with a cache reservoir. reagent (401, 402) and at least one flow cell channel, wherein the cache reservoir (108, 1903) has a volume that is at least 30% of the volume of a flow cell channel, wherein the cache reservoir (108, 1903) has sufficient volume to allow an amount of liquid reagent to flow from the flow cell to the cache reservoir (108, 1903) so that liquid reagent from the flow cell is not directed back to the reagent reservoir. (401, 402) after contacting the cell of flow and wherein one or more of the cache reservoirs (108, 1903) are in fluid communication with a pump configured to move liquid reagent from the cache reservoir (108, 1903) to the flow cell and from the flow cell back to the reservoir cache (108, 1903), in which the reagent input to the flow cell and the reagent output from the flow cell occur through the same orifice of the flow cell; and a sensing device operatively associated with the fluid system (100) and configured to detect nucleic acid characteristics in the flow cell.
    [0002]
    2. System according to claim 1, CHARACTERIZED by the fact that at least one valve (102, 109) is configured to differentially direct liquid reagent from the flow cell back to the cache reservoir (108, 1903) or to the waste reservoir (109).
    [0003]
    3. System according to claim 1, CHARACTERIZED by the fact that said cache reservoir (108) is configured to reduce fluid mixing within the cache reservoir, thus maintaining a liquid reagent gradient along the length of the reservoir from the proximal end to the flow cell to the distal end to the flow cell.
    [0004]
    4. System according to claim 1, CHARACTERIZED by the fact that said cache reservoir comprises a plurality of mixing elements configured to promote fluid mixing within the cache reservoir and, optionally, wherein the mixing elements comprise static characteristics in the cache reservoir or on an inner surface of the cache reservoir, or where the mixing elements comprise baffle elements.
    [0005]
    5. System according to claim 1, CHARACTERIZED by the fact that said cache reservoir comprises a channel of non-cylindrical shape.
    [0006]
    6. System according to claim 1, CHARACTERIZED by the fact that the manifold (101) is configured to deliver reagent from a first reagent reservoir to a first valve through a first channel and from the first reservoir of reagent to a second valve through a second channel.
    [0007]
    7. System according to claim 1, CHARACTERIZED by the fact that the detection apparatus comprises a plurality of microfluorimeters, in which each of the microfluorimeters comprises an objective configured for wide-field image detection in an image plane in a x and y dimension.
    [0008]
    8. Method of reusing reagents CHARACTERIZED in that it comprises: a) providing a reagent manifold assembly (101) comprising a manifold body having a solid material and a plurality of channels (107) configured to supply liquid reagents from reagent reservoirs (401, 402) of a reagent cartridge (400) to an inlet of a flow cell; b) extracting a liquid reagent from a reagent reservoir (401, 402) to a cache reservoir (108, 1903), the reservoir cache (108, 1903) being in fluid communication with the reagent reservoir (401, 402) and at least one channel of a flow cell, the cache reservoir (108, 1903) having a volume that is at least 30% of the volume. of the at least one channel, wherein the cache reservoir (108, 1903) has a volume sufficient to allow an amount of liquid reagent to flow from the flow cell to the cache reservoir (108, 1903) so that the liquid reagent in the cell flow is not directed back to the reagent reservoir (401, 402) after contacting the flow cell, and wherein one or more of the cache reservoirs (108, 1903) is in fluid communication with a pump configured to move the liquid reagent from the cache reservoir (108, 1903) to the flow cell and from the flow cell back to the cache reservoir (108, 1903), where reagent input to the flow cell and reagent output from the flow cell occurs via from the same flow cell orifice; c) transporting the reagent (1906) from the cache reservoir (108, 1903) to the at least one flow cell channel; d) transporting at least 30% of the reagent in the cell channel flow to the cache reservoir (108, 1903) such that the liquid reagent from the flow cell is not directed back to the reagent reservoir (401, 402) after contacting the flow cell; and e) repeat steps c) and d) to achieve reuse of the liquid reagent in the flow cell.
    [0009]
    9. Method according to claim 8, CHARACTERIZED by the fact that the flow cell reagent that is not transported to the cache reservoir (108, 1903) in step c) is transported to the waste reservoir.
    [0010]
    10. Method according to claim 8, CHARACTERIZED by the fact that the transport in one or both of steps c) and d) is performed by means of a valve (1911) that fluidly connects the cache reservoir (108, 1903) and the flow cell.
    [0011]
    11. Method according to claim 8, CHARACTERIZED by the fact that the liquid reagent comprises a reagent to perform nucleic acid sequencing, a polymerase, nucleotide or mixture of different nucleotides.
    [0012]
    12. Method according to claim 8, CHARACTERIZED by the fact that the reagent has flowed back (1906) to the cache reservoir (108, 1903) in step d) is mixed with similar reagent (1907) in the cache reservoir (108 , 1903), thus forming a reactant mixture comprising similar reactants from a plurality of repetitions of steps c) and d).
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    法律状态:
    2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-11-03| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2021-04-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-06-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/08/2014, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201361863795P| true| 2013-08-08|2013-08-08|
    US61/863.795|2013-08-08|
    PCT/US2014/050052|WO2015021228A1|2013-08-08|2014-08-07|Fluidic system for reagent delivery to a flow cell|
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