Temperature control for nucleic acid and other analyte analysis

Abstract

The present disclosure provides methods and devices for sequencing nucleic acids. For example, an analytical detection device is provided, comprising: (a) a stage configured to support a flow cell; (b) a detector configured to observe a detection channel of the flow cell when the flow cell is supported by the stage; (c) a plurality of fluid delivery channels, each fluid delivery channel fluidly connecting a reservoir to the detection channel of the flow cell; and (d) a first heater configured to heat the plurality of fluid delivery channels.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 782,565, filed on Dec. 20, 2018, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to the detection of chemical and biological analytes and has particular applicability to nucleic acid sequencing. Background Art

[0004] Accurate sequence determination of template nucleic acid strands is important for molecular diagnosis. Identifying individual nucleotide bases from alternatives at known positions can be used as a basis for analyzing single nucleotide polymorphisms (i.e., "SNPs"). SNPs, in turn, can be used to determine an individual's phenotype, such as susceptibility to a disease or a tendency to have a desired trait. Detecting genetic variations in a patient can indicate the efficacy of certain drug treatments for the patient or the risk of adverse side effects when treating the patient with certain drugs.

[0005] Commercially available nucleic acid sequencing platforms have greatly increased our understanding of the genetic basis of actionable traits. Improvements in sequencing biochemistry and detection hardware continue. However, despite their widespread use in research laboratories, the cost of currently available sequencing platforms inhibits their adoption in the clinic. Similarly, sequencing platforms are relatively slow in providing diagnostic or prognostic answers that meet the expectations of patients and the doctors treating them within a time frame. Summary of the Invention

[0006] The present disclosure provides an apparatus for performing an analytical procedure such as determining a nucleic acid sequence. The apparatus includes: (a) a stage configured to support a flow cell; (b) a detector configured to observe a detection channel of the flow cell when the flow cell is supported by the stage; (c) a plurality of fluid delivery channels, each of which fluid - connects a reservoir to the detection channel of the flow cell; and (d) a first heater configured to heat the plurality of fluid delivery channels.

[0007] The present disclosure also provides an apparatus for nucleic acid sequencing, comprising: (a) a stage in contact with a flow cell, wherein the flow cell comprises at least one detection channel, and wherein the detection channel is configured to accommodate a nucleic acid array; (b) a detector configured to observe the nucleic acid array in the detection channel; (c) a plurality of reservoirs storing reagents for sequencing the nucleic acid array; (d) a plurality of fluid delivery channels, wherein the fluid delivery channels fluidly connect the plurality of reservoirs to the detection channel of the flow cell; (e) a first heater that transfers heat to the plurality of fluid delivery channels; and (f) a second heater that transfers heat to the detection channel of the flow cell.

[0008] The present disclosure also provides a method for performing an analytical process such as determining a nucleic acid sequence. The method may comprise the steps of: (a) providing an analytical apparatus having a flow cell, a fluid system, and a detection system, wherein the flow cell contains an analyte array, such as nucleic acid, in a detection channel, the fluid system comprises fluid delivery channels that fluidly connect reservoirs to the detection channel of the flow cell, and the detection system observes signals from the analyte array; (b) transferring a liquid reagent from a reservoir to a heated region of the fluid delivery channel, whereby the liquid reagent is heated; (c) contacting the heated liquid reagent with the analyte array by transferring the heated liquid reagent to the detection channel; (d) detecting signals from the analyte array by the detection system. In a nucleic acid sequencing configuration, the liquid reagent is a reagent for sequencing nucleic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shows a thermally conductive aluminum having a plurality of grooves for heating fluid tubes.

[0010] Figure 2 Shows a block diagram of an exemplary nucleic acid sequencing system.

[0011] Figure 3 Shows a graph of the relationship between water temperature and position in a flow cell based on a one-layer thermal model.

[0012] Figure 4 Shows a graph of the relationship between water temperature and position in a flow cell at various time points during and after starting water flow in the flow cell based on a one-layer thermal model.

[0013] Figure 5 Shows a graph of the relationship between water temperature and position in a flow cell based on a three-layer thermal model.

[0014] Figure 6 Shows a graph of the relationship between water temperature and position in a flow cell at various time points during and after starting water flow in the flow cell based on a three-layer thermal model.

[0015] Figure 7Shows a test device for evaluating the thermal performance of preheated fluid before entering the flow cell.

[0016] Figure 8A Shows tabular results obtained from evaluating the thermal characteristics of fluid before entering the flow cell; Figure 8B Shows a graph of results obtained from evaluating the thermal properties of fluid before entering the flow cell.

[0017] Figure 9 Shows a test device for evaluating the thermal performance of preheated fluid when flowing through the flow cell.

[0018] Figure 10A Shows tabular results obtained from evaluating the thermal properties of fluid flowing through the flow cell; Figure 10B Shows a graph of results obtained from evaluating the thermal properties of fluid flowing through the flow cell.

[0019] Figure 11 Shows a perspective view of an assembly of several components of a nucleic acid sequencing device.

[0020] Figure 12 Shows a top view of a routing manifold, a pipette array, a rotary valve, and a conduction heater.

[0021] Figure 13 Shows a bottom view of a routing manifold, a pipette array, a rotary valve, and a conduction heater.

[0022] Figure 14A Shows a perspective view of the fluid connection between a nucleic acid sequencing system and a flow cell; Figure 14B Shows the same perspective view, but with the connector disconnected.

[0023] Figure 15 Shows a front view of the sequencing device 1000, and the arrow with hatching indicates the direction of heat conduction. Detailed Description

[0024] Many analytical procedures are temperature-sensitive. In such cases, temperature fluctuations that do not exceed those experienced in the typical laboratory surroundings can have an adverse effect on the results obtained from the analytical procedure. For analyses that use biological components (such as enzymes), the optimal temperature can be elevated compared to the ambient temperature of a typical laboratory. For example, many enzymes present in mammals have optimal activity at or near the body temperature of the mammal (i.e., about 36 °C to 40 °C). Many useful enzymes (such as polymerases) are derived from thermophiles or have been engineered to operate at higher temperatures. Such enzymes can be used in analytical procedures where the temperature is between 50 °C and 80 °C. Temperature changes from these elevated ranges to the ambient temperature of a typical laboratory can cause significant changes in the activity of reagents used in important analytical procedures (such as clinical diagnostics or prognostic tests). Therefore, it is important to regulate the temperature of the analytical procedure or device to obtain high-quality results.

[0025] For some analytical devices or procedures, a conflicting issue is the instability of reagents at the operating temperature of the device or procedure. In many cases, it is desirable or even necessary to store the reagents at a reduced temperature prior to use. For example, thermophilic polymerases or other reagents used at elevated temperatures in an analytical procedure can be stored at room temperature (about 25 °C) or lower to minimize inactivation. The reagents can then be heated in the reaction vessel where the analytical procedure is carried out.

[0026] For some analytical procedures, reagents at a lower temperature can be added to a container at a high temperature and the reagents can be allowed to equilibrate until the reagents reach the high temperature. In fact, there are a variety of heating devices and reaction vessels available for heating the reaction mixture relatively quickly, such as Peltier heaters used for polymerase chain reaction (PCR). However, heating the reagents takes time, and the effect of an extended incubation time on the analytical procedure can be detrimental.

[0027] Devices that perform procedures involving multiple reagent replacements present a unique set of challenges. The nucleic acid sequencing process provides an example. Many sequencing procedures are cyclic such that when each nucleotide in the sequence is detected, reagents are released from a container. In addition, each cycle typically includes multiple different sub-steps, each of which involves delivering a different reagent to the container. Although sequencing reactions typically occur at elevated temperatures, many sequencing reagents are unstable at elevated temperatures and thus the sequencing reagents are maintained at a lower temperature in a reservoir. Commercially available sequencing platforms utilize a heated stage to support the flow cell or other container where the sequencing reaction takes place, which has been shown to provide satisfactory results.

[0028] The present disclosure is at least partially based on the observation that a temperature difference between a reservoir storing sequencing reagents and a heated flow cell, in which the sequencing reagents must be equilibrated to different temperatures for reactions or detection to occur, can have an adverse effect on nucleic acid sequencing results. Such adverse effects include, for example, base errors due to side reactions or incomplete sequencing reactions occurring during the period in which the reagents equilibrate to the flow cell temperature; inconsistent results observed at different locations in the flow cell due to inconsistent heating of the flowing fluid reagent (e.g., the temperature in the region of the flow cell near the fluid inlet is different from the temperature in the region closest to the outlet); and anomalous results due to temperature gradients that occur between reagents on the surface of the flow cell in contact with a heater and the same type of reagent in the internal space of the flow cell located away from the flow cell surface.

[0029] Accordingly, the present disclosure provides fluid systems, methods, and processes that can reduce sequencing time, reduce sequencing costs, reduce reagent volume, and also provide related advantages. For example, there is provided an apparatus for performing an analytical procedure, such as determining a nucleic acid sequence. The apparatus includes (a) a stage configured to support a flow cell; (b) a detector configured to observe a detection channel of the flow cell when the flow cell is supported by the stage; (c) a plurality of fluid delivery channels, each of which fluidly connects a reservoir to the detection channel of the flow cell; and (d) a first heater configured to heat the plurality of fluid delivery channels.

[0030] The apparatus or method of the present disclosure may utilize a flow cell. As used herein, a "flow cell" is a reaction chamber that includes one or more channels that direct fluid to a detection region. The detection region may be functionally coupled to a detector such that reactions occurring in the channel can be observed. For example, a flow cell may contain a templated nucleic acid molecule attached to a surface, to which nucleotides and auxiliary reagents are repeatedly applied and washed away. The flow cell may include a transparent material that allows imaging of the sample after the desired reaction has occurred. For example, the flow cell may include a glass or plastic slide carrying a detection channel through which polymerase, dNTPs, and other fluid components can be pumped. The glass or plastic within the channel may be modified with one or more templated nucleic acid molecules to be sequenced. An external imaging system may be placed to detect molecules at a detection region in the detection channel or on a surface in the detection channel. Exemplary flow cells, their fabrication, and methods of use are described in U.S. Patent Application 16 / 141,896 (corresponding U.S. Patent Application Publication No. 2019 / 0055598 A1), U.S. Patent Application Publication No. 2010 / 0111768 A1, or 2012 / 0270305 A1, or WO 05 / 065814, each of which is incorporated herein by reference.

[0031] In the methods or devices of the present disclosure, a flow cell may include a solid support to which one or more target analytes or reagents are attached. A particularly useful solid support is a solid support having a series of sites. As used herein, the term "array" refers to a collection of molecules attached to one or more solid supports such that the molecules at one site can be distinguished from the molecules at other sites. An array may include different molecules, each located at a different addressable site on the solid support. Alternatively, an array may comprise individual solid supports, each serving as a site bearing a different molecule, where the different molecules can be identified based on the position of the solid support on the surface to which it is attached or based on the position of the solid support in a liquid such as a fluid flow. The molecules of the array can be, for example, nucleotides, nucleic acid primers, nucleic acid templates, primed nucleic acid templates, or nucleases such as polymerases, ligases, exonucleases, or combinations thereof.

[0032] As used herein, when referring to an array, the term "site" refers to the position in the array where a particular molecule is present. A site can contain only one molecule or can contain several molecules of the same species (i.e., a collection of those molecules). Alternatively, a site can include a population of molecules of different species (e.g., a population of ternary complexes having different template sequences). The sites of an array are typically discrete. The discrete sites can be contiguous or can have gaps between each other. Arrays useful herein can have sites, for example, that are less than 100 microns, 50 microns, 10 microns, 5 microns, 1 micron, or 0.5 microns. Alternatively or additionally, an array can have sites that are separated by at least 0.5 microns, 1 micron, 5 microns, 10 microns, 50 microns, or 100 microns. The area of the sites can all be less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron, or less.

[0033] As used herein, the term "solid support" refers to a rigid matrix that is insoluble in aqueous liquids. The matrix can be non-porous or porous. The matrix can optionally be capable of absorbing liquids (e.g., due to pores), but will generally have sufficient rigidity such that the matrix does not swell substantially upon absorption of a liquid and does not shrink substantially upon removal of the liquid by drying. Non-porous solid supports are typically impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrenes, and copolymers of styrene with other materials, polypropylenes, polyethylenes, polybutylenes, polyurethanes, polytetrafluoroethylene TM , cycloolefins, polyimides, etc.), nylon, ceramics, resins, Zeonor TM , silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glasses, fiber optic bundles, and polymers.

[0034] Arrays have the advantage of facilitating multiplex detection. For example, different reagents or analytes (such as cells, nucleic acids, proteins, candidate small molecule therapeutic agents, etc.) can be attached to the array by the attachment of each different analyte to a specific site on the array. Exemplary array substrates available include, but are not limited to, BeadChip available from Illumina, Inc. (San Diego, California). TM Arrays such as those described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570 or 7,622,294, or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Other examples of commercially available arrays that can be used include, for example, Affymetrix GeneChip TM arrays. According to some embodiments, dot arrays can also be used. An exemplary dot array is CodeLink available from Amersham Biosciences TM arrays. Another useful array is an array manufactured using an inkjet printing method (such as SurePrint TM technology available from Agilent Technologies).

[0035] Other useful arrays include arrays used in nucleic acid sequencing applications. For example, amplicons (commonly referred to as clusters) comprising ligated genomic fragments or arrays for generating such amplicons may be particularly useful. Examples of arrays and methods of making them that can be modified for use herein include those described in Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO91 / 06678, WO 04 / 018497 or WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,211,414, 7,315,019, 7,329,492 or 7,405,281, or U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0036] The distance between sites of the array is less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm. In certain embodiments, the area of the sites of the array can each be greater than about 100 nm 2 、250 nm 2 、500 nm 2 、1 μm 2 、2.5 μm 2 、5 μm 2 、10 μm 2 、100 μm 2 or 500 μm 2Alternatively or additionally, the area of the sites of the array can each be less than about 1 mm 2 , 500 μm 2 , 100 μm 2 , 25 μm 2 , 10 μm 2 , 5 μm 2 , 1 μm 2 , 500 nm 2 or 100 nm 2 . In fact, the size of the sites can be in the range between an upper limit and a lower limit selected from the above examples. The array can have sites at any of a variety of densities, including for example at least about 10 sites / cm 2 , 100 sites / cm 2 , 500 sites / cm 2 , 1,000 sites / cm 2 , 5,000 sites / cm 2 , 10,000 sites / cm 2 , 50,000 sites / cm 2 , 100,000 sites / cm 2 , 1,000,000 sites / cm 2 , 5,000,000 sites / cm 2 or higher. The devices or methods described herein can be used to detect the array at a resolution sufficient to distinguish sites or site separations at the above densities.

[0037] Although several aspects of the devices and methods of the present disclosure for detecting an analyte attached to a solid support in a flow cell have been exemplified herein, it should be understood that the analyte need not be attached to a solid support and can be detected in solution phase in the flow cell. Additionally, it is not necessary to use a flow cell, or even configure the flow cell for optical detection. Instead, compositions and methods known to those skilled in the art for performing those detection modalities can be used to configure the flow cell for alternative detection modalities.

[0038] Several configurations of the present device and method utilize the analyte in the optical detection flow cell.Therefore, the flow cell may include one or more channels, each of which has at least one transparent window, such as an optically transparent window.In a particular embodiment, the window may be transparent for radiation within a specific spectral range, including but not limited to one or more of X-rays, ultraviolet rays (UV), visible light (VIS), infrared rays (IR), microwaves and radio wave radiation.In some cases, the analyte is attached to the inner surface of the window.Alternately or additionally, one or more windows may provide a view of the internal matrix to which the analyte is attached.Exemplary flow cells and physical features of flow cells that can be used for the method or device set forth herein are described in U.S. Patent Application Publication No. 2010 / 0111768 A1, WO 05 / 065814 or U.S. Patent Application Publication No. 2012 / 0270305 A1, each of which is incorporated herein by reference as a whole.

[0039] The flow cell can be made of a material with a relatively high thermal conductivity, for example, to allow heat to be efficiently transferred between the contents of the flow cell and a heater or cooler external to the flow cell. Thus, the thermal conductivity of the flow cell material can be at least 1 Watt per meter Kelvin (W / (m·K), 10 W / (m·K), 100 W / (m·K), 1000 W / (m·K), or more. Alternatively, the flow cell material can have a relatively low thermal conductivity, for example, to help protect the contents of the flow cell from cooling or heating due to temperature gradients on the walls of the flow cell. Thus, the flow cell material can have a thermal conductivity less than 1 W / (m·K). For example, the thermal conductivity can be at most 1 W / (m·K), 0.1 W / (m·K), 0.01 W / (m·K), or less.

[0040] The flow cell can have one or more detection channels. The detection channel can be closed to the atmosphere (or other surrounding environment), for example, forming a tube or tunnel inside the flow cell structure. The detection channel can have any of a variety of cross-sectional shapes, including, for example, circular, elliptical, triangular, square, rectangular, polyhedral or other closed shapes. The cross-section of the detection channel can be uniform over its entire length. For example, a detection channel with a circular cross-section that is uniform over the entire channel length will have a cylindrical shape, while a detection channel with a circular cross-section that increases or decreases over the length of the channel will have a conical or funnel shape. The cross-sectional area of ​​the detection channel can be at least about 1 μm 2 , 10μm 2 , 100μm 2 , 1mm 2 , 10mm 2 or 100mm 2 Alternatively or additionally, the cross-sectional area of ​​the detection channel may be at most about 100 mm2 、 10 mm 2 、 1 mm 2 、 100 μm 2 、 10 μm 2 、 1 μm 2 or less. The volume of the detection channel in the flow cell can be at least about 1 nL, 10 nL, 100 nL, 1 μL, 10 μL, 100 μL, 1 mL, 10 mL or more. Alternatively or additionally, the volume of the detection channel in the flow cell is at most about 10 mL, 1 mL, 100 μL, 10 μL, 1 μL, 100 nL, 10 nL, 1 nL or less.

[0041] In some configurations, the flow cell is a fixed component of the fluid system, e.g., specialized tools and / or specialized training are required to remove it. Alternatively, the flow cell can be a removable component of the fluid system. For example, the device of the present disclosure can include a stage configured to facilitate the placement and removal of the flow cell. Thus, the flow cell can be a consumable component dedicated to a first analytical test and then removed and replaced with a second flow cell for a second analytical test.

[0042] Multiple analytes can be present in the flow cell. Exemplary analytes include, but are not limited to, the analytes set forth herein or in the references cited herein. Particularly useful analytes are involved in the nucleic acid sequencing process. Thus, the flow cell can contain one or more nucleic acids (e.g., primers, templates, or primer - templates), polymerases, polymerase inhibitors, polymerase co - factors, nucleotides, nucleic acid binding proteins, nucleotide de - blockers, etc. In some configurations, a flow cell is provided that includes a stable ternary complex immobilized inside the flow cell, where the stable ternary complex includes a polymerase, a primed template nucleic acid, and the next correct nucleotide for the template.

[0043] Fluid reagents can be transferred to the flow cell through a fluid system that includes at least one fluid delivery channel. In many configurations, several different fluid reagents will be delivered to the flow cell. Thus, the fluid system can include multiple fluid delivery channels. A single fluid delivery channel can be dedicated to delivering one type of fluid reagent (e.g., from a single reservoir), or a single fluid delivery channel can be configured to deliver more than one different type of fluid reagent (e.g., a single fluid delivery channel can deliver fluids from two or more different reservoirs).

[0044] The fluid delivery channels can be made of any of a variety of materials known in the art of fluidics. Generally, a material that is inert to the reagents, solvents, and other fluid components that will contact the material during transfer is preferably selected. For example, in the context of nucleic acid sequencing or other analytical methods, the material can be inert to the fluid reagents described herein or in the references cited herein.

[0045] In some configurations, the material for the fluid delivery channels is porous enough to allow gas to be transported through the material (e.g., for degassing purposes), but not porous enough to allow liquid to be transported through the material. Alternatively, a material that does not allow gas to pass through can be selected. The material can be selected based on its ability (or inability) to accommodate the passage of a particular gas, such as one or more of oxygen, nitrogen, argon, or air.

[0046] The devices and methods of the present disclosure are particularly applicable to small volume fluid systems, such as microfluidic or mesofluidic systems. In a microfluidic system, the inner diameter or width of the channels can range from about 10 μm to about 1 mm. In a mesofluidic system, the inner diameter or width of the channels can range from just over 1 mm to about 10 cm. If desired, the devices or methods of the present disclosure can be applied to higher volume systems, such as macrofluidic systems.

[0047] In certain configurations, the fluid delivery channels will be heated or cooled. The material for the fluid delivery channels can be selected to have favorable properties under the heating or cooling conditions used. Exemplary properties of interest include, but are not limited to, the chemical stability of the material, the structural integrity of the material, the flexibility of the material, the porosity for degassing, limited expansion, shrinkage within the temperature range experienced, etc. The material can be rigid or flexible to suit a particular configuration.

[0048] The material for one or more fluid delivery channels can also be selected to have high thermal activity to allow heat to be efficiently transferred from the heater to the fluid in the fluid delivery channels. Thus, the thermal conductivity of the flow cell material can be at least 1 watt per meter kelvin (W / (m·K)), 10 W / (m·K), 100 W / (m·K), 1000 W / (m·K), or higher. Alternatively, the flow cell material can have a relatively low thermal conductivity, e.g., to help insulate the contents of the flow cell from cooling or heating due to temperature gradients across the flow cell walls. Thus, the flow cell material can have a thermal conductivity of less than 1 W / (m·K). For example, the thermal conductivity can be at most 1 W / (m·K), 0.1 W / (m·K), 0.01 W / (m·K), or lower.

[0049] Exemplary materials that can be used for the fluid delivery channels include, but are not limited to, silicone, such as Silcon TM, Silbrade TM or Tygon TM ; fluoropolymers such as perfluoroalkoxy (PFA), Teflon TM or polytetrafluoroethylene (PTFE); polyetheretherketone (PEEK); polyetherimide polyethylene; polypropylene; polyurethane; nylon; polyvinyl chloride (PVC); metals such as copper or aluminum; carbon fiber, etc.

[0050] The device of the present disclosure may further include a heater configured to transfer heat to one or more fluid delivery channels. The heater may be positioned to heat at least a portion of the fluid delivery channel that is upstream of the flow cell. In this way, the fluid in the fluid delivery channel will be heated before being delivered to the flow cell. Typically, the fluid will come from a reservoir at a lower temperature. For example, the reservoir may hold the fluid at room temperature (about 25°C) or a cooled temperature (below 25°C, 20°C, 10°C, or 5°C). The fluid delivery channel may be heated to bring the fluid in the channel to a temperature higher than that of the reservoir. For example, the set point of the heater for the fluid delivery channel should be at least 30°C, 40°C, 50°C, 60°C, 70°C, or higher than the temperature of the fluid in the reservoir. The set point of the heater for the fluid delivery channel may be lower than a specific maximum value, such as a maximum of 100°C, 80°C, 70°C, 60°C, 50°C, 40°C, 30°C, or lower. The set point of the heater for the fluid delivery channel may be an intermediate value between the set point of the container and the set point of the flow cell connected to the container through the fluid delivery channel. It will be understood that the reservoir may be subject to the ambient temperature, or the temperature of the reservoir may be controlled by a heater or a cooler. The heater or cooler for the reservoir may have a set point within the values or ranges exemplified herein for the heater of the fluid delivery channel.

[0051] One or more fluid delivery channels can be heated by a thermal conductor such that heat is transferred through physical contact between the heat source and the channel. Alternatively or additionally, a flow cell can be heated by a thermal conductor such that heat is transferred through physical contact between the heat source and the detection channel in the flow cell. The conductive material of the heat source can be a solid, liquid, or gel. Exemplary solid materials from which a thermal conductor can be made include, but are not limited to, graphene, diamond, aluminum, steel, lead, copper, gold, silver, or metal alloys such as aluminum alloy. Exemplary liquid materials that can be made into a thermal conductor include, but are not limited to: liquid metals such as mercury; oils; alcohols such as methanol, glycerol, n-propanol, or n-butanol; glycols such as ethylene glycol; or water. These liquids can also be circulated through the fluid delivery channel to be heated by convection. Exemplary gel materials that can be made into a thermal conductor include, but are not limited to, those composed of epoxy resin, silicone, urethane, acrylate, alumina, boron nitride, zinc oxide, or aluminum nitride. Commercially available thermal greases or thermal gels can also be used.

[0052] The fluid delivery channel to be heated can pass through the conductive material of the heater such that the entire outer perimeter of the portion of the channel passing through the conductive material is in contact with the material. In this way, the fluid delivery channel forms a tunnel through the conductive material of the heater. Alternatively, the conductive material can be positioned to contact less than all sides (or less than the entire outer perimeter) of the channel passing through the conductive material. The solid or gel material used to conduct heat to the fluid delivery channel can be shaped to provide the desired amount of contact with the fluid delivery channel. For example, the solid or gel material can have a groove through which the channel passes, a ridge between which the channel passes, etc.

[0053] Figure 1 An aluminum thermal conductor with twelve grooves is shown. The grooved plate is configured to heat a 12-inch length of a silicone tube having an inner diameter of 1 / 32 inch and an outer diameter of 5 / 32 inch. The shape or size of the grooves can be modified to accommodate channels of different sizes or shapes, including but not limited to the shapes or sizes set forth herein. A single tube can pass through each groove such that approximately 180 degrees of the outer diameter of the tube is in direct contact with the aluminum. The other 180 degrees of the outer diameter of the tube that is not in contact with the metal can be in contact with the surrounding atmosphere. In a similar configuration, the grooves and the tube can be mounted to contact or conductively heat at least 10%, 25%, 50%, 75%, 90%, 99% or more of the outer diameter (or the local perimeter of a non-cylindrical channel) of the channel. Alternatively or additionally, the grooves and the tube can be mounted to contact or conductively heat at most 99%, 90%, 75%, 50%, 25%, 10% or less of the outer diameter (or the local perimeter of a non-cylindrical channel) of the channel. In some configurations, the entire outer surface of the channel is in contact with the heater.

[0054] The grooved plate can be heated by contact with a heating element. For example, it can be heated by four 3-inch by 3-inch 24VDC heating padsFigure 1 A grooved plate. The grooved plate configuration allows for a relatively large contact area for heat conduction while allowing gas to be transmitted through the remaining outer surface of the tube. In this way, the fluid in the tube can be degassed while being heated. Degassing can be facilitated by placing at least a portion of the fluid delivery channel (e.g., a heated portion or a portion downstream of the heat source) in a vacuum jacket or an air flow jacket. The vacuum or gas flow can serve to dissipate the gas leaving through the porous wall of the channel, thereby pulling the gas from the interior of the fluid delivery channel through the porous material. According to the law of mass action, removing the gas from the environment outside the channel can cause the gas to pass through the channel wall.

[0055] One or more fluid delivery channels can be heated by a radiator or a convection heater to transfer heat without direct contact between the heat source and the channels. Alternatively or additionally, a flow cell can be heated by a radiator or a convection heater to transfer heat without physical contact between the heat source and the detection channels in the flow cell.

[0056] Another option for heating the fluid delivery channel is to use a Joule heater instead of a heating pad. The Joule heater can have one or more heating elements in contact with the fluid delivery channel. For example, the element can extend parallel to the fluid delivery channel. The heating element of the Joule heater can be present in the grooved plate, for example, forming part of the base of the plate or part of the ridge defining the groove, or the element can be used in place of the groove.

[0057] The fluid delivery channel and its heater can be isolated to minimize heat loss. For example, the heating block, the heating element, and the fluid delivery channel can be surrounded by an insulating material to prevent excess heat from dissipating from the preheater. This can provide the benefits of reducing power consumption and thermally isolating the preheater from surrounding components.

[0058] One or more fluid delivery channels can be heated by a convection heater so that heat is transferred from the heated fluid (i.e., liquid or gas) moving past the outer wall of the detection channel. The convection heater can transfer heat from the circulating fluid to the outer wall of the fluid delivery channel in contact with the fluid. Similarly, one or more detection channels in a flow cell can be heated by a convection heater so that heat is transferred from the heated fluid (i.e., liquid or gas) moving past the outer wall of the detection channel. The convection heater can transfer heat from the circulating fluid to the outer wall of the detection channel in contact with the fluid. The fluid used for convective heating can be selected from the exemplary liquids for conductive heating above. Any of a variety of gases can be used for convective heating, including, for example, inert gases such as argon, nitrogen, helium, or neon, mixed gases such as air, and other gases.

[0059] One or more fluid delivery channels can be heated by a heat sink so that heat is transferred from a heat source adjacent to the channel. Thermal radiation is particularly useful when heating the fluid delivery channel by a vacuum (e.g., through a vacuum jacket) or by a gas (e.g., through an air flow jacket).

[0060] The heater for the fluid delivery channel can be thermally isolated from one or more other components of the apparatus described herein. For example, the flow cell can be insulated such that the outer surface of the flow cell does not directly receive heat from the heater used to heat the fluid delivery channel. In this configuration, the flow cell may be heated due to the entry of fluid heated by the fluid delivery channel heater, however, heat will not be transferred to the outside of the flow cell unless by an indirect means (e.g., through the fluid inside the flow cell). The heater for the fluid delivery channel can be thermally isolated from other components of the analytical apparatus such as a computer processor, reagent container, detector, electronic device, etc. Thus, the heater and the fluid delivery channel to be heated can be present in a chamber isolated from one or more chambers in which the other components are located. Similarly, the heater for the flow cell can be thermally isolated from one or more other components of the apparatus described herein. For example, one or more fluid delivery channels can be isolated so that they do not directly receive heat from the heater used to heat the flow cell.

[0061] Optionally, the heater that directly heats the fluid delivery channel can be configured to also directly heat another component of the apparatus described herein. For example, the heater can be configured to directly heat the fluid delivery channel and the flow cell. The heater can heat multiple system components by the same mechanism (e.g., conduction, convection, or radiation). Continuing with the previous example, the heater can physically contact the fluid delivery channel and the flow cell to transfer heat to both by conduction. For example, the fluid delivery channel can pass along a first side of a heated block and the flow cell can be placed on a second side of the block, or the flow cell can be placed on the same other side of the block as the fluid delivery channel. Alternatively, two different components can receive heat from the same heater by the same mechanism. For example, the heater can be in direct contact with the fluid delivery channel to transfer heat by conduction and the heater can be in the vicinity of the flow cell to transfer heat by radiation or convection. Thus, when heated, the outer surface (or a portion thereof) of the fluid delivery channel and the outer surface (or a portion thereof) of the flow cell can be present in the same chamber. Conversely, the heater can be in direct contact with the flow cell to transfer heat by conduction and the heater can be near one or more fluid delivery channels to transfer heat by radiation or convection. In some configurations, radiant or conductive heat can be transferred to a valve such as a rotary valve.

[0062] The heater can be configured to heat a defined internal volume of the fluid delivery channel. In the case where fluid remains in the delivery channel, the volume of the heated fluid can be substantially equal to the volume inside the heated channel. The internal volume of the heated fluid delivery channel can be less than, equal to, or greater than the internal volume of the detection channel in the flow cell to which the heated fluid will be delivered. For example, the internal volume of the fluid channel can be at least 10%, 50%, 90%, 100%, or at least 2 times, 3 times, 5 times, 10 times or more larger than the volume of the detection channel. Alternatively or additionally, the internal volume of the fluid channel is at most 10 times, 5 times, 3 times or 2 times larger than the volume of the detection channel, or at most 100%, 90%, 50%, 10% or less smaller than the volume of the detection channel. The heater can be configured to heat an equivalent internal volume of one or more fluid delivery channels. Alternatively, the fluid reagents used in the methods or devices described herein can vary with respect to the volume of fluid to be heated. Thus, the fluid delivery channels can differ in the internal volume to be heated. Optionally, a first subset of the fluid delivery channels can be heated, and a second subset of the fluid delivery channels can be substantially isolated or separated from any or all dedicated heat sources.

[0063] When heated, the fluid does not have to remain in the fluid delivery channel. Instead, the fluid flow rate can be adjusted based on the set point temperature of the heater and other characteristics of the heater and fluid system to achieve the desired temperature of the fluid when it enters the detection channel of the flow cell. Whether the fluid is stationary or flowing when heated, the device can be configured to heat a fluid volume equal to at least 10%, 50%, 90%, 100%, 2 times, 3 times, 5 times, 10 times or more of the volume of the detection channel. Alternatively or additionally, the device can be configured to heat a fluid volume equal to at most 10 times, 5 times, 3 times, 2 times, 100%, 90%, 50%, 10% or less of the volume of the detection channel. The heater can be configured to heat an equivalent volume of one or more fluid delivery channels. Alternatively, the fluid reagents used in the methods or devices described herein can vary with respect to the volume of fluid to be heated. Any one of the results can be achieved by adjusting one or more of the internal volume of the heated channel, the temperature of the heater, the thermal conductivity of the fluid transfer channel, and the flow rate of the fluid flowing through the heated portion of the channel.

[0064] The temperature of the fluid delivery channels can be regulated, for example, by a thermostat. The thermostat can be configured to measure the temperature of the heating component of the heater (e.g., a grooved plate, a solid phase block, or a liquid bath), the surface of one or more fluid delivery channels, or the fluid passing through the heated portion of the fluid delivery channel. The temperature of the fluid can be detected in the heated portion of the fluid delivery channel or at a downstream point of the heated portion. In some configurations, multiple temperature detectors can be used, for example, placed in the heated portion of the channel, at the outlet of the heated portion, and at one or more locations downstream of the heated portion. One or more temperature detectors can be upstream of the heated portion. Thermostats that are particularly useful employ thermocouples, such as type K or type J thermocouples. Exemplary temperature sensors and exemplary arrangements in fluid systems are set forth in Example II below. Such sensors can be used in a thermostat to regulate the temperature of the fluid delivery channel heater.

[0065] Fluid pressure can be regulated in the devices or methods of the present disclosure. A pressure sensor can be configured to detect pressure and provide a feedback loop to regulate the pressure to a desired level. The pressure sensor can be placed in a location similar to that described herein for the temperature sensor. For example, the pressure sensor can be placed downstream or upstream of the fluid delivery channel, downstream or upstream of the fluid delivery channel heater, downstream or upstream of the flow cell, or upstream of the waste reservoir.

[0066] The devices of the present disclosure can include one or more reservoirs. The reservoirs can be open to the atmosphere and can be accessed by lowering a series of pipettes into the reservoirs. The reservoirs can be made of any of a variety of materials, including but not limited to those set forth herein with respect to the fluid delivery channels, flow cells, or arrays. The reservoirs are typically fully enclosed. This can provide the advantage of avoiding contamination. If desired, the reservoirs can be pressurized, for example, to inhibit the degassing of reagents, which would otherwise result in the formation of unwanted bubbles in the downstream fluid components. Pressurization can also provide the advantage of driving the fluid from the reservoir to the flow cell. Screw-cap bottles are particularly useful, but other chambers can also be used. The reservoirs can be permanently fixed to the device, placed separately or removed from a larger device, or combined into a jet cartridge or canister that allows for convenient placement or removal of multiple reservoirs in a batch at one time. The reservoirs can contain the reagents described herein or those described in the references cited herein for sequencing processes or other analytical processes.

[0067] Each reservoir may have a dedicated fluid delivery channel. As described herein, one or more fluid delivery channels may be heated prior to transferring the fluid to the flow cell. Using a fluid delivery channel dedicated to a particular fluid reagent, such as by dedicating to a single container, may provide the following advantages: minimizing detrimental cross-reactions upstream of the flow cell and allowing for personalized control of flow characteristics (such as flow rate, total volume heated, or total volume delivered to the flow cell) or heating characteristics (such as heating rate or final temperature). One or more reservoirs may share a fluid delivery channel that will be heated prior to transferring the fluid to the flow cell. For example, when uniform flow characteristics or heating characteristics are used across several fluid reagents, such a configuration may provide efficient fluid handling.

[0068] Preheating of the reagent may occur in a common fluid delivery channel. The common fluid delivery channel may connect multiple reservoirs via a manifold, for example. In this way, heating of the fluid delivery channel may occur downstream of the manifold. This configuration may be useful when heating can be achieved quickly, in which case the heated portion of the common fluid delivery channel may be selected to be located directly upstream of the flow cell. In an alternative configuration, heating does not occur at the common fluid delivery channel but rather locally at a portion of the fluid system located upstream of the common fluid delivery channel, manifold, or rotary valve.

[0069] Multiple different fluid delivery channels may be combined to enter the detection channel of the flow cell using a manifold. The manifold may be placed downstream of the fluid delivery channel heater and upstream of the detection channel inlet. Fluid flow from one or more fluid delivery lines to the detection channel may be controlled by a valve. In a particular configuration, a rotary valve may serve to select one of the multiple fluid delivery channels to direct fluid flow to the detection channel. Other valves that may be used include, for example, ball valves, diaphragm valves, throttle valves, butterfly valves, pinch valves, solenoid valves, etc.

[0070] Optionally, the valve or manifold may be heated. The heater may be of the type described herein with respect to heating the fluid delivery channel or the flow cell. The set point of the valve or manifold heater may be equal to, higher than, or lower than the set point of the upstream fluid delivery channel heater. The set point of the valve or manifold heater may be equal to, higher than, or lower than the set point of the downstream flow cell heater. The set point of the valve or manifold heater may be between the set point of the upstream fluid delivery channel heater and the set point of the downstream flow cell heater. In this way, the three heaters may establish a temperature gradient that increases or decreases with the flow direction. For one or more steps of a particular reaction or analysis process, the configuration may be selected to achieve the desired final temperature in the flow cell.

[0071] The device of the present disclosure may include a pump configured to drive fluid through a fluid delivery channel, flow cell, or other component. An injection pump may be particularly useful. In addition to injection pumps, other types of devices may also be used to drive fluid, including, for example, positive or negative pressure, peristaltic pumps, diaphragm pumps, piston pumps, gear pumps, or Archimedes screws. The pump may be configured to apply a positive fluid displacement (e.g., by positive pressure) to push fluid from a reservoir, fluid delivery channel, or other fluid component into the flow cell. Alternatively, the pump may be configured to apply a negative fluid displacement (e.g., by negative pressure) to pull fluid from the flow cell into other fluid components, such as a waste liquid container or a storage location for reagent recovery. Particularly useful pumps include, for example, those used in the sequencing platforms described herein or in the references cited herein.

[0072] The pump may be located upstream of the reservoir and the flow cell, between the reservoir and the flow cell, or downstream of the reservoir and the flow cell. When the pump is upstream of the reservoir and the flow cell, the pump will push fluid from the reservoir towards the flow cell. If the pump is located between the reservoir and the flow cell, the pump will draw liquid from the reservoir and push the fluid towards the flow cell. If the pump is downstream of the reservoir and the flow cell, the pump will draw fluid from the reservoir and flow it into the flow cell.

[0073] The device of the present disclosure may include a stage configured to support the flow cell. The stage may be configured to position the flow cell relative to the fluid components of the device and / or the detector components of the device. Optionally, the stage may be configured to move the flow cell, for example, to allow observation of the contents of the flow cell by scanning. The stage may be movable in one or more of the x, y, and z directions in a Cartesian coordinate system. For the purposes of the present disclosure, the z-axis will be the axis along which focus changes are achieved (i.e., the axis extending along the distance between the detector and the surface of the flow cell), the x-axis will be the scanning direction of the flow cell, and the y-axis will be orthogonal to the z and x axes. In addition to linear movement along the x, y, and z axes, the stage is also capable of rotating the flow cell. Rotation about the z-axis will be referred to as yaw, rotation about the x-axis will be referred to as roll, and rotation about the y-axis will be referred to as pitch. These dimensions and movements are shown in Figure 1 U.S. Patent Application 16 / 141,896, which is published as U.S. Patent Application Publication No. US 2019 / 0055598 A1 and is incorporated herein by reference. Any stage may be used, such as a stage common to a laboratory microscope or a nucleic acid sequencing platform. Examples of useful stages include those incorporated in the following references in the context of the detection device of a nucleic acid sequencer.

[0074] A particularly useful stage is configured, for example, to slide a flow cell along a reference surface using the apparatus and method disclosed in U.S. Patent Application 16 / 141,896 (the corresponding U.S. Patent Publication No. is US 2019 / 0055598 A1), which is incorporated herein by reference. Such apparatus and method can provide the benefit of avoiding the use of high-precision actuators that are adjustable in various translational and rotational directions. High-precision actuators increase the cost and complexity of the scanner, and such devices generally require trained technicians for daily maintenance. The flow cell stage proposed in U.S. Patent Application 16 / 141,896 avoids such problems by decoupling the mechanism for translating the flow cell relative to the detector from the mechanism for rotationally registering the flow cell relative to the detector. Decoupling translation from rotational registration reduces the tolerance stack of the translation mechanism in the detection apparatus and other apparatuses of the present disclosure.

[0075] Another advantage of using a flow cell stage in U.S. Patent Application 16 / 141,896 (the corresponding U.S. Patent Publication No. is US 2019 / 0055598 A1) is that the flow cell can be scanned more quickly. The increase in scanning speed depends largely on the function of the flow cell translation device, which is configured to move a mass smaller than that of a typical stage. A smaller mass takes less time to settle down compared to a larger mass moving the same distance. For example, as the required detection resolution increases, the time to wait for the flow cell to settle down before acquiring an image becomes increasingly important, because the movement of the flow cell must decay to such an extent that the average displacement experienced by the features of the object being observed is small enough to prevent substantial distortion of the image.

[0076] The flow cell stage may include a reference surface, a preload, and a scanning actuator. The preload is configured to urge the flow cell to contact the reference surface during a detection event. The reference surface forms a fixed structural loop with the detector. The scanning actuator is configured to slide the flow cell along the reference surface in a scanning dimension. Thus, the detection apparatus used in the apparatus of the present disclosure may include: (a) a flow cell having an inner cavity and walls, the walls having an inner surface and an outer surface, the inner surface contacting the inner cavity; (b) a reference surface forming a fixed structural loop with the detector; (c) a preload configured to urge the outer surface of the flow cell to contact an area on the reference surface; (d) a scanning actuator configured to slide the flow cell along the reference surface in a scanning dimension; (e) a transmitter configured to direct a signal from the inner surface or the inner cavity to the detector when the outer surface of the flow cell is urged by the preload to contact the reference surface.

[0077] As provided herein, a detection device may include: (a) a flow cell having a lumen and walls, the walls having an inner surface and an outer surface, the inner surface contacting the lumen, and the outer surface having a length l in a scan dimension x; (b) a reference surface; (c) a preload configured to urge the outer surface of the flow cell into contact with a region on the reference surface, optionally, a maximum length of the contact region in the scan dimension x may be shorter than the length l; (d) a scan actuator configured to slide the flow cell along the reference surface in the scan dimension x; (e) a detector; (f) an objective lens configured to direct radiation from the flow cell to the detector when the outer surface of the flow cell is urged into contact with the reference surface by the preload.

[0078] The flow cell may be scanned using the following steps: (a) translating the flow cell along a reference surface of a detection device, wherein the flow cell has a cavity and walls, the cavity including an analyte, and during translation, the reference surface contacts at least a portion of the flow cell, and the reference surface forms a fixed structural loop with the detector; and (b) detecting the analyte at different positions along the flow cell using the detector, wherein during detection, the flow cell is urged against the reference surface by the preload, thereby scanning the flow cell.

[0079] The device of the present disclosure may include a heater configured to heat a flow cell supported by a stage. The heater may be separate and independent from a heater for heating a fluid delivery channel. Alternatively, and as described above, the same heater may be used to heat the flow cell and the fluid delivery channel. The flow cell heater may be selected from the same types used herein to heat fluid delivery channels, valves, or manifolds. The flow cell heater may be an integral part of the flow cell stage or a separate device that transfers heat to the flow cell when placed on the stage.

[0080] Optionally, a heating element may be integrated into the flow cell. For example, the flow cell may contain one or more thermal channels through which a heated fluid phase flows. Alternatively or additionally, the flow cell may contain a solid-phase heating element, such as a wire, coil, or filament. Optionally, the heating element may be located in an upstream region of the flow cell, e.g., adjacent to the entrance of a detection channel. In another option, heating elements may be placed along the length of one or more detection channels.

[0081] For one or more fluid delivery channels, the set point of the flow cell heater may be lower than, equal to, or higher than the set point of the heater. For example, the set point of the flow cell heater may have a set point of at least about 30°C, 40°C, 50°C, 60°C, 70°C, or higher. The set point of the flow cell heater may be lower than a specific maximum value, e.g., up to 100°C, 80°C, 70°C, 60°C, 50°C, 40°C, 30°C, or lower.

[0082] Relatively speaking, the set point of the fluid delivery channel heater can be higher than the set point of the flow cell heater. For example, it can be at least about 5°C, 10°C, 20°C, 30°C, or 50°C or higher. Alternatively or additionally, the set point for the fluid delivery channel heater can be at most about 50°C, 30°C, 20°C, 10°C, or 5°C higher than the set point for the flow cell heater.

[0083] Conversely, the set point of the flow cell heater can be higher than the set point of the fluid delivery channel heater. For example, it can be at least about 5°C, 10°C, 20°C, 30°C, or 50°C or higher. Alternatively or additionally, the set point of the flow cell heater can be at most about 50°C, 30°C, 20°C, 10°C, or 5°C higher than the set point of the fluid delivery channel heater.

[0084] Thus, the temperature of the fluid delivery channel can be the same as or different from the temperature of the flow cell. Optionally, the temperature of the fluid delivery channel is the same as the temperature of the flow cell. The temperature of the fluid delivery channel can be higher than or lower than the temperature of the flow cell. Additionally, the temperature of the fluid delivery channel and / or the flow cell can be higher than the temperature of the reservoir.

[0085] The temperature of the flow cell can be regulated, for example, by a thermostat. The thermostat can be configured to measure the temperature of the heating component of the heater (such as a heated stage), the surface of one or more regions of the flow cell (such as the regions through which one or more detection channels pass or across), or the fluid passing through the heated portion of the flow cell. The temperature of the fluid can be detected in the heated portion of the detection channel or at a point downstream of the heated portion. In some configurations, multiple temperature detectors can be used. For example, they can be placed in the heated portion of the flow cell, at the outlet of the heated portion, and at one or more positions downstream of the heated portion. Thermostats that use thermocouples, such as type K or type J thermocouples, are particularly useful. Exemplary temperature sensors and exemplary arrangements in the fluid system are set forth in Example II below. Such sensors can be used in the thermostat to regulate the temperature of the flow cell heater.

[0086] The devices described herein can employ components used in optical subsystems or nucleic acid sequencing systems to detect analytes in the flow cell. Several such detection devices are configured for optical detection, such as the detection of fluorescence signals. Examples of detection devices and their components that can be used here to detect the flow cell are described, for example, in U.S. Patent Application 16 / 141,896; U.S. Patent Application Publication No. 2010 / 0111768A1, or U.S. Patents 7,329,860, 8,951,781, or 9,193,996, each of which is incorporated herein by reference. Other detection devices include detection equipment commercially used for nucleic acid sequencing, such as those manufactured by Illumina TM , Inc. (such as HiSeq TM , MiSeqTM , NextSeq TM or NovaSeq TM system), Life Technologies TM (e.g., ABI PRISM TM or SOLiD TM system), Pacific Biosciences (e.g., a system using SMRT TM technology, such as Sequel TM or RS II TM system) or Qiagen (e.g., Genereader TM system). Other useful detectors are described in U.S. Patent Nos. 5,888,737, 6,175,002, 5,695,934, 6,140,489, or 5,863,722; or U.S. Patent Application Publication Nos. 2007 / 007991 A1, 2009 / 0247414 A1, or 2010 / 0111768; or WO2007 / 123744, each of which is hereby incorporated by reference in its entirety.

[0087] Particularly useful optical detection systems will use an objective lens having a numerical aperture of at least 0.1 and at most 0.9. Immersion objective lenses can be used to achieve a numerical aperture above 0.95, which will be described in further detail below. The objective lens can be configured to work with a detection system that can resolve features (e.g., nucleic acid sites) on a surface that are less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm apart. A detection system including the objective lens can be configured to resolve features on a surface having an area of less than about 1 mm 2 , 500 μm 2 , 100 μm 2 , 25 μm 2 , 10 μm 2 , 5 μm 2 , 1 μm 2 , 500 nm 2 or 100 nm 2 .

[0088] The optical system used in the devices or methods described herein can have a field of view of at least 0.1 mm 2 , 0.5 mm 2 , 1 mm 2 , 2 mm 2 , 3 mm 2 , 4 mm 2 or higher. Alternatively and / or additionally, the field of view can be configured to be at most 4 mm 2 , 3 mm 2 , 2 mm2 , 1 mm 2 , 0.5 mm 2 , 0.1 mm 2 or less.

[0089] In the methods or apparatuses described herein, the detector used to observe the flow cell need not be capable of optical detection. For example, the detector can be an electronic detector for detecting protons or pyrophosphate (e.g., see U.S. Patent Application Publication Nos. 2009 / 0026082 A1, 2009 / 0127589 A1, 2010 / 0137143 A1, or 2010 / 0282617 A1, each of which is incorporated herein by reference in its entirety, or the Ion Torrent commercially available from ThermoFisher of Waltham, Massachusetts TM systems), or the detector can be an electronic detector for detecting nanopores (e.g., Oxford Nanopore TM , Oxford, UK (such as the MinION TM or PromethION TM systems) commercialized nanopores), or those described in U.S. Patent 7,001,792; Soni & Meller, Clin. Chem. 53, 1996 - 2001 (2007); Healy, Nanomed. 2, 459 - 481 (2007); or Cockroft et al., J. Am. Chem. Soc. 130, 818 - 820 (2008), etc., each of which is incorporated herein by reference.

[0090] The control of system components (such as heaters, thermostats, pumps, or detectors) can be implemented using a general - purpose processor, a digital signal processor (DSP), an application - specific integrated circuit (ASIC), a field - programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic, discrete hardware components, or any combination thereof, designed to perform the functions described herein. The general - purpose processor can be a microprocessor, but alternatively, the processor can be any conventional processor, controller, microcontroller, or state machine. The processor can also be implemented as a combination of computing devices, e.g., a DSP and a microprocessor, multiple microprocessors, a combination of one or more microprocessors with a DSP core, or any other such configuration.

[0091] Optionally, the apparatus of the present disclosure may include a computer processing unit (CPU) configured to operate one or more system components described herein. The same or different CPUs may interact with the system to acquire, store, and process signals (e.g., signals detected by the methods described herein). In certain embodiments, the CPU may be used to determine the identity of a nucleotide present at a specific position in a template nucleic acid based on the signal. In some cases, the CPU will identify the nucleotide sequence of the template from the detected signals.

[0092] Useful CPUs may include, for example, personal computer systems, server computer systems, thin clients, fat clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics, network PCs, minicomputer systems, mainframe computer systems, smartphones, or one or more in a distributed cloud computing environment including any of the above systems or devices. The CPU may include one or more processors or processing units and may have a memory architecture including RAM and non-volatile memory. The memory architecture may further include removable / non-removable, volatile / non-volatile computer system storage media. Additionally, the memory architecture may include one or more readers for reading and writing to non-removable non-volatile magnetic media such as hard disk drives; disk drives for reading and writing to removable non-volatile disks; and / or optical disk drives for reading or writing from removable non-volatile optical disks such as CD-ROMs or DVD-ROMs. The CPU may also include various computer system readable media. Such media may be any available media accessible in a cloud computing environment, such as volatile and non-volatile media as well as removable and non-removable media.

[0093] The memory architecture of a processor used herein may include at least one program product having at least one program module implemented as executable instructions configured to control one or more components of the apparatus described herein or perform one or more parts of the methods described herein. For example, the executable instructions may include an operating system, one or more applications, other program modules, and program data. Generally, program modules may include routines, programs, objects, components, logic, data structures, etc., that perform specific tasks, such as controlling hardware to perform the steps described herein or processing signals detected by the apparatus or methods described herein.

[0094] Components of a computer processor can be coupled by an internal bus, which can be implemented as any one or more of several types of bus structures, including a memory bus or memory controller, a peripheral bus, an Accelerated Graphics Port, and a processor or local bus using any one of a variety of bus architectures. By way of example and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and Peripheral Component Interconnect (PCI) bus.

[0095] The CPU can optionally communicate with one or more external devices, such as a keyboard, a pointing device (e.g., a mouse), a display, such as a graphical user interface (GUI), or other devices that assist a user in interacting with the apparatus described herein. Similarly, the CPU can communicate with other devices (e.g., via a network card, a modem, etc.). Such communication can be carried out via an I / O interface. Additionally, the CPU of the system herein can communicate with one or more networks via a suitable network adapter, e.g., a local area network (LAN), a general wide area network (WAN), and / or a public network (e.g., the Internet).

[0096] Optionally, a proportional-integral-derivative (PID) controller can be used to regulate temperature, fluid flow rate, pressure, speed, and other process variables. The PID controller can be configured to use a control loop feedback mechanism to control the process variable. By way of a more specific example, the PID controller can be used to control a feedback loop that includes a temperature sensor or a pressure sensor used in the apparatus or method herein.

[0097] A bubble sensor can also be used, for example, to mitigate unwanted bubbles. An example of a bubble sensor configuration is to place the bubble sensor upstream of a rotary valve, and when bubbles are detected or when a threshold level of bubbles is reached, the valve can be activated to divert the bubbles to a flow cell or other container where the bubbles may have an adverse effect. In response to detecting bubbles, the fluid system can also respond by increasing the pressure on the fluid, decreasing the flow rate of the fluid, decreasing the temperature of the fluid, or taking other mitigation steps to remove or avoid bubbles.

[0098] However, not all bubbles are unwanted. In fact, when bubbles are desired, the apparatus provided herein can include a bubble sensor and a bubble generator. The bubble sensor can be used to detect and remove bubbles other than those with the desired bubble characteristics. Suitable bubbles for the provided apparatus, exemplary bubble generators, other hardware, and methods of sequencing nucleic acids using bubbles and performing other analytical procedures are set forth in U.S. Patent Application 16 / 700,422, which is incorporated herein by reference. For example, in a particular configuration, the bubble generator can be housed within the instrument connector 1110 (Figure 14A and 14B )。

[0099] The present disclosure provides a method for nucleic acid sequencing. The method includes the following steps: (a) providing an analysis device having a flow cell, a fluid system, and a detection system, wherein the flow cell contains an analyte array, such as nucleic acid, in a detection channel, the fluid system includes a fluid delivery channel that fluidly connects a reservoir to the detection channel of the flow cell, and the detection system observes signals from the analyte array; (b) transferring a liquid reagent from the reservoir to a heating region of the fluid delivery channel, whereby the liquid reagent is heated; (c) contacting the heated liquid reagent with the analyte array by transferring the heated liquid reagent to the detection channel; (d) detecting signals from the analyte array by the detection system. The sequencing reagent can be a fluid sequencing reagent, such as a liquid sequencing reagent.

[0100] The present disclosure provides methods and devices that are particularly useful for performing cyclic reactions. Each cycle can include delivering reagents for the reaction to a flow cell, where optionally a reaction or reaction product will be observed. Each cycle can further include heating or cooling a fluid reagent or a component of the fluid system using the devices or methods described herein. The methods and devices of the present disclosure are exemplified in the context of performing nucleic acid sequencing reactions. However, those skilled in the art will understand from the teachings herein how to modify the methods and devices for other cyclic reactions, such as nucleic acid synthesis reactions, peptide sequencing reactions, peptide synthesis reactions, combinatorial small molecule synthesis reactions, etc. However, the method need not be cyclic, but can be performed in a non-repetitive manner to observe a single reaction or phenomenon, such as real-time polymerase chain reaction (rtPCR), quantitative PCR (qPCR), binding assays, such as detecting an epitope using a labeled antibody, enzyme assays, nucleic acid hybridization assays, array-based genotyping, and RNA expression assays, etc.

[0101] Particularly useful sequencing reactions are Sequencing By Binding TM (SBB TM ) reactions, such as those described in commonly owned U.S. Patent Application Publication Nos. US 2017 / 0022553 A1, 2018 / 0044727 A1, 2018 / 0187245 A1, or 2018 / 0208983 A1, each of which is incorporated herein by reference. Generally, methods for determining the sequence of a template nucleic acid molecule can be based on the formation of a ternary complex (between a polymerase, a primed nucleic acid, and a cognate nucleotide) under specified conditions. The method can include a detection phase, followed by a nucleotide incorporation phase.

[0102] The detection phase can be carried out in a flow cell containing at least one primer-initiated template nucleic acid molecule. The reaction mixture can include the initiated template nucleic acid, a polymerase, and at least one nucleotide type. The interaction of the polymerase and the nucleotide with the initiated template nucleic acid molecule can be observed under conditions where the nucleotide is not covalently added to the primer. Using the observed interaction of the polymerase and the nucleotide with the initiated template nucleic acid molecule, the next base in each template nucleic acid can be identified. The interaction between the primer-template, polymerase, and nucleotide can be detected by a variety of schemes. For example, the nucleotide can contain a detectable label. Each nucleotide can have a distinguishable label relative to other nucleotides. Alternatively, some or all of the different nucleotide types can have the same label, and the nucleotide types can be distinguished based on the separate delivery of the different nucleotide types to the flow cell. In some embodiments, the polymerase can be labeled. The polymerase associated with different nucleotide types can have unique labels to distinguish the nucleotide types associated with them. Alternatively, the polymerase can have similar labels, and the different nucleotide types can be distinguished based on the separate delivery of the different nucleotide types to the flow cell. Detection can be carried out by scanning the flow cell using the apparatus or method set forth herein.

[0103] During the detection phase, correct and incorrect nucleotides can be distinguished by ternary complex stabilization. A variety of conditions and reagents are useful. For example, the primer can contain a reversible blocking moiety that prevents covalent attachment of the nucleotide; and / or a cofactor required for extension (e.g., a divalent metal ion) may be absent; and / or inhibitory divalent cations that inhibit polymerase-based primer extension can be present; and / or the polymerase present during the detection phase can have a chemical modification and / or a mutation that inhibits primer extension; and / or the nucleotide can have a chemical modification that inhibits incorporation, such as a 5’ modification that removes or alters the native triphosphate moiety.

[0104] The extension phase can then be carried out by creating conditions in the flow cell in which nucleotides can be added to the primers on each template nucleic acid molecule. In some embodiments, this involves removing the reagents used in the detection phase and replacing them with reagents that facilitate extension. For example, the detection reagents can be replaced with a polymerase and nucleotides capable of extension. Alternatively, one or more reagents can be added to the detection phase reaction to create extension conditions. For example, catalytic divalent cations can be added to a cation-deficient detection mixture, and / or polymerase inhibitors can be removed or disabled, and / or extendable nucleotides can be added, and / or a deblocking agent can be added to make the primer highly extendable, and / or an extendable polymerase can be added.

[0105] It should be understood that the apparatus and method of the present disclosure can be used to carry out any of a variety of nucleic acid sequencing reactions. Other exemplary sequencing methods are set forth below.

[0106] Sequencing-by-synthesis (SBS) techniques can be used. SBS generally involves iteratively increasing the enzymatic extension of a nascent primer by iterative addition of nucleotides to a template strand hybridized to the primer. Briefly, SBS can be initiated by contacting a target nucleic acid attached to a site in a flow cell with one or more labeled nucleotides, DNA polymerase, etc. Those sites that use the target nucleic acid as a template to extend the primer will incorporate a detectable labeled nucleotide. Detection can include scanning using the devices or methods set forth herein. Optionally, the labeled nucleotides can also include reversible termination characteristics that terminate further primer extension once the nucleotide has been added to the primer. For example, a nucleotide analogue having a reversible terminator moiety can be added to the primer such that subsequent extension does not occur until a deblocking agent is delivered to remove the moiety. Thus, for embodiments using reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection). Washing can be performed between different delivery steps. This cycle can be performed n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, WO 91 / 06678, WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019 or 7,405,281, and U.S. Patent Publication No. 2008 / 0108082 A1 describe exemplary SBS procedures, reagents, and detection components that can be readily adapted to be used with the methods or devices of the present disclosure, each of which is incorporated herein by reference. SBS methods commercially available from Illumina, Inc. (San Diego, CA) are also useful.

[0107] Some SBS embodiments include detecting protons released after incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use reagents and electrical detectors commercially available from ThermoFisher (Waltham, MA) or described in U.S. Patent Application Publication Nos. 2009 / 0026082 A1, 2009 / 0127589 A1, 2010 / 0137143 A1, or 2010 / 0282617 A1, each of which is incorporated herein by reference.

[0108] Other sequencing procedures can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when nucleotides are incorporated into a nascent primer hybridized to a template nucleic acid strand. (Ronaghi et al., Analytical Biochemistry 242(1), 84-9 (1996), Ronaghi, Genome Res. 11(1), 3-11 (2001), Ronaghi et al., Science 281(5375), 363 (1998), U.S. Patents 6,210,891, 6,258,568 and 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, the released PPi can be detected by converting it to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected by photons generated by luciferase. Thus, the sequencing reaction can be monitored by a luminescence detection system configured to scan a flow cell using the devices and methods set forth herein.

[0109] Sequencing-by-ligation reactions can also be used, including those described, for example, in Shendure et al. Science 309:1728-1732 (2005), U.S. Patents 5,599,675 and 5,750,341, each of which is incorporated herein by reference. Some embodiments may include sequencing-by-hybridization procedures, such as those described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988), Drmanac et al., Nature Biotechnology 16, 54-58 (1998), Fodor et al., Science 251(4995), 767-773 (1995) or WO 1989 / 10977, each of which is incorporated herein by reference. During sequencing-by-ligation and sequencing-by-hybridization, primers hybridized to a nucleic acid template undergo repeated extension cycles by oligonucleotide ligation. Typically, the oligonucleotides are fluorescently labeled and can be detected to determine the sequence of the template, for example, using the devices or methods set forth herein.

[0110] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase with a fluorophore and γ-phosphate labeled nucleotides or by interactions with zero-mode waveguides (ZMWs). Techniques and reagents for detecting fluorescence resonance energy transfer (FRET) and / or detecting zero-mode waveguides (ZMWs) for sequencing can be modified for use in the devices or methods described herein, such as those described in Levene et al., Science 299, 682-686 (2003), Lundquist et al., Opt. Lett. 33, 1026-1028 (2008), Korlach et al., Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), or U.S. Pat. Nos. 7,315,019, 8,252,911, or 8,530,164, the disclosures of each of which are incorporated herein by reference.

[0111] The steps of the sequencing method described above can be cycled. For example, the checking and extension steps of the SBB TM method can be repeated so that the next correct nucleotide is checked in each cycle (i.e., the next correct nucleotide is the nucleotide that binds correctly to the nucleotide in the template nucleic acid that is 5' to the base in the template that hybridizes to the 3' end of the hybridization primer), and subsequently, the next correct nucleotide is added to the primer. Any number of cycles of the sequencing method described herein can be performed, including, for example, at least 1, 2, 5, 10, 20, 25, 30, 40, 50, 75, 100, 150, or more cycles. Alternatively or additionally, no more than 150, 100, 75, 50, 40, 30, 25, 20, 10, 5, 2, or 1 cycle can be performed.

[0112] The nucleic acid template to be sequenced can be added to the flow cell using any of a variety of known methods. In some embodiments, a single nucleic acid molecule is to be sequenced. The nucleic acid molecule can be delivered to the flow cell and can optionally be attached to a surface in the flow cell or other container. Optionally, single molecule sequencing of the molecule is performed. Alternatively, multiple copies of the nucleic acid can be prepared, and the resulting population can be sequenced. For example, the nucleic acid can be amplified on a surface (e.g., the inner wall of the flow cell) using techniques further elaborated below.

[0113] Multiple different nucleic acid molecules (i.e., a population having multiple different sequences) can be sequenced. Optionally, the molecules can be attached to a surface in a flow cell or other container. The nucleic acids can be attached at unique sites on the surface, and individual nucleic acid molecules that can be spatially distinguished from each other can be sequenced in parallel. Alternatively, the nucleic acids can be amplified on the surface to produce multiple surface-attached clusters. The clusters can be spatially distinguished from one another and can be sorted in parallel.

[0114] Any of a variety of amplification techniques can be used in the methods described herein in a flow cell. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), bridge amplification, or random primer amplification (RPA). In certain embodiments, one or more primers for amplification can be attached to a surface in a flow cell. In such embodiments, extension of the surface-attached primers along the template nucleic acid will result in copies of the template being attached to the surface. A method of generating one or more sites on a solid support, where each site is linked to multiple copies of a particular nucleic acid template, can be referred to as a "clustering" method.

[0115] In a PCR embodiment, one or both primers for amplification can be attached to the surface. The use of two ligated primers is commonly referred to as bridge amplification because the double-stranded amplicon forms a bridge-like structure between two ligated primers located on either side of the replicated template sequence. For example, exemplary reagents and conditions for bridge amplification are described in U.S. Patent Nos. 5,641,658 or 7,115,400, U.S. Patent Publications 2002 / 0055100A1, 2004 / 0096853 A1, 2004 / 0002090 A1, 2007 / 0128624 A1, or 2008 / 0009420 A1, each of which is incorporated herein by reference. PCR amplification can also be performed with one of the amplification primers attached to the surface and the second primer present in solution. An exemplary form of using a combination of a solid-phase attached primer and a solution-phase primer is referred to as primer walking and can be performed as described in U.S. Patent No. 9,476,080, which is incorporated herein by reference. Another example is emulsion PCR, which can be performed as described in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145, or U.S. Patent Publications 2005 / 0130173 A1, 2005 / 0064460 A1, each of which is incorporated herein by reference.

[0116] The RCA technique can be used in the methods described herein. Exemplary reagents for RCA reactions and the principles by which RCA generates amplicons are described, for example, in Lizardi et al., Nat. Genet. 19:225-232 (1998) or U.S. Patent Publication 2007 / 0099208 A1, each of which is incorporated herein by reference. Primers for RCA can be in solution or attached to the surface of a flow cell.

[0117] The MDA technique can also be used in the methods of the present disclosure. For example, Dean et al., Proc Natl. Acad. Sci. USA 99:5261-66 (2002), Lage et al., Genome Research 13:294-307 (2003), Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, Walker et al., Nucl. Acids Res. 20:1691-96 (1992) or U.S. Patents 5,455,166, 5,130,238 or 6,214,587 describe some of the reagents and useful conditions for MDA, each of which is incorporated herein by reference. Primers for MDA can be in solution or attached to the surface of a flow cell.

[0118] The nucleic acid templates used in the methods or compositions herein can be DNA, such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), etc. RNA can also be used, such as mRNA, ribosomal RNA, tRNA, etc. Nucleic acid analogs can also be used as templates herein. Thus, mixtures of nucleic acids used herein can be derived from biological sources, synthetic sources, or amplification products. The primers used herein can be DNA, RNA, or analogs thereof.

[0119] Exemplary organisms from which nucleic acids can be derived include, for example, those from: mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates, humans or non-human primates; plants such as Arabidopsis thaliana, maize, sorghum, oats, wheat, rice, rapeseed or soybean; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila, mosquitoes, Drosophila melanogaster, bees or spiders; fish such as zebrafish; reptiles; amphibians such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi such as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharomyces cerevisiae or Schizosaccharomyces pombe; or Plasmodium falciparum. Nucleic acids can also be derived from prokaryotes such as bacteria, Escherichia coli, Staphylococcus or Mycoplasma pneumoniae; archaea; viruses such as hepatitis C virus or human immunodeficiency virus; or viroids. Nucleic acids can be from a homogeneous culture or population of the above organisms, or from an assemblage of several different organisms, for example in a community or ecosystem. Nucleic acids can be isolated using methods known in the art, including, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference. Cells, tissues, biological fluids, proteins and other samples can be obtained from these organisms and assayed using the devices or methods described herein.

[0120] Template nucleic acids can be obtained from preparation methods such as genomic isolation, genomic fragmentation, gene cloning, and / or amplification. The template can be obtained by amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), etc. Exemplary methods for isolating, amplifying, and fragmenting nucleic acids to produce a template for array analysis are set forth in U.S. Patent Nos. 6,355,431 or 9,045,796, each of which is incorporated herein by reference. Amplification can also be performed using the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.

[0121] The devices or methods of the present disclosure can employ a polymerase. The polymerase can be stored in a reservoir, flow through a fluid delivery channel where it can optionally be heated or cooled, and be provided to a flow cell for nucleic acid sequencing reactions or other analytical processes. Any of a variety of polymerases can be used in the methods described herein. Unless otherwise specified, references to a particular polymerase, such as those exemplified throughout the present disclosure, should be understood to include its functional variants. Particularly useful functions of the polymerase include using an existing nucleic acid as a template, forming a ternary complex, or catalyzing the polymerization of a nucleic acid strand. Polymerases can be classified based on structural homology, for example, classifying polymerases into families identified as A, B, C, D, X, Y, and RT. DNA polymerases in the A family include, for example, T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase γ, Escherichia coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus stearothermophilus Pol I. DNA polymerases in the B family include, for example, eukaryotic DNA polymerases α, δ, and ε, DNA polymerase ζ, T4 DNA polymerase, Phi29 DNA polymerase, and RB69 phage DNA polymerase. The C family includes, for example, the α subunit of Escherichia coli DNA polymerase III. Archaeal DNA polymerases in the B family include, for example, Vent, Deep Vent, Pfu, and 9°N (e.g., Therminator from New England BioLabs Inc., Ipswich, MA) TMDNA polymerase). The D family includes, for example, polymerases derived from the Euryarchaeota subdomain of archaea. DNA polymerases of the X family include, for example, eukaryotic polymerases Polβ, Polσ, Polλ, and Polμ, and Saccharomyces cerevisiae Pol 4. DNA polymerases of the Y family include, for example, Polη, Polι, Polκ, Escherichia coli Pol IV (DINB), and Escherichia coli Pol V (UmuD'2C). The RT (reverse transcriptase) family of DNA polymerases includes, for example, retroviral reverse transcriptases and eukaryotic telomerases. Exemplary RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase; eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and archaeal RNA polymerases.

[0122] Other examples of useful DNA polymerases include bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and phage DNA polymerases. Bacterial DNA polymerases include Escherichia coli DNA polymerases I, II, and III, IV, and V, the Klenow fragment of Escherichia coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase, and Sulfolobus solfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and and Revl polymerase (terminal deoxynucleotidyl transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerase, PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNA polymerases include thermostable and / or thermophilic DNA polymerases such as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Pyrococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilum DNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicus DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase D.Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase; Thermococcus celer DNA polymerase; Aeropyrum pernix DNA polymerase; and heterodimeric DNA polymerase DP1 / DP2. Engineered and modified polymerases can also be used in the disclosed techniques. For example, a modified version of the Therminator TM DNA polymerase from the hyperthermophilic marine archaeon Thermococcus 9°N (e.g., from New England BioLabs Inc.; Ipswich, MA) can be used.

[0123] Useful RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kll polymerase; eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and archaeal RNA polymerases.

[0124] Another useful polymerase is reverse transcriptase. Exemplary reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastosis virus, and telomerase reverse transcriptase that maintains eukaryotic chromosome telomeres.

[0125] Polymerases with inherent 3'-5' proofreading exonuclease activity can be used in certain applications of the methods and systems described herein. Polymerases substantially lacking 3'-5' proofreading exonuclease activity can also be used in certain configurations, e.g., in most sequencing systems and methods. The lack of exonuclease activity can be a wild-type characteristic or a characteristic conferred by variant or engineered polymerase structures. For example, the exo minus Klenow fragment is a mutant version of the Klenow fragment that lacks 3'-5' proofreading exonuclease activity. The Klenow fragment and its exo variants can be used in the methods or compositions described herein.

[0126] Polymerases that can be used in the methods or compositions described herein include naturally occurring polymerases and their modified variants, including but not limited to mutants, recombinants, fusions, genetically modified, chemically modified, synthetic, and analogs. Useful polymerases for ternary complex formation and detection are not limited to polymerases capable of catalyzing polymerization reactions. Optionally, useful polymerases will have the ability to catalyze polymerization reactions under at least one condition not used in the formation or examination of stable ternary complexes. Exemplary polymerases that can be used to form stable ternary complexes include, for example, the wild-type and mutant polymerases listed in U.S. Patent Application 2017 / 0314072 A1 or 2018 / 0155698 A1, each of which is incorporated herein by reference.

[0127] Polymerases that can be used to detect polymerases and contain exogenous labeled moieties (e.g., exogenous luminophores) can be useful in some embodiments. Optionally, the exogenous labeled moiety can be chemically linked to the polymerase, for example, using a free sulfhydryl or free amine moiety of the polymerase. The exogenous labeled moiety can also be linked to the polymerase by protein fusion. Exemplary labeled moieties that can be linked by protein fusion include, for example, green fluorescent protein (GFP), phycobiliproteins (e.g., phycocyanin and phycoerythrin), or wavelength-shifted variants of GFP or phycobiliproteins.

[0128] The devices or methods of the present disclosure can employ nucleotides. The nucleotides can be stored in a reservoir, flow through a fluid delivery channel, where they can optionally be heated or cooled, and be provided to a flow cell for nucleic acid sequencing reactions or other analytical processes. As needed, the nucleotides can be natural nucleotides, nucleotide analogs, or modified nucleotides to suit the particular application or configuration of the method. Such nucleotides can be present in ternary complexes or used in the sequencing methods described herein.

[0129] Optionally, the nucleotide analog has a nitrogenous base, a pentose sugar, and a phosphate group, where any part of the nucleotide can be modified, removed, and / or replaced compared to a natural nucleotide. The nucleotide analog can be a non-incorporable nucleotide (i.e., a nucleotide that cannot react with the 3'-oxygen of a primer to form a covalent bond). Such non-incorporable nucleotides include, for example, monophosphates and diphosphates. In another example, the nucleotide can contain a modification to the triphosphate group that renders the nucleotide non-incorporable. Examples of non-incorporable nucleotides can be found in U.S. Patent 7,482,120, which is incorporated herein by reference. In some embodiments, the non-incorporable nucleotides can subsequently be modified to become incorporable. Non-incorporable nucleotide analogs include, but are not limited to, α-phosphate modified nucleotides, α-β nucleotide analogs, β-phosphate modified nucleotides, β-γ nucleotide analogs, γ-phosphate modified nucleotides, or caged nucleotides. Other examples of nucleotide analogs are described in U.S. Patent 8,071,755, which is incorporated herein by reference.

[0130] The nucleotide analogs used in the methods or systems herein can include terminators that reversibly block the incorporation of subsequent nucleotides at the 3'-end of a primer. For example, U.S. Patent 7,544,794 and U.S. Patent 8,034,923 (the disclosures of these patents are incorporated herein by reference) describe reversible terminators where the 3'-OH group is 3'-ONH 2Partial substitution. Another type of reversible terminator is attached to the nitrogenous base of the nucleotide, for example, as described in U.S. Patent 8,808,989 (the disclosure of which is incorporated herein by reference). Other reversible terminators that can be similarly used in combination with the methods described herein include those described in the references cited elsewhere herein or in U.S. Patents 7,956,171, 8,071,755, and 9,399,798 (the disclosures of these documents are incorporated herein by reference). For example, an -O-azidomethyl moiety is particularly useful as a reversible terminator when present at the 3'-position of the nucleotide. In certain embodiments, the terminator moiety of the reversible process can be removed from the primer in a process referred to as "deblocking," thereby allowing subsequent nucleotide incorporation. Compositions and methods for deblocking are set forth in the references cited herein in the context of reversible terminators.

[0131] Alternatively, nucleotide analogs irreversibly block nucleotide incorporation at the 3'-end of a primer into which they have been incorporated. Irreversible nucleotide analogs include 2',3'-dideoxynucleotides (ddNTPs, such as ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs, which otherwise would participate in polymerase-mediated primer extension. Thus, the 3'-position has a hydrogen moiety instead of the native hydroxyl moiety. Nucleotides with irreversible termination can be particularly useful in genotyping applications or other applications where primer extension or sequential detection along the template nucleic acid is not required.

[0132] Nucleotide analogs used herein, for example, to participate in a stable ternary complex, need not include a protecting group (such as a reversible terminator) that prevents subsequent nucleotide incorporation at the 3'-end of the primer after the analog has been incorporated into the primer. Whether to use a nucleotide with a protecting group (such as a reversible terminator) for the extension step can be the case. It should be understood that, if desired, a protecting group can be present on the nucleotide analogs that participate in a stable ternary complex.

[0133] Nucleotides used herein, for example, to participate in forming a stable ternary complex can include exogenous labels. Exogenous-labeled nucleotides can include a reversible or irreversible terminator moiety, exogenous-labeled nucleotides can be non-incorporable, exogenous-labeled nucleotides can lack a terminator moiety, exogenous-labeled nucleotides can be incorporable, or exogenous-labeled nucleotides can be incorporable and non-terminated. Exogenous-labeled nucleotides can be particularly useful when used to form a stable ternary complex with an unlabeled polymerase.

[0134] Alternatively, the nucleotides used herein for, e.g., participating in the formation of a ternary complex may lack an exogenous label (i.e., the nucleotide may be "unlabeled"). Unlabeled nucleotides may include reversible or irreversible terminator moieties, unlabeled nucleotides may be non-incorporable, unlabeled nucleotides may lack a terminator moiety, non-labeled nucleotides may be incorporable, or non-labeled nucleotides may be incorporable and non-terminating. Unlabeled nucleotides may be useful when detecting a stable ternary complex using a label on the polymerase or when detecting without a label. Unlabeled nucleotides may also be used in the extension step of the methods described herein. It will be understood that the absence of a moiety or function in a nucleotide refers to a nucleotide that does not have such a function or moiety. However, it will also be understood that one or more functions or moieties described herein for nucleotides or their analogs may be specifically omitted in the methods or compositions set forth herein, or in other ways known in the art for nucleotides or their analogs.

[0135] Optionally, nucleotides (e.g., natural nucleotides or nucleotide analogs) are present in the mixture during or after the formation of the stable ternary complex. For example, there may be at least 1, 2, 3, 4, or more nucleotide types. Alternatively or additionally, there may be at most 4, 3, 2, or 1 nucleotide types. Similarly, one or more of the nucleotide types present may be complementary to at least 1, 2, 3, or 4 base types in the template nucleic acid. Alternatively or additionally, one or more of the nucleotide types present may be complementary to at most 4, 3, 2, or 1 base types in the template nucleic acid. Different base types can be identified by the presence of different exogenous labels on different nucleotides. Alternatively, two or more nucleotide types may have indistinguishable exogenous labels. However, in the latter format, different nucleotides can be distinguished, for example, as described in U.S. Patent Application 2018 / 0305749 A1 or U.S. Patent 9,951,385, either due to being delivered separately to the reaction vessel or due to the proposed encoding and decoding schemes, each of which is incorporated herein by reference.

[0136] The systems and methods of the present disclosure may employ detectable labels on reactants or products to be detected. In many cases, the label is an exogenous label added to a reactant or product (such as a polymerase, nucleic acid, or nucleotide). Examples of useful exogenous labels include, but are not limited to, radioactive label moieties, luminescent moieties, fluorophore moieties, quantum dot moieties, chromophore moieties, enzyme moieties, electromagnetically spin-labeled moieties, nanoparticle light-scattering moieties, and any of a variety of other signal-generating moieties known in the art. Suitable enzyme moieties include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Exemplary fluorophore labels include, but are not limited to, p-methylaminophenol; resorufin; coumarin; xanthene; acridine; fluorescein; rhodamine; gibberellin; anthocyanin; phthalaldehyde; naphthylamine; fluoroamine; benzodiazole; stilbene glycoside; pyrene; indole; boron azadipyrromethene; quinazolinone; eosin; erythrosin; malachite green; CY dyes (GE Biosciences), including Cy3 (and its derivatives), Cy5 (and its derivatives), and Cy7 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics), including DY-547, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY-777, and DY-782; fluorescein yellow; cascade blue; Texas red; BODIPY (boron dipyrromethene) (Molecular Probes) dyes, including BODIPY 630 / 650 and BODIPY 650 / 670; ATTO dyes (Atto-Tec), including ATTO 390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610, ATTO 635; ALEXA fluorophores including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) or any of its derivatives) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes (LiCor), including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester), and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec);MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIROAKS Red (Molecular Devices); SUNNYVALE Red (Molecular Devices); LIGHT CYCLER Red (Roche); EPOCH (Glen Research) dyes, including EPOCH REDMOND Red, EPOCH YAKIMA Yellow, EPOCH GIGHARBOR Green; Tokyo Green (M. Kamiya, et al., 2005 Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes, including CF647 and CF555 (Biotium), and other fluorophore labels known in the art, such as described in Joseph R. Lakowicz (ed.), Plenum Pub Corp, 2nd ed. (July 1999) "Principles of Fluorescence Spectroscopy" and the sixth edition of the "Molecular Probes Handbook" by Richard P. Hoagland.;

[0137] The label can be linked to a nucleotide, polymerase, or other molecule via a linker. The linker present in the nucleotide or polymerase can be, but need not be, cleavable. For example, the linker can be stable to the conditions used in the methods described herein such that the covalent structure of the linker does not change during any particular step or throughout the steps of the methods described herein.

[0138] The reactant or product may lack an exogenous label. For example, a stable ternary complex and all components involved in the stable ternary complex (e.g., polymerase, template nucleic acid, primer, and / or cognate nucleotide) can lack one, several, or all of the exogenous labels described herein or in the references cited and incorporated herein. In such embodiments, the ternary complex can be detected based on the intrinsic properties of the stable ternary complex, such as mass, charge, intrinsic optical properties, etc. Exemplary methods for detecting an unlabeled ternary complex are set forth in co-owned U.S. Patent Application 2017 / 0022553 A1, PCT Application No. PCT / US16 / 68916, or U.S. Patent Application 62 / 375,379 or 15 / 677,870, each of which is incorporated herein by reference.

[0139] The device or method of the present disclosure may further include a cooler for absorbing heat from the fluid delivery channel or flow cell. In addition to the heaters described herein, one or more coolers may be present (or used). For example, the cooler may be used in combination with the heater to achieve temperature regulation in both directions. The cooler may be used to lower the temperature of the fluid in the delivery channel, detection channel, rotary valve, or other fluid components, and the heater may be used to increase the temperature of such fluid components. Some configurations may use a heat transfer device, such as a Peltier device, that can act as both a heat source and a heat sink. Alternatively, one or more coolers may be present (or used) such that one or more of the heaters described herein are replaced by coolers. The cooler may be placed in the device where the heater is used as an example, but may be modified to accommodate different temperature effects.

[0140] Accordingly, the present disclosure provides a device for performing an analytical process such as determining a nucleic acid sequence. The device may include (a) a stage configured to support a flow cell; (b) a detector configured to observe a detection channel of the flow cell when the flow cell is supported by the stage; (c) a plurality of fluid delivery channels, wherein each fluid delivery channel fluidly connects a reservoir to the detection channel of the flow cell; and (d) a first cooler configured to cool the plurality of fluid delivery channels. Optionally, the stage includes a second cooler configured to cool the flow cell supported by the stage. Optionally, the device may include a thermostat configured to detect the temperature of the fluid delivery channels among the plurality of fluid delivery channels and selectively activate a first heater and a first cooler. As a further option, the device may include a second cooler configured to cool the flow cell supported by the stage. The device may further include a thermostat configured to detect the temperature of the flow cell and selectively activate a second heater and a second cooler.

[0141] The present disclosure further provides a method for performing an analytical procedure such as determining a nucleic acid sequence. The method includes the steps of: (a) providing an analytical device having a flow cell, a fluid system, and a detection system, wherein the flow cell contains an analyte array, such as nucleic acid, in a detection channel, the fluid system includes fluid delivery channels that fluidly connect reservoirs to the detection channel of the flow cell, and the detection system observes signals from the analyte array; (b) transferring a liquid reagent from a reservoir to a cooling region of the fluid delivery channel, whereby the liquid reagent is cooled; (c) contacting the frozen liquid reagent with the analyte array by transferring the frozen liquid reagent to the detection channel; (d) detecting signals from the analyte array by the detection system. In a nucleic acid sequencing configuration, the liquid reagent is a reagent for sequencing nucleic acid.

[0142] Methods and apparatus for preheating fluid reagents can be used in combination with methods and apparatus for delivering a mixed-phase fluid (such as a fluid foam, fluid slurry, or fluid emulsion) to a flow cell, as described, for example, in U.S. Patent Application 16 / 700,422, which claims priority to 62 / 774,998, each of which is incorporated herein by reference. The apparatus or method described herein can be used to modify a mixed-phase fluid apparatus or method such as those described in U.S. Patent Application 62 / 774,998 to heat the mixed-phase fluid prior to delivering it to the flow cell. Conversely, the apparatus or method set forth in U.S. Patent Application 16 / 700,422 or 62 / 774,998 can be used to modify the heating apparatus or method presented herein to produce a mixed-phase fluid.

[0143] Example 1

[0144] Thermal Model for a Nucleic Acid Sequencing System

[0145] This example provides a thermal model that shows the advantages of preheating fluid reagents that can be used in a Sequencing By Binding TM (SBBTM) system. The SBBTM system is configured for optically detecting fluorescently labeled ternary complexes formed on a series of primer nucleic acids in a glass flow cell. The ternary complex consists of a polymerase, one of the primed template nucleic acids, and the next correct nucleotide of the primed template nucleic acid.

[0146] Figure 2 A block diagram of an exemplary sequencing system 100 is shown. Sequencing reagents are stored at room temperature or cooled in a kit 160. Fluid reagents from each reservoir in the package are delivered to a manifold 122 through fluid delivery channels 131 that flow through a preheater 110. Manifold fluid delivery channels 132 deliver the preheated fluid to a rotary valve 120 that is heated by a heater 121. The rotary valve 120 is used to select a reagent from the manifold fluid delivery channels 132 for delivery through a common fluid delivery channel 133 to a flow cell 140. The common fluid delivery channel 133 is fluidly connected to a detection channel 143 through an inlet 141, and fluid exits the detection channel 143 through an outlet 142. An injection pump 150, which typically includes a selector valve, pumps fluid from the kit 160 through the flow cell 140 and through channels 134 and 135 into a waste reservoir 170.

[0147] A 1-layer thermal model was constructed. The 1-layer model assumes that the flow cell starts at a set temperature and that the fluid (water) has not yet started to be heated by the flow cell. The model assumes that the flow cell has an infinite heat capacity and provides an understanding of the effect of the temperature difference between the fluid and the injection into the flow cell. The relevant properties of the materials used in the model are shown in Table 1. The properties calculated based on these are shown in Table 2. The volume of water calculated to have the equivalent heat mass to the flow cell surface is 95 μl.

[0148] Table 1

[0149]

[0150] Table 2

[0151]

[0152] Figure 3 A graph showing the relationship between the water temperature and position in the flow cell based on the 1-layer model is shown. The results of the model show how long it takes for water to flow through the flow cell, assuming that the flow cell reaches temperature during flow and that the flow cell is not cooled by cold reagents. It is clear from the graph of the 1-layer model that the array in the flow cell will experience a temperature gradient in the first 2 cm of the flow cell. This can have an adverse effect on temperature-sensitive reactions.

[0153] Figure 4 Graphs showing the relationship between the water temperature and position in the flow cell at various time points during and after starting water flow in the flow cell based on the 1-layer model are shown. Water is a good substitute for sequencing reagents, which are typically provided in aqueous liquids. According to the 1-layer model, water reaches equilibrium with the flow cell temperature in approximately 0.2 seconds. The 1-layer model is accurate over a shorter time scale (e.g., early in the process directly after thorough incubation).

[0154] A three-layer thermal model was used to evaluate heat transfer through glass by water in the steady state during flow. Figure 5 A graph showing the relationship between the water temperature and position in the flow cell based on the 3-layer model is shown. The model assumes that flow and heat distribution are in a steady state and then shows the theoretical heat recovery after flow stops. By neglecting the heat capacity of the glass, the heat recovery curve is a better case than considering the thermal energy required to heat the glass when heating the fluid. It is clear from the graph of the 3-layer model that the array in the flow cell will experience a temperature gradient in the first 6 cm of the flow cell.

[0155] Figure 6Shows graphs of the relationship between water temperature and position in the flow cell at various time points during and after initiating water flow based on a 3 - layer model. The water did not fully equilibrate to the temperature of the flow cell within the 0.4 - second time period evaluated under the 3 - layer model. The 3 - layer model is well - suited for viewing flows that would deplete any heat in the glass during incubation. However, this model does not include heat conduction through the side of the flow cell opposite the proximal end of the heater.

[0156] The results of this model show that the inflow of room - temperature aqueous reagent into the flow cell will cause significant cooling of the array in the flow cell (for a flow cell maintained at 60 °C, the cooling level is about 10 °C). The results further show that it may take a certain amount of time (about tens of seconds) to re - heat the array in the flow cell. The results indicate that pre - heating the aqueous reagent will help improve the sequencing results. This pre - heating can also reduce the time and / or volume of the aqueous reagent required to achieve uniform results across the entire array and over time.

[0157] Example II

[0158] Thermal Testing of the Fluid Delivery Components of a Nucleic Acid Sequencing System

[0159] This example provides empirical testing to determine the thermal properties of a fluid delivery system used in a nucleic acid sequencing platform.

[0160] The testing was conducted on Figure 7 the test device 200 shown. The test device 200 includes four fluid lines 231 to 234 that pass through a pre - heater 210 and then connect to a rotary valve 220. The rotary valve 220 connects the four fluid lines 231 to 234 to a common line 230, which in turn connects to a flow cell (not shown). Thermocouples are used to measure the temperature at four positions: T1 detects the middle position of the pre - heater 210, T2 detects the position where the channel 231 exits the pre - heater 210, T3 detects the common line 230 approximately 2 cm from the connection to the rotary valve 220, and T4 detects the common line 230 approximately 10 cm from the connection to the rotary valve 220. Type - K thermocouples were used, but they can be replaced with Type - J thermocouples.

[0161] The set points for the pre - heater 210 and the rotary valve 220 were 60 °C. Before entering the device, the water temperature was 20 - 22 °C and the positive pressure was 15 PSI. All channels were made of silicone tubing with an inner diameter of 0.031 inches and an outer diameter of 5 / 32 inches (inner diameter 0.787 mm / outer diameter 3.968 mm). Measurements were taken from each thermocouple every 1 second.

[0162] The tabular results obtained from the test device 200 are shown in Figure 8A and the same results are in Figure 8BShown in. The data was collected from a single fluid delivery channel of the test device 200. The results show the effect of the rotary valve downstream of the preheater in the steady state and its effect on the T3 and T4 temperatures. The results show that setting the preheater to a temperature of 60 °C allows the fluid reagent to be delivered to the flow cell at a temperature of approximately 44 °C. The data shows that the culture medium in the preheater (which allows the fluid to reach the temperature) is offset by the heat transfer to the surrounding environment in the fluid delivery channel.

[0163] The test device is used to detect the temperature at different positions downstream of the valve. The test equipment was modified to relocate the thermocouples, as Figure 9 shown. Specifically, T1 is placed to detect the common pipeline 230 at the position leaving the heated rotary valve 220, T2 is placed at the connector of the flow cell inlet 24, T3 is placed at the inlet of the flow cell 240, and T4 is placed at the outlet of the flow cell 240, near the common pipeline 250, leaving the flow cell. The test device includes 12 fluid delivery channels, all sucking distilled water at 20 - 22 °C and flowing through 160 μL of liquid at the flow rates shown on the x-axis for each pulse.

[0164] The tabular results obtained from the modified test device are shown in Figure 10A and the same results are plotted in Figure 10B Using the fluid flowing through the twelve fluid transfer channels of the test device 200 to collect data. The results assume that each reagent reaches the temperature in the preheater after 1 minute (according to the results of Figure 8, which shows that the reagent will be heated to 60 °C in the preheater after approximately 20 seconds). The results show the difference in set points (60 °C and 70 °C), and show that when operating at a fast flow rate, the heat loss is reduced, which may be because less heat is lost to the ambient air in the pipeline. The fluid delivery channel between the preheater and the flow cell can be insulated to further reduce heat loss.

[0165] Example III

[0166] Nucleic acid sequencing device

[0167] This example description provides a device for nucleic acid sequencing, the device comprising: (a) a stage in contact with a flow cell, wherein the flow cell includes at least one detection channel, wherein the detection channel houses a nucleic acid array; (b) a detector configured to observe the nucleic acid array in the detection channel; (c) a plurality of reservoirs storing reagents for sequencing the nucleic acid array; (d) a plurality of fluid delivery channels, wherein the fluid delivery channels fluidly connect the plurality of reservoirs to the detection channel of the flow cell; (e) a first heater that transfers heat to the plurality of fluid delivery channels; and (f) a second heater that transfers heat to the detection channel of the flow cell.

[0168] Some components and functions of the nucleic acid sequencing system are further elaborated below. It should be understood that the system is exemplary. For example, as described elsewhere herein, one or more of the components described below may be omitted or replaced with other components. Other components described herein may be added to the exemplary system without necessarily replacing the components of the examples below.

[0169] Figure 11 A perspective view of an assembly of several components of the nucleic acid sequencing device 1000 is shown. The device 1000 is supported by a base 1009 and a frame 1010. The top middle region of the frame 1010 is configured for user access to the system to place the flow cell 1200 on the heating stage 1950. The stage 1950 provides a conductive surface for transferring heat from the heating element to the flow cell 1200. The stage includes a reference surface that is in direct contact with an area of the flow cell surface, thereby conducting heat to the area of the flow cell in contact with the heated reference surface. Other areas of the flow cell are not in direct contact with the stage but are heated by heat transferred from the stage surface through an air gap to the flow cell surface. The flow cell 1200 is pressed onto the stage 1950 by a preload 1951. In this way, the flow cell 1200 is positioned to be detected by the optical detector 1900. The flow cell 1200 is translated along the stage 1950 by a translation member 1955 and fixed to the reference surface on the stage 1950 to allow detection of an array of nucleic acids (or other assay samples) within the flow cell 1200. The components and operations of the preload 1951, the scanning system 1955, and the optical detector 1900 are set forth in U.S. Patent Application Publication No. 2019 / 0055596 A1, which is incorporated herein by reference. The system also includes a vent tube 1920 and a fan 1921 for exhausting heat generated by the optical detector.

[0170] In Figure 11 's view, the fluid connection between the routing manifold 1500 and the flow cell 1200 has been removed and is shown in FIG. 14. When the reservoir is engaged by the pipette array 1300, Figure 11 Multiple reservoirs 1400 are shown. Drawers 1402 and 1403 are also shown, which allow the user to replace the liquid in the reservoirs. The reservoirs can be filled with reagents for the nucleic acid sequencing process. In the context of nucleic acid sequencing, exemplary reagents include, but are not limited to, polymerase, nucleotides, and other reagents listed elsewhere herein or in the references cited herein. Optionally, the nucleotides and / or polymerase can be labeled, for example, with a luminescent exogenous label, such as those described herein or in the references cited herein. Also visible in Figure 11 are conductive heaters 1202 and 1203, which are positioned to heat the pipette manifold as described below.

[0171] Figure 11 Also shown is a nucleic acid sequencing device 1000, which also includes peristaltic pumps 1050 and 1051 configured to apply a fluid displacement force (e.g., positive pressure, positive displacement, etc.) at a location in a fluid delivery component between a plurality of reservoirs 1400 and an instrument connector Figure 14A and 14B ) to convey fluid from the fluid delivery component to the flow cell 1200. In the exemplary device 1000, the pump can be configured to apply a fluid displacement force to the fluid delivery component at a location between the rotary valve and the instrument connector component to convey fluid from the fluid delivery component to the flow cell.

[0172] Figure 12 A top view of a routing manifold 1500 fluidly connected to pipette manifolds 1302 and 1303 is shown. The manifold is composed of polyetherimide . Pipettes are attached to the pipette manifolds such that liquid aspirated by the pipettes from the reservoirs is transferred through dedicated channels in the pipette manifolds to the routing manifold 1500. For example, pipette manifold 1303 includes pipette attachment points 1320, 1321, and 1323, which are fluidly connected within pipette manifold 1303 to fluid lines 1324, 1325, and 1326, respectively. Fluid lines 1324, 1325, and 1326 are heated by a conductive heater 1203. Fluid lines 1324, 1325, and 1326 are connected via fluid line 1521 to corresponding lines in routing manifold 1500 to deliver reagents to a rotary valve 1560 (see Figure 13 ). Fluid line 1521 is heated by a conductive heater 1204. Thus, the fluid aspirated from the reservoirs via pipette attachment points 1320, 1321, and 1323 is heated by conductive heaters 1203 and 1204 in the path to the rotary valve 1560. Conductive heaters 1202, 1203, and 1204 are polyimide heaters with silicone insulators (also known as Kapton heaters).

[0173] Pipette manifold 1303 also includes pipette attachment points 1310, 1311, and 1313, which are fluidly connected within pipette manifold 1303 to fluid lines 1314, 1315, and 1316, respectively. Fluid lines 1314, 1315, and 1316 are not in direct contact with the conductive heater. Fluid lines 1314, 1315, and 1316 are connected via a connector 1511 to corresponding lines in routing manifold 1500 to deliver reagents to the rotary valve 1560. Fluid line 1511 is heated by a conductive heater 1204. The fluid lines are channels drilled in an Ultem TM block. In this way, most of the block is heated and the walls of the channels transfer heat to the fluid therein. Figure 12Also shown is a connector 1510 on the routing manifold 1500 that is connected to a corresponding connector on the pipette manifold 1302 such that fluid aspirated through the pipette can be directed to the rotary valve 1550. All pipette connection points and fluid lines in the pipette manifold 1302 are heated by contact with the conductive heater 1202.

[0174] The rotary valves 1550 and 1560 are visible in Figure 13 which shows a bottom view of the routing manifold 1500. Also visible in Figure 13 are the pipette arrays 1300 and 1301 which include pipettes attached to the pipette manifold 1302 and the pipette manifold 1303 respectively. Figure 12 Also shown is an outlet port 1178 that connects the effluent of the rotary valve 1560 ( Figure 13 ) to the instrument connector 1110, a fluid inlet port 1176 connects the outlet of the flow cell 1200 to a waste tank, and an optional gas outlet port 1177 delivers gas from a compressed air source to the instrument connector 1110 ( Figure 14A and 14B ). Similar fluid ports exist for the rotary valve 1550. When a bubble generator is used to introduce bubbles from a reservoir into a liquid reagent, the gas outlet port 1177 can be utilized. Compared to non-foamy liquids, the generated foam can be delivered to the flow cell to provide various advantages such as reducing the consumption of reagent per unit of flow cell and more rapidly exchanging different reagents. Exemplary bubble generators, other hardware, and methods for sequencing nucleic acids and performing other analytical procedures using foam are described in U.S. Patent Application 16 / 700,422, which is incorporated herein by reference. For example, in a particular configuration, the bubble generator can be housed within the instrument connector 1110 ( Figure 14A and 14B ).

[0175] Figure 14A Shows a perspective view of the fluid connection between the nucleic acid sequencing system 1000 ( Figure 11 ) and the flow cell 1200. Figure 14B Shows the same perspective view, but the connectors are disconnected and slightly displaced. The instrument connector 1110 engages with the instrument connection port 1172. The instrument connection port 1172 includes a fluid inlet 1176 ( Figure 14B ), a liquid outlet 1178 ( Figure 14B ), and an optional gas outlet 1177 ( Figure 14B)。The instrument connector 1110 is fluidly connected to the flow cell connector 1180 through flexible tubes 1191 and 1192. The flow cell connector 1180 engages with the flow cell port 1190. The flow cell connector 1180 provides a fluid inlet for the first detection channel in the flow cell 1200 and a fluid outlet for the second detection channel in the flow cell 1200. The instrument connector 1110 is configured to fluidly connect the flexible tube 1191 to the channel inlet of the flow cell and connect the flexible tube 1192 to the channel outlet of the flow cell. The instrument connector 1110 can be manually engaged with the instrument connection port 1172 because the connector 1110 has a compressible hook 1120 suitable for a complementary latch ( Figure 14B )。Similarly, the flow cell connector 1180 can be manually engaged with the flow cell connection port 1190 because the connector 1180 has a compressible hook suitable for a complementary latch. Note that a second connection can be made from the instrument connection port 1141 to the flow cell port 1140 using a connector similar to that illustrated for the instrument connection port 1172 to the flow cell port 1190. The second connection can include a fluid inlet for the second detection channel in the flow cell 1200 and a fluid outlet for the first detection channel in the flow cell 1200. In this way, the fluid will flow through the two detection channels of the flow cell 1200 in opposite directions.

[0176] Figure 14A and 14B Peristaltic pumps 1050 and 1051 are also shown. Figure 14A and Figure 14B Peristaltic pump 1050 is shown, which has a rotor that contacts the flexible tube 1193 to apply positive pressure upstream of the instrument connector 1110. The flexible tube 1193 passes through the hole 1130 ( Figure 14A ) of the instrument connector 1110, then over the rotor of the peristaltic pump 1050, and then into the hole 1131 ( Figure 14A ) of the instrument connector 1110. A preload 1951 is also shown, which pushes the flow cell 1200 to contact the reference surface on the stage 1950. When the flow cell 1200 is pushed onto the stage 1950, the flow cell is aligned with the detection optics 1900 ( Figure 11 ), and the flow cell is heated by heat transfer through the surface of the stage 1950.

[0177] Figure 15 A front view of the sequencing device 1000 is shown and includes a hatched arrow indicating the direction of hot air circulation. Hot air from the heating element is transferred through the convection outlet 1210 and circulated into the inlet 1211. The hot convection heats the rotary valves 1560 and 1550 and exits through 1212. Then the heat is circulated into the inlet 1213. The hot convection provides an alternative way to heat the fluid components as an alternative to Figures 11 to 13An alternative or supplement to the conduction heater shown. Figure 15 Also shown are peristaltic pumps 1050 and 1051, flow cell 1200, stage 1950, preloading 1951, scanning system 1955, and exhaust pipe 1920.

[0178] As shown in this example, a nucleic acid sequencing device may include three heating spaces. The first space is a fluid delivery channel heated by conduction and / or convection. The second space is an air space surrounding various fluid components (such as connectors, fluid delivery channels, and flow cells). The third space is a flow cell stage heated by direct contact with a reference surface on the stage and in proximity to other surfaces on the stage.

[0179] Example 4

[0180] Nucleic acid sequencing device, optional configurations

[0181] The nucleic acid sequencing device may be configured as described in the following numbered configurations. This example provides:

[0182] Configuration 1. A device for nucleic acid sequencing, comprising: (a) a stage in contact with a flow cell, wherein the flow cell includes at least one detection channel, and the detection channel is for accommodating a nucleic acid array; (b) a detector configured to observe the nucleic acid array in the detection channel; (c) a plurality of reservoirs storing reagents for sequencing the nucleic acid array; (d) a plurality of fluid delivery channels fluidly connecting the plurality of reservoirs to the detection channel of the flow cell; (e) a first heater that transfers heat to the plurality of fluid delivery channels; and (f) a second heater that transfers heat to the detection channel of the flow cell.

[0183] Configuration 2. The device according to Configuration 1, optionally, wherein the first heater includes a heat conductor in contact with the plurality of fluid delivery channels.

[0184] Configuration 3. The device according to Configuration 2, optionally, wherein the heat conductor of the first heater includes a surface having a plurality of grooves, and each fluid delivery channel includes a tube passing through one of the plurality of grooves.

[0185] Configuration 4. The device according to Configuration 2, optionally, wherein the heat conductor of the first heater includes a solid phase block, and the fluid delivery channel passes through the solid phase block.

[0186] Configuration 5. The device according to Configuration 2, optionally, wherein the heat conductor of the first heater includes a liquid bath, and each fluid delivery channel includes a tube passing through the liquid bath.

[0187] Configuration 6. The apparatus as described in any one of the foregoing configurations, optionally, wherein the temperature of the first heater is set higher than the temperature of the second heater.

[0188] Configuration 7. The apparatus as described in Configuration 6, optionally, wherein the set point of the first heater is higher than the temperature of the plurality of reservoirs.

[0189] Configuration 8. The apparatus as described in Configuration 7, optionally, wherein the set point of the second heater is higher than the temperature of the plurality of reservoirs.

[0190] Configuration 9. The apparatus as described in any one of the foregoing configurations, optionally, wherein the second heater includes a heat conductor in contact with the stage.

[0191] Configuration 10. The apparatus as described in any one of the foregoing configurations, optionally, wherein the second heater includes a heat conductor in contact with the flow cell.

[0192] Configuration 11. The apparatus as described in any one of the foregoing configurations, optionally, wherein the first heater transfers heat to a volume of each fluid delivery channel that is at least equal to the volume of the detection channel.

[0193] Configuration 12. The apparatus as described in any one of the foregoing configurations, optionally, further comprising a convection heater that transfers heat to the fluid delivery channels.

[0194] Configuration 13. The apparatus as described in Configuration 12, optionally, wherein the temperature of the convection heater is set higher than the temperature of the second heater.

[0195] Configuration 14. The apparatus as described in Configuration 12, optionally, wherein the temperature of the convection heater is set higher than the temperature of the plurality of reservoirs.

[0196] Configuration 15. The apparatus as described in any one of the foregoing configurations, optionally, wherein the volume of the detection channel is at most 10 ml.

[0197] Configuration 16. The apparatus as described in Configuration 15, optionally, wherein the volume of the detection channel is at most 1 ml.

[0198] Configuration 17. The apparatus as described in any one of the foregoing configurations, optionally, further comprising a thermostat configured to detect the temperature of a fluid delivery channel among the plurality of fluid delivery channels and selectively activate the first heater.

[0199] Configuration 18. The apparatus as described in any one of the foregoing configurations, optionally, further comprising a thermostat configured to detect the temperature of the flow cell or the detection channel of the flow cell and selectively activate the second heater.

[0200] Configuration 19. The device according to any one of the foregoing configurations, optionally, wherein the detector comprises an optical detector.

[0201] Configuration 20. The device according to Configuration 19, optionally, wherein the detection channel comprises an optically transparent window for displaying the nucleic acid array.

[0202] Configuration 21. The device according to any one of the foregoing configurations, optionally, further comprising a valve configured to control the flow of reagents through a fluid delivery channel among a plurality of fluid delivery channels.

[0203] Configuration 22. The device according to Configuration 21, optionally, wherein the valve comprises a rotary valve.

[0204] Configuration 23. The device according to Configuration 21 or 22, optionally, wherein the valve is located upstream of the flow cell and downstream of the first heater.

[0205] Configuration 24. The device according to Configuration 23, optionally, further comprising a heater configured to heat the valve.

[0206] Configuration 25. The device according to Configuration 23, optionally, wherein the first heater, the second heater or the convection heater is configured to heat the valve.

[0207] Configuration 26. The device according to any one of the foregoing configurations, optionally, further comprising a pump configured to move fluid from a reservoir to the flow cell.

[0208] Configuration 27. The device according to any one of the foregoing configurations, optionally, wherein the stage comprises a reference surface, a preloading and a scanning actuator. Optionally, wherein the preloading is configured to urge the flow cell into contact with the reference surface. Optionally, wherein the reference surface and the detector form a fixed structural ring. Optionally, wherein the scanning actuator is configured to slide the flow cell along the reference surface in a scanning dimension.

[0209] Configuration 28. The device according to Configuration 27, optionally, wherein the second heater comprises a radiation surface adjacent to the reference surface, and the radiation surface is configured not to contact the flow cell.

[0210] Configuration 29. The device according to any one of the foregoing configurations, optionally, wherein the first heater comprises a convection heater.

[0211] Configuration 30. The device according to any one of the foregoing configurations, optionally, wherein the flow cell is removable from the stage.

[0212] Configuration 31. The device according to any one of the foregoing configurations, optionally, wherein the array comprises at least 1×10 3different nucleic acids.

[0213] Configuration 32. The apparatus according to Configuration 31, optionally, wherein the nucleic acid is immobilized at a site in the array, and wherein the area of each site is less than 25 square micrometers.

[0214] Configuration 33. The apparatus according to Configuration 32, optionally, wherein the array comprises at least 1×10 3 sites.

[0215] Configuration 34. The apparatus according to Configuration 33, optionally, wherein the array further comprises a plurality of ternary complexes, and wherein each ternary complex comprises the nucleic acid of the array, a polymerase, and the next correct nucleotide for the nucleic acid.

[0216] Configuration 35. The apparatus according to any of the foregoing configurations, optionally, wherein the cross-sectional area of the detection channel is at most 100 mm 2 .

[0217] Configuration 36. The apparatus according to any of the foregoing configurations, optionally, wherein one of the plurality of reservoirs contains a polymerase.

[0218] Configuration 37. The apparatus according to any of the foregoing configurations, optionally, wherein one of the plurality of reservoirs contains nucleotides.

[0219] Configuration 38. The apparatus according to Configuration 37, optionally, wherein the nucleotides comprise one or both of a reversible terminator moiety and an exogenous label.

[0220] Throughout this application, various publications, patents, and / or patent applications have been cited. The entire disclosures of these documents are incorporated herein by reference.

[0221] The term "comprising" is intended to be open-ended herein, including not only the recited elements but also any other elements.

[0222] As used herein, the term "each", when used in reference to a collection of items, is intended to identify a single item in the collection, but not necessarily every item in the collection. Exceptions may exist if expressly disclosed or the context otherwise clearly dictates.

[0223] Multiple embodiments have been described. However, it will be understood that various modifications can be made. Accordingly, other embodiments are within the scope of the appended claims.

Claims

1. A method for nucleic acid sequencing, characterized in that, the method comprises: (a) providing a sequencing device, the sequencing device comprising a flow cell, a fluid system, a flow cell heater, a fluid delivery channel heater, a reservoir, and a detection system, wherein, the flow cell comprises a nucleic acid array in a detection channel, the flow cell is heated by the flow cell heater to heat the nucleic acid array, the fluid system comprises the fluid delivery channel heater and a fluid delivery channel, the fluid delivery channel fluidly connects the reservoir to the detection channel of the flow cell, the reservoir holds the fluid at a temperature below 25 °C, and the detection system observes signals from the nucleic acid array; (b) transferring a fluid sequencing reagent from the reservoir to a heated region of the fluid delivery channel, whereby the fluid sequencing reagent is heated; (c) contacting the heated fluid sequencing reagent with the nucleic acid array by transferring the heated fluid sequencing reagent to the detection channel; and (d) sequencing the nucleic acid by detecting signals from the nucleic acid array by the detection system.

2. The method according to claim 1, characterized in that: (i) the temperature of the fluid delivery channel is the same as the temperature of the flow cell; (ii) the temperature of the fluid delivery channel is higher than the temperature of the flow cell; (iii) the temperature of the fluid delivery channel is lower than the temperature of the flow cell; or (iv) the temperature of the fluid delivery channel is higher than the temperature of the reservoir; optionally, the temperature of the flow cell is higher than the temperature of the reservoir.

3. The method as claimed in claim 1, characterized in that, the flow cell is heated by a heat conductor.

4. The method as claimed in claim 1, characterized in that, the fluid delivery channel is heated by a heat conductor.

5. The method according to claim 4, characterized in that, the heat conductor comprises a surface having a plurality of grooves, and the fluid delivery channel comprises a tube passing through one of the plurality of grooves.

6. The method according to claim 4, characterized in that, the heat conductor comprises a solid block, and the fluid delivery channel passes through the solid block.

7. The method as claimed in claim 1, characterized in that, the volume of the fluid sequencing reagent heated in the fluid delivery channel is equal to the volume of the detection channel.

8. The method as claimed in claim 1, characterized in that, the signal comprises an optical signal, and the detector comprises a photodetector.

9. The method as claimed in claim 1, characterized in that, step (d) comprises translating the flow cell along a reference surface of the detection system, wherein the reference surface contacts a part of the flow cell during the translation, and the reference surface forms a fixed structural ring with the detector, so as to detect signals from the heated nucleic acid array by the detection system.

10. The method according to claim 9, characterized in that, the method further comprises applying a preload during detection to urge the flow cell to contact the reference surface.

11. The method as claimed in claim 1, characterized in that, The sequencing device further includes a convective heater that transfers heat to the fluid delivery channel.

12. The method according to claim 11, wherein, the temperature of the convective heater is set to be higher than the temperature of the fluid delivery channel.

13. The method according to claim 11, wherein, the setpoint temperature of the convective heater is higher than the temperature of the reservoir.

14. The method according to claim 1, wherein, the volume of the detection channel is at most 10 ml.

15. The method according to claim 14, wherein, the volume of the detection channel is at most 1 ml.

16. The method according to claim 1, wherein, the sequencing device further includes a valve configured to control the flow of the fluid sequencing reagent through the fluid delivery channel.

17. The method according to claim 16, wherein, the valve includes a rotary valve.

18. The method according to claim 17, wherein, the sequencing device further includes a heater configured to heat the valve.

19. The method according to claim 1, wherein, The array contains at least 1×10 3 different ones of said nucleic acids.

20. The method according to claim 19, wherein, the nucleic acid is fixed at a site in the array, and the area of each site is less than 25 square microns.

21. The method according to claim 20, wherein, The array comprises at least 1×10 3 such sites.

22. The method according to claim 21, wherein, the array further comprises a plurality of ternary complexes, each ternary complex comprising the nucleic acid of the array, a polymerase, and the next correct nucleotide of the nucleic acid.

23. The method according to any one of claims 1 to 22, wherein, The cross-sectional area of the detection channel is at most 100 mm 2 .

24. A device for nucleic acid sequencing, wherein, the device includes: (a) a stage in contact with a flow cell, the flow cell including at least one detection channel, the detection channel including a nucleic acid array; (b) a detector configured to observe the nucleic acid array in the detection channel; (c) a plurality of reservoirs containing reagents for sequencing the nucleic acid array, the plurality of reservoirs holding the reagents at a temperature below 25 °C; (d) a plurality of fluid delivery channels, wherein each fluid delivery channel fluidly connects the plurality of reservoirs to the detection channel of the flow cell; and (e) a first heater that transfers heat to the plurality of fluid delivery channels; (f) a second heater that transfers heat to the detection channel of the flow cell.

25. The device according to claim 24, wherein, the first heater includes a heat conductor in contact with the plurality of fluid delivery channels.

26. The device according to claim 24, wherein, the device further includes a valve configured to control the flow of the reagent through the fluid delivery channel among the plurality of fluid delivery channels, and further includes a heater configured to heat the valve.

27. The device according to any one of claims 24 to 26, wherein, the first heater includes a convection oven.

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