DNA sequencing system and application thereof

Abstract

The present disclosure provides flow cell devices, systems, and methods for facilitating and performing DNA sequencing assays with reduced system complexity and cost, significant cost savings, and reduced levels of contamination. The sequencing systems described herein allow for simultaneous processing of multiple flow cells such that sequencing and imaging steps or multiple sequencing methods can be performed in parallel using a single sequencing system.

Description

Cross-reference to related applications

[0001] This application claims priority and benefit to U.S. Provisional Application No. 63 / 515,816, filed July 26, 2023, and U.S. Provisional Application No. 63 / 666,463, filed July 1, 2024, the contents of each of which are incorporated herein by reference in their entirety. Background Technology

[0002] In next-generation sequencing (NGS) systems, flow cell devices are used to immobilize template nucleic acid molecules derived from biological samples, and then repeated streams of sequencing reagents are introduced to attach labeled nucleotides to specific positions in the template sequence. A series of signals from the labels are detected and decoded to reveal the corresponding nucleotide sequence of the template molecule, for example, an immobilized and / or amplified nucleic acid template molecule attached to the surface of the flow cell.

[0003] Typical NGS systems allow fluid and thermal communication from the system to the flow cell apparatus and the sample fixed thereon during sequencing, while the sample remains in a fixed position relative to the optics of the sequencing system. However, maintaining the flow cell in a fixed position relative to the optics of the sequencing system during steps not involving imaging leads to inefficient use of the optics and reduces the efficiency of the NGS system. Therefore, there is a need in the art for compositions, systems, and methods that allow for the parallel and asynchronous processing of multiple samples or portions of samples. Using the compositions, systems, and methods of this disclosure, imaging and non-imaging steps for different samples can occur simultaneously, or different samples can undergo different methods concurrently, thereby reducing idle time, increasing flexibility, and increasing efficiency. Summary of the Invention

[0004] This paper describes a flexible and scalable sequencing system for sequencing nucleic acids. The sequencing systems and methods described herein advantageously enable more efficient use of optical systems with minimal idle time (e.g., waiting for fluid application). The systems and methods described herein advantageously allow imaging of a sample while sequencing reactions in another sample occur in parallel, thereby increasing the throughput of existing sequencing systems. The systems and methods described herein advantageously separate the sample being imaged from fluid and / or thermal communication, thereby simplifying system architecture and enabling a more compact size than existing systems. The systems and methods described herein also advantageously enable independent fluid and / or thermal communication with a variety of samples, thereby allowing users to image samples using different reagents or sequencing protocols and potentially combine them within a single sequence run.

[0005] This disclosure provides a sequencing system comprising: an optical system 2020 including an objective lens; an xy stage 2010 configured to hold a sample to be imaged thereon and move the sample relative to the objective lens in the xy plane, wherein the sample is fixed on one or more flow cell devices; a nesting group 2050 configured to provide fluid and thermal communication to the sample when one or more flow cell devices are coupled to the nesting group; and a movement mechanism 2040 optionally including a movable arm configured to move one or more flow cell devices between the xy stage 2010 and the nesting group 2050 during sequence execution.

[0006] In some embodiments, the xy stage 2010 is automatically actuated by a first actuator with a first spatial precision.

[0007] In some embodiments, the movable arm is automatically actuated by a second actuator with a second spatial precision. In some embodiments, the first actuator, the second actuator, or both are controlled by one or more hardware processors of the sequencing system.

[0008] In some embodiments, the sequencing system further includes a housing configured to hold one or more of the optical system 2020, the xy stage 2010, the nesting group 2050, and the moving mechanism 2040 within the housing.

[0009] In some embodiments, the movable arm is automatically actuated to move in three dimensions (3D). In some embodiments, movement in each of the three dimensions is performed with one or more predetermined spatial precisions.

[0010] In some embodiments, when the flow cell device is fixed on the xy stage 2010, the sequencing system lacks fluid or thermal communication with one or more flow cell devices at or near the xy stage 2010.

[0011] In some embodiments, each of the one or more flow cell devices includes an open landing area configured to openly receive fluid from the nested group 2050. In some embodiments, the flow cell device includes a plurality of microfluidic channels, and the nested group 2050 is configured to allow independent and simultaneous fluid communication with each of the plurality of microfluidic channels. In some embodiments, the flow cell device includes a plurality of microfluidic channels, and the nested group 2050 is configured to allow independent and sequential fluid communication with each of the plurality of microfluidic channels. In some embodiments, the flow cell device includes a plurality of microfluidic channels, and the nested group 2050 is configured to allow independent and non-cross-contamination fluid communication with each of the plurality of microfluidic channels.

[0012] In some embodiments, the xy stage 2010 is actuated to move a predetermined distance in the xy plane. In some embodiments, the predetermined distance is based on the distance between two adjacent microfluidic channels of the flow cell device.

[0013] In some embodiments, the nesting group 2050 is configured to enable fluid and thermal communication with one or more flow cell devices. In some embodiments, the nesting group 2050 is configured to enable fluid and thermal communication with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 flow cell devices when each of the flow cell devices is in a locked position with the nesting group 2050. In some embodiments, the nesting group 2050 is configured to hold each of the flow cell devices in an unlocked position and a locked position, in which the flow cell device can be removed from the nesting group 2050, and in the locked position, the flow cell device is spatially registered to the nesting group 2050, fixedly coupled to the nesting group 2050, and allows for sealed fluid and thermal communication between the nesting group and the flow cell devices.

[0014] In some embodiments, the flow cell device is coupled to the carrier 2051. In some embodiments, a movable arm is configured to move the carrier 2051 and the flow cell device together. In some embodiments, the carrier 2051 is configured to be spatially registered to a nested group in a locked position. In some embodiments, the nested group 2050 includes one or more fasteners. In some embodiments, the one or more fasteners utilize magnetic force. In some embodiments, the one or more fasteners include rare-earth magnets, electromagnetic coils, or both. In some embodiments, the one or more fasteners are controlled by one or more processors to switch between an open phase and a closed phase. In some embodiments, the one or more fasteners lack mechanical fasteners.

[0015] In some embodiments, a movable arm is configured to move one or more flow cell devices between the xy stage 2010 and the nesting group 2050 with a first spatial accuracy. In some embodiments, the movable arm includes a gripper configured to grip the carrier 2051 when it is in a decoupled position relative to the nesting group 2050 or when it is in a decoupled position relative to the xy stage 2010. In some embodiments, the movable arm includes a horizontal arm mechanically supported by a vertical arm. In some embodiments, the movable arm includes an upper arm, a joint, a forearm, a wrist, and a gripper attached to the forearm. In some embodiments, the movable arm is configured to move in six degrees of freedom. In some embodiments, the gripper is movably attached to either the horizontal arm or the vertical arm. In some embodiments, the gripper is configured to move in 3D. In some embodiments, the movement mechanism 2040 includes a plurality of tracks, each track connecting the carrier 2051 coupled to the nesting group to the xy stage 2010. In some embodiments, the gripper is configured to hold the flow cell device carrier via friction, electromagnetic force, or magnetic force.

[0016] In some embodiments, the carrier 2051 includes one or more sensors. In some embodiments, the xy stage 2010 includes one or more sensors. In some embodiments, the nested group 2050 includes one or more sensors. In some embodiments, one or more sensors are configured to provide feedback to a processor that facilitates the positioning of the carrier 2051 relative to the xy stage 2010, the optical system 2020, or the nested group 2050.

[0017] In some embodiments, the moving mechanism 2040 includes one or more belt conveyors.

[0018] In some embodiments, the plurality of tracks include one or more actuators configured to actuate one or more of the plurality of tracks to move the corresponding carrier 2051 to the xy stage 2010.

[0019] In some embodiments, the xy stage 2010 is configured to be actuated to move to a 3D position with a second spatial accuracy. In some embodiments, the second spatial accuracy is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the first spatial accuracy.

[0020] In some embodiments, the xy stage 2010 includes fasteners configured to removably secure the flow cell assembly to it. In some embodiments, the fasteners include one or more clamps.

[0021] In some embodiments, each carrier 2051 includes a coupling location in which the carrier 2051 is removably attached to the xy stage 2010. In some embodiments, each carrier 2051 includes a decoupling location in which the carrier 2051 is removable from the xy stage 2010.

[0022] In some embodiments, the xy stage 2010 includes one or more pumps configured to extract fluid from a flow cell device when the corresponding carrier 2051 is coupled to the xy stage 2010. In some embodiments, the xy stage 2010 includes a heating device, a cooling device, or both. In some embodiments, the xy stage 2010 is coupled to a mechanical decoupling device configured to isolate the xy stage from vibrations or mechanical disturbances outside the xy stage 2010.

[0023] In some embodiments, the nesting group 2050 includes one or more fasteners, each fastener configured to secure a corresponding carrier 2051 to the nesting group 2050. In some embodiments, each fastener includes one or more clamps. In some embodiments, the one or more clamps are actuated by magnetic or electromagnetic force or pressure.

[0024] In some embodiments, the nested group 2050 includes one or more pumps configured to extract fluid from the flow cell device when the corresponding carrier 2051 is coupled to the nested group 2050.

[0025] In some embodiments, each carrier 2051 includes a decoupling location in which the carrier 2051 can be removed from the nesting group 2050. In some embodiments, each carrier 2051 includes a coupling location in which the flow cell device carrier is removably attached to the nesting group 2050 and is in sealed fluid communication with the nesting group 2050.

[0026] In some embodiments, the nested group 2050 includes a 3D moving device configured to position the carrier 2051 relative to the nested group with a third spatial precision. In some embodiments, the third spatial position is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the first spatial precision.

[0027] In some embodiments, the carrier 2051 includes an opening at its surface configured to receive a flow cell device therein. In some embodiments, the carrier 2051 includes one or more fluid paths that are in sealed fluid communication with the flow cell device when it is removably attached to the carrier 2051. In some embodiments, the carrier 2051 includes a pump configured to pull or push fluid between the flow cell device and the carrier 2051. In some embodiments, the carrier 2051 includes a valve positioned between a fluid path connected to the flow cell device and a port opening of the carrier 2051, wherein the valve is in an open position when the flow cell device is in a coupled position with the carrier 2051 and in a closed position when the flow cell device is in a decoupled position. In some embodiments, the carrier 2051 includes a port opening having a connector configured to allow sealed fluid communication between the carrier 2051 and a corresponding nested module when the connector is in a connected position. In some embodiments, the carrier 2051 includes a wire having an electrical connector configured to allow electrical communication between the carrier 2051 and a power source. In some embodiments, carrier 2051 includes a battery, a sensor, or both, and wherein the battery or sensor is connected to an electrical connector via a wire.

[0028] In some embodiments, the nested group 2050 includes one or more reagent containers. In some embodiments, the one or more reagent containers are disposable.

[0029] In some embodiments, the moving mechanism 2040 is configured to immerse the flow cell device into at least some of one or more reagent containers.

[0030] In some embodiments, the nested assembly 2050 further includes a cooler, a heater, or both. In some embodiments, the cooler or heater is configured to control the temperature of each sample fixed to one or more flow cell devices. In some embodiments, the cooler or heater includes one or more of the following: a fan configured to blow cold or hot air; a microwave, an infrared light source, and an electromagnetic wave source.

[0031] In some embodiments, the sequencing system further includes a beam collector configured to absorb at least some of the excitation light generated by the optical system. In some embodiments, the sequencing system further includes a beam collector configured to prevent at least some of the excitation light from reaching the imaging sensor of the optical system. In some embodiments, the beam collector is offset from the flow cell device by a gap region. In some embodiments, the beam collector contacts the flow cell device with a predetermined latching force. In some embodiments, the beam collector contacts the xy stage with a predetermined damping force. In some embodiments, the predetermined damping force is configured to reduce the predetermined latching force such that the net force on the flow cell device can be customized within a predetermined range.

[0032] This disclosure provides a sequencing method comprising: (a) moving a first flow cell device from a nested assembly 2050 to an xy stage 2010, wherein the first flow cell device includes a first sample fixed thereon; (b) moving the xy stage 2010 and the first sample thereon relative to an objective lens of an optical system of a sequencing system; (c) imaging the first sample fixed on the first flow cell device on the xy stage using an optical system 2020; (d) moving the first flow cell device from the xy stage 2010 to the nested assembly 2050; and (e) simultaneously allowing fluid and... (f) moving the second flow cell device from the nested assembly 2050 to the xy stage 2010, wherein the second flow cell device includes a second sample fixed thereon; (g) moving the xy stage 2010 and the second sample thereon relative to the objective lens of the optical system 2040 of the sequencing system; (h) imaging the second sample fixed on the second flow cell device on the xy stage 2010 using the optical system 2040; (i) moving the first flow device from the xy stage 2010 to the nested assembly 2050; and (j) simultaneously allowing fluid and thermal communication between the nested assembly 2050 and the first flow cell device during one or more of (f)-(i).

[0033] This disclosure provides a sequencing method comprising: (a) moving a first flow cell device from a nested assembly 2050 to an xy stage 2010, wherein the first flow cell device includes a first sample fixed thereon; (b) moving the xy stage 2010 and the first sample thereon relative to an objective lens of an optical system 2020 of a sequencing system; (c) imaging the first sample fixed on the first flow cell device on the xy stage 2010 using the optical system 2020; (d) moving the first flow cell device from the xy stage 2010 to the nested assembly 2050; (e) simultaneously allowing fluid and thermal communication between the nested assembly 2050 and a second flow cell device during one or more of (a)-(d); and (f) moving a second flow cell device from the nested assembly 2050 to the xy stage 2010, wherein the second flow cell device includes a second sample fixed thereon.

[0034] In some embodiments, the sequencing method further includes repeating operations (a)-(e). In some embodiments, the sequencing method further includes repeating operations (f)-(j). In some embodiments, the sequencing method further includes repeating operations (a)-(j) a certain number of times. In some embodiments, the number of repeats is in the range of 1 to 500.

[0035] In some embodiments, allowing fluid communication between the nested assembly 2050 and the first flow cell device includes: reversibly fastening the flow cell device to the carrier 2051 via one or more fasteners to enable sealed fluid communication between the flow cell device and the carrier 2051; and reversibly fastening the carrier 2051 to the nested assembly 2050 via one or more fasteners to enable sealed fluid communication between the nested assembly 2050 and the carrier 2051 and to enable physical contact with a heat dissipation element.

[0036] In some embodiments, (a) moving the first flow cell device from the nested group 2050 to the xy stage 2010 is within a first flow cycle of the sequencing run, and (f) moving the first flow cell device from the nested group 2050 to the xy stage 2010 is within a second flow cycle of the sequencing run, which is different from the first flow cycle.

[0037] In some embodiments, each of operations (a)-(b) and (d)-(g) is completed within less than 0.5 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds.

[0038] In some embodiments, allowing simultaneous fluid and thermal communication between the nested group 2050 and the first flow cell device during one or more of (a)-(d) includes: rotating one or more fasteners to an open phase to enable sealing for fluid communication and physical contact for thermal communication. In some embodiments, allowing simultaneous fluid and thermal communication between the nested group and the first flow cell device during one or more of (a)-(d) includes: immersing the flow cell device in at least some of one or more reagent containers in a predetermined sequence. Attached Figure Description

[0039] The novel features of the invention are specifically set forth in the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description of exemplary embodiments that utilize the principles of this disclosure, and in the accompanying drawings: Figure 1 A block diagram of a computer-implemented system for performing operations in DNA sequencing and sequencing analysis, according to some embodiments, is shown.

[0040] Figure 2 This is a schematic diagram illustrating an exemplary sequencing system 2000 according to some embodiments, in which the sequencing system includes an optical system 2020 including an objective lens, an xy stage 2010, a nesting group 2050, a flow cell device carrier 2051, and a moving mechanism 2040 (e.g., a movable arm) surrounded by a housing 2030.

[0041] Figure 3 This is a schematic diagram illustrating an exemplary nested group module according to some embodiments.

[0042] Figures 4A to 4B This illustrates some embodiments. Figure 3 Two schematic diagrams of an exemplary nested group module.

[0043] Figures 5A to 5B These are two schematic diagrams illustrating exemplary flow cell apparatuses according to some embodiments.

[0044] Figure 6 An exemplary nested group with reagent containers is shown according to some embodiments. Figure 6 In this system, each reagent container is at the appropriate temperature for a given reaction. The reagent containers can be part of a disposable (dry) instrument.

[0045] Figure 7 An exemplary flow pool apparatus with an open landing area is shown according to some embodiments.

[0046] Figure 8Block diagrams of a computer system for fluid control and for performing sequencing and sequencing analysis, according to some embodiments, are shown.

[0047] Figure 9 This is a schematic diagram illustrating an exemplary linear single-stranded library molecule (900) comprising: a surface-pinned primer binding site (920); an optional left unique identifier sequence (980); a left index sequence (960); a forward sequencing primer binding site (940); an insert region with a target sequence (910); a reverse sequencing primer binding site (950); a right index sequence (970); and a surface-capture primer binding site (930).

[0048] Figure 10 This is a schematic diagram illustrating an exemplary linear single-stranded library molecule (900) comprising: a surface-pinned primer binding site (920); a left index sequence (960); a forward sequencing primer binding site (940); an insert region having a target sequence (910); a reverse sequencing primer binding site (950); a right index sequence (970); an optional right unique recognition sequence (990); and a surface-capture primer binding site (930).

[0049] Figure 11 These are schematic diagrams illustrating various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of a multivalent molecule with a "starburst" or "helix-skelter" configuration. Middle (Class II): Schematic diagram of a multivalent molecule with a dendritic macromolecular configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by the reaction of streptavidin with a 4-arm or 8-arm PEG-NHS containing biotin and dNTPs. Nucleotide units are designated as 'N', biotin as 'B', and streptavidin as 'SA'.

[0050] Figure 12 This is a schematic diagram of an exemplary multivalent molecule containing a universal nucleus attached to multiple nucleotide arms.

[0051] Figure 13 This is a schematic diagram of an exemplary multivalent molecule comprising a dendritic macromolecular core attached to multiple nucleotide arms.

[0052] Figure 14 A schematic diagram of an exemplary multivalent molecule is shown, comprising a nucleus attached to multiple nucleotide arms, wherein the nucleotide arms include biotin, spacers, linkers, and nucleotide units.

[0053] Figure 15 This is a schematic diagram of an exemplary nucleotide arm that includes a nuclear attachment portion, a spacer, a linker, and a nucleotide unit.

[0054] Figure 16 The chemical structures of exemplary spacers (top) and various exemplary connectors, including 11-atom connectors, 16-atom connectors, 23-atom connectors and N3 connectors (bottom), are shown.

[0055] Figure 17 The chemical structures of various exemplary connectors (including connectors 1-9) are shown.

[0056] Figure 18 The chemical structures of various exemplary linkers that connect to / attach to nucleotide units are shown.

[0057] Figure 19 The chemical structures of various exemplary linkers that connect to / attach to nucleotide units are shown.

[0058] Figure 20 The chemical structures of various exemplary linkers that connect to / attach to nucleotide units are shown.

[0059] Figure 21 The chemical structures of various exemplary linkers that connect to / attach to nucleotide units are shown.

[0060] Figure 22 The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide unit is connected to the linker via a propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base.

[0061] Figure 23 A schematic diagram of an example flow cell apparatus is shown, wherein the support comprises alternating layers of a glass substrate and a hydrophilic coating covalently or non-covalently adhered to the glass, and the support further comprises chemically reactive functional groups that act as attachment sites for oligonucleotide primers.

[0062] Figures 24A to 24B Each of the following schematic diagrams illustrates an exemplary embodiment of a nested module that is part of a nested group 2050, which receives the carrier 2051 when the carrier 2051 is coupled to the nested group 2050.

[0063] Figure 25 This is a schematic diagram of the movement of carrier 2051 between optical system 2020 and nesting group 2050.

[0064] Figure 26A It shows Figure 25 A schematic diagram of the xy stage 2010 of the optical system.

[0065] Figure 26B It shows Figure 25 A schematic diagram of nested group 2050 in the diagram.

[0066] Figures 27A to 27C Schematic diagrams of exemplary embodiments of the moving mechanism 2040 associated with the optical system 2020 and the nesting group 2050 are shown respectively.

[0067] Figure 28 A series of schematic diagrams illustrating exemplary embodiments of the sequencing system are shown.

[0068] Figure 29 A schematic diagram of an exemplary embodiment of a flow cell apparatus carrier is shown.

[0069] Figure 30 A schematic diagram of an exemplary embodiment of a flow cell device carrier 2051 moving between an optical system 2020 and a nesting group 2050 is shown.

[0070] Figure 31 A schematic diagram of an exemplary embodiment of a flow pool device carrier 2051 in a coupling position with a nested group 2050 for fluid communication is shown.

[0071] Figures 32A to 32B Each of the above illustrates an exemplary embodiment of a flow cell device carrier 2051 positioned in a coupling position with the xy stage 2010 for imaging. Detailed Implementation

[0072] This article describes systems and apparatuses for analyzing various nucleic acid sequences, such as those from amplified nucleic acid arrays in a flow cell or from immobilized nucleic acid arrays. The systems and apparatuses described herein can also be used for applications such as sequencing in comparative genomics, tracking gene expression, microRNA sequence analysis, epigenomics, and aptamer and phage display library characterization, as well as other sequencing applications. The systems and apparatuses described herein encompass various combinations of optical, mechanical, fluidic, thermal, electrical, and computational devices / aspects.

[0073] The advantages of the disclosed flow cell apparatus, fluid control apparatus, system, and method include, but are not limited to: flexible and scalable system throughput and flexible system adaptation to different sequencing applications; reduced device and system manufacturing / maintenance complexity and cost; reduced optics idle time; and separation of fluid and thermal communication from the sample during imaging, thereby reducing potential interference with imaging results.

[0074] Some publicly disclosed design features of capillary flow cell devices, flow cell device carriers, and systems include, but are not limited to: open dispensing tips in the fluid control device and open landing areas on the flow cell device to allow open delivery of reagents without the complexity and cost of traditional tubing, and to enable the system's flexibility to adapt to different sequencing applications; moving mechanisms (e.g., movable arms) that allow the sample to move relative to the optics to maximize the use of the optical system and reduce the time required to complete sequence runs; fluid and thermal connectivity that is confined to nested groups but not present on the xy stage, thereby reducing system size and eliminating potential interference during imaging; and a combination of coarse and efficient sample movement via the movable arm and fine movement and adjustment of the sample relative to the objective or dispensing tip to ensure accurate and efficient alignment of the sample for imaging and fluid application.

[0075] While the disclosed flow cell apparatus, systems, and methods are described primarily in the context of their application in nucleic acid sequencing, various aspects of the disclosed apparatus and systems can be used not only for nucleic acid sequencing but also for any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis application. It should be understood that the different aspects of the disclosed apparatus and systems can be understood individually, collectively, or in combination with each other.

[0076] sequencing system In some embodiments, flow cell apparatuses and systems are disclosed herein that can be used to perform or facilitate DNA sequencing analysis using a sequencing system. Sequencing systems can utilize a variety of sequencing technologies, including but not limited to those disclosed herein.

[0077] definition The headings provided herein are not intended to limit the various aspects of this disclosure, which are to be understood by referring to the specification as a whole. Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, terms related to the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are terms well-known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY2000). Also see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with the laboratory procedures and techniques described herein is that well-known and commonly used in the art.

[0078] Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless explicitly and definitively limited to one referent, the singular forms “a / an” and “the”, as well as the singular use of any word, contain multiple referents.

[0079] It should be understood that the use of alternative terms (e.g., "or") is considered to refer to one or both of the alternatives or any combination thereof.

[0080] As used herein, the term “and / or” should be considered to refer to a specific disclosure of each of the specified features or components, whether or not they are together with another. For example, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). Similarly, the term “and / or” as used herein, such as “A, B and / or C”, is intended to cover each of the following aspects: “A, B and C”; “A, B or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0081] As used herein and in the appended claims, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, are intended to be non-limiting, such that one or more items in the list do not exclude other items that may be substituted for or added to the listed items. It should be understood that whenever an aspect is described herein with the language “comprising,” other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0082] As used herein, the terms “about,” “approximately,” and “substantially” mean a value or composition within an acceptable range of error for a particular value or composition, as determined by one of ordinary skill in the art, depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about,” “approximately,” or “substantially” may mean within one or more standard deviations. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, specifically with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about,” “approximately,” or “substantially” should be assumed to be within an acceptable range of error for said particular value or composition. Furthermore, in the case of providing ranges and / or subranges of values, the range and / or subrange may include the endpoints of the range and / or subrange.

[0083] The term "glass" refers to silica-based materials, including silicates, borosilicates, fused silica, fused silica, glass, quartz, or lead glass.

[0084] As used herein, the term "colony" refers to a nucleic acid library molecule that can be cloned and amplified in solution or on a support to produce an amplicon that can serve as a template molecule for sequencing. For example, a linear library molecule can be circularized to generate a circularized library molecule, and a circularized library molecule can be cloned and amplified in solution or on a support to generate a tandem molecule. Tandem molecules can serve as nucleic acid template molecules that can be sequenced. Tandem molecules are sometimes referred to as communities. In some embodiments, a community comprises a nucleotide chain.

[0085] As used herein, the term "clonal amplification" and its variations refer to a nucleic acid template molecule that has undergone one or more amplification reactions in solution or on a support. In the case of amplifying template molecules in solution, the resulting amplicons can be distributed on a support. Prior to amplification, the template molecule typically contains the target sequence and at least one universal adaptor sequence, i.e., a sequence common to all template molecules in the reaction or from a particular sample. In some embodiments, clonal amplification includes the use of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridging amplification, isothermal bridging amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding (SSB) protein-dependent amplification, or any combination thereof.

[0086] The terms “peptide,” “polypeptide,” and “protein,” as used herein, and other related terms, are used interchangeably and refer to polymers of amino acids and are not limited to any particular length. Polypeptides can include both natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not undergone post-translational modifications, such as proteolytic cleavage, cleavage due to ribosome jumping, hydroxylation, methylation, esterification, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass natural and artificial proteins, protein fragments and polypeptide analogues (such as mutant proteins, variants, chimeric proteins, and fusion proteins), and proteins that are post-translationally or otherwise covalently or non-covalently modified.

[0087] As used herein, the term "sequencing" and its variations include obtaining sequence information from a nucleic acid strand by generally determining the identity of at least some nucleotides (including their nucleobase components) within a nucleic acid template molecule. While in some embodiments, "sequencing" of a given region of a nucleic acid template molecule includes identifying each or every nucleotide within the sequenced region, in some embodiments, "sequencing" includes methods that determine the identity of only some nucleotides in the region, without determining or incorrectly determining the identity of some nucleotides. Any suitable sequencing method can be used. In exemplary embodiments, sequencing may include label-free or ion-based sequencing methods. In some embodiments, sequencing may include labeled or dye-based or fluorescence-based nucleotide sequencing methods. In some embodiments, sequencing may include polymerase cloning-based sequencing or bridge sequencing methods. In some embodiments, sequencing includes a massively parallel sequencing platform employing synthetic sequencing, hybridization sequencing, or combined sequencing procedures. Examples of massively parallel synthesis sequencing programs include polymerase community sequencing, pyrosequencing (e.g., from Life Sciences; U.S. Patents 7,211,390, 7,244,559, and 7,264,929), chain terminator sequencing (e.g., from Illumina; U.S. Patent 7,566,537; Bentley 2006 Current Opinion Genetics and Development 16:545-552; and Bentley et al., 2008 Nature 456:53-59), ion-sensitive sequencing (e.g., from IonTorrent), probe-anchor ligation sequencing (e.g., Complete Genomics), DNA nanosphere sequencing, and nanopore DNA sequencing. Examples of single-molecule sequencing include Heliscope single-molecule sequencing and single-molecule real-time (SMRT) sequencing from Pacific Biosciences (Levene et al., 2003 Science 299(5607):682-686; Eid et al., 2009 Science 323(5910):133-138; U.S. Patent Nos. 7,170,050, 7,302,146, and 7,405,281). Examples of hybridization sequencing include SOLiD sequencing (e.g., from Life Technologies; WO 2006 / 084132). Examples of conjugation sequencing include Omniome sequencing (e.g., U.S. Patent No. 10,246,744).

[0088] As used herein, the term "polymerase" and its variants include any enzyme capable of catalyzing the polymerization of nucleotides (including analogs thereof) into nucleic acid chains. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, a polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has chain displacement activity. A polymerase may include, but is not limited to: naturally occurring polymerases and any subunits and truncated forms thereof, mutant polymerases, variant polymerases, recombinant, fusion, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. In some embodiments, a polymerase may be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase may be expressed in prokaryotes, eukaryotes, viruses, or bacteriophages. In some embodiments, a polymerase may be a post-translational modified protein or a fragment thereof. Polymerases can originate from prokaryotes, eukaryotes, viruses, or bacteriophages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0089] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization performed by a template-dependent DNA polymerase. DNA polymerase fidelity is typically measured by the error rate (the frequency of incorporation of inaccurate nucleotides, i.e., nucleotides that are not complementary to the template nucleotide). The accuracy or fidelity of DNA polymerization is maintained by the polymerase activity and 3'-5' exonuclease activity of the DNA polymerase.

[0090] As used herein, the term "binding complex" refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, wherein the nucleic acid duplex includes a nucleic acid template molecule that hybridizes with a nucleic acid primer. In the binding complex, the free nucleotide or nucleotide unit may or may not bind to the 3' end of the nucleic acid primer at a position opposite to a complementary nucleotide in the nucleic acid template molecule. A "ternary complex" is an example of a binding complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, wherein the free nucleotide or nucleotide unit binds to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) at a position opposite to a complementary nucleotide in the nucleic acid template molecule.

[0091] As used in this article, "nucleotide unit" or "nucleotide moiety" refers to a nucleotide ( For exampleNucleotide units (dATP, dTTP, dGTP, dCTP, or dUTP) or their analogues, comprising a base, a sugar, and at least one phosphate ester group. Nucleotide units can be attached to multivalent molecules used in the sequencing reactions described herein. Generally, all nucleotide units attached to the same multivalent molecule will have the same identity (…). For example (All A, all T, all C, or all G), although those skilled in the art will understand that it may be advantageous for a multivalent molecule to contain nucleotide units of different identities.

[0092] The term "retention time" and related terms refer to the length of time a binding complex remains stable without any dissociation of its components, wherein the components of the binding complex include a nucleic acid template and primer, a polymerase, nucleotide units of a multivalent molecule, or free (e.g., unconjugated) nucleotides. Nucleotide units or free nucleotides may be complementary or non-complementary to nucleotide residues in the template molecule. Nucleotide units or free nucleotides may bind to the 3' end of the nucleic acid primer at a position opposite to the complementary nucleotide residues in the nucleic acid template molecule. Retention time indicates the stability of the binding complex and the strength of the binding interactions. Retention time can be measured by observing the onset and / or duration of the binding complex (e.g., by observing a signal from a labeled component of the binding complex). For example, a labeled nucleotide or a labeled reagent containing one or more nucleotides may be present in the binding complex, thus allowing a signal from the label to be detected during the retention time of the binding complex. An exemplary label is a fluorescent label. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that cause dissociation between the polymerase, template molecule, primer, and / or nucleotide units or any of the nucleotides. For example, dissociation conditions include contacting the bound complex with any or any combination of detergent, EDTA, and / or water.

[0093] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as used herein, and other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, wherein the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids contain naturally occurring nucleoside bonds, such as phosphodiester bonds. Nucleic acids contain non-natural nucleoside bonds, including thiophosphate, sulfur-containing phosphate, or peptide nucleic acid (PNA) bonds. Nucleic acids may also contain mixtures of natural and non-natural nucleoside bonds. In some embodiments, nucleic acids comprise one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0094] As used herein, the term "primer" and related terms refer to a natural or synthetic oligonucleotide capable of hybridizing with a DNA and / or RNA polynucleotide template to form a double-stranded molecule. Primers can have any length, but typically range from 4 to 50 nucleotides. Typical primers include a 5' end and a 3' end. The 3' end of a primer may contain a 3'OH portion that acts as the initiation site for nucleotide polymerization in a polymerase-mediated primer extension reaction. Alternatively, the 3' end of a primer may lack the 3'OH portion or may contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-mediated reaction. Any nucleotide or more along the length of the primer may be labeled with a detectable reporter gene portion. Primers may be in solution (e.g., soluble primers) or may be immobilized to a support (e.g., capture primers).

[0095] The terms “template nucleic acid,” “template polynucleotide,” “target nucleic acid,” “target polynucleotide,” “template strand,” and other variations refer to the nucleic acid strand that serves as the basis for generating complementary nucleic acid strands in a nucleic acid molecule. The template nucleic acid can be single-stranded or double-stranded, or may have single-stranded or double-stranded portions. The sequence of the template nucleic acid can be partially or completely complementary to the sequence of the complementary strand. The template nucleic acid can be obtained from naturally occurring sources, in recombinant forms, or chemically synthesized to include any type of nucleic acid analogue. The template nucleic acid can be linear, circular, or other forms. The template nucleic acid may include an insert region having an insert sequence also referred to herein as the target sequence. The template nucleic acid may also include at least one adaptor sequence. The template nucleic acid can be a tandem of two or tandem copies having the target sequence and at least one adaptor sequence. The insert region can be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloned or amplified cDNA, RNA (such as precursor mRNA or mRNA), oligonucleotides, whole genomic DNA obtained from fresh-frozen paraffin-embedded tissue, needle biopsy, cell-free circulating DNA, or any type of nucleic acid library. The insert region can be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, viruses, cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic samples, sweat, semen, environmental samples, biofilms, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insert region can be isolated from any organ, including the head, neck, brain, mammary gland, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid gland, pituitary gland, thymus, skin, heart, larynx, or other organs. The insert region can be isolated from multiple cells or a single cell. The template nucleic acid can undergo nucleic acid analysis (including sequencing and compositional analysis).

[0096] When used to refer to nucleic acid molecules, the terms "hybridize," "hybridizing," "hybridization," or other related terms refer to the hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes the hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridized molecule with double-stranded regions. Hybridization can include Watson-Crick or Hoogstein binding to form double-stranded nucleic acids or double-stranded regions within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be completely complementary or partially complementary. Complementary nucleic acid strands do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing or can be other forms of base pairing interactions. Double-stranded nucleic acids can contain mismatched base-paired nucleotides.

[0097] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and at least one phosphate ester group. Canonical or non-canonical nucleotides are used in accordance with this terminology. In some embodiments, phosphate esters include monophosphate, diphosphate, or triphosphate esters, or corresponding phosphate ester analogs. In some embodiments, a nucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate ester groups. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar.

[0098] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants or analogues thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Husstein hydrogen bonds with suitable complementary bases. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethylene adenine, N... 6 -Δ 2 -Isopentenyladenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -Methyladenine, guanine (G), isoguanine, N 2 -Dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6-Methylguanine; 7-deazo-purine, such as 7-deazoadenine (7-deazoaden-A) and 7-deazoguanine (7-deazo-G); pyrimidine, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole, such as nitroindole and 4-methylindole; pyrrole, such as nitropyrrole; muscarin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated, and acylated base moieties; etc. Additional exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385–394, CRC Press, Boca Raton, Fla.

[0099] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), an acyclic moiety (Martinez et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7: 3013-3016), and other sugar moieties (Joeng et al., 1993 J. Med. Chem. 36: 2627-2638; Kim et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284: 2118-2124; and US Patent No. 5,558,991). The sugar moiety includes: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0100] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, including phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0101] When used to refer to nucleic acids, the terms "extend," "extending," "extension," and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3'OH end of the nucleic acid chain, resulting in the extension of the nucleic acid chain. Nucleotide incorporation can be performed using native nucleotides and / or nucleotide analogs. Typically, but not always, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, which involves primer extension catalyzed by DNA polymerase or RNA polymerase.

[0102] The terms "reporter moiety," "reporter moieties," or related terms refer to compounds that produce or cause the production of a detectable signal. A reporter moiety is sometimes referred to as a "tag." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemical, mass spectrometry, Raman spectroscopy, haptens, affinity tags, atoms, or enzymes. A reporter moiety produces a detectable signal caused by a chemical or physical change, such as heat, light, electricity, pH, salt concentration, enzymatic activity, or proximity events. Proximity events include two reporter moieties coming close to each other, associating with each other, or binding to each other. Those skilled in the art are well aware of selecting reporter moieties such that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing for the monitoring of the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties with spectrally distinct emission profiles or with minimal overlap spectral emission profiles can be selected. The reporter gene portion may be linked to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases) or supports (e.g., surfaces) in an operative manner.

[0103] The reporter gene portion (or marker) includes a fluorescent marker or fluorophore. Exemplary fluorescent portions that can be used as fluorescent markers or fluorophores include, but are not limited to, fluoresceins and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthol fluorescein, isothiocyanate fluorescein, NHS-fluorescein, iodoacetamide-fluorescein, maleimide fluorescein, SAMSA-fluorescein, aminothiourea fluorescein, hydrazine methylthioacetamide fluorescein; rhodamine and rhodamine derivatives, such as TRITC, TMR, rhodamine sulfonyl chloride, Texas red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, rhodamine sulfonyl chloride, rhodamine sulfonyl hydrazine, Texas red sulfonyl chloride, Texas red hydrazine; coumarins and coumarin derivatives, such as AMCA, AMCA-NHS, AMCA-sulfonyl-NHS, AMCA-HPDP, DCIA, AMCE-hydrazine; and BODIPY. TM and derivatives such as BODIPY TM FL C3-SE, BODIPY TM 530 / 550 C3, BODIPY TM 530 / 550 C3-SE, BODIPY TM 530 / 550 C3 hydrazide, BODIPY TM 493 / 503 C3 hydrazide, BODIPY TM FLC3 acylhydrazide, BODIPY TM FL IA, BODIPY TM 530 / 551 IA, Br-BODIPY TM 493 / 503, Cascade Blue® and its derivatives, such as Cascade Blue acetyltriazine, Cascade Blue® cadaverine, Cascade Blue® ethylenediamine, Cascade Blue® hydrazide; Lucifer Yellow and its derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH; anthocyanins and their derivatives, such as indolonyl anthocyanin dyes, benzoindolonyl anthocyanin dyes, pyridiniumyl anthocyanin dyes, thiazolonyl anthocyanin dyes, quinolinelonyl anthocyanin dyes, imidazolonyl anthocyanin dyes; Cy 3, Cy 5; lanthanide chelates and their derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT; europium chelates, terbium chelates; Alexa Fluor® dyes, DyLight® dyes, Atto dyes, LightCycler® Red dyes, CAL Flour dyes; JOE and its derivatives; Oregon Green. TMDyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, diphenylethylene, DEG dyes, NR dyes, near-infrared dyes, and other dyes known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edition, Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd edition, or derivatives thereof, or any combination thereof. Anthocyanin dyes may be present in sulfonated or non-sulfonated forms and consist of two pseudoindole, benzoindoline, pyridinium, thiazoline, and / or quinolinium groups separated by a polymethystylene bridge between two nitrogen atoms.Commercially available anthocyanin fluorophores include, for example, Cy3 (which may contain 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{ ...3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2- [Oxypyrrolidone-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfonyl-1,3-dihydro-2H-indole-2-ylide]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-5-sulfonate), Cy5 (which may contain 1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indole-2-ylide) 1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfonylindole-2-yl)penta-1,3-diene-1-yl)-3,3-dimethyl-3H-indole-1-on-5-sulfonate) and Cy7 (which may contain 1-(5-carboxypentyl) Cy2 is an oxazole derivative rather than a pseudoindole, and the benzo-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0104] In some embodiments, the reporting portion may be a fluorescence resonance energy transfer (FRET) pair, allowing for multiple classifications within a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0105] The terms “connected,” “joined,” “attached,” and variations thereof encompass any type of fusion, binding, adhesion, or association between any combination of compounds or molecules used in a particular procedure that possesses sufficient stability to withstand such fusion. The procedure may include, but is not limited to: transient binding of nucleotides; nucleotide incorporation; deblocking; washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and so on. In some embodiments, such bonds occur intramolecularly, for example, by joining the ends of single-stranded or double-stranded linear nucleic acid molecules together to form a cyclic molecule. In some embodiments, such bonds may occur between combinations of different molecules, or between molecules and non-molecules, including, but not limited to: bonds between nucleic acid molecules and solid surfaces; bonds between proteins and detectable reporter gene portions; bonds between nucleotides and detectable reporter gene portions; and so on. Some examples of the key can be found in Hermanson, G., “Bioconjugate Techniques”, 2nd edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0106] As used herein, the terms “operably linked” and “operably conjoined” or related terms refer to the juxtaposition of components. Juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond conjoining the two components comprises a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can be linked together, wherein the first nucleic acid component can confer function to the second nucleic acid component. For example, a bond between a primer-binding sequence and a target sequence forms a nucleic acid library molecule having a portion that can bind to a primer. In another instance, a transgene (e.g., a nucleic acid encoding a polypeptide or a target nucleic acid sequence) can be linked to a vector, wherein the bond allows the transgene sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgene expression. In some embodiments, the vector includes at least one host cell regulatory sequence, which includes a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signaling sequence, and so on. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of transgene expression. In some cases, components can be non-covalently linked together. Those skilled in the art will understand that components can be operably linked without direct physical linkage.

[0107] The term "adaptor" and related terms refer to an oligonucleotide that can be operatively linked (added to) a target polynucleotide, wherein the adaptor confers function on the co-linked adaptor-target molecule. The adaptor may comprise DNA, RNA, chimeric DNA / RNA, or the like. The adaptor may include at least one ribonucleoside residue. The adaptor may be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. The adaptor may be configured in a linear, stem-loop, hairpin, or Y-shaped form. The adaptor may be of any length, including 4 to 100 nucleotides or longer. The adaptor may have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' and 3' overhangs. The 5' end of a single-stranded adaptor or one strand of a double-stranded adaptor may have a 5' phosphate group or lack a 5' phosphate group. The adaptor may include a 5' tail that has not hybridized to the target polynucleotide (e.g., a tailed adaptor), or the adaptor may be tailless. The adaptor may include a sequence complementary to at least a portion of a primer as described herein (such as an amplification primer, sequencing primer, or capture primer (e.g., a soluble or immobilized capture primer)). The adaptor may include a random sequence or a degenerate sequence. The adaptor may include a random sequence ( For example(NNN), or may lack a random sequence. The adaptor may include at least one inosine residue. The adaptor may include at least one thiophosphate, thiophosphate, and / or phosphoramide bond. The adaptor may include a barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex assays. The adaptor may contain a unique identification sequence (e.g., a unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify the nucleic acid molecule to which the adaptor is appended. The unique identification sequence may include a random sequence ( For example (NNN) or may lack a random sequence. In some embodiments, a unique recognition sequence may be used to increase error correction and accuracy, reduce the false positive variant recognition rate, and / or increase the sensitivity of variant detection. The adaptor may include at least one restriction enzyme recognition sequence, including any combination of any one or more of the following groups: type I, type II, type III, type IV, Hs, or type IIB.

[0108] The terms "universal sequence," "universal adaptor sequence," and related terms refer to a sequence common to two or more polynucleotide molecules within a nucleic acid molecule. For example, an adaptor having the same universal sequence can conjugate with multiple polynucleotides, resulting in a population of co-conjugated molecules carrying the same universal adaptor sequence. Examples of universal adaptor sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or support-fixed capture primers).

[0109] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated herein by reference in its entirety. In the event of any conflict between the terminology used herein and that used in the incorporated references, the terminology used herein shall prevail.

[0110] sequencing system Figure 1 A block diagram of a computer-implemented system 100 for performing sequencing and sequencing analysis according to one or more embodiments disclosed herein is shown. System 100 has a sequencing system 110 including a flow cell 112, a sequencer 114, an imager 116, a data storage device 122, and a user interface 124. Sequencing system 110 may optionally be connected to a cloud 130 (e.g., coupled to a server, computing device, database, etc.). Sequencing system 110 may include one or more of the following: a dedicated processor 118, an integrated circuit (e.g., a field-programmable gate array (FPGA)) 120, and a computer system 126.

[0111] In some embodiments, flow cell device 112 is configured to capture DNA fragments and form DNA sequences for base recall by imaging of flow cell device 112 by imager 116. Flow cell 112 may include as described herein. Figure 7 The support described herein may be a solid support. As disclosed herein, the support may include a surface coating. The surface coating may be a polymer coating as disclosed herein. The surface coating may be disposed on the surface of one or more channels of the flow cell apparatus 112. Different or identical surfaces may be placed on the surface of the inlet of the flow cell apparatus 112.

[0112] In some embodiments, the flow cell device 112 may include a plurality of patches thereon (e.g., portions, locations, regions, segments, etc.) configured to be imaged by the imager 116, and each patch may be divided into a plurality of subpatches. In some embodiments, the subpatches may be organized in a grid. Each subpatch may include a plurality of clusters or communities thereon (e.g., a collection of DNA molecules, such as tandem template molecules disclosed herein). In some embodiments, the flow cell device 112 may include a plurality of patches ranging from about 1 patch to about 2000 patches, about 100 patches to about 1500 patches, or about 200 patches to about 500 patches, including all ranges and subranges therebetween. In some embodiments, each patch may be divided into a plurality of subpatches ranging from about 2 subpatches to about 200 subpatches, about 10 subpatches to about 100 subpatches, or about 20 subpatches to about 50 subpatches, including all ranges and subranges therebetween. In some embodiments, sub-patterns may be organized in a grid that may have M×N sub-patterns. As a non-limiting example, flow cell device 112 may have 424 patches, and each patch may be divided into 6 x 9 grids, thus including 54 sub-patterns. In some embodiments, imager 116 may be configured to acquire one or more images of multiple patches, subsets of multiple patches, and / or subsets of multiple sub-patterns (hereinafter “flow cell images”). Flow cell images disclosed herein may include images comprising signals (e.g., fluorescence levels) of multiple clusters or communities. Flow cell images may include one or more signal patches or one or more signal sub-patterns. In some embodiments, flow cell images may be images including all patches and approximately all signals thereon. Flow cell images may be acquired from channels using imager 116 during (i) an imaging cycle or (ii) a sequencing cycle. In some embodiments, each patch may include millions of communities or clusters. As a non-limiting example, a patch may include about 1 to 10 million clusters or communities. Each community may be a collection of many copies of DNA molecules.

[0113] Further details of the flow cell apparatus 112 and its functional and structural components are presented herein in conjunction with figures. Figure 7 To make it public.

[0114] The sequencer 114 can be configured to flow a reagent mixture onto a flow cell. Such a reagent mixture includes a nucleotide mixture, polymerase, reagents for adding or removing nucleotide cleavage inhibitors between nucleotide addition steps, and other steps for forming DNA molecules suitable for sequencing applications on flow cell 112. Nucleotides may have attached fluorescent elements (also referred to as “tags” or “parts”) that emit light or energy at wavelengths indicating the type of nucleotide. Each type of fluorescent element may correspond to a specific nucleotide base (e.g., A, G, C, T). The fluorescent elements may emit light at visible wavelengths. In some embodiments, the sequencer 114 and flow cell device 112 can be configured to perform various sequencing methods disclosed herein or known in the art, such as affinity-while-sequencing, binding-while-sequencing, or synthesis-while-sequencing.

[0115] For example, each nucleotide base can be assigned a color. Different types of nucleotides can have different colors. For example, adenine (A) can be red, cytosine (C) can be blue, guanine (G) can be green, and thymine (T) can be yellow. The color or wavelength of the fluorescent element for each nucleotide can be selected so that the nucleotides can be distinguished from each other based on the wavelength of the light emitted by the fluorescent element.

[0116] Imager 116 can be configured to capture an image of flow cell 112 after each flow step. In one embodiment, imager 116 may include a camera, such as a CMOS or CCD camera, configured to capture digital images. The camera may be configured to capture images of the wavelengths of fluorescent elements that bind to nucleotides. These images may be referred to as flow cell images.

[0117] In some embodiments, imager 116 may include one or more optical systems disclosed herein. The optical systems may be configured to capture optical signals from the flow cell and generate a corresponding digital image. The digital image may then be used for base calling. In some embodiments, the optical systems and / or flow cell 112 may be coupled to one or more moving mechanisms configured to position the flow cell 112 relative to imager 116. In some embodiments, flow cell 112 and / or sequencer 114 may be coupled to one or more moving mechanisms to position the flow cell 112 relative to one or more dispensers of the sequencer. In some embodiments, sequencer 114 may include a nested group configured to receive and hold one or more flow cells 112 during a sequencing cycle. In some embodiments, the moving mechanism may move the flow cell 112 from the nested group to imager 116 for imaging cycles. In some embodiments, when flow cell 112 is positioned on or near a nested group, the flow cell and / or a portion of the nested group may transition to a first locking (e.g., registration) configuration such that the flow cell 112 is in fluid communication with the nested group. In some embodiments, flow cell 112 may be configured to transition from a locked configuration to an unlocked configuration for movement to imager 116. In some embodiments, flow cell 112 may transition from an unlocked configuration to a second locked configuration, allowing flow cell 112 to be positioned relative to imager 116 (e.g., without mechanical, fluid, or thermal disturbance). In some embodiments, multiple flow cells 112 may be located simultaneously in sequencing system 110. For example, a first flow cell may undergo an imaging cycle while a second flow cell undergoes a sequencing cycle. This allows sequencing system 110 to increase sequencing efficiency and achieve high sample throughput. In some embodiments, imager 116 (e.g., any part of the optical system) may be fluidly isolated from sequencer 114 to prevent fluid exposure to imager 116 and / or improve image quality. In some embodiments, the moving mechanism may include at least one of a movable arm and / or an xy stage. In some embodiments, the processors of sequencing system 110 (e.g., dedicated processor 118, FPG 120, and / or the CPU of computer system 126) may each be configured to run different tasks, allowing sequencing and imaging to be performed simultaneously.

[0118] In one embodiment, images of the flow cell can be captured in groups, where each image in the group is taken at a wavelength or spectrum that matches or includes only one of the fluorescent elements. In another embodiment, images can be captured as a single image capturing all wavelengths of the fluorescent element.

[0119] The resolution of imager 116 controls the level of detail in the flow cell image, including pixel size. This resolution is critical in existing systems because it controls the accuracy of the point-finding algorithm in identifying community centers. In some embodiments, the image resolution of the flow cell image disclosed herein can be from about 10 nanometers (nm) to 900 nm, including all ranges or subranges therein. In some embodiments, the image resolution of the flow cell image can be between about 10 nm and about 900 nm, between about 10 nm and about 500 nm, between about 10 nm and about 200 nm, between about 20 nm and about 500 nm, between about 20 nm and about 200 nm, or any range or subrange therein. One way to improve the accuracy of point-finding is to improve the resolution of imager 116 or to improve the processing of the image captured by imager 116. Detection of community centers in pixels other than those detected by the point-finding algorithm can be performed. Suitable point-finding algorithms are known to those skilled in the art. These methods can allow for improved accuracy in community center detection without increasing the resolution of imager 116. The resolution of the imager 116 can even be lower than that of existing systems with comparable performance, which can reduce the cost of the sequencing system 110.

[0120] The image quality of the flow cell image can control the accuracy of base calling. The imager 116 disclosed herein can increase the accuracy of base calling. Alternatively, processing of the images captured by the imager 116 can produce better image quality, thereby increasing the accuracy of base calling using the system disclosed herein.

[0121] After a base call is executed, a processor (e.g., a dedicated processor 118, FPGA 120, computer system 126, or a combination thereof) may optionally perform additional processing and / or analysis of the base call results. In some embodiments, after a base call is executed, sequencing reads (processed and / or raw) may be output from the system to an external device (e.g., cloud 130 and / or computer system 400). The sequencing reads herein may include forward reads (R1), reverse reads (R2), or both. The sequencing reads herein may be any ordered sequence of A, T, C, and G bases.

[0122] In some embodiments, sequencing reads may be transmitted (e.g., directly or indirectly) to computer system 126 for subsequent analysis, such as, for example, adaptor trimming or phasing.

[0123] These sequencing analysis methods, including preliminary and / or secondary analyses, can advantageously be executed in parallel within computer system 126 without interfering with or delaying the existing sequencing workflow of system 100. The results of the sequencing analysis can be used to generate sequencing results for users. Some or all of the sequencing process operations can advantageously be performed by FPGA, and data can be transferred between the CPU and FPGA to reduce the total operation time of methods that do not use FPGA operations.

[0124] The operations or actions disclosed herein may be performed by a dedicated processor 118, FPGA 120, computing system 126, or a combination thereof. One or more operations or actions (e.g., methods) disclosed herein may be performed by a dedicated processor 118, FPGA 120, computing system 126, or a combination thereof. In some embodiments, which operations or actions will be performed by the dedicated processor 118, FPGA 120, computing system 126, or a combination thereof may be determined based on one or more of the following: computation time for a particular operation, complexity of computation in a particular operation, need for data transfer between hardware devices, and / or a combination thereof.

[0125] The computing system 126 may include one or more general-purpose computers that provide interfaces for running various programs on operating systems such as Windows™ or Linux™. Such operating systems typically offer users a great deal of flexibility.

[0126] In some embodiments, the dedicated processor 118 may not be a general-purpose processor, but rather a custom processor with specific hardware or instructions for executing method steps. The dedicated processor 118 can directly run specific software without an operating system. The lack of an operating system reduces overhead, but at the cost of limited flexibility that the dedicated processor 118 can execute. The dedicated processor 118 can use a custom programming language, which can be designed to operate more efficiently than software running on a general-purpose computer. This can increase the speed of step execution and allow for real-time processing.

[0127] In some embodiments, FPGA 120 can be configured to perform the sequencing analysis methods described herein. The FPGA is programmed to be hardware that will only perform specific tasks. Software steps can be translated into hardware components using a special programming language. Once the FPGA is programmed, the hardware directly processes the digital data provided to it without running software. Instead, the FPGA uses logic gates and registers to process digital data. Because the operating system requires no overhead, the FPGA typically processes data faster than a general-purpose computer. Similar to the dedicated processor 118, this comes at the cost of flexibility.

[0128] The lack of software overhead also allows the FPGA 120 to operate faster than the dedicated processor 118, although this may depend on the exact processing to be performed and the specific FPGA 120 and dedicated processor 118.

[0129] A group of FPGAs 120 can be configured to execute processing steps in parallel. For example, a plurality of FPGAs 120 can be configured to perform processing steps targeting a selected region of an image, a set of images, a sub-tile, or one or more images. In some embodiments, each FPGA 120 can execute a corresponding step or sub-step of a processing step simultaneously, thereby reducing the time required to process data. This can allow near real-time completion of processing steps. Further discussion of the use of FPGAs is provided below.

[0130] Real-time execution of processing steps allows system 100 to use less storage because data can be processed upon receipt rather than stored for subsequent analysis. This offers advantages over conventional systems that store data before processing, which may require more storage and / or access and connectivity to computer systems located in the cloud 130. In some embodiments, data storage device 122 is used to store information used for or obtained from sequencing analysis. For example, DNA sequences determined after adaptor trimming may be stored in data storage device 122. Compressed and / or uncompressed sequencing data may be stored in data storage device 122. FASTQ files may also be stored in data storage device 122.

[0131] User interface 124 can be used by users to operate the sequencing system or access data stored in data storage 122 or computer system 126.

[0132] Computer system 126 can control the general operation of the sequencing system and can be coupled to user interface 124. In some embodiments, computer system 126 can perform one or more steps in sequencing analysis, such as base calling, adaptor trimming, demultiplexing, phasing, etc. In some embodiments, computer system 126 may be structurally and / or functionally similar to computer system 800, such as... Figure 8 As described in more detail below. Computer system 126 may include information configured to store operations of sequencing system 110, such as configuration information, instructions for operating sequencing system 110, or user information. Computer system 126 may be configured to transfer information between sequencing system 110 and cloud 130. For example, computer system 126 may be configured to receive base call results from dedicated processor 118 and / or FPGA and send the base call results to cloud 130 for storage and / or further analysis.

[0133] As discussed above, sequencing system 110 may have a dedicated processor 118, an FPGA 120, or a computer system 126. Sequencing system 110 may use one, two, or all of these elements to perform the necessary processing described above. In some embodiments, when these elements are present together, processing tasks are separated among them. FPGA 120 may be used to perform some or all of the sequencing analysis operations, while computer system 126 may perform other processing functions for sequencing system 110. Processing distribution across dedicated processor 118, FPGA 120, and / or general-purpose processors (e.g., in computer system 126) can enable parallel processing and / or increase the efficiency of processing steps. For example, complex processing steps may be assigned to dedicated processor 118 and / or FPGA 120, while processing for general operation of system 110 is performed by computer system 126.

[0134] Those skilled in the art will understand that various combinations of these elements can allow for different system embodiments that balance processing efficiency and speed with the cost of the processing elements. The cloud 130 can be a network, a server, a remote storage device, or some other remote computing system isolated from the sequencing system 110. Connection to the cloud 130 can allow access to data stored outside the sequencing system 110 or allow for updates to the software within the sequencing system 110.

[0135] Flow pool device In some embodiments, flow cell devices and systems are disclosed herein that can be used to perform or facilitate DNA sequencing analysis. The flow cell devices described herein can be used to immobilize template nucleic acid molecules derived from biological samples and then introduce a repetitive stream of sequencing reagents (e.g., sequencing-by-binding, sequencing-by-synthesis, and / or sequencing-by-affinity) to attach labeled nucleotides or labeled multivalent molecules to specific sites in the template sequence. A series of labeled signals are detected and decoded to reveal the nucleotide sequence of the template molecule, e.g., immobilized and / or amplified nucleic acid template molecules attached to the surface of the flow cell.

[0136] Figure 7 An exemplary embodiment of a flow cell device 200 is shown. The flow cell device 200 may include a support (e.g., as a surface) on which one or more substrates are disposed, a plurality of channels (not shown), an inlet (not shown), and an outlet (not shown). In some embodiments, liquid (280) is dispensed from a dispenser into the flow cell device 200.

[0137] In some embodiments, the support 210 of the flow cell apparatus 200 disclosed herein may be configured to define or receive one or more channels and / or one or more substrates. In some embodiments, the support 210 may be solid, i.e., robust and stable in shape. At least a portion of the support 210 may be transparent, allowing light to pass through from an imager ( Figure 1 The light transmitted by the light source (116) can travel through the transparent part of the support 210 and reach the sample located on the flow cell device 200.

[0138] Support 210 may include or receive one or more substrates. When the flow cell assembly 200 is placed in a sequencing system (e.g., sequencing system 110) for imaging, the top substrate may be closer along the z-direction to the camera of an imager (e.g., imager 116) than the bottom substrate. The bottom substrate may be closer to the xy stage of sequencing system 110 than the top substrate to fix and support the flow cell 200 during sequencing.

[0139] In some embodiments, the flow cell device 200 may further include an intermediate substrate located between the top substrate and the bottom substrate.

[0140] Each substrate may have a predetermined thickness. In some embodiments, any or all substrates may have different thicknesses. In some embodiments, each substrate may have a uniform thickness along the z-direction. In some embodiments, each substrate may have a uniform thickness along the z-direction in at least a portion of the substrate. For example, the portion with a uniform thickness may cover the channel or imaging region of the flow cell device.

[0141] In some embodiments, the top substrate and / or bottom substrate may have a first thickness, and the intermediate substrate may have a second thickness less than the first thickness. In some embodiments, the top and / or bottom substrates may have a thickness of about 0.2 mm to about 5 mm, including all ranges and subranges therebetween. In some embodiments, the top and / or bottom substrates may have a thickness of about 0.6 mm to about 3 mm, including all ranges and subranges therebetween. In some embodiments, the top and / or bottom substrates may have a thickness of about 0.8 mm to about 2 mm, including all ranges and subranges therebetween. In some embodiments, the top and / or bottom substrates may have a thickness of about 0.8 mm to about 1.5 mm, including all ranges and subranges therebetween.

[0142] In some embodiments, the intermediate substrate may have a thickness of about 40 μm to 200 μm, including all ranges and subranges therein. In some embodiments, the intermediate substrate may have a thickness of about 40 μm to 150 μm, including all ranges and subranges therein. In some embodiments, the intermediate substrate may have a thickness of about 40 μm to 70 μm, including all ranges and subranges therein.

[0143] In some embodiments, the intermediate substrate may have a thickness of about 80 μm to 120 μm, including all ranges and subranges therebetween.

[0144] In some embodiments, the substrate may be formed in an elongated shape extending along the y-axis on the surface of the support 210, the substrate being disposed on the support. In some embodiments, the substrate may have various shapes, such as rectangular, square, elliptical, etc.

[0145] In some embodiments, one or more substrates may be planar or substantially planar. In some embodiments, one or more substrates do not contain any curvature perceptible to the naked eye, such that one or more substrates may have planar surfaces. However, in some embodiments, the substrates do not necessarily have to be planar. Alternatively, some or all of one or more substrates may also be curved.

[0146] In some embodiments, the support 210 or one or more substrates may comprise glass or plastic. In some embodiments, the support or one or more substrates may be all glass or all plastic. In some embodiments, the support or one or more substrates may comprise tape, such as pressure-sensitive adhesive (PSA) tape.

[0147] The substrate may define one or more channels 250 of the flow cell device 200 (e.g., extending longitudinally along the top surface of the flow cell device 200). The channels 250 may allow fluids (e.g., liquids or gases) to flow through them.

[0148] The gas described herein may include one type of gas or a combination of different types of gases. In some embodiments, the gas comprises air. The gas may comprise dry air. In some embodiments, the gas comprises one or more inert gases, such as argon or nitrogen. In some embodiments, the gas comprises one or more reactive gases.

[0149] The reagents described herein for sequencing may include liquids. In some embodiments, air bubbles larger than a predetermined size may be removed from the reagents (e.g., to improve the accuracy and / or reproducibility of reactions performed in a flow cell apparatus, enhance the sharpness of images captured by the optical systems of this disclosure, and / or enhance the transmission of fluorescence used during excitation). In some embodiments, a first reagent is configured to wet a first coating on the surface of one or more channels 250. In some embodiments, a second reagent is configured to rewet the surface of one or more channels 250 after the surface has been at least partially dried through an air gap.

[0150] In some embodiments, channel 250 may include a microfluidic channel. In some embodiments, the gap or height of channel 250 defined between the top inner surface and the bottom inner surface of the substrate is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm along the z-direction, or any range therebetween. In some embodiments, the gap or height of channel 250 does not exceed about 100 μm. In some embodiments, the gap or height of channel 250 does not exceed about 60 μm, 50 μm, or 40 μm.

[0151] In some embodiments, the length of the channel 250 along the y-direction is approximately 120 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, or 30 mm, or any range thereof. In some embodiments, the length of the channel 250 does not exceed approximately 100 μm. In some embodiments, the length of the channel 250 does not exceed approximately 80 mm, 75 mm, 70 mm, 65 mm, 60 mm, 55 mm, or 50 mm.

[0152] In some embodiments, the width of the channel 250 along the x-direction is approximately 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 10 mm, 15 mm, 8 mm, or 5 mm, or any range thereof. In some embodiments, the length of the channel 250 does not exceed approximately 10 mm or approximately 7 mm. In some embodiments, the width of the channel 250 does not exceed approximately 40 mm, 35 mm, 30 mm, 25 mm, 20 mm, or 15 mm.

[0153] In some embodiments, the distance between two adjacent channels 250 or the distance from the edge of channel 250 to the edge of flow pool device 200 along the x-axis is about 0.5 mm to about 15 mm. In some embodiments, the distance between two adjacent channels 250 or the distance from the edge of channel 250 to the edge of flow pool device along the x-axis is about 1 mm to about 5 mm.

[0154] In some embodiments, the flow cell device 200 may have more than one channel 250, and all channels 250 may have a uniform size and shape. In some embodiments, the flow cell device 200 may have channels 250 with different sizes and / or shapes.

[0155] The flow cell device 200 may include one or more inlets and one or more outlets. Channels 250 may run from their respective inlets or extend to their respective outlets, thereby allowing fluid communication from the respective inlet to the respective outlet. Sequencing reagents may be introduced into the flow cell device 200 through the inlets, flow through each of the channels 250 and interact with the sample located therein, and then exit from the outlets.

[0156] The size and shape of the inlet and outlet can be customized to suit a variety of sequencing applications.

[0157] The flow cell apparatus, fluid control apparatus, and system may include an open landing area on which reagent 280 can flow. Figure 7 A mobile device with an open landing area is shown.

[0158] In some embodiments, the open landing area may be formed at least partially by a corresponding inlet. The open landing area may be located on the bottom substrate. For example, the open landing area may be formed by a gap in a corresponding region of the intermediate substrate and the top substrate, or by an extension or hole through a corresponding region of the intermediate substrate and the top substrate. In some embodiments, each channel 250 may be coupled to a corresponding open landing area. The open landing area may be fluidly connected to its corresponding channel. In some embodiments, the open landing area is sealed. In some embodiments, the open landing area is open to external or atmospheric gases. In some embodiments, the open landing area is exposed to ambient air or a gaseous atmosphere, such as the gaseous atmosphere surrounding the system and apparatus.

[0159] Nested groups and movable arms In some embodiments, the sequencing system 2000 disclosed herein may include an optical system 2020 including an objective lens; an xy stage 2010 configured to hold a sample to be imaged thereon and to move the sample relative to the objective lens in the xy plane, wherein the sample is fixed on one or more flow cell devices, such as... Figure 2As shown. The sequencing system 2000 may further include a nested group 2050 configured to provide fluid and thermal communication to the sample when one or more flow cell devices are coupled to the nested group 2050; and a moving mechanism 2040 (e.g., a movable arm) configured to move one or more flow cell devices between the xy stage 2010 and the nested group 2050 during sequence execution. The nested group 2050 may include one or more identical nested modules. As used herein, a “nested module” refers to one or more regions of the nested group configured to receive the carrier 2051 and provide fluid and thermal fluid communication with the carrier 2051 and / or the flow cell devices disposed therein.

[0160] In some embodiments, the sequencing system 2000 includes a housing (not shown) configured to hold one or more of the following: an optical system, an xy stage 2010, a nesting assembly 2050, a vector 2051, and a movement mechanism 2040 including a movable arm. In other words, the nesting assembly 2050, the movement mechanism 2040, and the vector 2051 may be positioned within an overall control housing of the sequencing system. Alternatively, one or more of the structural elements, such as the nesting assembly and the movable arm, may be at least partially positioned outside the housing of the sequencing system 2000.

[0161] The xy stage in this document can be equivalent to a translation stage and / or sample stage in a sequencing system. The xy stage can be automatically actuated by an actuator (e.g., a first actuator) with a first spatial accuracy. Various actuators can be used herein, such as motors, cams, or gear systems. In some embodiments, the xy stage can be manually actuated (e.g., manually actuated by a user). In some embodiments, the xy stage, for example... Figure 2 and Figure 27AThe xy stage 2010 includes a base and a movable stage coupled thereon. In some embodiments, the xy stage 2010 can be actuated to move in three-dimensional space (3D). In some embodiments, the xy stage 2010 can be moved. In some embodiments, the xy stage 2010 is actuated to move a predetermined distance, for example, to focus a sample relative to an objective lens. The predetermined distance can be in 3D. In some embodiments, the predetermined distance can be along the xy plane. In some embodiments, the predetermined distance is based on the distance between two adjacent microfluidic channels of the flow cell device. In some embodiments, the xy stage 2010 is actuated to move from a first position (e.g., a predetermined start position) to a second position (e.g., a predetermined stop position). For example, the xy stage 2010 can be actuated to move between two different spatial positions (e.g., a start position and a stop position) such that each of the two microfluidic channels of the same flow cell device can be positioned relative to an objective lens for imaging. In some embodiments, the xy stage 2010 is actuated to move a sample relative to an objective lens for imaging. The XY stage 2010 can move in 3D from a predetermined start position and stop position at various speeds and / or spatial accuracy (e.g., ±0.1 mm, ±0.100 μm). The time required to move the sample to the predetermined position relative to the objective lens can be from 0.5 seconds to 10 seconds, including all ranges and subranges therein. The time required to move the sample to the predetermined position relative to the objective lens can be from 0.5 seconds to 3 seconds, including all ranges and subranges therein. Various speeds can be used to optimize the time and accuracy of sample movement. For example, the speed can be in the range of 0.001 mm / s to 2 m / s, such as between 0.001 mm / s to 100 cm / s, 0.01 mm / s to 50 cm / s, or 0.1 mm / s to 50 mm / s.

[0162] In some embodiments, the xy stage 2010 in the sequencing system 2000 differs from the sample stage and / or translation stage of existing sequencing systems in that, when the flow cell apparatus is fixed on the xy stage 2010, the xy stage 2010 (or vicinity of the xy stage 2010) is not in fluid or thermal communication with one or more flow cell apparatuses. In existing systems, fluid and thermal communication (e.g., fluid or heat exchange) or connections (fluid paths and physical contact) may exist when the sample is fixed on the xy stage, even during sample imaging. Conversely, the sequencing system 2000 disclosed herein lacks fluid and thermal communication (e.g., fluid or heat exchange) or connections (fluid paths and physical contact) when the sample is on the xy stage, and the sample has no fluid and / or thermal communication or connection.

[0163] In some embodiments, the xy stage 2010 can be coupled to a motion mount for precise positioning. For example, magnetic contacts of the motion mount can engage via inductive magnetic force to load a flow cell device and disengage to release that flow cell device for loading the next flow cell device. Compared to existing systems, the sequencing system 2000 with the motion mechanism 2040, nesting group 2050, and xy stage 2010 can produce higher imaging reliability, manufacturing robustness, and flexibility because imaging is completely isolated from fluid and thermal communication. Movement of the xy stage 2010 (e.g., along the x-axis) can be achieved using a cam system, which reduces cost and increases speed and flexibility.

[0164] In some embodiments, the xy stage 2010 may be coupled to one or more sensors to provide feedback on the alignment of the flow cell apparatus relative to the xy stage 2010 and / or the optical system. Various sensors may be used, such as visible light sensors, audio sensors, other optical sensors such as infrared sensors, pressure and / or force sensors. Feedback from one or more sensors may be sent to a processor of the sequencing system 2000 or a processor external to the sequencing system 2000, and this feedback may be used to fine-tune the positioning of the flow cell apparatus relative to the xy stage 2010 and / or the optical system, aligning it with the objective lens for imaging. For example, an audio sensor may be used to detect sound waves reflected from the carrier 2051 to determine the distance between the carrier 2051 and the aligned imaging position (e.g., to determine the difference between the actual position of the carrier 2051 and the desired position of the carrier 2051). As another example, a camera may be used to detect whether the carrier is positioned at the aligned imaging position.

[0165] In some embodiments, the xy stage 2010 may include a hardware processor that is separate from and external to the processor of the sequencing system 2000. The hardware processor of the xy stage 2010 may be connected to one or more sensors, such that the xy stage 2010 is not connected to the processor of the sequencing system (e.g., one or more dedicated processors 118, FPGA 120, processor of computer system 126) to process feedback from the one or more sensors and generate instructions for actuators (e.g., tilting motors) to move the xy stage 2010 to a desired 3D position. The movement may be performed with predetermined precision (e.g., ±0.1 mm, ±0.100 μm). In some embodiments, the xy stage 2010 may include a hardware processor that is separate from and external to the processor of the sequencing system, enabling autonomous or semi-autonomous movement of the xy stage 2010 based on feedback from one or more sensors.

[0166] In some embodiments, the xy stage does not include any hardware processor external to the sequencing system's processor. One or more sensors may communicate with one or more of the processors in the sequencing system 2000 (e.g., one or more dedicated processors 118, FPGA 120, or the processor of computer system 126) to process feedback from the one or more sensors and generate instructions for actuators (e.g., tilting devices, motors) to move the xy stage 2010 to a desired 3D position.

[0167] In some embodiments, the motion of the xy stage can be linear. For example, the xy stage 2010 can move along the x, y, or any linear axis in 3D. In some embodiments, the motion of the xy stage is non-linear, such as rotation in 3D.

[0168] In some embodiments, the optical system 2020 can move along one or more directions in 3D. In some embodiments, the optical system 2020 can be coupled to a motion mount for precise positioning in 3D. In some embodiments, the optical system 2020 can move with predetermined spatial accuracy and a predetermined speed range. For example, the xy stage 2010 can move linearly along the x-axis, and the optical system (e.g., at least the objective lens) can move linearly along the y-axis, and the combination of movement of the optical system and the xy stage 2010 can allow the sample to be positioned at a predetermined location relative to the objective lens for imaging. As another example, the xy stage 2010 or one of the optical systems can move non-linearly, such as rotating about a predetermined starting point, and the xy stage 2010 or the other of the optical system can move linearly, and the combination of movement of the optical system and the xy stage 2010 can allow the sample to be positioned at a predetermined location relative to the objective lens for imaging. The xy stage 2010 and the optical system can move relative to each other in multiple directions to precisely position the sample relative to the objective lens.

[0169] In some embodiments, the movable arm can be automatically actuated by an actuator (e.g., a second actuator) with a second spatial precision. The first actuator, the second actuator, or both can be controlled by one or more hardware processors of the sequencing system 2000 (e.g., processors similar to one or more dedicated processors 118, FPGA 120, or computer system 126). The movable arm can be automatically actuated to move in three dimensions (3D). The movement of the movable arm in each of the three dimensions can have the same or different spatial precision. The movable arm can move at various speeds and spatial precisions. In some embodiments, the speed and precision of the movable arm are determined by the user. The time required to move a sample from the nested group 2050 to the xy stage 2010 can be between 0.5 seconds and 10 seconds, including all ranges and subranges in between. The time required to move a sample from the nested group 2050 to the xy stage 2010 can be between 0.5 seconds and 3 seconds, including all ranges and subranges in between. The movable arm of the moving mechanism 2040 can move at various speeds, for example, speeds can be in the range of 1 mm / s to 2 m / s, including all ranges and subranges therebetween. As a further example, the speed range can be from 1 mm / s to 500 cm / s, between 10 mm / s and 50 cm / s, or between 1 cm / s and 100 cm / s.

[0170] In some embodiments, the movable arm of the moving mechanism 2040 is configured to move one or more flow cell devices between two spatial locations in three dimensions (e.g., between the xy stage 2010 and the nesting group 2050) with a first spatial accuracy. For example, the movable arm may be configured to move the flow cell device from the nesting group 2050 to the xy stage 2010 to image a portion of the flow cell device and / or, after imaging, move the flow cell device from the xy stage 2010 back to the nesting group 2050.

[0171] Figures 27A to 27C Different exemplary embodiments of a sequencing system including the movable arm disclosed herein are shown. Figures 27A to 27CAs shown, the sequencing system may include one or more flow cell devices in a nested group 2050 comprising corresponding flow cell carrier devices 2051 located at a first position relative to an optical system 2020 including an objective lens. The nested group 2050 may be positioned at a distance from the objective lens such that there is separation between the optical system and fluid and / or temperature changes as reagents flow through the flow cell device. In some embodiments, each flow cell carrier 2051 may be coupled to a corresponding xy stage 2010, and a moving mechanism 2040 may pick up the carrier 2051 (e.g., using the corresponding xy stage 2010), move the carrier 2051 to a second position near the objective lens, and position the carrier 2051 at the second position. In some embodiments, the sequencing system may include an xy stage 2010 positioned near the objective lens, and the moving mechanism 2040 may be configured to move the flow cell carrier 2051 onto the xy stage 2010 for imaging.

[0172] The movable arm may include a gripper, for example Figure 27A 2043, which is configured to grasp or otherwise hold carrier 2051. In some embodiments, the movable arm and carrier 2051 can be configured to switch between a coupled state and an uncoupled state, which in Figures 30 to 31 Further description is provided below. In the coupled state, the movable arm can be securely coupled to the flow cell apparatus and its carrier 2051. When coupled, the movable arm can securely move the carrier, for example, between the xy stage 2010 (for imaging) and the nesting group 2050. The movable arm can be configured to transition to an uncoupled state, wherein the carrier 2051 is removed from the movable arm when the flow cell apparatus and its carrier 2051 are at a target position. The gripper 2043 of the movable arm can be configured to receive and / or hold the carrier 2051 when it is in a decoupled position relative to the nesting group or when it is in a decoupled position relative to the xy stage. In some embodiments, the movable arm includes a horizontal arm 2041 (e.g., a rod, support, track, extension, etc.) coupled to a vertical arm 2042 (e.g., a rod, support, track, extension, etc.) (e.g., mechanically supported by the vertical arm), such as... Figure 27A As shown. The horizontal arm 2041, vertical arm 2042, and gripper 2043 can move in 3D relative to the housing of the sequencing system or any other reference point of the sequencing system, such that gripper 2043 can grip the flow cell carrier 2051 and move it between the nested group 2050 and the xy stage 2010. In some embodiments, the horizontal arm 2041 and / or the vertical arm 2042 can be positioned above the nested group 2050, and gripper 2043 can face downwards. Figure 27BAs shown, the movable arm of the moving mechanism 2040 can be coupled to the surface of a sequencing system on which the nested assembly 2050 and the xy stage 2010 are disposed. In some embodiments, the base of the movable arm can be secured to the surface of the sequencing system, and the movable arm 2040 can include one or more joints at which the movable arm can be bent and / or rotated. In some embodiments, the movable arm can include one or more of an upper arm (e.g., a first portion), joints, a forearm (e.g., a second portion), a wrist, and a gripper attached to the forearm, such as... Figure 27B As shown. The upper arm and forearm can be coupled to each other via a first joint, and the gripper can be coupled to the forearm via a second joint. Some or all of the components of the movable arm can move individually relative to the housing of the sequencing system or any other reference point of the sequencing system. In such embodiments, the movable arm can move in 3D with 6 degrees of freedom by combining the movement of one or more of the components of the movable arm.

[0173] Figures 30 to 31 An exemplary embodiment of the movable arm disclosed herein is illustrated. The movable arm may include, for example... Figure 30 The coupling state is shown, in which the movable arm is firmly coupled to the flow cell device 200 and its carrier 2051. When the movable arm is in the coupled state, it can firmly move the carrier 2051, for example, between the xy stage 2010 (for imaging) and the nesting assembly 2050. The movable arm may also include an uncoupled state, such as... Figure 31 As shown, the flow pool device 200 and its carrier 2051 are removed from the movable arm. In some embodiments, the width of the gripper of the movable arm in the coupled state may be greater than the width of the gripper in the uncoupled state. For example, the gripper in the uncoupled state may be in an intermediate position, the width of which is less than the width of the carrier 2051, and may be configured to extend to accommodate the width of the carrier 2051, such that the gripper applies a clamping force on the carrier 2051 to securely hold the carrier 2051 between one or more extensions of the gripper (e.g., fingers, claws).

[0174] In some embodiments, the movable arm includes one or more arm elements that are movable relative to each other. For example... Figures 30 to 31 As shown, the horizontal arm 2041 can support two different forearms 2041 to move at least along a horizontal plane (e.g., the xy plane). In some embodiments, the two different forearms 2041 can also move along a z-axis perpendicular to the horizontal plane.

[0175] In some embodiments, the gripper 2043 may use various mechanisms to grip or otherwise hold the carrier 2051 and move the carrier with it. For example, the gripper 2043 may include, Figures 27A to 27BThe finger-like structure is shown. As another example, gripper 2043 may lack any finger-like structure. In some embodiments, the gripper may use friction, magnetism, or electromagnetic force to grip or otherwise hold the carrier 2051 such that it will move the carrier with it.

[0176] In some embodiments, the gripper is movably attached to a horizontal or vertical arm. In some embodiments, the gripper is configured to move in 3D relative to the housing of the sequencing system or any other reference point of the sequencing system.

[0177] In some embodiments, the moving mechanism 2040 includes multiple tracks, such as... Figure 27C As shown. Each track can connect a carrier 2051 coupled to the nested group 2050 to the xy stage 2010. In some embodiments, the carrier 2051 can be actuated by a motor or other actuator to move along the track between the xy stage 2010 and the nested group 2050. Figures 27A to 27B Compared to the embodiments in the text, the track allows the flow pool device to move in 3D with less flexibility, but it can be simpler, its movement less prone to change, or more compact due to the predetermined layout of the travel track.

[0178] In some embodiments, the moving mechanism 2040 includes one or more belt conveyors that can function similarly to tracks to move the carrier between the xy stage 2010 and the nesting group 2050.

[0179] Figure 25 An exemplary layout of the moving mechanism 2040, nesting group 2050, and optical system (including objectives) relative to carrier 2051 is shown. In some embodiments, carrier 2051 may have at least two docking positions, namely fluid position 2503 and imaging position 2502. Multiple flow cell carriers may be located in a single sequencing system, and multiple carriers may share a common optical system. In fluid position 2503, dispenser 2070 may dispense fluids such as library solutions, samples, reagents, wash buffers, etc., onto an open landing area of ​​the flow cell device disposed in carrier 2051. Dispenser 2070 may include various embodiments of dispensing tips, such as pipette tips, and removable cartridges for holding different reagents therein and in fluid communication with the dispensing tip.

[0180] In some embodiments, the movement of the movable arm (e.g., at the gripper or distal tip of the movable arm) can be linear, such that the carrier 2051 moved by the moving mechanism 2040 also moves linearly. For example, the movable arm can move the carrier 2051 and the sample along the x or y axis in the xy plane. In some embodiments, the movement of the movable arm can be non-linear, such as rotation in 3D, and the carrier 2051 carried by the gripper of the movable arm also moves correspondingly non-linearly.

[0181] The samples disclosed herein may include a variety of samples to be sequenced on a sequencing system. The samples described herein may be 2D or 3D samples, including in situ samples such as cells and / or tissues. Fluid can be extracted from the flow cell apparatus, for example, using an extraction pump, which can be connected via a connector (e.g., Figure 24A and 25 Connection 1006 is connected to carrier 2051 and flow tank. Connection 1006 may be a quick-connect connection with gaskets or other sealing components to prevent leakage and enable sealed fluid communication. In some embodiments, the extraction pump is coupled to the nesting assembly. In some embodiments, the extraction pump is coupled to the flow tank.

[0182] In imaging position 2502, the optical system can perform imaging and may have optional fluid communication with the flow cell device if needed.

[0183] The moving mechanism 2040 can be used to move the flow pool device carrier between docking positions. For example, the moving mechanism 2040 may include a movable arm, a belt conveyor, a roller conveyor, a track system, or a pick-and-place robot.

[0184] In some embodiments, the sequencing system may include a plurality of flow cell carriers, each having its own fluid line 1001 connected to one or more pumps. The moving mechanism 2040 may use algorithms to move and manage the movement of the plurality of carriers 2051 to prevent the plurality of fluid lines 1001 from becoming tangled or otherwise interfering with each other.

[0185] In some embodiments, the xy stage 2010 is configured to be actuated to move to a 3D position with a predetermined spatial accuracy (e.g., a second spatial accuracy), which differs from the spatial accuracy of the moving mechanism 2040. The second spatial accuracy can be higher than the first spatial accuracy of the moving mechanism 2040. In some embodiments, the second spatial accuracy can be 2, 4, 5, 6, 8, 10, 15, or more times the first spatial accuracy. For example, the first spatial accuracy can be 1 mm, and the second spatial accuracy can be 0.05 mm. Having different spatial accuracies can advantageously enable the moving mechanism 2040 to make coarse movements to translate the sample onto the stage and to finely adjust the spatial position relative to the objective lens by the xy stage 2010 for focusing and imaging.

[0186] In some embodiments, the xy stage 2010 may include one or more fasteners configured to removably secure the flow cell assembly thereto. A variety of fasteners may be used herein. As a non-limiting example, the fasteners may include one or more clamps using mechanical force (e.g., snap-fit, friction fit), magnetic force, or electromagnetic force.

[0187] In some embodiments, each carrier 2051 includes a coupling position in which the carrier 2051 is removably attached and secured to the xy stage 2010 by fasteners. In the coupling position, the carrier 2051 is in sealed fluid communication with the xy stage 2010. In some embodiments, each carrier 2051 includes a decoupling position in which the carrier 2051 is removable from the xy stage 2010 and not secured to the xy stage. In the decoupling position, the carrier 2051 is not in fluid communication with the xy stage 2010. Additionally, the carrier 2051 may include one or more valves or other fluid plugs that prevent fluid leakage from within the carrier 2051 to the outside of the carrier 2051.

[0188] In some embodiments, the xy stage 2010 does not include a pump or fluid path that can be connected to and fluidly communicated with a flow cell device coupled thereto. The absence of a pump or fluid path on the xy stage 2010 simplifies the sequencing system and makes it more cost-effective. Furthermore, separating the fluid from the xy stage 2010 and the optics advantageously eliminates or minimizes contamination or leakage during imaging.

[0189] In some embodiments, the xy stage 2010 may include one or more pumps configured to deliver fluid to or extract fluid from the flow cell device when the corresponding carrier 2051 is coupled to the xy stage 2010. In some embodiments, the xy stage 2010 may include one or more valves or plugs that can eliminate or reduce possible leakage from the flow cell device when the flow cell device is coupled to the xy stage 2010.

[0190] In some embodiments, the xy stage 2010 may be configured to control the temperature of the xy stage 2010, the carrier 2051, and / or the flow cell device. In some embodiments, the xy stage 2010 may include a heating device, a cooling device, or both. In some embodiments, the xy stage 2010 may be configured to maintain the temperature of the carrier 2051 and / or the flow cell device at a predetermined temperature and / or within a predetermined temperature range. In some embodiments, the xy stage 2010 may include one or more temperature sensors. The one or more temperature sensors may monitor the temperature of the xy stage 2010, the carrier 2051, and / or the flow cell device and send signals to a processor coupled to the xy stage 2010 to adjust (e.g., the temperature of the heating device and / or cooling device) to achieve the predetermined temperature. Various thermal devices and sensors may be used herein.

[0191] In some embodiments, the xy stage 2010 is coupled to a mechanical decoupler configured to isolate the xy stage 2010 from external vibrations or other mechanical disturbances. Suitable mechanical decouplers are known in the art, and especially Including air-floating platforms.

[0192] Figure 32A An exemplary embodiment of the xy stage 2010 coupled to a mechanical decoupling device or mechanical isolator 2014 is shown. The mechanical isolator 2014 can prevent or otherwise minimize external mechanical disturbances (e.g., vibrations) from reaching the flow cell apparatus 200 and / or the optical system 2020, such that disturbances outside the flow cell apparatus 200 do not interfere with imaging and / or cause undesirable movement of the sample fixed to the flow cell apparatus 200. Various mechanical isolators 2014 can be used to isolate external mechanical disturbances.

[0193] In some embodiments, the xy stage 2010 may include one or more mechanisms for capturing stray light from the optical system 2020. In some embodiments, the xy stage 2010 includes one or more beam collector devices (e.g., Figures 32A to 32B(2503 in the original text) is configured to capture energy from a light source in the optical system, preventing excitation light from returning to the optical system (e.g., an image sensor) and generating noise signals in the flow cell image. In some embodiments, the beam collector device captures excitation light traveling from the optical system through the flow cell device 200 and then to the beam collector device. In some embodiments, the beam collector device captures emitted light from the sample and traveling to the beam collector device. In some embodiments, the beam collector device may include a laser beam collector device. In some embodiments, the beam collector device may include various components configured to absorb photon energy, such as graphite, tungsten, concrete, marble, etc.

[0194] In some embodiments, the optical system 2020 or some structural elements of the optical system (e.g., objectives) may be positioned above the flow cell device 200, such as... Figures 32A to 32B As shown in the figure. In some embodiments, the optical system 2020 may be positioned in various different locations relative to the flow cell device 200, and should not be limited to Figures 32A to 32B The embodiments shown are illustrated. For example, the optical system 2020 or some structural elements of the optical system (e.g., objectives) may be positioned below the flow cell device 200, while the xy stage 2010 may be positioned above or to the side of the flow cell device 200.

[0195] In some embodiments, the xy stage 2010 may include one or more mounting elements 2509 configured to secure the carrier 2051 to the xy stage 2010. Figure 32A A schematic diagram illustrating an exemplary embodiment of the xy stage 2010 and the carrier 2051 is shown. In some embodiments, the mounting element 2509 may include a precision motion mount. In some embodiments, the mounting element 2509 may be configured to precisely maintain the position of the carrier 2051 relative to the xy stage 2010. In some embodiments, the mounting element 2509 may be secured to the xy stage 2010 via various fixing elements such as magnetic latches.

[0196] In some embodiments, mounting element 2509 may include Maxwell coupling or Kelvin coupling. In some embodiments, mounting element 2509 may include precision motion mounting elements. In some embodiments, mounting element 2509 has a total of fewer than 10, 9, 8, 7, 6, or 5 contact points with the xy stage 2010. In some embodiments, mounting element 2509 has a total of fewer than 6, 5, 4, or 3 contact points with the xy stage 2010. In some embodiments, mounting element 2509 includes various geometries that satisfy accurate constraint design principles. For example, in some embodiments, the number of constraint points of mounting element 2509 is equal to the number of degrees of freedom of the flow pool device carrier 2051 to be constrained. As an example, mounting element 2509 may include three elements, each having a spherical surface respectively supported on a concave tetrahedron, a V-groove pointing to the tetrahedron, and a flat surface of the xy stage 2010. The tetrahedron provides three contact points, the V-groove provides two contact points, and the flat surface provides one contact point, for a total of six contact points with the xy stage 2010. As another example, the xy stage 2010 may include three V-grooves, and the mounting element 2509 includes three elements, each having a curved surface configured to be positioned on a corresponding groove. Each of the three V-grooves provides two contact points with the corresponding mounting element 2509, for a total of six contact points. In some embodiments, the mounting element 2509 is configured to control the position of the carrier 2051 with an accuracy from 0.1 nm to 0.1 mm, including all ranges and subranges therein. In some embodiments, the mounting element 2509 is configured to control the position of the carrier with an accuracy from 1 nm to 1 μm, including all ranges and subranges therein. In some embodiments, the mounting element 2509 is configured to control the position of the carrier 2051 with an accuracy from 0.1 μm to 0.1 mm, including all ranges and subranges therein. In some embodiments, the mounting element 2509 is configured to control the position of the carrier 2051 with an accuracy from 1 μm to 0.1 mm, including all ranges and subranges therein.

[0197] In some embodiments, when the flow cell device 200 and its carrier 2051 are in the imaging position, a gap region 2508 may exist between the flow cell device 200 and the xy stage 2010. The gap region 2508 lacks any heater / cooler connection or fluid connection to the flow cell device 200 or its carrier 2051. The gap region 2508 is maintained to reduce mechanical and thermal stresses that may be applied to the flow cell device 200 from heaters / coolers or fluid paths, such that flow cell images can be acquired with less thermal or mechanical disturbance when the flow cell device 200 is in the imaging position. Therefore, the gap region 2508 can improve the image quality of the flow cell image by reducing mechanical disturbances (e.g., vibration, swaying, twisting, etc. of the flow cell device 200) and by reducing thermal disturbances (e.g., non-uniform temperature of the flow cell device 200, excess or stray infrared energy, etc.). The height of the air gap (e.g., along the z-axis) can be predetermined. The height of the air gap can be adjusted by adjusting the relative position of the flow cell device 200 and the xy stage 2010. The air gap can have a height from 0.01 mm to 1 cm, including all ranges and subranges therein. The air gap can have a height from 0.1 mm to 5 cm, including all ranges and subranges therein. The air gap can have a height from 1 mm to 50 cm, including all ranges and subranges therein.

[0198] Figure 32B Another embodiment of the xy stage 2010, also known as the imaging pedestal 2010, is shown. In this embodiment, the xy stage 2010 includes a beam collector 2503. The beam collector 2503 can be in direct contact with the flow cell device 200, the carrier 2051, or both. In some embodiments, the beam collector 2053 can contact the flow cell device 200 and / or the carrier 2051 with a minimum contact force or any force within a predetermined force range. It is not desirable to be bound by theory, but it is considered that a minimum contact force with the beam collector reduces mechanical stress and lowers the likelihood of heat or vibration transmission. Contact with the predetermined force can be maintained by balancing a latching force 2504b to be only slightly greater than a damping force 2504c, both of which can be applied to the beam collector 2503 via various mechanisms. For example, the latching force 2504b can be applied via one or more magnetic latches located in the flow cell device 200, the carrier 2051, and / or the xy stage 2010. The magnetic latch can pull the carrier 2051 thereby toward the flow cell device 200 toward the xy stage 2010 and / or the xy stage 2010 toward the carrier 2051.

[0199] In some embodiments, the beam collector 2503 can generate a damping force 2504c that pulls the beam collector away from the flow cell assembly 200 and the carrier 2051. In some embodiments, the damping force 2504c can be generated using a biasing mechanism (e.g., a spring-deformable member, a flexible material, etc.). In some embodiments, the damping force 2504c can counteract the latching force 2504b. The net effect of the damping force 2504c and the latching force 2504b can be used to minimize the amount of force applied to the flow cell 200. For example, the total force combining the latching force 2504b and the damping force 2504c can be adjusted by selecting various combinations of magnets and springs.

[0200] In some embodiments, the latching force 2504b and the damping force 2504c can be within a predetermined range. In some embodiments, the net force of the combined latching force 2504b and the damping force 2504c can be within a predetermined range. In some embodiments, the net force can be in the range from -0.001 Newtons (N) to 0.001 N (e.g., a positive force upward along the z-axis toward the flow cell device), including all ranges and subranges therein. In some embodiments, the net force can be in the range from -0.01 N to 0.01 N (e.g., a positive force upward along the z-axis toward the flow cell device), including all ranges and subranges therein. In some embodiments, the net force can be in the range from -0.05 N to 0.05 N (e.g., a positive force upward along the z-axis toward the flow cell device), including all ranges and subranges therein. In some embodiments, the net force can be in the range from -0.5 N to 0.5 N (e.g., a positive force upward along the z-axis toward the flow cell device), including all ranges and subranges therein. In some embodiments, the net force can be in the range of -10 N to 10 N (e.g., a positive force along the z-axis toward the flow cell apparatus), including all ranges and subranges therein.

[0201] Figure 26AA schematic diagram of an example of an xy stage 2010 is shown. The xy stage 2010 can be used to secure the carrier 2051 using a coupling mechanism 2012 (e.g., a clamp, fastener, bolt, anchor, rivet, etc.). The xy stage 2010 may include a temperature controller (e.g., a heating and / or cooling device) 2013 optimized to maintain a predetermined temperature for incubation during library preparation, imaging, etc. The xy stage 2010 may include a position adjustment device 2014, such as a tilting device, which positions the carrier 2051 relative to a 3D position with a predetermined accuracy (e.g., a first accuracy) for imaging. In some embodiments, the xy stage 2010 is motorized or otherwise actuated by an actuator 2015 for translation and / or rotation, thereby positioning the carrier 2051 in 3D for imaging. In some embodiments, the xy stage 2010 is actuated via actuator 2015 to move to a position with relatively low but sufficient accuracy to bring the sample to the vicinity of a predetermined location (e.g., an imaging location). The position adjustment device 2014 then fine-tunes the sample position with higher accuracy to the predetermined location with even higher accuracy. The combination of movement of the xy stage 2010 (and the sample) relative to the housing or any other reference point and movement of the sample relative to the xy stage 2010 allows the sample to be precisely and reliably positioned at a predetermined 3D location for imaging.

[0202] In some embodiments, the xy stage 2010, the moving mechanism (e.g., moving mechanism 2040, not shown), or both are actuated to move with relatively low precision, but can bring the sample to the vicinity of a predetermined position, for example, within a few millimeters of the predetermined position. Then, a position adjustment device 2014 located on the xy stage 2010 fine-tunes the sample position with higher precision to the predetermined position with even higher precision. In some embodiments, the combination of movement of the stage (and sample) relative to the housing or any other reference point and movement of the sample relative to the stage allows the sample to be precisely and reliably positioned at a predetermined 3D location for imaging.

[0203] As a non-limiting example, when the carrier 2051 is coupled to the nesting group 2050 for fluid application to the corresponding flow cell, the moving mechanism 2040 can be prevented from moving the carrier 2051 away from the nesting group 2050. The xy stage 2010 can be moved to a position between the optical system 2020 and the nesting group 2050 to await the sample and save some travel distance and time for the moving mechanism to carry the sample. After fluid application is completed, the moving mechanism 2040 can be allowed to move the carrier 2051 to the xy stage 2010, and then the xy stage 2010 can be moved back to the position of the optical system 2020 for imaging the sample. In this particular embodiment, the position adjustment device 2014 can then fine-tune the position of the sample relative to the objective lens for imaging, while the xy stage 2010 and the moving mechanism 2040 can move the sample with lower spatial accuracy, optionally at a potentially higher speed to save time if necessary. This arrangement, which moves with varying spatial precision, can effectively reduce system complexity, save manufacturing costs, improve the robustness of sequencing systems, and reduce the time users spend performing sequencing runs.

[0204] In some embodiments, at least some components of the optical system 2020, the xy stage 2010, or both are mounted on a vibration isolator that mechanically decouples the rest of the instrument for the purpose of improving image quality. This isolation can advantageously allow imaging with minimal motion disturbance and can also facilitate fluid distribution and chemical processing without external motion disturbance. In some embodiments, the flow cell device is coupled to a carrier, and a movable arm is configured to move the carrier and the flow cell device together. When the flow cell device is on the xy stage 2010, the carrier 2051 can remain fixedly coupled to the flow cell device. In some embodiments, the optical system 2020 can move linearly or non-linearly in 3D. For example, the optical system 2020 or at least a portion thereof can move along the x, y, or any other linear axis in 3D. As another example, at least a portion thereof can rotate in 3D about the z-axis or other axes in 3D. For example, the xy stage 2010 can move linearly along the x-axis, and the optical system (e.g., at least the objective lens) can move linearly along the y-axis. The combination of the movement of the optical system and the xy stage 2010 allows the sample to be positioned at a predetermined position relative to the objective lens for imaging. As another example, one of the xy stage 2010 or the optical system 2020 can move non-linearly, such as rotating about a predetermined starting point, and the other of the xy stage 2010 or the optical system 2020 can move linearly. The combination of the movement of the optical system and the xy stage allows the sample to be positioned at a predetermined position relative to the objective lens for imaging.

[0205] In some embodiments, the nested group 2050 of the sequencing system can hold flow cell devices thereon and prepare flow cell devices and samples thereon for imaging during sequencing runs.

[0206] Nested groups can be fluidly connected to a variety of reagents and buffers, such as wash buffers and / or library loading buffers. Fluid communication between nested groups and reagent or solution containers can be via closed fluid paths or open fluid connections (where fluid can be dispensed openly).

[0207] In some embodiments, each nested module of the nested group 2050 includes a thermal and fluid interface with the flow pool device. The nested modules can be as follows: Figure 27A The diagram shows a linear distribution in an array or various other spatial distributions. Thermal incubation can take place within a chamber corresponding to the carrier of each flow cell device. One or more heat sinks can be positioned below each nested module, as shown below. Figure 3 and 4A As shown. The fluid interface may include a dispensing tip that is dispensed into an open orifice of the flow cell device or a plug that is coupled to a corresponding microfluidic channel of the flow cell device.

[0208] Each component in the flow cell apparatus may include an open landing area configured to openly receive fluid from the nested group. The flow cell apparatus includes multiple microfluidic channels, and the nested group is configured to allow fluid communication with each of the multiple microfluidic channels. Fluid communication from the nested group to each channel can be independent, thereby avoiding cross-contamination. For example, different pipette tips can be used to dispense different reagents into different channels via corresponding openings in the nested group. In some embodiments, fluid communication from the nested group to multiple channels can be simultaneous to reduce fluid communication time during sequential runs. In some embodiments, fluid communication from the nested group to multiple channels can be continuous to simplify the communication process and reduce the complexity and cost of the nested group.

[0209] Figures 24A to 24B An exemplary embodiment of the carrier 2051 described herein is shown. In some embodiments, for example, in Figure 24AThe carrier 2051 may include an opening 1004 on its surface configured to receive a flow cell device (not shown). When the flow cell device is removably attached to the carrier 2051, the carrier 2051 may further include one or more fluid paths 1001 in sealed fluid communication with the flow cell device. The carrier 2051 may include a pump 1003 configured to draw or push fluid between the flow cell device and the carrier 2051. The flow cell device carrier may include a port opening 1006 with a connector configured to allow sealed fluid communication between the carrier 2051 and a corresponding nested module when the connector is in the connected position. The carrier 2051 may include a valve 1005 positioned between the fluid path connected to the flow cell device and the port opening of the carrier 1006, wherein the valve is in an open position when the flow cell device is in a coupled position with the carrier (e.g., not during imaging); and in a closed position when the flow cell device is in a decoupled position (e.g., during imaging). The carrier 2051 may include a wire 1007 having an electrical connector 1008 configured to enable electrical communication between the carrier 2051 and a power source. The carrier 2051 may include an onboard battery and / or sensor 1009 electrically connected to one or more of the following components: pump 1003, port 1006, and fastener (e.g., fastener 2012). One or more of the battery, sensor, pump, and fastener may be connected to an external power source via wires using the electrical connector 1008.

[0210] Figures 5A to 5B and Figure 29 An exemplary embodiment of the carrier 2051 is shown. Figure 29 In the illustrated embodiment, carrier 2051 may not include valves or similar structures that function similarly. In this particular embodiment, the open landing area for aspiration and / or the outlet for fluid extraction may be oriented upwards, thus facing away from the direction of gravity. Furthermore, the fluid port (e.g., similar to port 1006) may also face upwards. This arrangement helps prevent fluid loss without requiring valves on carrier 2051. The upward-facing port can function equivalently to the fluid extraction port of fluid port 1006. In this embodiment, gripper coupler 2056 may be located on the carrier to help a gripper (e.g., a gripper with a movable arm) securely grip carrier 2051 as it moves from the fluid station to the imaging station. Gripper coupler 2056 may be mechanical, electromechanical, or magnetic.

[0211] In some embodiments, one or more fasteners may include one or more positioning references. Figure 29 It is shown that one or more lateral positioning references and / or center positioning references can be used to securely position the flow cell relative to the xy stage (not shown) or nested group (not shown).

[0212] In some embodiments, the carrier 2051 may include an electronic chip 2055 embedded within the carrier to identify the flow cell, such as a sequence number or any other unique ID, and any associated sequencing information such as sequencing cycles, as well as assay identifiers. The electronic chip 2055 may be electrically connected to a processor of the sequencing system. The electronic chip 2055 may be electrically connected to a power source, such as a battery on the carrier 2051 or a power outlet external to the carrier.

[0213] In some embodiments, the carrier 2051 may be equipped with one or more sensors to provide feedback on the alignment of the carrier 2051 relative to the xy stage and / or optical system. Various sensors may be used, such as cameras, audio sensors, light sensors, thermal sensors, radio frequency sensors, pressure sensors, and / or force sensors. Feedback from one or more sensors may be sent to the processor of the sequencing system, and this feedback may be used to fine-tune the positioning of the carrier 2051 and the flow cell device therein relative to the xy stage and / or optical system, such that the sample is aligned with the objective lens for imaging. For example, an audio sensor may be used to detect sound waves reflected from the carrier to see how far the carrier is from the aligned imaging position. As another example, a camera may be used to detect whether the carrier is positioned at the aligned imaging position.

[0214] In some embodiments, the vector 2051 may include a hardware processor that is separate from and external to the sequencing system's processor. The hardware processor of the vector 2051 may communicate with one or more sensors, allowing the vector 2051 to move to a desired 3D position (e.g., relative to a nesting group) without needing to communicate with the sequencing system's processor to process feedback from the sensors. The movement may be performed with predetermined precision. In some embodiments, the vector 2051 may include a hardware processor that is separate from and external to the sequencing system's processor, enabling autonomous or semi-autonomous movement of the vector 2051 based on feedback from one or more sensors. A separate processor for the vector 2051 can also help distribute computing power and increase the operating speed of the sequencing system.

[0215] In some embodiments, the carrier 2051 may not include a hardware processor external to the sequencing system's processor. One or more sensors may be connected to the sequencing system's processor to process feedback from the one or more sensors and generate instructions for actuators (e.g., tilting devices, motors) to move the flow cell carrier to a desired 3D position.

[0216] In some embodiments, for example, in Figure 24BIn this configuration, pump 1003 is located outside the carrier 2051 and may or may not move with the carrier 2051. Having an external pump 1003 advantageously allows the carrier 2051 to be simpler, more compact, and lighter, thus making it easier to move by a moving mechanism. In some embodiments, Figure 24A Other components, such as the battery, may be located outside the carrier 2051. In some embodiments, the fluid path may include conduits, such as flexible or semi-flexible conduits, and allow for permanent connection to the pump 1003.

[0217] In some embodiments, the nested group 2050 is configured to enable fluid and thermal communication with one or more flow cell devices. In some embodiments, the nested group is configured to enable fluid and thermal communication with a variety of numbers of flow cell devices. For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 flow cell devices. Figure 2 A nested group configured to hold three flow cell devices is shown.

[0218] Nested group 2050 is configured to hold each of the flow cell devices in an unlocked position, in which the flow cell devices can be removed from the nested group. Nested group 2050 is configured to hold the flow cell devices in a locked position, in which the flow cell devices are spatially registered to the nested group, fixedly coupled to the nested group, and capable of sealed fluid and thermal communication between the nested group and the flow cell devices. Nested group 2050 is configured to transition between the unlocked and locked positions (e.g., via a moving mechanism) when a flow cell device is positioned on or near a portion of the nested group. In some embodiments, nested group 2050 may automatically transition from the unlocked to the locked position when a flow cell device is positioned on or near a portion of the nested group. In some embodiments, sequencing (e.g., reagent flow) may be blocked until the flow cell device transitions to the locked position in nested group 2050, as described below.

[0219] The carrier 2051 is configured to be spatially registered to the nested group in a locked position, thereby spatially registering the corresponding flow cell device therein to the nested group 2050. Figures 5A to 5B A top view and an exploded view of the flow cell device coupled to the corresponding carrier 2051 are shown. In this embodiment, pins embedded in the frame of carrier 2051 are configured to clamp the components of carrier 2051 and / or the flow cell device together. Ribs in the frame of carrier 2051 can push the flow cell device to a registration position relative to carrier 2051, and thus to a registration position relative to nesting group 2050.

[0220] The nested assembly 2050 may include one or more fasteners. The one or more fasteners may use various mechanisms to secure the carrier and the corresponding flow cell device to the nested assembly 2050. The one or more fasteners may use magnetic force. Figure 3 and Figures 4A to 4B An exemplary embodiment of a nested group is shown, wherein one or more fasteners 2052 (see also) Figure 26B The fastener may include multiple magnets. For example, each magnet may be a rare-earth magnet, an electromagnetic coil, or both. In some embodiments, one or more fasteners may be opened or closed. For example, one or more fasteners may be controlled by one or more processors to switch between an open phase and a closed phase. In some embodiments, one or more fasteners may lack a mechanical fastener that can be actuated by a physical actuator such as a motor, clamp, spring, etc. One or more fasteners 2052 may be pushed by pin 2057 to ensure that they are in a locked position, such as... Figure 4B As shown. One or more fasteners, individually or in combination with the fastener, ensure that the flow pool device and its corresponding carrier 2051 are in a locked position relative to the nesting group 2050, such that they are spatially registered to the nesting group 2050, and in such a locked position, sealed fluid communication and thermal communication (e.g., physical contact with a heat sink) are possible.

[0221] In some embodiments, the nesting group 2050 may include one or more fasteners, each fastener configured to removably fasten and secure a corresponding carrier to the nesting group. Various fasteners may be used herein. For example, each fastener may include one or more clamps. The one or more clamps may be actuated by different forces, such as magnetic or electromagnetic forces.

[0222] In some embodiments, the nesting assembly 2050 may include one or more pumps configured to enable fluid communication with a flow cell device when a corresponding carrier 2051 is coupled to the nesting assembly 2050. Each flow cell device carrier may include a coupling position in which the carrier 2051 is removably attached and secured to the nesting assembly 2050 via fasteners and is in sealed fluid communication with the nesting assembly. Each carrier 2051 may include a decoupling position in which the flow cell device carrier can be removed from the nesting assembly 2050. In the decoupling position, the flow cell device carrier is not in fluid communication with the nesting assembly 2050, and fluid within the carrier 2051 is sealed to prevent leakage. The seal may be achieved by one or more valves, for example... Figure 24A 1005 in the middle.

[0223] The nested assembly 2050 may include a 3D moving device configured to position the carrier 2051 relative to the rest of the nested assembly 2050 with a third spatial accuracy, while the carrier 2051 remains coupled to the nested assembly 2050. The third spatial position may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more times the first spatial accuracy of the movable arm.

[0224] In some embodiments, the nested assembly 2050 may include a 3D moving device configured to position the carrier 2051 relative to the dispensing tip while maintaining coupling to the nested assembly with a fourth spatial accuracy. The movement can advantageously fine-tune the position of the open landing area of ​​the flow cell device relative to the dispensing tip to ensure safe and precise fluid application to the flow cell device. The fourth spatial position may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more times the first spatial accuracy of the movable arm. In some embodiments, the 3D moving device may actuate the carrier 2051 to translate in 3D or rotate about an axis in 3D. For example, the 3D moving device may be a piezoelectric stage or actuator that can move along the z-axis, tilt, or pitch within a predetermined angle range.

[0225] In some embodiments, the nesting group 2050 may be equipped with one or more sensors to provide feedback on the alignment of the carrier 2051 relative to the nesting group 2050. Various sensors may be used, such as cameras, audio sensors, light sensors, thermal sensors, radio frequency sensors, pressure sensors, and / or force sensors. Feedback from one or more sensors may be sent to the processor of the sequencing system, and this feedback may be used to fine-tune the positioning of the flow cell device carrier and the flow cell device therein relative to the nesting group 2050, such that the sample is aligned to be firmly coupled to the nesting group 2050. For example, an audio sensor may be used to detect sound waves reflected from the carrier to see how far the carrier 2051 is from its aligned position relative to the nesting group 2050. As another example, a camera may be used to detect whether the carrier 2051 is positioned at its aligned position relative to the nesting group 2050 (and / or dispenser 2070).

[0226] It is worth noting that the sensor may be equipped with one or more structural elements of the sequencing system, such as the xy stage 2010, the moving mechanism, the carrier 2051, and the nesting group 2050, to facilitate efficient and accurate positioning of the carrier 2050 relative to the xy stage 2010 and / or the optical system 2020, and to facilitate efficient and accurate positioning of the carrier 2051 relative to the nesting group 2050.

[0227] Alternatively, the nested assembly 2050 may not include such a 3D movement device for moving the carrier 2051 relative to the rest of the nested assembly 2050 or the dispensing tip. Instead, the dispenser 2070 may be equipped with a 3D movement device configured to move the dispensing tip in 3D while the nested assembly 2050 and the carrier 2051 remain fixed relative to a reference point. Thus, the dispensing tip and the open landing area are aligned relative to each other for precise and secure fluid application.

[0228] Figure 26B A schematic diagram of a nested group 2050 having a nested group module 2051 is shown. The nested group module disclosed herein corresponds to a flow pool device carrier (e.g., carrier 2051) docked within the nested group. Although not shown in this embodiment, more than one nested group module or an equivalent carrier 2051 may be included in the same nested group.

[0229] The nested group can independently secure each carrier 2051 using clamping mechanisms 2052. In some embodiments, the flow pool device may include an open landing area that receives open dispensing by dispenser 2070, such as... Figure 27A As shown. In such embodiments, the nested assembly may include a pump and optional valves to hermetically engage with the flow cell carrier and extract fluid, such as waste, from the flow cell when the carrier is in a coupled position relative to the nested assembly.

[0230] Figure 31 A schematic diagram of a nested group 2050 having a nested group module 2051 is shown. In this particular embodiment, the nested group module, or equivalently referred to herein as the carrier 2051, is in a locked position with respect to the nested group 2050. Figure 31 As shown, in some embodiments, the flow pool device 200 may include a flow pool frame 2092 that surrounds at least a portion of the flow pool device 200, such as one or more edges of the flow pool device 200 and some regions of the flow pool device 200 along the xy plane. The flow pool frame 2092 may include various materials, including but not limited to metals, plastics, silicone, and rubber. In some embodiments, the flow pool frame 2092 is configured to hold one or more gaskets 2093 in position relative to the flow pool device 200. The gaskets 2093 may facilitate sealed fluid communication between the flow pool device 200 and the nesting assembly 2050. In some embodiments, the gaskets 2093 may be connected to a fluid manifold 2095 of the nesting assembly 2050. The fluid manifold 2095 may be in fluid communication with one or more fluid lines 1001 connected to one or more pumps.

[0231] In some embodiments, carrier 2051 may include one or more fasteners or mating features 2094 that can be coupled to mating features in nested assembly 2050 for securely coupling the carrier to nested assembly 2050. Various mechanical elements may be used as fasteners or mating features, such as clamps, clips, bolts, magnets, snap-fit ​​parts, latches, adhesives, etc. Exemplary mating feature 2094 may include alignment pawls and magnetic latches. The mating features may be configured to maintain a reliable (e.g., sealing and alignment) connection from flow pool gasket 2093 to fluid manifold 2095. The mating features may also be configured to maintain a reliable (e.g., sealing and alignment) connection from flow pool device 200 to heater and / or cooler 2053 of nested assembly 2050.

[0232] Similar to the XY stage (not shown), the nested assembly 2050 may include heating and / or cooling devices 2053 optimized for developing fluid and measurement applications for chemical processing. In some embodiments, the nested assembly may further include a temperature controller (e.g., a cooler, a heater, or both). The cooler or heater is configured to control the temperature of each sample fixed to one or more flow cell devices. The cooler or heater may include various sources for heating or cooling the samples. For example, the cooler or heater may include at least one of a fan, microwave, infrared light source, and / or electromagnetic wave source configured to blow cold or hot air. In some embodiments, the temperature controller of the nested assembly may be structurally and / or functionally similar to the temperature controller of the XY stage.

[0233] like Figure 26B As shown, similar to the xy stage, the nested assembly 2050 may include a position adjustment device 2054, such as a tilting device, which positions the carrier 2051 to a 3D position with a predetermined accuracy (e.g., a first accuracy) for imaging. The position adjustment device 2054 described herein is configured to travel along the z-axis, tilt and / or pitch within a predetermined angle range for rapid and accurate multi-axis positioning. The tilt or pitch can be about an axis in 3D, such as about the x, y, or other axes in the xy plane. The tilt or pitch angle can be within a variety of ranges. For example, the tilt or pitch angle can be greater than 0 but less than 0.001 mrad, 0.01 mrad, 0.1 mrad, 1 mrad, 10 mrad, 100 mrad, or 200 mrad.

[0234] In some embodiments, such as those using in situ samples, a fluid station or at least a portion thereof may be located outside the housing of the sequencing system to facilitate sample preparation. In some embodiments, a dispenser (e.g., dispenser 2070) and a nesting module may be located (at least partially) outside the housing of the sequencing system, allowing them to be monitored during sample preparation.

[0235] In some embodiments, the nested assembly 2050 includes one or more reagent containers. In some embodiments, the one or more reagent containers may be disposable. In some embodiments, a moving mechanism (e.g., a movable arm) is configured to immerse the flow cell device into at least some of the one or more reagent containers. For example, as... Figure 6 As shown, the nested assembly can include three different reagent containers for each flow cell device, and a moving mechanism can carry the flow cell device and immerse it in each container for a predetermined duration and temperature. Some of the reagent containers can hold wash buffer to reduce cross-contamination between buffers. For example, the first flow cell device can be immersed in reagent container A and then moved to the xy stage for imaging, then immersed in reagent container B for washing, and then immersed in reagent container C for allowing a second type of reagent to flow into the microfluidic channel.

[0236] In some embodiments, the sequencing system may not include a movement mechanism comprising a movable arm that moves the flow cell device carrier between a nested group (e.g., nested group 2050) and an optical system (e.g., optical system 2020). In such alternative embodiments, the optical system may be movable relative to the nested group to position the flow cell at a predetermined position relative to the objective for imaging. For example, the optical system may be movable linearly along the y-axis, and the nested group or the flow cell carrier decoupled from the nested group may be movable linearly along the x-axis, and their combined movement allows the sample to be positioned for imaging under the objective. As another example, the optical system may be fixed relative to the housing of the sequencing system or another reference point, and the xy stage and / or flow cell device carrier may be movable linearly in the xy plane to allow the sample to be positioned for imaging under the objective. As yet another example, the optical system may be fixed relative to the housing of the sequencing system or another reference point, and the xy stage and / or flow cell device carrier may be movable non-linearly in the xy plane, e.g., rotated about the z-axis, and position the flow cell device carrier relative to the fixed optical system for imaging.

[0237] It is worth noting that the movable arm, xy stage, nested assembly, and flow cell device carrier described herein can be independently actuated into linear or nonlinear movements, such that the combination of their movements can position the sample in 3D relative to the objective lens on the flow cell for imaging. In some embodiments, a dispenser that holds a volume of reagent and dispenses it into the flow cell device moves together with the nested assembly, such that it remains fixed relative to the nested assembly, to ensure that appropriate fluid is applied to the flow cell when the flow cell carrier is coupled to the nested assembly.

[0238] This document discloses sequencing methods using the sequencing system described herein. Sequencing methods may include one or more of the operations disclosed herein. The methods disclosed herein may include some or all of the operations disclosed herein. These operations may be performed in, but are not limited to, the order described herein.

[0239] This method can be performed by one or more hardware processors disclosed herein. In some embodiments, the processor may include one or more of the following: a processing unit, an integrated circuit, or a combination thereof. For example, a processing unit may include a central processing unit (CPU) and / or a graphics processing unit (GPU). An integrated circuit may include a chip such as a field-programmable gate array (FPGA). In some embodiments, the processor may include a computer system 126.

[0240] In some embodiments, some or all of the operations in the method may be performed by an FPGA (e.g., FPGA 120). In an embodiment, when some operations are performed by the FPGA, data following the FPGA's performance of the operations may be transferred from the FPGA to a CPU (e.g., the CPU of computer system 126) so that the CPU can use such data to perform subsequent operations in method 500. Similarly, data may also be transferred from the CPU to the FPGA for processing by the FPGA. In some embodiments, all operations in method 500 may be performed by the CPU. Alternatively, operations performed by the CPU may be performed by other processors (such as dedicated processors) or a GPU. In some embodiments, all operations in method 500 may be performed by an FPGA.

[0241] In some embodiments, the method herein may include: (a) moving a first flow cell device from a nested group to an xy stage, wherein the first flow cell device includes a first sample fixed thereon; (b) moving the xy stage and the first sample thereon relative to an objective of an optical system of the sequencing system; (c) imaging the first sample fixed on the first flow cell device on the xy stage using the optical system; (d) moving the first flow cell device from the xy stage to the nested group; and (e) allowing (e.g., simultaneously allowing) fluid and thermal communication between the nested group and a second flow cell device during one or more of (a)-(d).

[0242] In some embodiments, operations (a) through (d) occur during the same flow cycle of the sequence run. In some embodiments, operation (e) occurs within the same flow cycle as operations (a) through (d). Operations can be repeated in each individual cycle of the sequence run. For example, an operation can be repeated at least once in each cycle. As another example, some of the operations can be repeated more than once in a single cycle. For example, moving the flow cell apparatus to the xy stage can occur within the same cycle after each application of reagent to the flow cell apparatus.

[0243] In some embodiments, some of the operations can occur immediately after the preceding operations are completed to avoid wasting time during the execution of the sequence. For example, immediately after the first reagent is applied to the first sample, even while imaging the second sample, the movable arm can move the first sample to a position close to the xy stage and objective lens, thereby allowing the first sample to be quickly moved to the imaging position after the second sample has been imaged.

[0244] Despite Figure 2 An exemplary embodiment is depicted as a movable arm, but may include more than one movable arm, and each arm may move one or more corresponding flow cell devices to improve system efficiency and throughput and reduce the idle time of the imaging system.

[0245] In some embodiments, the method further includes (f) moving a second flow cell device from a nested group to an xy stage, wherein the second flow cell device includes a second sample fixed thereon; (g) moving the xy stage and the second sample thereon relative to an objective of the optical system of the sequencing system; (h) imaging the second sample fixed on the second flow cell device on the xy stage using the optical system; (i) moving a first flow cell device from the xy stage to a nested group; and (j) allowing (e.g., simultaneously allowing) fluid and thermal communication between the nested group and the first flow cell device during one or more of (f)-(i).

[0246] In some embodiments, the sequencing method further includes repeating operations (a)-(e). Each repeat of operations (a) to (e) may occur within a single cycle or in different flow cycles running across sequences. In some embodiments, the sequencing method further includes repeating operations (f)-(j). Each repeat of operations (f) to (j) may occur within a single cycle or in different flow cycles running across sequences. Each repeat of operations (f) to (j) may occur after operations (a) to (e).

[0247] In some embodiments, the sequencing method further includes repeating operations (a)-(j) a certain number of times for a nested group that holds two different flow cell devices. Similar operations may also be repeated a certain number of times for an additional flow cell device to which the nested group is configured to be held. In some embodiments, the number of repetitions is in the range of 1 to 500, including all ranges and subranges therein. In some embodiments, the number of repetitions corresponds to the number of cycles within a sequence run.

[0248] In some embodiments, allowing fluid communication between the nested assembly and the first flow cell device in operation (f) may include reversibly fastening the flow cell device (e.g., flow cell device 200) to a carrier (e.g., carrier 2051) via one or more fasteners to enable sealed fluid communication between the flow cell device and the carrier; and reversibly fastening the carrier to the nested assembly via one or more fasteners to enable sealed fluid communication between the nested assembly and the carrier and to enable physical contact with the heat dissipation element. The one or more fasteners between the carrier and the flow cell device may include screws, pins, mechanical clamps, or other magnetic structures.

[0249] In some embodiments, the operation (a) of moving the first flow cell device from the nested group to the xy stage is performed within a first flow cycle of the sequence, and the operation (f) of moving the first flow cell device from the nested group to the xy stage is performed within a second flow cycle different from the first flow cycle. In some embodiments, the operation (f) that simultaneously allows fluid and thermal communication between the nested group and the first flow cell device during one or more of (a)-(e) includes rotating one or more fasteners to an open phase to enable sealing fluid communication and physical contact for thermal communication. For example, turning on the power to the electromagnetic coil.

[0250] In some embodiments, operation (f), which allows fluid and thermal communication between the nested group and the first flow cell device during one or more of (a)-(e), may include immersing the first flow cell device in at least some of one or more reagent containers in a predetermined order.

[0251] In some embodiments, each of operations (a)-(b) and (d)-(g) is completed within less than 0.5 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds.

[0252] Computer System Various embodiments of the method may, for example, use one or more computer systems (such as...) Figure 8The computer system 800 shown herein may be used for implementation. For example, one or more computer systems 800 may be used to implement any of the embodiments discussed herein, as well as combinations and sub-combinations thereof.

[0253] Computer system 800 may include one or more hardware processors 404. Hardware processor 804 may be a central processing unit (CPU), a graphics processing unit (GPU), or a combination thereof. Processor 804 may be connected to a bus or communication infrastructure 806.

[0254] Computer system 800 may also include user input / output devices 803, such as a display, keyboard, pointing device, etc., and the computer system can communicate with communication infrastructure 406 through user input / output interface 802. User input / output device 803 can be coupled to... Figure 1 User interface 124.

[0255] One or more processors in processor 804 may be graphics processing units (GPUs). In one embodiment, a GPU may be a processor designed to process dedicated electronic circuitry for mathematically intensive applications. The GPU may have a parallel architecture that is effective for: parallel processing of large blocks of data (such as common mathematically intensive data for computer graphics applications, images, videos, vector processing, array processing, etc.) and cryptography (including brute-force attacks), generating cryptographic hashes or hash sequences, solving partial hash reversal problems, and / or producing results of other proof-of-work computations for applications such as some blockchain-based applications. With its general-purpose computing capabilities on a graphics processing unit (GPGPU), the GPU may be particularly useful, at least in the areas of image recognition and machine learning described herein.

[0256] Additionally, one or more processors in processor 404 may include a coprocessor or other logical implementations for accelerating cryptographic computations or other specialized mathematical functions, including hardware-accelerated cryptographic coprocessors. Such an acceleration processor may further include an instruction set for using the coprocessor and / or other logic to facilitate such acceleration.

[0257] The computer system 800 may also include a data storage device, such as main memory (primary memory) 808, for example, random access memory (RAM). Main memory 808 may include one or more levels of cache. Main memory 808 may store control logic (i.e., computer software) and / or data therein.

[0258] The computer system 800 may also include one or more secondary data storage devices or secondary storage units 810. The secondary storage unit 810 may include, for example, a primary storage drive 812 and / or a removable storage device or drive 814. The primary storage drive 812 may be, for example, a hard disk drive or a solid-state drive. The removable storage drive 814 may be a floppy disk drive, a magnetic tape drive, an optical disk drive, an optical storage device, a magnetic tape backup device, and / or any other storage device / drive.

[0259] The removable storage drive 814 can interact with the removable storage unit 818.

[0260] Removable storage unit 818 may include a computer-usable or readable storage device on which computer software and / or data are stored. The software may include control logic. The software may include instructions executable by hardware processor 804. Removable storage unit 818 may be a floppy disk, magnetic tape, optical disc, DVD, optical storage disc, and / or any other computer data storage device. Removable storage drive 814 may read segments from and / or write to removable storage unit 818.

[0261] Auxiliary storage 810 may include other components, means, parts, tools, or other methods for allowing computer system 800 to access computer programs and / or other instructions and / or data. Such devices, means, parts, tools, or other methods may include, for example, removable storage unit 822 and interface 820. Examples of removable storage unit 822 and interface 820 may include a program box and box interface (such as those found in video game devices), a removable storage chip (such as EPROM or PROM) and associated slot, a memory stick and USB port, a memory card and associated memory card slot, and / or any other removable storage unit and associated interface.

[0262] Computer system 800 may further include a communication or network interface 824. Communication interface 824 enables computer system 800 to communicate and interact with any combination of external devices, external networks, external entities, etc. (individually and collectively referred to by reference numeral 828). For example, communication interface 824 may allow computer system 800 to communicate with external or remote device 828 via communication path 826, which may be wired and / or wireless (or a combination thereof) and may include any combination of LAN, WAN, network, etc. Control logic and / or data may be transmitted to and from computer system 800 via communication path 826. In some embodiments, communication path 826 is a connection to cloud 130, such as... Figure 1 As depicted in the figure. External devices, etc., referred to by reference numeral 828 in the figure may be devices, networks, entities, etc. in the cloud 130.

[0263] The computer system 800 may also be any one or any combination of the following: a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smartphone, a smartwatch or other wearable device, an appliance, a part of the Internet of Things (IoT) and / or an embedded system, to name just a few non-limiting examples.

[0264] It should be understood that the framework described herein can be implemented as a method, process, apparatus, system, or article of art, such as a non-transitory computer-readable medium or device. For illustrative purposes, this framework can be described in the context of a publicly available or at least accessible distributed ledger to an untrusted third party. One example of a modern use case is a blockchain-based system. However, it should be understood that this framework can also be applied to other settings where sensitive or confidential information may need to pass through the hands of an untrusted third party, and this technology is by no means limited to distributed ledger or blockchain applications.

[0265] Computer system 800 can be a client or server, accessing or hosting any application and / or data through any delivery mode, including but not limited to remote or distributed cloud computing solutions; on-premises or field-deployed software (e.g., “field-deployed” cloud-based solutions); “as-a-service” models (e.g., Content as a Service (CaaS), Digital Content as a Service (DCaaS), Software as a Service (SaaS), Managed Software as a Service (MSaaS), Platform as a Service (PaaS), Desktop as a Service (DaaS), Framework as a Service (FaaS), Backend as a Service (BaaS), Mobile Backend as a Service (MBaaS), Infrastructure as a Service (IaaS), Database as a Service (DBaaS), etc.); and / or hybrid models that include any combination of the foregoing instances or other services or delivery modes.

[0266] Any applicable data structures, file formats, and schemas may be derived from, but are not limited to, the following standards: JavaScript Object Notation (JSON), Extensible Markup Language (XML), YAML, Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), MessagePack, XML User Interface Language (XUL), or any other functionally similar representation, either individually or in combination. Alternatively, proprietary data structures, formats, or schemas may be used individually or in combination with known or open standards.

[0267] Any related data, files, and / or databases may be stored, retrieved, accessed, and / or transmitted in human-readable formats such as numerical, text, graphical, or multimedia formats, further including various types of markup languages ​​and other possible formats. Alternatively or in combination with the formats described above, data, files, and / or databases may be stored, retrieved, accessed, and / or transmitted in binary, encoded, compressed, and / or encrypted formats or any other machine-readable format.

[0268] Interfaces or interconnections between various systems and layers can take any number of mechanisms, such as any number of protocols, programming frameworks, layout schemes, or application programming interfaces (APIs), including but not limited to the Document Object Model (DOM), Discovery Service (DS), NSUserDefaults, Web Services Description Language (WSDL), Messaging Exchange Pattern (MEP), Web Distributed Data Exchange (WDDX), Web Hypertext Application Technology Working Group (WHATWG) HTML5 web messaging, Representational State Transfer (REST or RESTful web services), Extensible User Interface Protocol (XUP), Simple Object Access Protocol (SOAP), XML Schema Definition (XSD), XML Remote Procedure Call (XML-RPC), or any other open or proprietary mechanism that can achieve similar functionality and results.

[0269] Such interface connections or interconnections can also utilize Uniform Resource Identifiers (URIs), which may further include Uniform Resource Locators (URLs) or Uniform Resource Names (URNs). Other forms of uniform and / or unique identifiers, locators, or names may be used, alone or in combination with those forms as described above.

[0270] Any of the above protocols or APIs can interface with or be implemented in any programming language (procedural, functional, or object-oriented) and can be compiled or interpreted. Non-limiting examples include C, C++, C#, Objective-C, Java, Scala, Clojure, Elixir, Swift, Go, Perl, PHP, Python, Ruby, JavaScript, WebAssembly, or virtually any other language, as well as any other library or pattern in any type of framework, runtime environment, virtual machine, interpreter, stack, engine, or similar mechanism, including but not limited to Node.js, V8, Knockout, jQuery, Dojo, Dijit, OpenUI5, AngularJS, ExpressJS, Backbone.js, Ember.js, DHTMLX, Vue, React, Electron, and many other non-limiting examples.

[0271] In some embodiments, a tangible non-transitory device or article of manufacture that includes a tangible non-transitory computer-usable or readable medium having control logic (software) stored thereon may also be referred to herein as a computer program product or program storage device. This includes, but is not limited to, computer system 800, main memory 808, secondary memory 810 and removable storage units 818 and 822, and tangible articles of manufacture embodying any combination thereof. When executed by one or more data processing devices (such as computer system 800), such control logic can cause such data processing devices to operate as described herein.

[0272] Based on the teachings contained herein, it will be helpful to those skilled in the art to use methods different from those described herein. Figure 8 It will be apparent from the data processing apparatus, computer system, and / or computer architecture shown herein that embodiments of this disclosure can be made and used. Specifically, embodiments may operate in conjunction with software, hardware, and / or operating system implementations other than those described herein.

[0273] Imaging system Figure 1 The imager 116 may include one or more optical systems 2020. This document further discloses optical system design guidelines and high-performance fluorescence imaging methods and systems that provide improved optical resolution and image quality for fluorescence imaging-based genomics applications. The disclosed optical imaging system designs offer a larger field of view, increased spatial resolution, improved modulation transfer, contrast-to-noise ratio and image quality, higher spatial sampling frequencies, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., images from different fields of view), and improved imaging system duty cycle, thus enabling higher throughput image acquisition and analysis.

[0274] In some cases, improvements in imaging performance, such as for two-sided (flow cell) imaging applications, can be achieved by using an electro-optic phase plate in conjunction with an objective lens to compensate for optical aberrations caused by fluid layers on the upper (near) and lower (far) inner surfaces of the separated flow cell. In some cases, this design approach can also compensate for vibrations introduced, for example, by a motion-driven compensator that moves into or out of the optical path depending on which surface of the flow cell being imaged.

[0275] In some cases, improvements in imaging performance, such as for two-sided (flow cell) imaging applications, involve the use of thick flow cell walls (e.g., wall (or coverslip) thickness > 700 µm) and fluid channels (e.g., fluid channel height or thickness of 50–200 µm), even when using commercially available off-the-shelf objectives, can be achieved by using a tube lens design that corrects for optical aberrations caused by the combination of thick flow cell walls and / or an intervening fluid layer with the objective.

[0276] In some cases, improvements in imaging performance, such as for multi-channel (e.g., two-color or four-color) imaging applications, can be achieved by using multiple tube lenses (one tube lens per imaging channel), where each tube lens design has been optimized for the specific wavelength range used in that imaging channel.

[0277] The exemplary embodiments disclosed herein may include a fluorescence imaging system comprising: a) at least one light source configured to provide excitation light within one or more specified wavelength ranges; b) an objective lens configured to collect fluorescence generated from a specified field of view of a sample plane when the sample plane is exposed to the excitation light, wherein the numerical aperture of the objective lens is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, or at least 0.9, or a numerical aperture value falling within the range defined by at least two of the foregoing values; wherein the working distance of the objective lens is at least 400 micrometers (μm), at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1000 μm, or a working distance falling within the range defined by at least two of the foregoing values; and wherein the area of ​​the field of view is at least 0.1 mm². 2 At least 0.2mm 2 At least 0.5 mm 2 At least 0.7 mm 2 At least 1 mm 2 At least 2 mm 2 At least 3 mm 2 At least 5 mm 2 Or at least 10 mm 2 Or a field of view falling within the range defined by at least two of the aforementioned values; and c) at least one image sensor, wherein fluorescence collected by the objective lens is imaged onto the image sensor, and wherein the pixel size of the image sensor is selected such that the spatial sampling frequency of the fluorescence imaging system is at least twice the optical resolution of the fluorescence imaging system.

[0278] In some embodiments, the numerical aperture may be at least 0.75. In some embodiments, the numerical aperture is at least 1.0. In some embodiments, the working distance is at least 850 μm. In some embodiments, the working distance is at least 1,000 μm. In some embodiments, the working distance is between 500 μm and 7,000 μm, between 100 μm and 5,000 μm, or between 500 μm and 2,000 μm. In some embodiments, the field of view area may be at least 2.5 mm². 2 In some embodiments, the area of ​​the field of view can be at least 3 mm. 2 In some embodiments, the area of ​​the field of view may be 0.5 mm. 2 With 10 mm 2 Between, 1 mm 2 With 10 mm 2 Between, 1 mm 2 With 5 mm 2 Between, 1.5 mm 2 With 5 mm 2 Between, 2 mm 2 With 5 mm 2 Between or 2.5mm 2 With 3 mm 2In some embodiments, the spatial sampling frequency may be at least 2.5 times the optical resolution of the fluorescence imaging system. In some embodiments, the spatial sampling frequency may be at least 3 times the optical resolution of the fluorescence imaging system. In some embodiments, the spatial sampling frequency is between 2 and 10 times the optical resolution, between 2 and 5 times the optical resolution, or between 2.5 and 3 times the optical resolution. In some embodiments, the system may further include an XYZ translation stage, such that the system is configured to acquire a series of two or more fluorescence images in an automated manner, wherein each image in the series is or can be acquired for a different field of view. In some embodiments, the position of the sample plane may be adjusted simultaneously in the X, Y, and Z directions to match the position of the objective focal plane between acquiring images of different fields of view. In some embodiments, the time required for simultaneous adjustment in the X, Y, and Z directions may be less than 0.3 seconds, less than 0.4 seconds, less than 0.5 seconds, less than 0.7 seconds, or less than 1 second, or a time falling within the range defined by any two of the foregoing. In some embodiments, the system further includes an autofocus mechanism configured to adjust the focal plane positioning before acquiring images of different fields of view if an error signal indicates that the positioning difference between the focal plane and the sample plane in the Z direction is greater than a specified error threshold. In some embodiments, the specified error threshold is 100 nm or greater. In some embodiments, the specified error threshold is 50 nm or less. In some embodiments, the system includes three or more image sensors, and the system is configured to image fluorescence in each of three or more wavelength ranges onto different image sensors. In some embodiments, the positioning difference between the focal plane and the sample plane of each of the three or more image sensors is less than 100 nm. In some embodiments, the positioning difference between the focal plane and the sample plane of each of the three or more image sensors is less than 50 nm. In some embodiments, the total time required for repositioning the sample plane, adjusting the focal length (if necessary), and acquiring images is less than 0.4 seconds per field of view. In some embodiments, the total time required for repositioning the sample plane, adjusting the focal length (if necessary), and acquiring images is less than 0.3 seconds per field of view.

[0279] This document also discloses a fluorescence imaging system for biplane imaging of a flow cell, comprising: a) an objective lens configured to collect fluorescence generated within a designated field of view of a sample plane within the flow cell; b) at least one tubular lens positioned between the objective lens and at least one image sensor, wherein the at least one tubular lens is configured to correct an imaging performance metric of the combination of the objective lens, the at least one tubular lens, and the at least one image sensor when imaging the inner surface of the flow cell, and wherein the wall thickness of the flow cell is at least 700 μm (e.g., between 500 µm and 3,000 µm, between 700 µm and 2,000 µm, between 700 µm and 1,500 µm, between 1,000 µm and 2,000 µm, between 2,000 µm and 3,000 µm, or any range thereof), and the gap between the upper inner surface and the lower inner surface is at least 50 μm (e.g., between 50 µm and 1,000 µm, between 50 µm and 200 µm, between 100 µm and 3,000 µm, or any range thereof). (Any range between µm and 500 µm, between 100 µm and 200 µm or in between); wherein for imaging the upper inner surface or the lower inner surface of the flow cell, the imaging performance measure is substantially the same, without moving the optical compensator into or out of the optical path between the flow cell and at least one image sensor, without moving one or more optical elements of the tube lens along the optical path, and without moving one or more optical elements of the tube lens into or out of the optical path.

[0280] In some embodiments, the objective lens may be a commercially available microscope objective lens. The selection of a suitable objective lens will be within the knowledge of those skilled in the art. In some embodiments, the numerical aperture of the commercially available microscope objective lens may be at least 0.3. In some embodiments, the working distance of the objective lens may be at least 700 μm. In some embodiments, the objective lens may be calibrated to compensate for a coverslip thickness (or flow cell wall thickness) of 0.17 mm or greater or less than 0.17 mm. In some embodiments, the optical system may be calibrated to compensate for the coverslip thickness, flow cell thickness, or distance between desired focal planes. In some embodiments, the calibration may be performed by inserting a calibration optics device, such as a lens or optical assembly, into the optical path of the optical system. In some embodiments, the calibration may be performed without inserting a calibration optics device, such as a lens or optical assembly, into the optical path of the optical system. In some embodiments, the fluorescence imaging system may further include an electro-optic phase plate positioned adjacent to the objective lens and between the objective lens and the tube lens, wherein the electro-optic phase plate can provide correction for optical aberrations caused by fluid filling the gap between the upper and lower inner surfaces of the flow cell. In some embodiments, the at least one tubular lens may be a compound lens comprising three or more optical components. In some embodiments, the at least one tubular lens is a compound lens comprising four optical components, which may include one or more of the following: a first asymmetric convex-convex lens, a second convex-planar lens, a third asymmetric concave-concave lens, and a fourth asymmetric convex-concave lens, which may be present in the order listed above or in any alternative order. In some embodiments, the at least one tubular lens is configured to correct the imaging performance metric of the combination of an objective lens, at least one tubular lens, and at least one image sensor when imaging the inner surface of a flow cell with a wall thickness of at least 1 mm. In some embodiments, the at least one tubular lens is configured to correct the imaging performance metric of the combination of an objective lens, at least one tubular lens, and at least one image sensor when imaging the inner surface of a flow cell having a gap of at least 100 μm. In some embodiments, the at least one tubular lens is configured to correct the imaging performance metric of the combination of an objective lens, at least one tubular lens, and at least one image sensor when imaging the inner surface of a flow cell with a gap of at least 200 µm. In some embodiments, the system includes a single objective lens, two tube lenses, and two image sensors, with each of the two tube lenses designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the system includes a single objective lens, three tube lenses, and three image sensors, with each of the three tube lenses designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the system includes a single objective lens, four tube lenses, and four image sensors, with each of the four tube lenses designed to provide optimal imaging performance at a different fluorescence wavelength.In some embodiments, the objective lens or the at least one tube lens is designed to optimize the modulation transfer function over the medium to high spatial frequency range. In some embodiments, the imaging performance metric includes measurements of one or more modulation transfer functions (MTF), defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof, at a specified spatial frequency. In some embodiments, the difference in imaging performance metrics for imaging the upper and lower inner surfaces of the flow cell is less than 10%. In some embodiments, the difference in imaging performance metrics for imaging the upper and lower inner surfaces of the flow cell is less than 5%. In some embodiments, using at least one tube lens provides at least an equivalent or better improvement in imaging performance metrics for bi-lateral imaging compared to a conventional system including an objective lens, a motion-actuated compensator, and an image sensor. In some embodiments, using at least one tube lens provides at least a 10% improvement in imaging performance metrics for bi-lateral imaging compared to a conventional system including an objective lens, a motion-actuated compensator, and an image sensor.

[0281] This article discloses an illumination system for imaging-based solid-phase genotyping and sequencing applications, comprising: a) a light source; and b) a liquid light guide configured to collect light emitted by the light source and deliver it to a designated illumination field on a support surface containing tethered biomacromolecules.

[0282] In some embodiments, the lighting system further includes a focusing lens. In some embodiments, the area of ​​the designated illumination field is at least 2 mm. 2 In some embodiments, the light delivered to the designated illumination field has a uniform intensity across the designated field of view of the imaging system used to acquire an image of the support surface. In some embodiments, the area of ​​the designated field of view is at least 2 mm. 2 In some embodiments, when the coefficient of variation (CV) of light intensity is less than 10%, the light delivered to the designated illumination field has a uniform intensity across the designated field of view. In some embodiments, when the coefficient of variation (CV) of light intensity is less than 5%, the light delivered to the designated illumination field has a uniform intensity across the designated field of view. In some embodiments, the speckle contrast value of the light delivered to the designated illumination field is less than 0.1. In some embodiments, the speckle contrast value of the light delivered to the designated illumination field is less than 0.05.

[0283] Those skilled in the art will understand that, in some cases, the disclosed optical systems, imaging systems, or modules may be standalone optical systems designed for imaging sample or substrate surfaces. In some cases, they may include one or more processors or computers. In some cases, they may include one or more software packages providing instrument control and / or image processing functions. In some cases, in addition to optical components such as light sources (e.g., solid-state lasers, dye lasers, diode lasers, arc lamps, halogen tungsten lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, optical filters, optical bandpass filters, apertures, and image sensors (e.g., complementary metal-oxide-semiconductor (CMOS) image sensors and cameras, charge-coupled device (CCD) image sensors and cameras, etc.), they may also include mechanical and / or optomechanical components, such as XY translation stages, XYZ translation stages, piezoelectric focusing mechanisms, etc. In some cases, they may serve as modules, components, subassemblies, or subsystems of larger systems designed for genomics applications (e.g., gene testing and / or nucleic acid sequencing applications). For example, in some cases, they can serve as modules, components, subassemblies, or subsystems of a larger system, which further includes opaque and / or other environmental control housings, temperature control modules, fluid control modules, fluid dispensing robots, pick-and-place robots, one or more processors or computers, one or more local and / or cloud-based software packages (e.g., instrument / system control software packages, image processing software packages, data analysis software packages), data storage modules, data communication modules (e.g., Bluetooth, WiFi, intranet, or Internet communication hardware and related software), display modules, or any combination thereof.

[0284] Methods for sequencing This disclosure provides methods for sequencing immobilized or non-immobilized nucleic acid template molecules. The method can be operated in a sequencing system 110, such as a sequencer 114. In some embodiments, the immobilized nucleic acid template molecule comprises a plurality of nucleic acid template molecules having one copy of a target sequence. In some embodiments, a nucleic acid template molecule having one copy of the target sequence can be generated by bridging amplification using a linear library molecule. In some embodiments, the immobilized nucleic acid template molecule comprises a plurality of nucleic acid template molecules, each having two or more tandem copies of the target sequence (e.g., tandem template molecules). In some embodiments, a nucleic acid template molecule comprising a tandem template molecule can be generated by rolling circle amplification of a circularized library molecule. In some embodiments, the non-immobilized nucleic acid template molecule comprises a circular molecule. The sequencing method can employ a soluble (e.g., non-immobilized) sequencing polymerase or a sequencing polymerase immobilized to a support. Those skilled in the art will be able to select a polymerase suitable for the various sequencing methods disclosed herein.

[0285] In some embodiments, the sequencing reaction employs a detectably labeled nucleotide analog. In some embodiments, the sequencing reaction employs a two-stage sequencing reaction, which includes a first stage that binds a detectably labeled multivalent molecule (see [link to documentation]). Figures 11 to 15 The sequencing reaction then proceeds to the second stage, which involves the incorporation of nucleotide analogs, as described in further detail below. In some embodiments, the sequencing reaction uses unlabeled nucleotide analogs. In some embodiments, the sequencing reaction uses phosphate-labeled nucleotides.

[0286] In some embodiments, the immobilized tandem template molecule comprises a tandem repeat unit (also referred to as an insert region) of the target sequence and any adaptor sequence. For example, the tandem repeat device comprises: (i) a left universal adaptor sequence (920) having a binding sequence for a first surface primer (e.g., a surface immobilization primer), (ii) a left universal adaptor sequence (940) having a binding sequence for a first sequencing primer (e.g., a forward sequencing primer), (iii) the target sequence (910), (iv) a right universal adaptor sequence (950) having a binding sequence for a second sequencing primer (e.g., a reverse sequencing primer), (v) a right universal adaptor sequence (930) having a binding sequence for a second surface primer (e.g., a surface capture primer), and (vii) a left index sequence (960) and / or a right index sequence (970), which may be sample index sequences. In some embodiments, the tandem repeat unit further comprises a left unique identifier sequence (980) and / or a right unique identifier sequence (990). In some embodiments, the tandem repeat unit further comprises a binding sequence of at least one compacted oligonucleotide. Figure 9 and 10Exemplary embodiments of a single unit of a linear library molecule or a tandem template molecule are shown.

[0287] Immobilized tandem template molecules can self-collapse into compact nucleic acid nanospheres. Binding sites on the tandem template molecules during the RCA reaction contain one or more compacted oligonucleotides that can further compact the size and / or shape of the nanospheres. An increase in the number of tandem repeat units in each tandem template molecule increases the number of sites along the tandem template molecule for hybridization with multiple sequencing primers (e.g., sequencing primers with a universal sequence), which act as multiple initiation sites for the polymerase-catalyzed sequencing reaction. When the sequencing reaction employs detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., multivalent molecules with nucleotide units), the signal intensity increases for each tandem template molecule due to signals emitted by the nucleotides or nucleotide units involved in the parallel sequencing reaction along the tandem template molecule. Multiple portions of a given tandem template molecule can be sequenced simultaneously. Furthermore, multiple binding complexes can form along a specific tandem template molecule, each containing a sequencing polymerase that binds to the template / primer duplex and the multivalent molecule, wherein these multiple binding complexes remain stable without dissociation, resulting in increased retention time, which increases signal intensity and reduces imaging time.

[0288] Methods for sequencing using nucleotide analogs This disclosure provides a method for sequencing any immobilized template molecule described herein, the method comprising the step (a): contacting a sequencing polymerase with (i) a nucleic acid template molecule and (ii) nucleic acid sequencing primers, wherein the contact is performed under conditions suitable for binding the sequencing polymerase to the nucleic acid template molecule hybridizing to the nucleic acid primers, wherein the nucleic acid template molecule hybridizing to the nucleic acid primers forms a nucleic acid double strand. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase capable of binding and incorporating nucleotide analogs. Exemplary polymerases are described, for example, in U.S. Patent No. 11,891,241, the contents of which are incorporated herein by reference in their entirety.

[0289] In some embodiments of the sequencing method, sequencing primers (e.g., first and / or second sequencing primers) include a 3' extendable end or a 3' non-extendable end. In some embodiments, multiple nucleic acid template molecules include amplified template molecules (e.g., template molecules amplified in a clonal manner). In some embodiments, multiple nucleic acid template molecules include one copy of a target sequence. In some embodiments, multiple nucleic acid molecules include two or more tandem copies of a target sequence (e.g., tandem template molecules). In some embodiments, multiple nucleic acid template molecules contain the same target sequence. In some embodiments, each nucleic acid template molecule in the multiple nucleic acid template molecules contains a different target sequence. In some embodiments, multiple nucleic acid primers are in solution or immobilized to a support, such as support 210 of the flow cell apparatus disclosed herein. In some embodiments, when multiple nucleic acid template molecules and / or multiple nucleic acid primers are immobilized to a support, they bind to a first sequencing polymerase to generate multiple immobilized first complex polymerases. In some embodiments, multiple nucleic acid template molecules and / or nucleic acid primers are immobilized to support 10 2 -10 15 Multiple different sites. In some embodiments, the binding of multiple template molecules and nucleic acid primers to multiple first sequencing polymerases generates 10 different sites immobilized on a support. 2 -10 15 Multiple first complex polymerases at different sites. In some embodiments, multiple immobilized first complex polymerases on the support are immobilized to predetermined or random sites on the support. In some embodiments, the multiple immobilized first complex polymerases are fluidly connected to each other to allow a solution of reagents (e.g., enzymes including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow onto the support using the sequencing system 110 described herein, such that the multiple immobilized complex polymerases on the support react with the reagent solution in a large-scale parallel manner.

[0290] In some embodiments, the method for sequencing further comprises step (b): contacting a sequencing polymerase with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the sequencing polymerase which binds to a nucleic acid duplex and suitable for incorporating a polymerase-catalyzed nucleotide, wherein the incorporation of the polymerase-catalyzed nucleotide causes the sequencing primer to extend by one nucleotide. In some embodiments, the sequencing polymerase is contacted with the plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain-terminating portion at a sugar 2' or 3' position. In some embodiments, the chain-terminating portion can be removed from the sugar 2' or 3' position to convert the chain-terminating portion to an OH or H group. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain-terminating portion. In some embodiments, at least one nucleotide of the plurality of nucleotides is labeled with a detectable reporter gene portion (e.g., a fluorophore) that emits a detectable signal. In some embodiments, the detectable reporter gene portion comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleobase. In some embodiments, the fluorophore is attached to a nucleobase with a cleavable / removable adapter. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to a nucleotide may correspond to the identity of a nucleobase (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) and thus to a corresponding complementary nucleobase in the nucleic acid template molecule, allowing for the detection and identification of nucleobases in the nucleic acid template molecule. When the incorporated chain-terminating nucleotide is detectably labeled, step (b) may further include detecting a signal emitted from the incorporated chain-terminating nucleotide. In some embodiments, step (b) further includes identifying the nucleobase of the incorporated chain-terminating nucleotide.

[0291] In some embodiments, the method for sequencing further includes step (c): removing the chain-terminating portion from the incorporated chain-terminating nucleotide to produce an extendable 3'OH group. In some embodiments, step (c) further includes removing a detectable marker from the incorporated chain-terminating nucleotide. In some embodiments, the sequencing polymerase remains bound to a template molecule that hybridizes with a sequencing primer that extends one nucleobase.

[0292] In some embodiments, the method for sequencing further includes step (d): repeating steps (b) and (c) at least once.

[0293] Two-stage method for sequencing nucleic acids This disclosure provides a two-stage method for sequencing any of the immobilized template molecules described herein. In some embodiments, the first stage typically includes conjugating a multivalent molecule with a complex polymerase to form a multivalent complex polymerase and detecting the multivalent complex polymerase.

[0294] In some embodiments, the first stage includes step (a): contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid sequencing primers, wherein the contact is performed under conditions suitable for binding the plurality of first sequencing polymerases with the plurality of nucleic acid template molecules and the plurality of nucleic acid primers, thereby forming a plurality of first complex polymerases, each of the first complex polymerases comprising a first sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In some embodiments, the first polymerase comprises a recombinant mutant sequencing polymerase.

[0295] In some embodiments, sequencing primers comprise oligonucleotides having a 3' extendable end or a 3' non-extendable end. In some embodiments, multiple nucleic acid template molecules comprise amplified template molecules (e.g., template molecules amplified in a clonal manner). In some embodiments, multiple nucleic acid template molecules comprise one copy of a target sequence. In some embodiments, multiple nucleic acid molecules comprise two or more tandem copies of a target sequence (e.g., tandem template molecules). In some embodiments, nucleic acid template molecules among multiple nucleic acid template molecules comprise the same target sequence. In some embodiments, individual nucleic acid template molecules among multiple nucleic acid template molecules comprise different target sequences. In some embodiments, multiple nucleic acid template molecules and / or multiple nucleic acid primers are in solution or immobilized to a support, such as support 210 of the flow cell apparatus disclosed herein. In some embodiments, when multiple nucleic acid template molecules and / or multiple nucleic acid primers are immobilized to a support, they bind to a first sequencing polymerase to generate multiple immobilized first complex polymerases. In some embodiments, multiple nucleic acid template molecules and / or nucleic acid primers are immobilized to support 10 2 -10 15 Multiple different sites. In some embodiments, the binding of multiple template molecules and nucleic acid primers to multiple first sequencing polymerases generates 10 different sites immobilized on a support. 2 -10 15Multiple first complex polymerases at different sites. In some embodiments, multiple immobilized first complex polymerases on the support are immobilized to predetermined or random sites on the support. In some embodiments, the multiple immobilized first complex polymerases are fluidly connected to each other to allow a solution of reagents (e.g., enzymes including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow onto the support using the sequencing system 110 described herein, such that the multiple immobilized complex polymerases on the support react with the reagent solution in a large-scale parallel manner.

[0296] In some embodiments, the method for sequencing further includes step (b): contacting a plurality of first complex polymerases with a plurality of multivalent molecules to form a plurality of multivalent complex polymerases (e.g., a binding complex). In some embodiments, each of the plurality of multivalent molecules includes a nucleus attached to a plurality of nucleotide arms. In some embodiments, each nucleotide arm is attached to a nucleotide (e.g., a nucleotide unit) (e.g., Figures 11 to 15 In some embodiments, the contact in step (b) is performed under conditions suitable for binding complementary nucleotide units of a multivalent molecule to at least two of a plurality of first complex polymerases, thereby forming a plurality of multivalent complex polymerases. In some embodiments, the conditions are adapted to inhibit the complementary nucleotide unit polymerase-catalytic incorporation into the primers of the plurality of multivalent complex polymerases. In some embodiments, the plurality of multivalent molecules comprises at least one having a plurality of nucleotide arms (e.g., Figures 9-14 The plurality of multivalent molecules comprises at least one multivalent molecule having a nucleotide analog (e.g., a nucleotide analog unit) attached to each nucleotide arm, wherein the nucleotide analog includes a chain termination portion located at the 2' and / or 3' positions of the sugar. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule having multiple nucleotide arms, each nucleotide arm having a nucleotide unit lacking a chain termination portion attached. In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules is labeled with a signaling detectable reporter gene portion. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, the contact in step (b) is performed in the presence of at least one noncatalytic cation comprising strontium, barium, and / or calcium.

[0297] In some embodiments, the method for sequencing further includes step (c): detecting a plurality of multivalent-complex polymerases. In some embodiments, detection includes detecting a signal emitted by a multivalent molecule bound to a complex polymerase, wherein complementary nucleotide units of the multivalent molecule bind to a primer but inhibit the incorporation of complementary nucleotide units. In some embodiments, the multivalent molecule is labeled with a detectable reporter gene portion to allow detection. In some embodiments, the labeled multivalent molecule comprises a fluorophore attached to the nucleus, linker, and / or nucleotide units of the multivalent molecule.

[0298] In some embodiments, the sequencing method further includes step (d): identifying the nucleobases of complementary nucleotide units that bind to a plurality of first complex polymerases, thereby determining the identity of the corresponding nucleobases in the nucleic acid template molecule and thus determining the sequence of the nucleic acid template molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter gene portion corresponding to a specific nucleotide unit attached to a nucleotide arm to allow identification of complementary nucleotides (e.g., nucleotide bases adenine, guanine, cytosine, thymine, or uracil) in the nucleic acid molecule that bind to a plurality of first complex polymerases.

[0299] In some embodiments, the method for sequencing further includes step (e): dissociating multiple multivalent complex polymerases and removing multiple first sequencing polymerases and the multivalent molecules bound thereto, while retaining multiple nucleic acid duplexes.

[0300] The second stage of a two-stage sequencing method typically involves nucleotide incorporation. In some embodiments, the method for sequencing further includes step (f): contacting a plurality of retained nucleic acid duplexes from step (e) with a plurality of second sequencing polymerases, wherein the contact is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second complex polymerases, each comprising a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerases comprise recombinant mutant sequencing polymerases.

[0301] In some embodiments, the plurality of first sequencing polymerases in step (a) have an amino acid sequence that is 100% identical to the amino acid sequence of the plurality of second sequencing polymerases in step (f). In some embodiments, the plurality of first sequencing polymerases in step (a) have an amino acid sequence that is different from the amino acid sequence of the plurality of second sequencing polymerases in step (f).

[0302] In some embodiments, the method for sequencing further comprises step (g): contacting a plurality of second complex polymerases with a plurality of nucleotides, wherein the contact is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second complex polymerases, thereby forming a plurality of nucleotide complex polymerases. In some embodiments, the contact in step (g) is performed under conditions suitable for promoting the catalytic incorporation of the bound complementary nucleotide polymerase into a primer of the nucleotide complex polymerase, thereby extending the sequencing primer by one nucleotide. In some embodiments, the incorporation of the nucleotide into the 3' end of the sequencing primer in step (g) comprises a primer extension reaction. In some embodiments, the contact in step (g) is performed in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises native nucleotides (e.g., non-analogous nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides comprises removable 2' and / or 3' chain termination portions. In some embodiments, the plurality of nucleotides comprises non-removable 2' and / or 3' chain termination portions. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is linked to a nucleotide base with a linker that may be cleavable / removable from the base or may not be removable from the base. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleobase (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of the nucleobase.

[0303] In some embodiments, when the multiple nucleotides in step (g) are detectably labeled, the method for sequencing further includes step (h): detecting the labeled nucleotides incorporated into the primers of the nucleotide complex polymerase. In some embodiments, the multiple nucleotides are labeled with a detectable reporter gene portion to allow detection. In some embodiments, when the multiple nucleotides in step (g) are not labeled, the detection in step (h) is omitted.

[0304] In some embodiments, when the multiple nucleotides in step (g) are detectably labeled, the method for sequencing further includes step (i): identifying the bases of the nucleotides incorporated into the primers of the nucleotide complex polymerase based on the detection label, as described above. In some embodiments, the identification of the incorporated nucleotides in step (i) can be used to confirm the identity of complementary nucleotides of the multivalent molecules bound to the multiple first complex polymerases in step (d). In some embodiments, the identification in step (i) can be used to determine the sequence of the nucleic acid template molecule. In some embodiments, when the multiple nucleotides in step (g) are not labeled, the identification in step (i) is omitted.

[0305] In some embodiments, the method for sequencing further includes step (j): removing the chain termination portion from the incorporated nucleotide when step (g) is performed by contacting a plurality of second complex polymerases with a plurality of nucleotides comprising at least one nucleotide having a 2' and / or 3' chain termination portion.

[0306] In some embodiments, the sequencing method further includes step (k): repeating steps (a)-(j) at least once, for example at least 5, 10, 15, 20, 25, 30, 40, 50, 70, 100, 120, 150, or 200 times. In some embodiments, the sequence of the nucleic acid template molecule can be determined by detecting and identifying a multivalent molecule that binds to the sequencing polymerase but is not incorporated into the 3' end of the primer at steps (c) and (d). In some embodiments, the sequence of the nucleic acid template molecule can be determined (or confirmed) by detecting and identifying nucleotides incorporated into the 3' end of the primer at steps (h) and (i).

[0307] In some embodiments of the sequencing method, a plurality of first complex polymerases are bound to a plurality of multivalent molecules to form at least one affinity complex, and the method includes the steps of: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a tandem template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and (b) binding a second nucleic acid primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the second multivalent molecule binds to the second sequencing polymerase, including the first and second binding complexes of the same multivalent molecule forming an affinity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. The tandem template molecule comprises a tandem repeat sequence of a target sequence and at least one universal sequencing primer binding site. The first nucleic acid primer and / or the second nucleic acid primer may bind to the sequencing primer binding site along the tandem template molecule. Exemplary multivalent molecules include Figures 11 to 14 As shown, and an exemplary single unit of the tandem template molecule is as follows Figures 9 to 10 As shown.

[0308] In some embodiments of the sequencing method, the method includes binding a plurality of first complex polymerases to a plurality of multivalent molecules to form at least one affinity complex. The method includes the steps of: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different portions of a tandem template molecule to form at least first and second complex polymerases on the same tandem template molecule; and (b) contacting the plurality of multivalent molecules with at least the first and second complex polymerases on the same tandem template molecule under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first and second complex polymerases, wherein at least a first nucleotide unit of the single multivalent molecule binds to the first complex polymerase, the first complex polymerase containing a first primer that hybridizes to a first portion of the tandem template molecule, thereby forming a first binding complex (e.g., a first ternary complex), and wherein the single multivalent molecule... At least a second nucleotide unit of the molecule binds to a second complex polymerase comprising a second primer that hybridizes to a second portion of the tandem template molecule, thereby forming a second binding complex (e.g., a second ternary complex), wherein the contact is performed under conditions suitable for inhibiting binding, whereby the first and second nucleotide units are polymerase-catalyzed incorporation into the first and second binding complexes, and wherein the first and second binding complexes binding to the same multivalent molecule form an affinity complex; and (c) detecting the first and second binding complexes on the same tandem template molecule; (d) identifying the first nucleotide unit in the first binding complex to determine the sequence of the first portion of the tandem template molecule, and identifying the second nucleotide unit in the second binding complex to determine the sequence of the second portion of the same tandem template molecule. In some embodiments, the plurality of sequencing polymerases comprise any wild-type or mutant sequencing polymerase described herein or known in the art (e.g., in U.S. Patent No. 11,859,241). In some embodiments, the tandem template molecule comprises a tandem repeat sequence of a target sequence and at least one universal sequencing primer binding site. A plurality of nucleic acid primers may bind to the sequencing primer binding site along the tandem template molecule. An exemplary multivalent molecule is shown in Figures 9 to 12 middle.

[0309] sequencing by binding This disclosure provides a method for sequencing any of the immobilized template molecules described herein using a sequencing system, wherein the sequencing method includes a sequencing-by-binding (SBB) procedure using unlabeled chain-terminating nucleotides. In some embodiments, the sequencing-by-binding (SBB) method includes the following steps: (a) causing the initiated template nucleic acid molecule (e.g., having...) The above text(a) Sequentially contacting the single or multiple copies of the target sequence with at least two separate mixtures under conditions stabilizing the ternary complex, wherein each of the at least two separate mixtures contains a polymerase and a nucleotide, thereby sequentially contacting such that the initiated template nucleic acid is contacted with nucleotides of the first, second, and third base types homologous in the template under conditions stabilizing the ternary complex; (b) examining at least two separate mixtures to determine whether a ternary complex has formed; and (c) identifying the next correct nucleotide for the initiated template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first, second, or third base type, and wherein the next correct nucleotide is classified as a nucleotide homolog of the fourth base type based on the absence of a ternary complex in step (b); (d) adding the next correct nucleotide to the primer of the initiated template nucleic acid molecule after step (b) to produce an extended primer; and (e) repeating steps (a) through (d) at least once for the primer template nucleic acid including the extended primer. Exemplary binding sequencing methods are described in U.S. Patent Nos. 10,246,744 and 10,731,141 (the contents of which are incorporated herein by reference in their entirety).

[0310] Methods for sequencing using phosphate-labeled nucleotides This disclosure provides a method for sequencing using a sequencing system, the method using an immobilized sequencing polymerase bound to an unimmobilized template molecule, wherein a sequencing reaction is performed with phosphate-labeled nucleotides. In some embodiments, the sequencing method includes the step (a): providing a support to which multiple sequencing polymerases are immobilized. In some embodiments, the sequencing polymerase includes a persistent DNA polymerase. In some embodiments, the sequencing polymerase includes a wild-type or mutant DNA polymerase, including, for example, Phi29 DNA polymerase. In some embodiments, the support includes multiple individual compartments, and the sequencing polymerase is immobilized to the bottom of the compartments. In some embodiments, the individual compartments include a light-permeable silica bottom. In some embodiments, the individual compartments include a silica bottom configured with a nanophotonic confinement structure comprising pores in a metal coating (e.g., an aluminum coating). In some embodiments, the pore size in the metal coating is, for example, about 70 nm. In some embodiments, the height of the nanophotonic confinement structure is about 100 nm. In some embodiments, the nanophotonic confinement structure includes a zero-mode waveguide (ZMW). In some embodiments, the nanophotonic confinement structure contains a liquid.

[0311] In some embodiments, the sequencing method further includes step (b): contacting multiple immobilized sequencing polymerases with multiple single-stranded circular nucleic acid template molecules and multiple oligonucleotide sequencing primers under conditions suitable for binding of a single immobilized sequencing polymerase to a single single-stranded circular template molecule and suitable for hybridization of a single sequencing primer to a single single-stranded circular template molecule, thereby generating multiple polymerase / template / primer complexes. In some embodiments, a single sequencing primer hybridizes to a universal sequencing primer binding site on a single-stranded circular template molecule.

[0312] In some embodiments, the sequencing method further includes step (c): contacting a plurality of polymerase / template / primer complexes with a plurality of phosphate-ester-tagged nucleotides, each phosphate-ester-tagged nucleotide comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and a phosphate ester chain comprising 3-20 phosphate ester groups, wherein the terminal phosphate ester group is attached to a detectable reporter gene portion (e.g., a fluorophore). The first, second, and third phosphate ester groups may be referred to as α, β, and γ phosphate ester groups. In some embodiments, the specific detectable reporter gene portion attached to the terminal phosphate ester group corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleobases. In some embodiments, the plurality of polymerase / template / primer complexes are contacted with the plurality of phosphate-ester-tagged nucleotides under conditions suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase is capable of binding to a complementary phosphate-ester-tagged nucleotide and incorporating a complementary nucleotide to a nucleotide in the template molecule. In some embodiments, the polymerase-catalyzed nucleotide incorporation reaction cleaves between α-phosphate groups and β-phosphate groups, thereby releasing a polyphosphate chain linked to a fluorophore.

[0313] In some embodiments, the sequencing method further includes step (d): detecting a fluorescent signal emitted by a phosphate-labeled nucleotide, which is bound by a sequencing polymerase and incorporated into the end of a sequencing primer. In some embodiments, step (d) further includes identifying the phosphate-labeled nucleotide bound by the sequencing polymerase and incorporated into the end of the sequencing primer.

[0314] In some embodiments, the sequencing method further includes step (d): repeating steps (c)-(d) at least once. In some embodiments, the sequencing method using phosphate-labeled nucleotides can be performed according to the methods described in U.S. Patent Nos. 7,170,050, 7,302,146, and / or 7,405,281.

[0315] Sequencing polymerase This disclosure provides methods for sequencing nucleic acid molecules, wherein any sequencing method described herein employs at least one type of sequencing polymerase and multiple nucleotides, or employs at least one type of sequencing polymerase and multiple nucleotides and multiple multivalent molecules. In some embodiments, the sequencing polymerase is capable of incorporating a complementary nucleotide to a nucleotide in a template molecule. In some embodiments, the sequencing polymerase is capable of binding to a complementary nucleotide unit of a multivalent molecule to a nucleotide in a template molecule. In some embodiments, the multiple sequencing polymerases include recombinant mutant polymerases.

[0316] Examples of suitable polymerases for sequencing nucleotides and / or multivalent molecules include, but are not limited to: Klenow DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaea; Yellowstone subterranean archaea provisional species; Hadesarchaea archaea; Archaea of ​​the phylum Gastromycota; Archaea of ​​the class Thermoplasmata; Thermococcus polymerases, such as Thermococcus litoralis; Bacteriophage T7 DNA polymerase; Human α, δ, and ε DNA polymerases; Bacteriophage polymerases, such as T4, RB69, and phi29 Bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; E. coli DNA polymerases IIIα and ε; 9°N polymerase; reverse transcriptases, such as HIV M or O type reverse transcriptases; avian myeloblastoma virus reverse transcriptases; Moloney mouse leukemia virus (MMLV) reverse transcriptases; or telomerases. Other non-limiting examples of DNA polymerases include those from various archaea genera (such as *Aerothermos*, *Archaeglobus*, *Thiococcus*, *Firecoccus*, *Firecoccus*, *Firecoccus*, *Thermophyton*, *Staphylococcus*, *Thermophyton*, etc., or variants thereof), including such polymerases as known in the art, such as 9°N, VENT®, DEEP VENT®, THERMINATOR. TM Polymerases such as Pfu, KOD, Pfx, Tgo, and RB69 are described. Other polymerases are described, for example, in U.S. Patent No. 11,891,241, the contents of which are incorporated herein by reference in their entirety.

[0317] Nucleotides This disclosure provides methods for sequencing nucleic acid template molecules, wherein any of the sequencing methods described herein employs at least one nucleotide or at least one or more nucleotides. A nucleotide comprises a base, a sugar, and at least one phosphate ester group. In some embodiments, at least one nucleotide of the plurality of nucleotides comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate ester groups (e.g., 1 to 10 phosphate ester groups). The plurality of nucleotides may comprise at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides may comprise a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, at least one nucleotide of the plurality of nucleotides is not a nucleotide analog. In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a nucleotide analog.

[0318] In some embodiments, in any of the methods described herein for sequencing nucleic acid molecules, at least one nucleotide of a plurality of nucleotides comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, at least one nucleotide of the plurality is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, including phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0319] In some embodiments, in any of the methods described herein for sequencing nucleic acid molecules, at least one nucleotide of a plurality of nucleotides includes a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion may inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides containing the chain-terminating portion into the nascent chain during primer extension reactions. In some embodiments, the chain-terminating portion is linked to a 3' sugar localization, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain-terminating portion may be removed / cleaved from the 3' sugar localization to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide group, amine group, amide group, ketone group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, silyl, or acetal group. In some embodiments, the chain-terminating portion may be cleaved / removed from the nucleotide, for example by reacting the chain-terminating portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl groups of the chain-terminating portion may be cleaved with tetra(triphenylphosphine)-palladium(O) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl groups of the chain-terminating portion may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, sulfur, and disulfide groups of the chain-terminating portion may be cleaved with phosphine or with a thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the carbonate group of the chain-terminating portion may be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating portion of the urea and silyl group can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the chain-terminating portion can be cleaved / removed with nitrous acid. In some embodiments, the chain-terminating portion can be cleaved / removed using a solution comprising nitrite (e.g., a combination of nitrite and an acid such as acetic acid, sulfuric acid, or nitric acid). In some other embodiments, the solution may include an organic acid.

[0320] In some embodiments, at least one nucleotide of the plurality of nucleotides includes a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion includes an azide, an azide group, or an azidemethyl group. In some embodiments, the chain-terminating portion includes a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the chain-terminating portion azide, azide group, and an azidemethyl group can be cleaved / removed by a phosphine compound. In some embodiments, the phosphine compound includes a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP) or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent includes 4-dimethylaminopyridine (4-DMAP). In some embodiments, a chain-terminating portion comprising one or more of 3'-O-amino, 3'-O-aminomethyl, 3'-O-methylamino, or derivatives thereof can be cleaved with nitrous acid using a mechanism utilizing nitrous acid or using a solution containing nitrous acid. In some embodiments, a chain-terminating portion comprising one or more of 3'-O-amino, 3'-O-aminomethyl, 3'-O-methylamino, or derivatives thereof can be cleaved using a solution containing nitrite. In some embodiments, the nitrite can be combined with or contacted with an acid such as acetic acid, sulfuric acid, or nitric acid. In some additional embodiments, the nitrite can be combined with or contacted with an organic acid (e.g., formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, etc.). In some embodiments, the chain-terminating portion comprises a 3'-acetal portion that can be cleaved with a palladium deblocking agent (e.g., Pd(0)).

[0321] In some embodiments, the nucleotide includes a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'- Uncle Butyl, 3'-fluorenylmethoxycarbonyl, 3' Uncle Butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, 3-O-benzyl and 3'-O-benzyl, 3-acetal moiety or derivative thereof.

[0322] In some embodiments, the plurality of nucleotides comprises at least one nucleotide labeled with a detectable reporter gene portion. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base with a cleavable / removable linker. In some embodiments, at least one nucleotide in the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleotide base.

[0323] In some embodiments, a cleavable linker on a nucleotide base (e.g., a linker that attaches a nucleotide to a detectable reporter gene portion) comprises a cleavable portion including: alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide group, amine group, amide group, ketone group, isocyanate group, phosphate group, thioyl, disulfide group, carbonate group, urea group, or silyl. In some embodiments, the cleavable linker can be cleaved / removed from the nucleobase by reacting the cleavable portion with a chemical agent, pH change, light, or heat. In some embodiments, the cleavable portions of alkyl, alkenyl, alkynyl, and allyl can be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the cleavable portions of aryl and benzyl can be cleaved with H2 Pd / C. In some embodiments, cleavable amines, amides, ketones, isocyanates, phosphates, sulfur, and disulfides may be cleaved with phosphine or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, cleavable carbonates may be cleaved with potassium carbonate (K₂CO₃) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, cleavable ureas and silyl groups may be cleaved with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0324] In some embodiments, the cleavable linker on the nucleotide base comprises a cleavable portion including an azide, an azide group, or an azide methyl group. In some embodiments, the cleavable portion of the azide, azide group, and azide methyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bis(sulfotriphenylphosphine) (BS-TPP) or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0325] In some embodiments, in any method described herein for sequencing nucleic acid molecules, the chain termination portion (e.g., at the 2' and / or 3' positions of the sugar) and the cleavable linker on the nucleotide base have the same or different cleavable portions. In some embodiments, the chain termination portion linked to the base (e.g., at the 2' and / or 3' positions of the sugar) and the detectable reporter gene portion can be chemically cleaved / removed with the same chemical agent. In some embodiments, the chain termination portion linked to the base (e.g., at the 2' and / or 3' positions of the sugar) and the detectable reporter gene portion can be chemically cleaved / removed with different chemical agents.

[0326] Multivalent molecules This disclosure provides methods for sequencing nucleic acid template molecules, wherein any of the sequencing methods described herein employs at least one monovalent molecule. In some embodiments, the multivalent molecule comprises a plurality of nucleotide arms attached to the nucleus and having any configuration, including starburst, spiral ladder, or bottle brush configurations (e.g., Figure 11 In some embodiments, the multivalent molecule comprises: (1) a nucleus; and (2) a plurality of nucleotide arms comprising (i) a nucleus attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the nucleus is attached to the plurality of nucleotide arms, the spacer is attached to the linker, and the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate ester group, and the linker is attached to the nucleotide unit via a base. In some embodiments, the linker comprises an aliphatic chain or an oligomeric glycol chain, wherein both linker chains have 2 to 6 subunits. In some embodiments, the linker further comprises an aromatic portion. An exemplary nucleotide arm is shown in Figure 15 In the middle. An exemplary multivalent molecule is shown in Figures 11 to 14 In the middle. An exemplary spacer is shown in Figure 16 (At the top) and an exemplary connector is shown in Figure 16 (Bottom) and Figure 17 In the middle. An exemplary nucleotide attached to the adapter is shown in Figures 18 to 21 middle. Figure 22An exemplary biotinylated nucleotide arm is shown in the figure.

[0327] In some embodiments, the multivalent molecule includes a nucleus attached to a plurality of nucleotide arms, wherein the plurality of nucleotide arms have the same type of nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0328] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, each arm comprising a nucleotide unit. The nucleotide unit comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of multivalent molecules may comprise a type of multivalent molecule having a type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of multivalent molecules may comprise a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture comprise nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0329] In some embodiments, the nucleotide unit comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically linked to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analog, which includes phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0330] In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, wherein each nucleotide arm comprises a nucleotide unit that is a nucleotide analog having a chain termination portion (e.g., a blocking portion) at a sugar 2' position, a sugar 3' position, or both sugar 2' and 3' positions. In some embodiments, the nucleotide unit comprises a chain termination portion (e.g., a blocking portion) at a sugar 2' position, a sugar 3' position, or both sugar 2' and 3' positions. In some embodiments, the chain termination portion may inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain termination portion is linked to a 3' sugar position, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain termination portion may be removed / cleaved from the 3' sugar position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide group, amine group, amide group, ketone group, isocyanate group, phosphate group, thioyl, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the chain-terminating portion may be cleaved / removed from the nucleotide unit, for example by reacting the chain-terminating portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl groups of the chain-terminating portion may be cleaved with tetrakis(triphenylphosphine)-palladium(O) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl groups of the chain-terminating portion may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, sulfur, and disulfide groups of the chain-terminating portion may be cleaved with phosphine or with thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating carbonate portion can be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating urea and silyl groups can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0331] In some embodiments, the nucleotide unit comprises a chain termination portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain termination portion comprises an azide, an azide group, or an azidemethyl group. In some embodiments, the chain termination portion comprises a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the chain termination portion azide, azide group, and an azidemethyl group can be cleaved / removed by a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP) or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0332] In some embodiments, the nucleotide unit includes a chain termination portion, which is free from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'- Uncle Butyl, 3'-fluorenylmethoxycarbonyl, 3' Uncle Butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate and 3-O-benzyl or their derivatives.

[0333] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the nucleotide arms include spacer regions, linkers, and nucleotide units, and wherein the core, linkers, and / or nucleotide units are labeled with a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, all fluorophores on a single multivalent molecule are identical, i.e., have the same excitation and emission spectra. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotide bases.

[0334] In some embodiments, at least one nucleotide arm of the multivalent molecule has a nucleotide unit attached to a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion is attached to a nucleotide base. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to the identity of the nucleotide base of the nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotide bases.

[0335] In some embodiments, the core of the multivalent molecule comprises an avidin-like or streptavidin-like moiety, and the core attachment portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type moiety comprising avidin protein, and any derivative, analog, or other non-natural form of avidin that can bind to at least one biotin moiety. Other forms of the avidin moiety include natural and recombinant avidin and streptavidin, as well as derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety comprises a deglycosylated form of avidin, derived from... chain Molds (For example, Streptomyces avidin Streptavidin produced by bacteria, and its derivative forms, such as N-acylavidin, including N-acetyl, N-phthaloyl, and N-succinylavidin, as well as commercially available products EXTRAVIDIN® and CAPTAVIDIN. TM , NEUTRAVIDIN and NEUTRALITE AVIDIN.

[0336] In some embodiments of the sequencing method described herein, the method may include forming a binding complex comprising (i) a polymerase, a nucleic acid template molecule that forms a double strand with the primer, and nucleotides, or the binding complex comprising (ii) a polymerase, a nucleic acid template molecule that forms a double strand with the primer, and nucleotide units of a multivalent molecule. In some embodiments, the residence time of the binding complex is greater than about 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, or 1 second. In some embodiments, the residence time of the binding complex is greater than about 0.1 to 0.25 seconds, or about 0.25 to 0.5 seconds, or about 0.5 to 0.75 seconds, or about 0.75 to 1 second, or about 1 to 2 seconds, or about 2 to 3 seconds, or about 3 to 4 seconds, or about 4 to 5 seconds, and / or the method is performed at or may be performed at temperatures of 15°C or higher, 20°C or higher, 25°C or higher, 35°C or higher, 37°C or higher, 42°C or higher, 55°C or higher, 60°C or higher, 72°C or higher, or 80°C or higher, or within the range defined by any of the foregoing. The binding complex (e.g., a ternary complex) may remain stable before being subjected to conditions that cause dissociation between the polymerase, template molecule, primer and / or nucleotide unit or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with any or any combination of detergent, EDTA, and / or water. In some embodiments, this disclosure provides a method in which the binding complex is deposited onto, attached to, or hybridized to a surface that exhibits a contrast-to-noise ratio greater than 20 in a detection step. In some embodiments, this disclosure provides a method in which contact is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide unit is complementary to the next base of the template nucleic acid, and destabilize the binding complex when the nucleotide or nucleotide unit is not complementary to the next base of the template nucleic acid.

[0337] Compacted oligonucleotides This disclosure provides methods for preparing nucleic acid template molecules for sequencing, such as the tandem template molecules described herein, which comprise compacted oligonucleotides. Suitable compacted oligonucleotides are known in the art and are described, for example, in WO2024040058A1, the contents of which are incorporated herein by reference in their entirety.

[0338] Exemplary compacted oligonucleotides comprise single-stranded linear oligonucleotides and compacted oligonucleotides having a 5' region capable of hybridizing with a first portion of a tandem template molecule, and a 3' region capable of hybridizing with a second portion of a tandem template molecule (e.g., the same tandem molecule). In some embodiments, hybridization of the compacted oligonucleotides with the respective tandem template molecules causes the tandem molecule to collapse or fold into DNA nanospheres, which are more compact in shape and size compared to non-collapsed DNA molecules. A dot image of the DNA nanospheres can be represented as a Gaussian spot, and the size can be measured as full width at half maximum (FWHM). Smaller spot sizes, as indicated by a smaller FWHM, are generally associated with a refined image of the spots. In some embodiments, the FWHM of the DNA nanosphere spots can be about 10 μm or smaller. The DNA nanospheres can be compact nucleic acid structures with a smaller full width at half maximum (FWHM) compared to a tandem molecule that has not collapsed / folded into a DNA nanosphere.

[0339] In some embodiments, the compacted oligonucleotide comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotide can be of any length, including 20-150 nucleotides, 30-100 nucleotides, or 40-80 nucleotides.

[0340] In some embodiments, the compacted oligonucleotide comprises a 5' region and a 3' region, and optionally an intermediate region between the 5' and 3' regions. The intermediate region can be of any length, for example, about 2-20 nucleotides in length. The intermediate region comprises a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intermediate region may also contain a non-homogeneous sequence.

[0341] The 5' region of the compacted oligonucleotide can be fully or partially complementary to the first part of the tandem template molecule along its length. The 3' region of the compacted oligonucleotide can be fully or partially complementary to the second part of the tandem template molecule along its length. The 5' region of the compacted oligonucleotide can hybridize with the first universal sequence portion of the tandem template molecule. The 3' region of the compacted oligonucleotide can hybridize with the second universal sequence portion of the tandem molecule. The 5' and 3' regions of the compacted oligonucleotide can hybridize with the tandem to pull the distal portions of the tandem together, thereby compacting the tandem to form DNA nanospheres.

[0342] The 5' region of a compacted oligonucleotide can have the same sequence as the 3' region. The 5' region of a compacted oligonucleotide can have a different sequence from the 3' region. The 3' region of a compacted oligonucleotide can have a sequence opposite to the 5' region.

[0343] Support and low nonspecific coating In some embodiments, the flow cell apparatus described herein may include a support 210, such as a solid support as disclosed herein. This disclosure provides sequencing compositions and methods employing a support containing a plurality of oligonucleotide surface primers immobilized thereon. In some embodiments, the support is passivated with a low-nonspecific binding coating. The surface coatings described herein exhibit very low nonspecific binding to reagents such as dyes, nucleotides, enzymes, and nucleic acid primers commonly used in nucleic acid capture, amplification, and sequencing workflows. These surface coatings exhibit low background fluorescence signal or high contrast-to-noise (CNR) ratio compared to conventional surface coatings.

[0344] Low nonspecific bonding coatings include one or more layers ( Figure 23 In some embodiments, multiple surface primers are immobilized to a low-nonspecific binding coating. In some embodiments, at least one surface primer is embedded within the low-nonspecific binding coating. The low-nonspecific binding coating enables improved nucleic acid hybridization and amplification performance. Generally, the support comprises a substrate (or support structure), one or more layers of covalently or non-covalently attached low-binding chemically modified layers (e.g., silane layers, polymer membranes), and one or more covalently or non-covalently attached surface primers for tethering single-stranded nucleic acid library molecules to the support. In some embodiments, the formulation of the coating (e.g., the chemical composition of one or more layers, the coupling chemistry for crosslinking the one or more layers with the support and / or with each other, and the total number of layers) can be varied such that nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the coating is minimized or reduced relative to a comparable monolayer. The formulation of the coating described herein can be varied such that nonspecific hybridization on the coating is minimized or reduced relative to a comparable monolayer. The formulation of the coating can be varied such that nonspecific amplification on the coating is minimized or reduced relative to a comparable monolayer. The coating formulation can be varied to maximize a specific amplification rate and / or yield on the coating. In some of the cases disclosed herein, suitable amplification levels are achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles.

[0345] The support structure comprising one or more chemically modified layers (e.g., layers of low nonspecifically binding polymers) can be standalone or integrated into another structure or component. For example, in some embodiments, the support structure may include one or more surfaces within an integrated or assembled flow cell device as described herein. The support structure may include one or more surfaces within a microplate format (e.g., the bottom surface of a well in a microplate). In some embodiments, the support structure includes the inner surface of a capillary (such as an inner lumen surface). In some embodiments, the support structure includes the inner surface of a capillary etched into a planar chip (such as an inner lumen surface).

[0346] The attachment chemistry used to graft a first chemically modified layer onto the surface of a support will generally depend on both the material from which the surface is made and the chemical properties of the layer. In some embodiments, the first layer may be covalently attached to the surface. In some embodiments, the first layer may be non-covalently attached to the support (e.g., adsorbed onto the support) through non-covalent interactions between the support and the molecular components of the first layer (such as electrostatic interactions, hydrogen bonding, or van der Waals interactions). In either case, the support may be treated prior to the attachment or deposition of the first layer. Any of a variety of surface preparation techniques known to those skilled in the art can be used to clean or treat surfaces. For example, glass or silicon surfaces may be acid-washed using a Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2), alkaline-treated in KOH and NaOH, and / or cleaned using oxygen plasma treatment methods.

[0347] The silane chemical composition is a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple connector molecules (e.g., linear hydrocarbon molecules of various lengths (such as C6, C12, C18 hydrocarbons) or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to produce any of the disclosed low-binding coatings include, but are not limited to: (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), any of a variety of PEG-silanes (e.g., including molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., including free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and the like.

[0348] Any of a variety of molecules known to those skilled in the art (including, but not limited to, amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof) may be used to generate one or more chemically modified layers on a support, wherein the selection of the components used may be varied to alter one or more properties of the layer, such as the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the layer, or the three-dimensional properties of the layer (i.e., “thickness”). Examples of polymers that may be used to generate one or more layers of low nonspecific binding material in any of the disclosed coatings include, but are not limited to, polyethylene glycol (PEG), streptavidin, polyacrylamide, polyesters, dextran, polylysine and polylysine copolymers, or any combination thereof, of various molecular weights and branched structures. Examples of conjugation chemistry that can be used to graft one or more layers (e.g., polymer layers) of a material onto a surface and / or crosslink the layers to each other include, but are not limited to: biotin-streptavidin interactions (or variants thereof), his-tag-Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylic acid ester conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxy resins, azides, hydrazides, alkynes, isocyanates, and silanes.

[0349] Low nonspecificity bonding surface coatings can be uniformly applied to a support. Alternatively, the surface coating can be patterned such that the chemically modified layer is confined to one or more discrete regions of the support. For example, photolithography can be used to pattern the coating to create an ordered array or random pattern of chemically modified regions on the support. Alternatively or in combination, contact printing and / or inkjet printing techniques can be used to pattern the coating. In some embodiments, the ordered array or random pattern of chemically modified regions may include at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions.

[0350] In some embodiments, the low nonspecific binding coating comprises a hydrophilic polymer that is nonspecifically adsorbed or covalently grafted onto a support. Passivation is typically performed using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyethylene oxide) or other hydrophilic polymers with different molecular weights and end groups chemically linked to the support, such as silanes. End groups distal to the surface may include, but are not limited to, biotin, methoxy ethers, carboxylic esters, amines, NHS esters, maleimides, and bissilanes. In some embodiments, two or more layers of a hydrophilic polymer (e.g., a linear polymer, a branched polymer, or a multibranched polymer) may be deposited on the surface. In some embodiments, the two or more layers may be covalently coupled to each other or internally crosslinked to improve the stability of the resulting coating. In some embodiments, surface primers with different nucleotide sequences and / or base modifications (such as surface capture primers and surface pinning primers described herein) (or other biomolecules, such as enzymes or antibodies) may be tethered to the resulting layers at various surface densities. In some embodiments, for example, both the surface functional group density and the surface primer concentration may be varied to obtain a desired range of surface primer densities. Furthermore, surface primer density can be controlled by diluting the surface primers with other molecules carrying the same functional groups. For example, amine-labeled surface primers can be diluted with amine-labeled polyethylene glycol in reactions with NHS-ester-coated surfaces to reduce the final primer density. Surface primers with linkers of varying lengths between the hybridization region and the surface attachment functional groups can also be applied to control surface density. Examples of suitable linkers include: poly-T and poly-A chains (e.g., 0 to 20 bases) at the 5' end of the primer, PEG linkers (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be tethered to the surface, and the fluorescent reads can then be compared with those of a dye solution of known concentration.

[0351] In some embodiments, the low nonspecific binding coating comprises a functionalized polymer coating covalently bound to at least a portion of the support via chemical groups on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM).

[0352] To scale primer surface density and add additional dimensions to hydrophilic or amphoteric coatings, supports comprising multilayer coatings of PEG and other hydrophilic polymers have been developed. Primer loading density on supports can be significantly increased by using hydrophilic and amphoteric surface layering methods (including, but not limited to, polymer / copolymer materials described below). Conventional PEG coating methods use monolayer primer deposition, which is typically reported for single-molecule applications but does not produce high copy numbers for nucleic acid amplification applications. As described herein, “layering” can be achieved using conventional crosslinking methods with any compatible polymer or monomer subunit, allowing for the sequential construction of surfaces comprising two or more highly crosslinked layers. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and copolymers of polylysine and PEG. In some embodiments, different layers can be linked together by any of a variety of conjugation reactions, including but not limited to: biotin-streptavidin binding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, and ionic interactions between positively charged and negatively charged polymers. In some embodiments, high primer density materials can be constructed in solution and subsequently layered onto a surface in multiple steps.

[0353] Examples of materials from which the support structure may be manufactured include, but are not limited to: glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic support structures are contemplated.

[0354] The support structure can be presented in any of a variety of geometries and sizes known to those skilled in the art, and can include any of a variety of materials known to those skilled in the art. For example, the support structure can be partially planar (e.g., including a microscope slide or the surface of a microscope slide). Generally, the support structure can be cylindrical (e.g., including a capillary or the inner surface of a capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., including the outer surface of an irregularly shaped non-porous bead or particle). In some embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be a solid, non-porous surface. In some embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be porous, such that the coating described herein penetrates the porous surface, and the nucleic acid hybridization and amplification reactions performed thereon can occur within the pores. In some embodiments, the support geometry includes one or more channels, inlets, and / or outlets as described herein.

[0355] The support structure comprising one or more chemically modified layers (e.g., layers of low nonspecifically binding polymers) can be standalone or integrated into another structure or component. For example, the support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may include one or more surfaces within a microplate format (e.g., the bottom surface of a hole in a microplate). In some embodiments, the support structure includes the inner surface of a capillary (such as an inner lumen surface). In some embodiments, the support structure includes the inner surface of a capillary etched into a planar chip (such as an inner lumen surface). In some embodiments, the support includes one or more inner surfaces of the flow cell device described herein.

[0356] As noted, the low-nonspecific binding supports of this disclosure exhibit reduced nonspecific binding to proteins, nucleic acids, and other components of hybridization and / or amplification formulations used for solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited on a given support surface can be assessed qualitatively or quantitatively. For example, by exposing the surface to a fluorescent dye (e.g., anthocyanins (such as Cy3 or Cy5, etc.), fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a set of standardized conditions, a specified washing protocol and fluorescence imaging can then be used as quantitative tools to compare nonspecific binding on supports comprising different surface formulations. In some embodiments, surfaces are exposed to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions. A specified rinsing protocol and fluorescence imaging can then be used as qualitative tools to compare nonspecific binding on supports comprising different surface formulations—provided that care has been taken to ensure that fluorescence imaging is linearly correlated (or predictably correlated) with the number of fluorophores on the support surface and that suitable calibration standards are used (e.g., where signal saturation and / or fluorophore self-quenching are not problematic). In some embodiments, other techniques known to those skilled in the art (e.g., radioisotope labeling and counting methods) can be used to quantitatively assess the extent of nonspecific binding exhibited by different support surface formulations of this disclosure.

[0357] Some surfaces disclosed herein exhibit specific to nonspecific binding ratios of fluorophores (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the ranges herein. Some surfaces disclosed herein exhibit specific to nonspecific fluorescence ratios of fluorophores (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the ranges herein.

[0358] The degree of nonspecific binding exhibited by the disclosed low-binding supports can be evaluated using a standardized protocol that involves contacting the surface with labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., under a standardized set of incubation and rinsing conditions, followed by detecting the amount of label remaining on the surface and comparing the resulting signal with an appropriate calibration standard. In some embodiments, the label may include a fluorescent label. In some embodiments, the label may include a radioisotope. In some embodiments, the label may include any other detectable label known to those skilled in the art. In some embodiments, the degree of nonspecific binding exhibited by a given support surface formulation can therefore be evaluated based on the number of nonspecifically bound protein molecules (or nucleic acid molecules or other molecules) per unit area. In some embodiments, the low-binding supports of this disclosure may exhibit less than 0.001 molecules per μm. 2 Less than 0.01 molecules per μm 2 Less than 0.1 molecules per μm 2 Less than 0.25 molecules per μm 2 Less than 0.5 molecules per μm 2 Less than 1 molecule per μm 2 Less than 10 molecules per μm 2 Less than 100 molecules per μm 2 or less than 1,000 molecules per μm 2 Nonspecific binding to proteins (or nonspecific binding to other specified molecules, such as anthocyanins (e.g., Cy3 or Cy5), fluorescein, coumarin, rhodamine, or other dyes disclosed herein)). Those skilled in the art will recognize that the given support surface of this disclosure can exhibit any position falling within this range (e.g., less than 86 molecules per μm). 2 Non-specific binding of ), for example, after contact with a 1 μM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate-buffered saline (PBS) buffer for 15 minutes, followed by three rinses with deionized water, some of the modified surfaces disclosed herein exhibited less than 0.5 molecules / μm. 2 Non-specific protein binding. Some of the modified surfaces disclosed in this paper exhibited less than 0.25 molecules per μm. 2Nonspecific binding of Cy3 dye molecules was observed. In independent nonspecific binding assays, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM 7-propynylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propynylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated in 384-well plates on a low-binding coated support at 37 °C for 15 min. Wash each well 2–3 times with 50 μL of deionized RNase-free / DNase-free water, and then 2–3 times with 25 mM ACES buffer (pH 7.4). Image the 384-well plate on a GETyphoon instrument using the Cy3, AF555, or Cy5 filter set specified by the manufacturer (depending on the dye assay performed) at a PMT gain setting of 800 and a resolution of 50–100 μm. For higher resolution imaging, images are collected on an Olympus IX83 microscope (e.g., an inverted fluorescence microscope) with total internal reflection fluorescence (TIRF) objectives (100×, 1.5 NA, Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome camera, an Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochrome camera), an illumination source (e.g., an Olympus 100W Hg lamp, an Olympus 75W Xe lamp, or an Olympus U-HGLGPS fluorescence source), and an excitation wavelength of 532 nm or 635 nm. (Olympus Corp., Center Valley, Pa.) Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, NY), including 405 nm, 488 nm, 532 nm, or 633 nm dichroic mirrors / beam splitters, and bandpass filters were selected to be 532 LP or 645 LP integrated with appropriate excitation wavelengths. Some of the modified surfaces disclosed herein exhibited less than 0.25 molecules per μm. 2The dye molecules bind nonspecifically. In some embodiments, the coated support is immersed in a buffer solution (e.g., 25 mMACES, pH 7.4) while images are acquired.

[0359] In some embodiments, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding to a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the ranges herein. In some embodiments, the surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence signal for a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the ranges herein.

[0360] Low-background surfaces conforming to the present disclosure may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific dye molecules attached to each nonspecifically adsorbed molecule, with a ratio of specific dye attachment (e.g., Cy3 attachment) to nonspecific dye adsorption (e.g., Cy3 dye adsorption). Similarly, when subjected to excitation energy, low-background surfaces conforming to the present disclosure that have attached fluorophores (e.g., Cy3) may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50:1, with a ratio of specific fluorescence signal (e.g., generated from Cy3-labeled oligonucleotides attached to the surface) to nonspecifically adsorbed dye fluorescence signal.

[0361] In some embodiments, the degree of hydrophilicity (or “wetability” to an aqueous solution) of the disclosed support surface may be assessed, for example, by measuring the water contact angle, wherein a small drop of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some embodiments, a static contact angle may be determined. In some embodiments, a forward or backward contact angle may be determined. In some embodiments, the water contact angles disclosed herein for hydrophilic low-binding support surfaces may range from about 0 degrees to about 30 degrees. In some embodiments, the water contact angles disclosed herein for hydrophilic low-binding support surfaces may not exceed 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed 40 degrees. Those skilled in the art will recognize that a given hydrophilic low-binding support surface of this disclosure may exhibit a water contact angle with values ​​at any location within this range.

[0362] In some embodiments, the hydrophilic surfaces disclosed herein help reduce washing time for bioassays, typically due to reduced nonspecific binding of biomolecules to low-binding surfaces. In some embodiments, a sufficient washing step can be performed in less than 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or less than 10 seconds. For example, a sufficient washing step can be performed in less than 30 seconds.

[0363] Some of the low-binding surfaces disclosed herein exhibit significant improvements in stability or durability under prolonged exposure to solvents and elevated temperatures, or under repeated cycles of solvent exposure or temperature variations. For example, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups or tethered biomolecules (e.g., oligonucleotide primers) on the surface, and monitoring the fluorescence signal before, during, and after prolonged exposure to solvents and elevated temperatures, or under repeated cycles of solvent exposure or temperature variations. In some embodiments, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours (or any combination of these percentages as measured within these time periods) over time periods of exposure to solvents and / or elevated temperatures. In some embodiments, the degree of change in fluorescence used to assess surface quality can be less than 1%, 2%, 3%, 4%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000 cycles of repeated exposure to solvent variations and / or temperature variations, respectively (or any combination of these percentages as measured by the range of cycles).

[0364] In some embodiments, the surfaces disclosed herein may exhibit a high ratio of specific signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 times the signal of adjacent non-population regions of the surface. Similarly, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 times the signal of adjacent amplified nucleic acid population regions of the surface.

[0365] In some embodiments, when used for nucleic acid hybridization or amplification applications to generate communities of hybridized or clonally amplified nucleic acid molecules (e.g., directly or indirectly labeled with fluorophores), the disclosed low-background surface fluorescence images exhibit a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250 or greater than 250.

[0366] One or more types of primers may be attached to or tethered to the surface of a support. In some embodiments, one or more types of adaptors or primers may comprise spacer sequences, adaptor sequences for hybridizing with target library nucleic acid sequences linked to the adaptor, forward amplification primers, reverse amplification primers, sequencing primers, surface capture primers, surface pinning primers, and / or molecular barcoding sequences or any combination thereof. In some embodiments, one primer or adaptor sequence may be tethered to at least one layer of the surface. In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, or more than ten different primer or adaptor sequences may be tethered to at least one layer of the surface. In some embodiments, the support comprises a plurality of primers tethered to the support, and all primers contain the same sequence, such as the same surface capture sequence. In some embodiments, the support comprises two or more primers tethered to the support, and each of the plurality of primers contains a primer containing the same sequence, which is different from the corresponding sequence in the other plurality of primers. As a non-limiting example, the first plurality of primers contains a first surface capture sequence, the second plurality of primers contains a second surface capture sequence, and the first capture sequence and the second capture sequence are not the same.

[0367] In some embodiments, the length of the tethered adaptor and / or primer sequence can range from about 10 nucleotides to about 100 nucleotides. In some embodiments, the length of the tethered adaptor and / or primer sequence can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the length of the tethered adaptor and / or primer sequence can be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. Any of the lower and upper limits described in this paragraph can be combined to form a range included in this disclosure; for example, in some embodiments, the length of the tethered adaptor and / or primer sequence can range from about 20 nucleotides to about 80 nucleotides. Those skilled in the art will recognize that the length of the tethered adaptor and / or primer sequence can have any value within this range, for example, about 24 nucleotides.

[0368] In some embodiments, the resulting surface density of primers (e.g., trapping primers) on the low-binding support surface of this disclosure can range from about 100 primer molecules per μm. 2 Approximately 100,000 primer molecules per μm 2 In some embodiments, the resulting surface density of primers on the low-binding support surface of this disclosure can be approximately 1,000 primer molecules per μm. 2 Approximately 1,000,000 primer molecules per μm 2 Within the range. In some embodiments, the surface density of the primers may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules per μm. 2 In some embodiments, the surface density of the primers can be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules per μm. 2 The lower and upper limits described in this paragraph can be combined to form a range included within this disclosure. For example, in some embodiments, the surface density of the primers can be approximately 10,000 molecules per μm. 2 Approximately 100,000 molecules per μm 2 Within this range. Those skilled in the art will recognize that the surface density of primer molecules can have any value within this range, for example, approximately 455,000 molecules / μm. 2In some embodiments, the surface density of the target library nucleic acid sequence initially hybridized with the adaptor or primer sequence on the support surface may be less than or equal to the surface density indicated by the surface density of the tethered primer. In some embodiments, the surface density of the target library nucleic acid sequence cloned and amplified with the adaptor or primer sequence on the support surface may span the same range as indicated by the surface density of the tethered primer.

[0369] The local density listed above does not exclude variations in density across the surface, such that the surface may include densities with, for example, 500,000 / μm. 2 The region of oligonucleotide density also includes at least a second region with substantially different local densities.

[0370] In some embodiments, fluorescence imaging techniques can be used to evaluate the performance of nucleic acid hybridization and / or amplification reactions using the disclosed reaction formulations and low-binding supports, where the contrast-to-noise ratio (CNR) of the image provides a key metric for evaluating amplification specificity and non-specific binding on the support. CNR is typically defined as: CNR = (Signal - Background) / Noise. The background term is generally considered to be the signal measured against a specific feature (diffraction-limited spot, DLS) in a designated region of interest (ROI). While signal-to-noise ratio (SNR) is generally considered a benchmark for overall signal quality, it can be demonstrated that in applications requiring rapid image capture (e.g., sequencing applications where cycle time must be minimized), an improved CNR can provide a significant advantage over SNR as a benchmark for signal quality, as illustrated in the examples below. At high CNRs, even slight improvements in CNR can drastically reduce the imaging time required to achieve accurate differentiation (and therefore, accurate base recall in the case of sequencing applications). Improved CNR in imaging data related to imaging integration time provides a method for more accurately detecting features such as clonal amplified nucleic acid colonies on support surfaces.

[0371] In most ensemble-based sequencing methods, background terms are typically measured as signals associated with the "mesenchymal" region. This excludes the "interstitial" background (B). 间隙 In addition to the "intrastitial" background (B) 内质 These background signals exist within regions occupied by amplified DNA colonies. The combination of these two background signals determines the achievable CNR and subsequently directly affects optical instrumentation requirements, architecture costs, reagent costs, runtime, cost / genome, and ultimately, accuracy and data quality for cyclic array-based sequencing applications. 间隙Background signals have multiple sources; some examples include: autofluorescence from consumable flow cells, nonspecific adsorption of detection molecules that generate spurious fluorescence signals (which may mask signals from the ROI), and the presence of nonspecific DNA amplification products (e.g., those generated from primer dimers). In typical next-generation sequencing (NGS) applications, this background signal in the current field of view (FOV) is averaged and subtracted over time. Signals generated from individual DNA colonies (i.e., (signal)-B (gap) in the FOV) produce classifiable, distinguishable features. In some embodiments, endoplasmic background (B (endoplasm)) can contribute confounding fluorescence signals that are not specific to the target but are present in the same ROI, thus making averaging and subtraction much more difficult.

[0372] Nucleic acid amplification on the low-binding coated support described herein can reduce the B (gap) background signal by reducing nonspecific binding, leading to improvements in specific nucleic acid amplification and reductions in nonspecific amplification (which can affect the background signal generated from both the gap and endoplasmic regions). In some embodiments, the disclosed low-binding coated support (optionally used in combination with the disclosed hybridization and / or amplification reaction formulations) can result in improvements in CNR that are superior to those achieved using conventional supports and hybridization, amplification, and / or sequencing protocols, by folds of 2, 5, 10, 100, 250, 500, or 1000. Although described herein in the context of using fluorescence imaging as a readout or detection mode, the same principles apply to the use of the disclosed low-binding coated support and nucleic acid hybridization and amplification formulations for other detection modes, including both optical and non-optical detection modes.

[0373] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0374] In some embodiments, the surface of the support may be substantially smooth. In some embodiments, the support may have a regular or irregular texture, including bumps, etched surfaces, holes, three-dimensional supports, or any combination thereof.

[0375] In some embodiments, the support comprises beads of any shape, including spherical, hemispherical, cylindrical, barrel-shaped, ring-shaped, disc-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or linear.

[0376] The support can be made of any material, including but not limited to: glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic substrates are contemplated.

[0377] In some embodiments, the surface of the support is coated with one or more compounds to create a passivation layer on the carrier. In some embodiments, the support comprises a low-nonspecificity binding surface, which can improve the nucleic acid hybridization and amplification performance on the support. Generally, the support is one or more layers of covalently or non-covalently attached low-binding chemically modified layers (e.g., silane layers, polymer films), and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize multiple nucleic acid template molecules to the support.

[0378] In some embodiments, the degree of hydrophilicity (or “wetting” to aqueous solutions) of the surface coating can be assessed, for example, by measuring the water contact angle, wherein a small water droplet is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some embodiments, a static contact angle can be determined. In some embodiments, a forward or backward contact angle can be determined. In some embodiments, the water contact angles disclosed herein for hydrophilic low-binding support surfaces can range from about 0 degrees to about 30 degrees. In some embodiments, the water contact angles disclosed herein for hydrophilic low-binding support surfaces can not exceed 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed 40 degrees. Those skilled in the art will recognize that a given hydrophilic low-binding support surface of this disclosure can exhibit a water contact angle with values ​​at any location within this range.

[0379] This disclosure provides multiple (e.g., two or more) nucleic acid template molecules immobilized to a support. In some embodiments, the multiple nucleic acid templates have the same sequence or different sequences. In some embodiments, individual nucleic acid template molecules from the multiple nucleic acid templates are immobilized to different sites on the support. In some embodiments, two or more individual nucleic acid template molecules from the multiple nucleic acid templates are immobilized to sites on the support. In some embodiments, the support includes multiple sites arranged in an array. The term "array" refers to a support comprising an array of multiple sites located at predetermined positions on the support...

Claims

1. A sequencing system comprising: Optical system 2020, which includes objective lenses; The xy stage 2010 is configured to hold a sample to be imaged thereon and move the sample relative to the objective lens in the xy plane, wherein the sample is fixed on one or more flow cell devices. Nested group 2050, which is configured to provide fluid and thermal communication to the sample when the one or more flow cell devices are coupled to the nested group; as well as The moving mechanism 2040 optionally includes a movable arm configured to move the one or more flow pool devices between the xy stage 2010 and the nesting group 2050 during sequential operation.

2. The sequencing system according to claim 1, wherein the xy stage 2010 is automatically actuated by a first actuator with a first spatial precision.

3. The sequencing system according to claim 1 or 2, wherein the movable arm is automatically actuated by a second actuator with a second spatial precision.

4. The sequencing system according to any one of the preceding claims, wherein the first actuator, the second actuator, or both are controlled by one or more hardware processors of the sequencing system.

5. The sequencing system according to any one of the preceding claims, wherein the sequencing system further comprises: A housing configured to hold one or more of the optical system 2020, the xy stage 2010, the nesting group 2050, and the moving mechanism 2040 within the housing.

6. The sequencing system according to any one of the preceding claims, wherein the movable arm is automatically actuated to move in three dimensions (3D).

7. The sequencing system according to any one of the preceding claims, wherein movement in each of the three dimensions is performed with one or more predetermined spatial precisions.

8. The sequencing system according to any one of the preceding claims, wherein when the flow cell device is fixed on the xy stage 2010, the sequencing system lacks fluid or thermal communication with the one or more flow cell devices at or near the xy stage 2010.

9. The sequencing system according to any one of the preceding claims, wherein each of the one or more flow cell devices includes an open landing region configured to openly receive fluid from the nested group 2050.

10. The sequencing system according to any one of the preceding claims, wherein the flow cell device comprises a plurality of microfluidic channels, and the nesting group 2050 is configured to allow independent and simultaneous fluid communication with each of the plurality of microfluidic channels.

11. The sequencing system according to any one of the preceding claims, wherein the flow cell device comprises a plurality of microfluidic channels, and the nesting group 2050 is configured to allow independent and sequential fluid communication with each of the plurality of microfluidic channels.

12. The sequencing system according to any one of the preceding claims, wherein the flow cell device comprises a plurality of microfluidic channels, and the nesting group 2050 is configured to allow independent and cross-contamination-free fluid communication with each of the plurality of microfluidic channels.

13. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 is actuated to move a predetermined distance in the xy plane.

14. The sequencing system according to any one of the preceding claims, wherein the predetermined distance is based on the distance between two adjacent microfluidic channels of the flow cell device.

15. The sequencing system according to any one of the preceding claims, wherein the nesting group 2050 is configured to enable fluid and thermal communication with the one or more flow cell devices.

16. The sequencing system according to any one of the preceding claims, wherein the nesting group 2050 is configured to enable fluid and thermal communication with at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 flow cell devices when each of the flow cell devices is in a locked position with the nesting group 2050.

17. The sequencing system according to any one of the preceding claims, wherein the nesting group 2050 is configured to hold each of the flow cell devices in an unlocked position and a locked position, wherein in the unlocked position the flow cell device is removable from the nesting group 2050, and in the locked position the flow cell device is spatially registered to the nesting group 2050, fixedly coupled to the nesting group 2050, and capable of sealed fluid and thermal communication between the nesting group and the flow cell device.

18. The sequencing system according to any one of the preceding claims, wherein the flow cell device is coupled to the vector 2051.

19. The sequencing system according to any one of the preceding claims, wherein the movable arm is configured to move the vector 2051 and the flow cell device together.

20. The sequencing system according to any one of the preceding claims, wherein the vector 2051 is configured to be spatially registered to the nested group in the locked position.

21. The sequencing system according to any one of the preceding claims, wherein the nested group 2050 includes one or more fasteners.

22. The sequencing system of claim 21, wherein the one or more fasteners use magnetic force.

23. The sequencing system of claim 21 or 22, wherein the one or more fasteners comprise rare-earth magnets, electromagnetic coils, or both.

24. The sequencing system according to any one of claims 21 to 23, wherein the one or more fasteners are controlled by one or more processors to switch between an on phase and an off phase.

25. The sequencing system according to any one of claims 21 to 24, wherein one or more of the fasteners lack mechanical fasteners.

26. The sequencing system according to any one of the preceding claims, wherein the movable arm is configured to move the one or more flow cell devices between the xy stage 2010 and the nesting group 2050 with a first spatial precision.

27. The sequencing system according to any one of the preceding claims, wherein the movable arm includes a gripper configured to grip the carrier 2051 when the carrier 2051 is in a decoupled position relative to the nesting group 2050 or when the carrier 2051 is in a decoupled position relative to the xy stage 2010.

28. The sequencing system according to any one of the preceding claims, wherein the movable arm comprises a horizontal arm mechanically supported by a vertical arm.

29. The sequencing system according to any one of the preceding claims, wherein the movable arm comprises an upper arm, a joint, a forearm, a wrist, and a gripper attached to the forearm.

30. The sequencing system according to any one of the preceding claims, wherein the movable arm is configured to move with six degrees of freedom.

31. The sequencing system according to any one of claims 27 to 30, wherein the gripper is movably attached to the horizontal arm or the vertical arm.

32. The sequencing system according to any one of claims 27 to 31, wherein the grasper is configured to move in 3D.

33. The sequencing system according to any one of the preceding claims, wherein the moving mechanism 2040 includes a plurality of tracks, each track connecting a carrier 2051 coupled to the nested group to the xy stage 2010.

34. The sequencing system according to any one of claims 27 to 32, wherein the gripper is configured to hold the flow cell device carrier via friction, electromagnetic force or magnetic force.

35. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises one or more sensors.

36. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 includes one or more sensors.

37. The sequencing system according to any one of the preceding claims, wherein the nested group 2050 includes one or more sensors.

38. The sequencing system according to any one of claims 35 to 37, wherein the one or more sensors are configured to provide feedback to the processor, the feedback facilitating the positioning of the carrier 2051 relative to the xy stage 2010, the optical system 2020, or the nesting group 2050.

39. The sequencing system according to any one of the preceding claims, wherein the moving mechanism 2040 comprises one or more belt conveyors.

40. The sequencing system according to any one of claims 33 to 39, wherein the plurality of orbits comprises one or more actuators configured to actuate one or more of the plurality of orbits to move the corresponding vector 2051 to the xy stage 2010.

41. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 is configured to be actuated to move to a 3D position with a second spatial precision.

42. The sequencing system of claim 41, wherein the second spatial precision is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the first spatial precision.

43. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 comprises: Fasteners configured to removably secure the flow cell device to it.

44. The sequencing system of claim 43, wherein the fastener comprises one or more clamps.

45. The sequencing system according to any one of the preceding claims, wherein each vector 2051 includes a coupling position in which the vector 2051 is removably attached to the xy stage 2010.

46. ​​The sequencing system according to any one of the preceding claims, wherein each vector 2051 includes a decoupling location in which the vector 2051 can be removed from the xy stage 2010.

47. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 includes one or more pumps configured to extract fluid from the flow cell apparatus when the corresponding vector 2051 is coupled to the xy stage 2010.

48. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 includes a heating device, a cooling device, or both.

49. The sequencing system according to any one of the preceding claims, wherein the xy stage 2010 is coupled to a mechanical decoupler configured to isolate the xy stage from vibrations or mechanical disturbances outside the xy stage 2010.

50. The sequencing system according to any one of the preceding claims, wherein the nesting group 2050 includes one or more fasteners, each fastener being configured to fasten a corresponding vector 2051 to the nesting group 2050.

51. The sequencing system according to any one of claims 21 to 50, wherein each fastener comprises one or more clamps.

52. The sequencing system of claim 51, wherein the one or more clamps are actuated by magnetic force, electromagnetic force, or pressure.

53. The sequencing system according to any one of the preceding claims, wherein the nested group 2050 includes one or more pumps configured to extract fluid from the flow cell device when the corresponding vector 2051 is coupled to the nested group 2050.

54. The sequencing system according to any one of the preceding claims, wherein each vector 2051 includes a decoupling location in which the vector 2051 can be removed from the nesting group 2050.

55. The sequencing system according to any one of the preceding claims, wherein each vector 2051 includes a coupling position in which the flow cell device vector is removably attached to the nesting group 2050 and is in sealed fluid communication with the nesting group 2050.

56. The sequencing system according to any one of the preceding claims, wherein the nesting group 2050 includes a 3D moving device configured to position the vector 2051 relative to the nesting group with a third spatial precision.

57. The sequencing system of claim 56, wherein the third spatial location is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the first spatial precision.

58. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: An opening on the surface of the carrier 2051 is configured to receive a flow pool device therein.

59. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: One or more fluid paths are in sealed fluid communication with the flow pool device when the flow pool device is removably attached to the carrier 2051.

60. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: A pump configured to pull or push fluid between the flow pool device and the carrier 2051.

61. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: A valve is positioned between the fluid path connected to the flow pool device and the port opening of the carrier 2051, wherein the valve is in an open position when the flow pool device is in the coupled position with the carrier 2051, and in a closed position when the flow pool device is in the decoupled position.

62. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: The port opening has a connector, which is configured to allow for sealed fluid communication between the carrier 2051 and the corresponding nested module when the connector is in the connected position.

63. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: The wire has an electrical connector and is configured to enable electrical connection between the carrier 2051 and the power source.

64. The sequencing system according to any one of the preceding claims, wherein the vector 2051 comprises: A battery, a sensor, or both, wherein the battery or the sensor is connected to the electrical connector via the wire.

65. The sequencing system according to any one of the preceding claims, wherein the nested group 2050 comprises one or more reagent containers.

66. The sequencing system according to any one of the preceding claims, wherein the one or more reagent containers are disposable.

67. The sequencing system of claim 65 or 66, wherein the moving mechanism 2040 is configured to immerse the flow cell device in at least some of the one or more reagent containers.

68. The sequencing system according to any one of the preceding claims, wherein the nested group 2050 further comprises: Cooler, heater, or both.

69. The sequencing system of claim 68, wherein the cooler or the heater is configured to control the temperature of each sample fixed on the one or more flow cell devices.

70. The sequencing system of claim 68 or 69, wherein the cooler or the heater comprises one or more of the following: a fan configured to blow cold or hot air; a microwave, an infrared light source, and an electromagnetic wave source.

71. The sequencing system according to any one of the preceding claims, further comprising a beam collector configured to absorb at least some of the excitation light generated by the optical system.

72. The sequencing system according to any one of the preceding claims, further comprising a beam collector configured to prevent at least some of the excitation light from reaching the imaging sensor of the optical system.

73. The sequencing system according to claim 71 or 72, wherein the beam collector is offset from the flow cell device by a gap region.

74. The sequencing system according to any one of claims 71 to 73, wherein the beam collector contacts the flow cell device with a predetermined latching force.

75. The sequencing system according to any one of claims 71 to 74, wherein the beam collector contacts the xy stage with a predetermined damping force.

76. The sequencing system of claim 75, wherein the predetermined damping force is configured to reduce the predetermined latching force such that the net force on the flow cell device can be customized within a predetermined range.

77. A sequencing method, comprising: (a) Moving a first flow cell device from nested group 2050 to xy stage 2010, wherein the first flow cell device includes a first sample fixed thereon; (b) Move the xy stage 2010 and the first sample thereon relative to the objective lens of the optical system of the sequencing system; (c) Image the first sample fixed on the first flow cell device on the xy stage using the optical system 2020; (d) Move the first flow device from the xy stage 2010 to the nesting group 2050; (e) During one or more of (a)-(d), fluid and thermal communication between the nested group 2050 and the second flow pool device is simultaneously permitted; (f) Moving the second flow cell device from the nested group 2050 to the xy stage 2010, wherein the second flow cell device includes a second sample fixed thereon; (g) Move the xy stage 2010 and the second sample thereon relative to the objective lens of the optical system 2040 of the sequencing system; (h) The second sample fixed on the second flow cell device on the xy stage 2010 is imaged using the optical system 2040; (i) Move the first flow device from the xy stage 2010 to the nesting group 2050; as well as (j) During one or more of (f)-(i), fluid and thermal communication between the nested group 2050 and the first flow pool device is simultaneously permitted.

78. A sequencing method, comprising: (a) Moving a first flow cell device from nested group 2050 to xy stage 2010, wherein the first flow cell device includes a first sample fixed thereon; (b) Move the xy stage 2010 and the first sample thereon relative to the objective lens of the optical system 2020 of the sequencing system; (c) Image the first sample fixed on the first flow cell device on the xy stage 2010 using the optical system 2020; (d) Move the first flow device from the xy stage 2010 to the nesting group 2050; (e) During one or more of (a)-(d), fluid and thermal communication between the nested group 2050 and the second flow pool device is simultaneously permitted; as well as (f) Move the second flow cell device from the nested group 2050 to the xy stage 2010, wherein the second flow cell device includes a second sample fixed thereon.

79. The method according to claim 77 or 78, wherein the sequencing method further comprises: Repeat operation (a)-(e).

80. The method according to claim 78 or 79, wherein the sequencing method further comprises: Repeat operation (f)-(j).

81. The method according to any one of claims 78 to 80, wherein the sequencing method further comprises: Repeat operation (a)-(j) a certain number of times.

82. The method according to any one of claims 79 to 81, wherein the number of repetitions is in the range of 1 to 500.

83. The method according to any one of claims 77 to 82, wherein allowing fluid communication between the nested group 2050 and the first flow cell device comprises: The flow pool device is reversibly fastened to the carrier 2051 via one or more fasteners so that a sealed fluid communication can be maintained between the flow pool device and the carrier 2051. as well as The carrier 2051 is reversibly fastened to the nesting assembly 2050 via one or more fasteners, such that a sealed fluid communication is possible between the nesting assembly 2050 and the carrier 2051, and that physical contact with the heat dissipation element is possible.

84. The method according to any one of claims 77 to 83, wherein (a) moving the first flow cell device from the nested group 2050 to the xy stage 2010 is within a first flow cycle of the sequencing run, and (f) moving the first flow cell device from the nested group 2050 to the xy stage 2010 is within a second flow cycle of the sequencing run, different from the first flow cycle.

85. The method according to any one of claims 77 to 84, wherein each of operations (a)-(b) and (d)-(g) is completed in less than 0.5 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds.

86. The method according to any one of claims 77 to 85, wherein each of operations (a)-(b) and (d)-(g) is completed in less than 0.5 seconds, 1 second, 2 seconds or 3 seconds.

87. The method according to any one of claims 77 to 86, wherein (e) simultaneously allowing fluid and thermal communication between the nested group 2050 and the first flow cell device during one or more of (a)-(d) comprises: Rotate the one or more fasteners to the open position to enable sealing for fluid communication and physical contact for thermal communication.

88. The method according to any one of claims 77 to 87, wherein (e) simultaneously allowing fluid and thermal communication between the nested group and the first flow pool device during one or more of (a)-(d) comprises: The flow cell apparatus is immersed in at least some of the one or more reagent containers in a predetermined sequence.

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