DNA sequencing system and its use

Abstract

This disclosure provides flow cell devices, systems, and methods for facilitating and performing DNA sequencing analysis, thereby reducing system complexity and cost, significantly saving product costs, and lowering contamination levels. The sequencing systems described herein enable the simultaneous processing of multiple flow cells, thereby allowing sequencing and imaging steps, or multiple sequencing methods, to be performed in parallel using a single sequencing system.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefits of U.S. Provisional Application No. 63 / 515,816, filed on 26 July 2023, and U.S. Provisional Application No. 63 / 666,463, filed on 1 July 2024, the contents of each of these applications being 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 introduce a repeating flow of sequencing reagents 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 nucleotide sequence of the corresponding template molecule (e.g., an immobilized and / or amplified nucleic acid template molecule attached to the surface of the flow cell).

[0003] A typical NGS system allows fluid and thermal communication from the system to the flow cell device and the sample(s) immobilized on it during sequencing, while the sample(s) remain fixed relative to the optical system of the sequencing system. However, keeping the flow cell fixed relative to the optical system of the sequencing system during steps that do not involve imaging results in inefficient use of the optical system and reduces the efficiency of the NGS system. Therefore, there is a need in the art for compositions, systems, and methods that enable the parallel asynchronous processing of multiple samples or parts of a sample. Using the compositions, systems, and methods of this disclosure, imaging and non-imaging steps on different samples can be performed simultaneously, or different samples can be subjected to different methods simultaneously, thereby reducing idling time, increasing flexibility, and improving efficiency. [Overview of the project]

[0004] This specification describes a sequencing system for sequencing nucleic acids that is flexible and scalable. The sequencing systems and methods described herein can advantageously achieve more efficient use of the optical system with the shortest idling time (e.g., fluid dosing waiting time). The systems and methods described herein can advantageously enable imaging of a sample while sequencing reactions on another sample are being carried out in parallel, thereby improving the throughput of existing sequencing systems. The systems and methods described herein can advantageously separate the sample being imaged from the fluid and / or thermal communications, thereby simplifying the system architecture and enabling a more compact size than existing systems. The systems and methods described herein can also advantageously enable independent fluid and / or thermal communications to various samples, thereby enabling the user to image samples using different reagents or sequencing protocols and, in some cases, combine them within a single sequencing run.

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

[0006] In some embodiments, the xy stage 2010 is automatically driven by a first actuator having a first spatial accuracy.

[0007] In some embodiments, the movable arm is automatically driven by a second actuator having a second spatial accuracy. In some embodiments, the first actuator, the second actuator, or both are controlled by one or more hardware processors of the array determination system.

[0008] In some embodiments, the array determination system further comprises a housing configured to hold within it one or more of the optical system 2020, xy stage 2010, nest bank 2050, and moving mechanism 2040.

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

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

[0011] In some embodiments, each of one or more flow cell devices includes an open landing region configured to openly receive fluid from a nest bank 2050. In some embodiments, the flow cell device comprises a plurality of microfluidic channels, and the nest bank 2050 is configured to allow fluid communication to each of the plurality of microfluidic channels independently and simultaneously. In some embodiments, the flow cell device comprises a plurality of microfluidic channels, and the nest bank 2050 is configured to allow fluid communication to each of the plurality of microfluidic channels independently and sequentially. In some embodiments, the flow cell device comprises a plurality of microfluidic channels, and the nest bank 2050 is configured to allow fluid communication to each of the plurality of microfluidic channels independently without cross-contamination.

[0012] In some embodiments, the xy stage 2010 is driven 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 in the flow cell device.

[0013] In some embodiments, the nest bank 2050 is configured to enable fluid and thermal communication with one or more flow cell devices. In some embodiments, the nest bank 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 flow cell device is in a locked position with respect to the nest bank 2050. In some embodiments, the nest bank 2050 is configured to hold each flow cell device in an unlocked position in which the flow cell device is removable from the nest bank 2050, and in a locked position in which the flow cell device is spatially registered with respect to the nest bank 2050, fixedly coupled to the nest bank 2050, and allows for sealed fluid and thermal communication between the nest bank and the flow cell device.

[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 aligned with respect to the nest bank in a locked position. In some embodiments, the nest bank 2050 comprises one or more fasteners. In some embodiments, one or more fasteners use magnetism. In some embodiments, one or more fasteners include rare earth magnets, electromagnetic coils, or both. In some embodiments, one or more fasteners are controlled by one or more processors to switch between on and off states. In some embodiments, one or more fasteners lack mechanical fasteners.

[0015] In some embodiments, the movable arm is configured to move one or more flow cell devices between the xy stage 2010 and the nest bank 2050 with a first spatial accuracy. In some embodiments, the movable arm includes a gripping section configured to grip the carrier 2051 when the carrier 2051 is in a decoupled position relative to the nest bank 2050 or when the carrier 2051 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, joints, forearm, wrist, and a gripping section attached to the forearm. In some embodiments, the movable arm is configured to move with six degrees of freedom. In some embodiments, the gripping section is movably attached to the horizontal arm or the vertical arm. In some embodiments, the gripping section is configured to move in 3D. In some embodiments, the movement mechanism 2040 includes multiple tracks, each track connecting the carrier 2051 coupled to the nest bank to the xy stage 2010. In some embodiments, the gripping portion 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 nest bank 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 nest bank 2050.

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

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

[0019] In some embodiments, the xy stage 2010 is configured to be driven 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 greater than the first spatial accuracy.

[0020] In some embodiments, the xy stage 2010 includes a fastener configured to removably secure a flow cell device thereto. In some embodiments, the fastener includes one or more clamps.

[0021] In some embodiments, each carrier 2051 includes a bonded position in which the carrier 2051 is removably attached to the xy stage 2010. In some embodiments, each carrier 2051 includes a detached position 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 the flow cell device when a 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 decoupler configured to isolate the xy stage from external vibrations or mechanical disturbances.

[0023] In some embodiments, the nest bank 2050 comprises one or more fasteners, each configured to fasten a corresponding carrier 2051 to the nest bank 2050. In some embodiments, each fastener includes one or more clamps. In some embodiments, one or more clamps are driven by magnetic or electromagnetic force or pressure.

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

[0025] In some embodiments, each carrier 2051 includes a detachable position in which the carrier 2051 is removable from the nest bank 2050. In some embodiments, each carrier 2051 includes a coupled position in which the flow cell device carrier is removablely attached to the nest bank 2050 and is in sealed fluid communication with the nest bank 2050.

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

[0027] In some embodiments, the carrier 2051 includes an opening on 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 the flow cell device is removably mounted to the carrier 2051. In some embodiments, the carrier 2051 includes a pump configured to draw or push fluid between the flow cell device and the carrier 2051. In some embodiments, the carrier 2051 includes a valve positioned between the fluid path connected to the flow cell device and the port opening of the carrier 2051, the valve being in an open position when the flow cell device is in a coupled position relative to the carrier 2051 and in a closed position when the flow cell device is in a discoupled 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 nesting module when the connector is in a coupled position. In some embodiments, the carrier 2051 includes electrical wiring having an electrical connector configured to allow electrical communication between the carrier 2051 and a power source. In some embodiments, the carrier 2051 includes a battery, a sensor, or both, and the battery or sensor is connected to an electrical connector via electrical wiring.

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

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

[0030] In some embodiments, the Nestbank 2050 further comprises a cooler, a heater, or both. In some embodiments, the cooler or heater is configured to control the temperature of each sample immobilized on 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 comprises a beam dump configured to absorb at least some of the excitation light generated by the optical system. In some embodiments, the sequencing system further comprises a beam dump configured to prevent at least some of the excitation light from reaching the imaging sensor of the optical system. In some embodiments, the beam dump is separated from the flow cell device by a gap zone. In some embodiments, the beam dump contacts the flow cell device with a predetermined locking force. In some embodiments, the beam dump contacts the xy stage with a predetermined damping force. In some embodiments, the predetermined damping force is configured to reduce the predetermined locking force so that the net force acting on the flow cell device is within a predetermined range.

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

[0033] This disclosure relates to a sequencing method comprising: (a) moving a first flow cell device from a nest bank 2050 to an xy stage 2010, wherein the first flow cell device includes a first sample immobilized thereon; (b) moving the xy stage 2010 and the first sample thereon relative to the objective lens of an optical system 2020 of a sequencing system; and (c) imaging the first sample immobilized on the first flow cell device on the xy stage 2010 using the optical system 2020. The present invention provides an array determination method comprising: (d) moving the first flow device from the xy stage 2010 to the nest bank 2050; (e) simultaneously enabling fluid and thermal communication between the nest bank 2050 and the second flow cell device during one or more of (a) to (d); and (f) moving the second flow cell device from the nest bank 2050 to the xy stage 2010, wherein the second flow cell device includes a second sample immobilized thereon.

[0034] In some embodiments, the sequence determination method further includes repeating operations (a) to (e). In some embodiments, the sequence determination method further includes repeating operations (f) to (j). In some embodiments, the sequence determination method further includes repeating operations (a) to (j) for a certain number of iterations. In some embodiments, the number of iterations is in the range of 1 to 500.

[0035] In some embodiments, enabling fluid communication between the nest bank 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 nest bank 2050 via one or more fasteners to enable sealed fluid communication between the nest bank 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 nest bank 2050 to the xy stage 2010 occurs within a first flow cycle of sequencing execution, and (f) moving the first flow cell device from the nest bank 2050 to the xy stage 2010 occurs within a second flow cycle of sequencing execution, distinct from the first flow cycle.

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

[0038] In some embodiments, enabling fluid and thermal communication between the nest bank 2050 and the first flow cell device simultaneously during one or more of (a) to (d) includes switching one or more fasteners to the ON state to enable physical contact for sealed fluid and thermal communication. In some embodiments, enabling fluid and thermal communication between the nest bank and the first flow cell device simultaneously during one or more of (a) to (d) includes immersing the flow cell device in at least some of one or more reagent containers in a predetermined order.

[0039] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are used, and to the appended drawings. [Brief explanation of the drawing]

[0040] [Figure 1] Block diagrams of computer implementation systems for performing operations in DNA sequencing and sequencing analysis are shown in several embodiments. [Figure 2] This is a schematic diagram of an exemplary sequencing system 2000 according to several embodiments, in which an optical system 2020 with an objective lens enclosed by a housing 2030, an xy stage 2010, a nest bank 2050, a flow cell device carrier 2051, and a moving mechanism 2040 (e.g., a movable arm) are included. [Figure 3] This is a schematic diagram showing an exemplary nest bank module according to several embodiments. [Figure 4A] Figure 3 is a schematic diagram showing an exemplary nest bank module according to several embodiments. [Figure 4B] Figure 3 is a schematic diagram showing an exemplary nest bank module according to several embodiments. [Figure 5A] This is a schematic diagram showing an exemplary flow cell device according to several embodiments. [Figure 5B] This is a schematic diagram showing an exemplary flow cell device according to several embodiments. [Figure 6] An exemplary nest bank with reagent containers according to several embodiments is shown. In Figure 6, each reagent container is at a temperature appropriate for a given reaction. The reagent containers can be part of a disposable (dry) apparatus. [Figure 7] The following describes exemplary flow cell devices having an open landing area according to several embodiments. [Figure 8] Block diagrams of computer systems for fluid control and for performing sequencing and sequencing analysis are shown according to several embodiments. [Figure 9]This is a schematic diagram showing an exemplary linear single-stranded library molecule (900) including a surface pinning primer binding site (920), an optional left-side unique identification sequence (980), a left-side index sequence (960), a forward sequencing primer binding site (940), an insertion region containing the sequence of interest (910), a reverse sequencing primer binding site (950), a right-side index sequence (970), and a surface capture primer binding site (930). [Figure 10] This is a schematic diagram showing an exemplary linear single-stranded library molecule (900) including a surface pinning primer binding site (920), a left index sequence (960), a forward sequencing primer binding site (940), an insertion region containing the sequence of interest (910), a reverse sequencing primer binding site (950), a right index sequence (970), an optional right identification sequence (990), and a surface capture primer binding site (930). [Figure 11] These are schematic diagrams of various exemplary configurations of polyvalent molecules. Left (Class I): Schematic diagram of a polyvalent molecule having a "starburst" or "helter-skelter" configuration. Center (Class II): Schematic diagram of a polyvalent molecule having a dendrimer configuration. Right (Class III): Schematic diagram of multiple polyvalent molecules formed by reacting streptavidin with 4-armed or 8-armed PEG-NHS and dNTPs containing biotin. Nucleotide units are represented as "N", biotin as "B", and streptavidin as "SA". [Figure 12] This is a schematic diagram of an exemplary polyvalent molecule, including a typical core attached to multiple nucleotide arms. [Figure 13] This is a schematic diagram of an exemplary polyvalent molecule containing a dendrimer core attached to multiple nucleotide arms. [Figure 14] A schematic diagram of an exemplary polyvalent molecule is shown, containing a core attached to multiple nucleotide arms, where the nucleotide arms include biotin, spacers, linkers, and nucleotide units. [Figure 15]This is a schematic diagram of an exemplary nucleotide arm, including the core attachment portion, spacer, linker, and nucleotide units. [Figure 16] The chemical structure of an exemplary spacer (top) and the chemical structures of various exemplary linkers, including 11-atom linkers, 16-atom linkers, 23-atom linkers, and N3 linkers (bottom), are shown. [Figure 17] The chemical structures of various exemplary linkers, including linkers 1-9, are shown. [Figure 18] The chemical structures of various exemplary linkers linked to / attached to nucleotide units are shown. [Figure 19] The chemical structures of various exemplary linkers linked to / attached to nucleotide units are shown. [Figure 20] The chemical structures of various exemplary linkers linked to / attached to nucleotide units are shown. [Figure 21] The chemical structures of various exemplary linkers linked to / attached to nucleotide units are shown. [Figure 22] The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide units are linked to the linker via a propargylamine bond at position 5 of the pyrimidine base or position 7 of the purine base. [Figure 23] A schematic diagram of one embodiment of a flow cell device is shown, in which the support comprises a glass substrate and alternating layers of hydrophilic coatings covalently or noncovalently bonded to the glass, and further comprises chemically reactive functional groups that serve as adhesion sites for oligonucleotide primers. [Figure 24A] A schematic diagram of an exemplary embodiment of a nest module, which is a part of the nest bank 2050 that receives carrier 2051, is shown when carrier 2051 is coupled to the nest bank 2050. [Figure 24B] A schematic diagram of an exemplary embodiment of a nest module, which is a part of the nest bank 2050 that receives carrier 2051, is shown when carrier 2051 is coupled to the nest bank 2050. [Figure 25]This is a schematic diagram of the movement of carrier 2051 between optical system 2020 and nest bank 2050. [Figure 26A] Figure 25 shows a schematic diagram of the xy stage 2010 of the optical system. [Figure 26B] Figure 25 shows a schematic diagram of NestBank 2050. [Figure 27A] A schematic diagram of an exemplary embodiment of the movement mechanism 2040 for the optical system 2020 and nest bank 2050 is shown. [Figure 27B] A schematic diagram of an exemplary embodiment of the movement mechanism 2040 for the optical system 2020 and nest bank 2050 is shown. [Figure 27C] A schematic diagram of an exemplary embodiment of the movement mechanism 2040 for the optical system 2020 and nest bank 2050 is shown. [Figure 28-1] A series of schematic diagrams illustrating exemplary embodiments of a sequencing system are shown. [Figure 28-2] A series of schematic diagrams illustrating exemplary embodiments of a sequencing system are shown. [Figure 29] A schematic diagram of an exemplary embodiment of a flow cell device carrier is shown. [Figure 30] A schematic diagram of an exemplary embodiment of the movement of the flow cell device carrier 2051 between the optical system 2020 and the nest bank 2050 is shown. [Figure 31] A schematic diagram of an exemplary embodiment of a nest bank 2050 for fluid communication and a flow cell device carrier 2051 located at the coupling position is shown. [Figure 32A] An exemplary embodiment of the XY stage 2010 for imaging and the flow cell device carrier 2051 in a coupled position is shown. [Figure 32B] An exemplary embodiment of the XY stage 2010 for imaging and the flow cell device carrier 2051 in a coupled position is shown. [Modes for carrying out the invention]

[0041] This specification describes systems and devices for analyzing different nucleic acid sequences from amplified nucleic acid arrays in a flow cell or from immobilized nucleic acid arrays. The systems and devices described herein can also be useful, for example, in sequencing for comparative genomics, gene expression tracking, microRNA sequencing, epigenomics, characterization of aptamer and phage display libraries, and other sequencing applications. The systems and devices described herein include various combinations of optical, mechanical, fluid, thermal, electrical, and computational devices / modes.

[0042] The advantages of the disclosed flow cell devices, fluid control devices, systems, and methods include, but are not limited to, flexible and scalable system throughput, flexible adaptation of the system to different sequencing applications, reduced complexity and cost of manufacturing and maintaining the devices and systems, reduced idling time of the optical system, and separation of fluid and thermal communications from the sample during imaging (thereby reducing potential interference to the imaging results).

[0043] Some disclosed design features of capillary flow cell devices, flow cell device carriers, and systems include, but are not limited to, open dispensing tips within the fluid control device and open landing areas on the flow cell device (allowing open reagent delivery without the complexity and cost of conventional tubing and enabling flexibility in the system to adapt to different sequencing applications), moving mechanisms (e.g., movable arms) (allowing the sample(s) to be moved relative to the optical system to maximize the use of the optical system and reduce the time required to complete the sequencing run), fluid and thermal communication (localized in the nest bank but not present in the xy stage, thereby reducing the size of the system and eliminating potential interference from it during imaging), and a combination of coarse and efficient movement of the sample by the movable arms and fine movement and fine adjustment of the sample relative to the objective lens or dispensing tip (to ensure accurate and efficient alignment of the sample(s) for imaging and fluid administration).

[0044] While the disclosed flow cell devices, systems, and methods are described primarily in the context of their use for nucleic acid sequencing applications, various embodiments of the disclosed systems and devices may be applicable not only to nucleic acid sequencing but also to any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. It should be understood that the different embodiments of the disclosed devices and systems can be understood individually, collectively, or in combination with each other.

[0045] Sequence determination system In some embodiments, what is disclosed herein are flow cell devices and systems that can be used to perform or facilitate DNA sequencing analysis using a sequencing system. The sequencing system may utilize a variety of sequencing techniques, including, but not limited to, those disclosed herein.

[0046] definition The titles provided herein are not limitations on the various aspects of this disclosure, which can be understood by referring to this entire specification. Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art. Generally, the terms relating to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization techniques described herein are well known and commonly used in the art. The techniques and procedures described herein are generally performed in accordance with conventional methods well known in the art, and are performed as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000). Also see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with the experimental procedures and techniques described herein, as well as the experimental procedures and techniques themselves, are well known and commonly used in the art.

[0047] Unless otherwise specified herein by context, singular terms include plural forms, and plural terms include singular forms. Unless explicitly and explicitly limited to a single reference, the use of the singular forms "a," "an," and "the," as well as the singular form of any of these words, includes plural references.

[0048] The use of alternative terms (e.g., "or") is understood to mean one or both of the alternative forms, or any combination thereof.

[0049] As used herein, the term “and / or” should be understood to mean a specific disclosure that each of a particular feature or component may or may not have the other. For example, as used herein in phrases such as “A and / or B,” the term “and / or” includes “A and B,” “A or B,” “A” (A only), and “B” (B only). In a similar manner, as used in phrases such as “A, B, and / or C,” the term “and / or” includes each of the following embodiments: “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 only); “B” (B only); and “C” (C only).

[0050] As used herein and in the appended claims, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations as used herein, are intended to be non-limiting so as not to exclude any other items that one or more items in a list may replace or add to the listed items. Wherever an aspect is described herein in the term “comprising,” it is understood that other similar aspects described in the terms “consisting of” and / or “essentially consisting of” are also provided.

[0051] As used herein, the terms “about,” “approximately,” and “substantially” refer to a value or composition that is within an acceptable error range for a particular value or composition, as determined by a person skilled in the art, and the acceptable error range depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measuring system. For example, “about,” “approximately,” or “substantially” may mean within one or more standard deviations per practice in the art. Alternatively, “about,” or “approximately,” may mean a range of up to 10% (i.e., ±10%) or more, depending on the limitations of the measuring system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean a value of up to one order of magnitude or up to five times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about,” “approximately,” and “substantially” should be understood to mean that it is within an acceptable error range for that particular value or composition. Also, where a range and / or subrange of a value is provided, the range and / or subrange may include the endpoint of the range and / or subrange.

[0052] The term "glass" refers to silica-based materials, including silicates, borosilicates, fused silica, fused quartz, glass, quartz, or lead glass.

[0053] As used herein, the term "polony" refers to a nucleic acid library molecule that can be clonally amplified in solution or on a support to produce an amplicon that can function as a template molecule for sequencing. For example, a linear library molecule can be cyclized to produce a cyclized library molecule, and the cyclized library molecule can be clonally amplified in solution or on a support to produce a concatemer molecule. The concatemer can function as a nucleic acid template molecule that can be sequenced. Concatemers are sometimes also called polony. In some embodiments, the polony includes a nucleotide chain.

[0054] As used herein, the term “clonally amplified” and its variants refer to nucleic acid template molecules subjected to one or more amplification reactions, either in solution or on a support. In the case of template molecules amplified in solution, the resulting amplicons can be distributed onto a support. Prior to amplification, the template molecule typically contains the sequence of interest and at least one universal adapter 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 substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), real-time SDA, bridge amplification, isothermal bridge 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.

[0055] The terms “peptide,” “polypeptide,” and “protein,” as well as other related terms used herein, are used synonymously and refer to polymers of amino acids, not limited to any particular length. Polypeptides may include natural and non-natural amino acids. Polypeptides may be recombinant or chemically synthesized. Polypeptides also include precursor molecules that have not yet undergone post-translational modifications such as proteolytic cleavage, ribosome skipping cleavage, hydroxylation, methylation, lipidization, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass natural and artificial proteins of protein sequences, protein fragments and polypeptide analogs (such as mutant proteins, variants, chimeric proteins, and fusion proteins), as well as proteins that have been modified post-translationally or otherwise covalently or non-covalently.

[0056] As used herein, the term “sequencing” and its variants typically involve obtaining sequence information from a nucleic acid chain by determining the identity of at least some nucleotides (including their nucleic acid base components) within a nucleic acid template molecule. In some embodiments, “sequencing” a given region of a nucleic acid template molecule involves identifying each and all nucleotides within the region to be sequenced, while in some embodiments, “sequencing” includes a method in which the identity of only some nucleotides within the region is determined, while the identity of some nucleotides remains undetermined or is incorrectly determined. Any preferred sequencing method may be used. In exemplary embodiments, sequencing may include label-free methods or ion-based sequencing methods. In some embodiments, sequencing may include labeled or dye-containing nucleotides, or fluorescence-based nucleotide sequencing methods. In some embodiments, sequencing may include Polony-based sequencing or bridge sequencing methods. In some embodiments, sequencing may include a massively parallel sequencing platform employing synthetic sequencing, hybridization sequencing, or conjugation sequencing procedures. Examples of sequencing procedures using massively parallel synthesis include Polony sequencing, pyrosequencing (e.g., U.S. Patent Nos. 7,211,390, 7,244,559, and 7,264,929 from 454 Life Sciences), strand terminator sequencing (e.g., U.S. Patent No. 7,566,537 from Illumina, 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 Ion Torrent), probe-anchored ligation sequencing (e.g., Complete Genomics), DNA nanoball sequencing, and nanopore DNA sequencing.Examples of single-molecule sequencing include Heliscope single-molecule sequencing from Pacific Biosciences and single-molecule real-time sequencing (SMRT) (Levene, et al., 2003 Science 299(5607):682-686, Eid, et al., 2009 Science 323(5910):133-138, U.S. Patents 7,170,050, 7,302,146, and 7,405,281). Examples of sequencing by hybridization include SOLiD sequencing (e.g., WO2006 / 084132 from Life Technologies). Examples of sequencing by binding include Omniome sequencing (e.g., U.S. Patent 10,246,744).

[0057] The term “polymerase” and its variants, as used herein, encompass any enzyme capable of catalyzing the polymerization of nucleotides (including their analogues) into nucleic acid chains. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase comprises one or more active sites, at which one or more active sites the catalysis of nucleotide bonding and / or nucleotide polymerization can occur. In some embodiments, a polymerase comprises other enzymatic activity, e.g., 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, spontaneous polymerases and any of their subunits and cleaved, mutant polymerases, variant polymerases, recombinant, fused or otherwise manipulated polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any of their analogues, derivatives, or fragments (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. In some embodiments, polymerases can be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases can be expressed in prokaryotes, eukaryotes, viruses, or phage organisms. In some embodiments, polymerases can be post-translationally modified proteins or fragments thereof. Polymerases can be derived from prokaryotes, eukaryotes, viruses, or phages. Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0058] As used herein, the term “fidelity” refers to the precision of DNA polymerization by template-dependent DNA polymerase. DNA polymerase fidelity is typically measured by the error rate (the frequency of incorporating inaccurate nucleotides, i.e., nucleotides not complementary to the template nucleotide). The precision or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3'-5' exonuclease activity of the DNA polymerase.

[0059] 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 polyvalent molecule, wherein the nucleic acid duplex includes a nucleic acid template molecule hybridized to a nucleic acid primer. In a binding complex, the free nucleotide or nucleotide unit may or may not be bound to the 3' end of the nucleic acid primer at a position opposite to the 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 polyvalent molecule, wherein the free nucleotide or nucleotide unit is bound (as part of the nucleic acid duplex) to the 3' end of the nucleic acid primer at a position opposite to the complementary nucleotide in the nucleic acid template molecule.

[0060] As used herein, “nucleotide unit” or “nucleotide moiety” refers to a nucleotide (e.g., dATP, dTTP, dGTP, dCTP, or dUTP) or analogue thereof, comprising a base, a sugar, and at least one phosphate group. Nucleotide units can be attached to polyvalent molecules used in sequencing reactions described herein. Generally, all nucleotide units attached to the same polyvalent molecule will have the same identity (e.g., all A, all T, all C, or all G), but it will be understood by those skilled in the art that there may be situations in which a polyvalent molecule containing nucleotide units of different identities is advantageous.

[0061] The term “duration” and related terms refer to the length of time during which a binding complex remains stable without any of its components dissociating, and which includes a nucleic acid template and nucleic acid primer, polymerase, nucleotide units of a polyvalent molecule, or free (e.g., non-conjugated) nucleotides. Nucleotide units or free nucleotides may be complementary or apart from nucleotide residues in the template molecule. Nucleotide units or free nucleotides may be bound to the 3' end of a nucleic acid primer at the opposite position from a complementary nucleotide residue in the nucleic acid template molecule. Duration indicates the stability of the binding complex and the strength of the binding interaction. Duration can be measured by observing the onset and / or duration of the binding complex, for example, 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 duration of the binding complex. One exemplary label is a fluorescent label. The binding complex (e.g., a ternary complex) remains stable until subjected to conditions that cause dissociation of the interactions between the polymerase, template molecule, primer, and / or nucleotide units or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with one of the following: a washing agent, EDTA, and / or water, or any combination thereof.

[0062] As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as well as other related terms, are used interchangeably and refer to polymers of nucleotides, none of which are limited to any particular length. Nucleic acids include recombinant or 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 nucleotide analogs not found in nature), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids contain polymers of nucleotides, where nucleotides contain native or unnative bases and / or sugars. Nucleic acids contain naturally occurring nucleoside bonds, e.g., phosphodiester bonds. Nucleic acids contain unnative nucleoside bonds, where unnative nucleoside bonds include phosphorothioates, phosphorothiolates, or peptide nucleic acid (PNA) bonds. Nucleic acids may also contain mixtures of native and unnative nucleoside bonds. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0063] As used herein, the term “primer” and related terms refer to either natural or synthetic oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form double-stranded molecules. Primers can have any length, but are typically in the range of 4 to 50 nucleotides. Typical primers include a 5' end and a 3' end. The 3' end of a primer may include a 3'OH moiety that functions as a nucleotide polymerization initiation site in polymerase-mediated primer extension reactions. Alternatively, the 3' end of a primer may lack a 3'OH moiety or may include a terminal 3' block group that inhibits nucleotide polymerization in polymerase-mediated reactions. Any one or more nucleotides along the length of the primer can be labeled with a detectable reporter moiety. Primers can be in solution (e.g., soluble primers) or immobilized on a support (e.g., capture primers).

[0064] "Template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template chain," and other variations refer to a nucleic acid chain that functions as a base nucleic acid molecule for generating a complementary nucleic acid chain. The template nucleic acid can be single-stranded or double-stranded, or it can 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 chain. The template nucleic acid can be obtained from naturally occurring sources or recombinant forms, or it can be chemically synthesized to contain nucleic acid analogs of either type. The template nucleic acid can be linear, cyclic, or in other forms. The template nucleic acid may contain an insertion region having an insertion sequence also referred to herein as the sequence of interest. The template nucleic acid may also contain at least one adapter sequence. The template nucleic acid may be a concatemer having two or tandem copies of the sequence of interest and at least one adapter sequence. The insertion region can be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, clones, amplifications, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole-genomic DNA obtained from fresh-frozen paraffin-embedded tissue, needle biopsies, cell-free circulating DNA, or any type of nucleic acid library. The insertion region can be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, viral cells, tissues, normal or diseased cells or tissues, bodily fluids including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, semen, environmental samples, biofilms, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemosynthesis methods. The insertion region can be isolated from any organ, including the head, neck, brain, breasts, ovaries, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testes, liver, lungs, kidneys, esophagus, pancreas, thyroid gland, pituitary gland, thymus, skin, heart, larynx, or other organs. The insertion region can be isolated from multiple cells or from a single cell.The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and compositional analysis.

[0065] When used in relation to nucleic acid molecules, the terms “hybridize,” “hybridizing,” or “hybridization,” or other related terms, refer to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridization molecule having a double-stranded region. Hybridization can include Watson-Crick or Hoogstein bonds to form a double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid, or a single nucleic acid, can be fully complementary or partially complementary. Complementary nucleic acid strands do not need to hybridize to each other over their entire length. Complementary base pairing can be standard AT or CG base pairing, or it can be other forms of base pairing interactions. Double-stranded nucleic acids can contain mismatched base-pairing nucleotides.

[0066] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of this term. In some embodiments, the phosphate includes monophosphate, diphosphate, or triphosphate, or the corresponding phosphate analogue. In some embodiments, the nucleotide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar.

[0067] Nucleotides (and nucleosides) typically consist of heterocyclic bases containing substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, which are commonly found in nucleic acids and include spontaneous, substituted, modified, or engineered variants or analogues thereof. The bases of a nucleotide (or nucleoside) can form Watson-Crick and / or Hoogstein hydrogen bonds with appropriate complementary bases. Exemplary bases include purines and pyrimidines, e.g., 2-aminopurine, 2,6-diaminopurine, adenine (A), etenoadenine, N 6 -Δ 2 -Isopentenyl adenine (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-deazapurine, e.g., 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, e.g., 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); indoles, e.g., nitroindole and 4-methylindole; pyrroles, e.g., nitropyrrole; nebularin; inosine; hydroxymethylcytosine; 5-methicytosine; bases (Y); and methylated, glycosylated, and acylated base moieties, etc., are included but not limited to these. Additional exemplary bases can be found in Fasman, 1989, “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0068] Nucleotides (and nucleosides) typically include sugar moieties, e.g., carbon-cyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (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 U.S. Patent No. 5,558,991). The sugar portion includes ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic, or other modified sugars.

[0069] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, the chain typically bonded to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain, within which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise a substituted side chain group comprising O, S, or BH3. In some embodiments, the chain comprises a phosphate group substituted with analogs comprising phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoramidite groups.

[0070] When used in relation to nucleic acids, the terms “extend,” “extend,” and other variations of “extend” refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides to the terminal 3'OH end of a nucleic acid chain, resulting in the elongation of the nucleic acid chain. Nucleotide incorporation can be performed with native nucleotides and / or nucleotide analogs. Typically, but not always, nucleotide incorporation occurs in a template-dependent manner. Any preferred method for elongating a nucleic acid molecule may be used, including primer elongation catalyzed by DNA polymerase or RNA polymerase.

[0071] The terms “reporter moiety” or “reporter moieties” or related terms refer to a compound that produces or causes to produce a detectable signal. A reporter moiety is often referred to as a “label.” Any suitable reporter moiety may be used, and suitable reporter moieties include luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, hapten, affinity tags, atoms, or enzymes. A reporter moiety produces a detectable signal resulting from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or proximity event). A proximity event includes two reporter moieties approaching each other, associating with each other, or binding to each other. It is well known to those skilled in the art that reporter moieties can be selected such that each 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 can be selected that have spectrally different emission profiles or minimum overlap spectral emission profiles. Reporter moieties can be bound (e.g., operably ligated) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerase or reverse transcriptase), or supports (e.g., surfaces).

[0072] The reporter portion (or label) contains a fluorescent label or fluorophore. Examples of fluorescent moieties that can function as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives, e.g., carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaptofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidefluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and rhodamine derivatives, e.g., TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazides, coumarins and coumarin derivatives, e.g., AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazides, BODIPY® and derivatives, e.g., BODIPY® FL C3-SE, BODIPY® 530 / 550 C3, BODIPY® 530 / 550 C3-SE, BODIPY® 530 / 550 C3 hydrazides, BODIPY® 493 / 503 C3 hydrazides, BODIPY® FL C3 hydrazides, BODIPY® FL IA, BODIPY® 530 / 551 IA, Br-BODIPY® 493 / 503, Cascade Blue® and derivatives, e.g., Cascade Blue® acetylazide, Cascade Blue® Cadaverine, Cascade Blue® Ethylenediamine, Cascade Blue® Hydrazide, Lucifer Yellow and derivatives, e.g., Lucifer Yellow Iodoacetamide, Lucifer Yellow CH, cyanines and derivatives, e.g., indolium-based cyanine dyes, benzo-indolium-based cyanine dyes,Pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives, e.g., BCPDA, TBP, TMT, BHHCT, BCOT, europium chelate, terbium chelate, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, e.g., Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New Examples include, but are not limited to, those described in York (1999) or Hermanson, Bioconjugate Techniques, 2nd Edition, or their derivatives, or any combination thereof. Cyanine dyes may exist in either sulfonated or unsulfonated forms and consist of two indorenine, benzoindolinium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3(this is 1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indole-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolinium,Or it may contain 1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indole-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indoleum-5-sulfonate), Cy5 (this is 1-(6-((2,5-dioxopyrrolidine (1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-ium, or 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)- 5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-ium-5-sulfonate (may contain these), and Cy7(1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indole-2-ylidene) The formula may include (e.g., heptate-1,3,5-trien-1-yl]-3H-indolinium, or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indole-2-ylidene)heptate-1,3,5-trien-1-yl]-3H-indolinium-5-sulfonate). "Cy" stands for "cyanine," and the first number specifies the number of carbon atoms between the two indolenin groups. Cy2, which is an oxazole derivative rather than indolenin, as well as the benzo derivatives Cy3.5, Cy5.5, and Cy7.5, are exceptions to this rule.

[0073] In some embodiments, the reporter portion may be a fluorescence resonance energy transfer (FRET) pair, thereby allowing multiple classifications to be performed under a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0074] The terms “linked,” “joined,” “attached,” and their variants include any kind of fusion, joining, adhesion, or association between any combination of compounds or molecules that are stable enough to withstand use in a particular procedure. Procedures may include, but are not limited to, transient nucleotide joining, nucleotide incorporation, deblocking, washing, removal, flow, detection, imaging, and / or identification. Such joining may include, for example, covalent bonds, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonds, bonds or associations involving van der Waals forces, and mechanical bonds. In some embodiments, such joining occurs within a molecule, 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 joining may occur between different combinations of molecules, or between molecules and non-molecules, including, but are not limited to, joining a nucleic acid molecule to a solid surface, joining a protein to a detectable reporter moiety, joining a nucleotide to a detectable reporter moiety, and so on. Some examples of coupling can be found, for example, in Hermanson, G., "Bioconjugate Techniques", Second Edition (2008), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998), and Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998).

[0075] As used herein, the terms “operably linked” and “operably linked,” or related terms, refer to the juxtaposition of components. Parallel components can be covalently linked together. For example, two nucleic acid components can be enzymatically ligated together, and the bond linking the two components together includes a phosphodiester bond. A first and second nucleic acid component can be linked together, and the first nucleic acid component can confer function to the second nucleic acid component. For example, ligation between a primer-binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to the primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) can be ligated into a vector, and ligation enables the expression or function of the transgene sequence contained within the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects the expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, the at least one host cell regulatory sequence comprising a promoter sequence, an enhancer, a transcription start sequence and / or a translation start sequence, a transcription termination sequence and / or a translation termination sequence, and a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of the expression of the transgene. In some cases, the components can be linked together non-covalently. It will also be understood by those skilled in the art that the components do not need to be directly and physically linked in order to be functionally linked.

[0076] The term "adapter" and related terms refer to an oligonucleotide that can operably ligate (attach) to a target polynucleotide, and the adapter confers function to the coligated adapter-target molecule. Adapters can include DNA, RNA, chimeric DNA / RNA, or analogues thereof. Adapters can contain at least one ribonucleoside residue. Adapters can be single-stranded or double-stranded, or have single-stranded and / or double-stranded portions. Adapters can be configured to be linear, stem-loop, hairpin, or Y-shaped. Adapters can be any length containing 4 to 100 or more nucleotides. Adapters can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5' and 3' overhang ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, may or may not have a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tailed adapter), or the adapter may be tailless. The adapter may include a sequence that is complementary to at least a portion of a primer described herein, e.g., an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter may include a random sequence or a degenerate sequence. The adapter may include a random sequence (e.g., NNN) or may not include a random sequence. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothiolate, and / or phosphoramidate bond. The adapter may include a barcode sequence, which may be used in a multiplex assay to distinguish polynucleotides (e.g., insert sequences) from different sample sources.The adapter may include a unique identification sequence (e.g., a unique molecular index, UMI, or unique molecular tag), which may be used to uniquely identify the nucleic acid molecule to which the adapter is attached. The unique identification sequence may include a random sequence (e.g., NNN) or may not include a random sequence. In some embodiments, the unique identification sequence may be used to increase error correction and accuracy, reduce the rate of false-positive variant calls, and / or increase the sensitivity of variant detection. The adapter may include at least one restriction enzyme recognition sequence, which may include one or more selected from the group consisting of type I, type II, type III, type IV, type Hs, or type IIB.

[0077] The terms "universal sequence," "universal adapter sequence," and related terms refer to sequences in nucleic acid molecules that are common to two or more polynucleotide molecules. For example, an adapter having the same universal sequence can be ligated to multiple polynucleotides, so that the group of co-ligated molecules possesses the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or support-immobilized capture primers).

[0078] All publications, patents, and patent applications referenced herein are incorporated herein by whole to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by whole. In the event of any conflict between terms used herein and terms used in incorporated references, the terms defined herein shall prevail.

[0079] Sequence determination system Figure 1 shows a block diagram of a computer implementation system 100 for performing sequencing and sequencing analysis according to one or more embodiments disclosed herein. System 100 has a sequencing system 110 including a flow cell device 112, a sequencer 114, an imager 116, data storage 122, and a user interface 124. The sequencing system 110 may optionally be connected to a cloud 130 (e.g., a server, computing device, database, etc.). The sequencing system 110 may include one or more of a dedicated processor 118, an integrated circuit (e.g., a field-programmable gate array (FPGA)) 120, and a computer system 126.

[0080] In some embodiments, the flow cell device 112 is configured to capture DNA fragments and form a DNA sequence for base calling from imaging of the flow cell device 112 by an imager 116. The flow cell 112 may include a support as disclosed herein with respect to Figure 7. The support may be a solid support. The support may include a surface coating thereon as disclosed herein. The surface coating may be a polymer coating as disclosed herein. The surface coating may be placed on the surface of one or more channels of the flow cell device 112. Different or identical surfaces may be placed on the inlet surface of the flow cell device 112.

[0081] In some embodiments, the flow cell device 112 may include a plurality of tiles (e.g., parts, locations, regions, sections, etc.) configured to be imaged by the imager 116, and each tile may be separated into a plurality of subtiles. In some embodiments, the subtiles may be organized in a grid. Each subtile may include a plurality of clusters or polony (e.g., a collection of DNA molecules such as concatemer template molecules disclosed herein) on it. In some embodiments, the flow cell device 112 may include a number of tiles in the range of about 1 tile to about 2000 tiles, about 100 tiles to about 1500 tiles, or about 200 tiles to about 500 tiles (including all ranges and subranges in between). In some embodiments, each tile may be divided into a number of subtiles in the range of about 2 subtiles to about 200 subtiles, about 10 subtiles to about 100 subtiles, or about 20 subtiles to about 50 subtiles (including all ranges and subranges in between). In some embodiments, the subtiles may be organized into a grid that may have M × N subtiles. As a non-limiting example, the flow cell device 112 may have 424 tiles, and each tile may be divided into a 6 × 9 grid, and therefore 54 subtiles. In some embodiments, the imager 116 may be configured to acquire one or more images of a plurality of tiles (hereinafter, "flow cell images"), a subset of a plurality of tiles, and / or a subset of a plurality of subtiles. The flow cell images disclosed herein may include images containing signals of a plurality of clusters or polony. A flow cell image may include one or more tiles of signals, or one or more subtiles of signals. In some embodiments, a flow cell image may be an image containing all tiles and substantially all signals on them. Flow cell images may be acquired from the channel using the imager 116 during (i) an imaging cycle or (ii) an array determination cycle. In some embodiments, each tile may contain millions of polony or clusters.As a non-limiting example, a tile can contain approximately 1 to 10 million clusters or polony. Each polony can be a collection of many copies of a DNA molecule.

[0082] Further details of the flow cell device 112 and its functional and structural elements are disclosed herein in relation to the figures (e.g., Figure 7).

[0083] The sequencer 114 may be configured to flow a mixture of reagents onto a flow cell. Such a mixture of reagents may include a nucleotide mixture, polymerase, reagents for adding or cleaving blockers from nucleotides during a nucleotide addition step, and reagents for performing other steps to form a DNA molecule suitable for sequencing applications on the flow cell 112. The nucleotides may have attached fluorescent elements (also referred to as "labels" or "parts") that emit light or energy of a wavelength 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 of visible wavelengths. In some embodiments, the sequencer 114 and the flow cell device 112 may be configured to perform various sequencing methods disclosed herein or known in the art (e.g., avidite sequencing, binding sequencing, or synthesis sequencing).

[0084] For example, each nucleotide base can be assigned a color. Different types of nucleotides can have different colors. For example, adenine (A) may be red, cytosine (C) may be blue, guanine (G) may be green, and thymine (T) may be yellow. The color or wavelength of the fluorescent element of each nucleotide may be selected based on the wavelength of light emitted by the fluorescent element, so that the nucleotides are distinguishable from one another.

[0085] The imager 116 may be configured to capture an image of the flow cell 112 after each flow step. In one embodiment, the imager 116 may include a camera configured to capture a digital image, such as a CMOS or CCD camera. The camera may be configured to capture an image at the wavelength of a nucleotide-bound fluorescent element. The image may be called a flow cell image.

[0086] In some embodiments, the imager 116 may include one or more optical systems disclosed herein. The optical system(s) may be configured to capture optical signals from a flow cell and generate a corresponding digital image thereof. The digital image can then be used for base calling. In some embodiments, the optical system and / or the flow cell 112 may be coupled to one or more moving mechanisms configured to position the flow cell 112 relative to the imager 116. In some embodiments, the flow cell 112 and / or the PLC 114 may be coupled to one or more moving mechanisms to position the flow cell 112 relative to one or more dispensers of the PLC. In some embodiments, the PLC 114 may include a nest bank configured to receive and hold one or more flow cells 112 during an array determination cycle. In some embodiments, the moving mechanism may move the flow cell 112 from the nest bank to the imager 116 for an imaging cycle. In some embodiments, a portion of the flow cell and / or nest bank may transition to a first locked (e.g., aligned) configuration when the flow cell 112 is positioned on or near the nest bank so that the flow cell 112 is in fluid communication with the nest bank. In some embodiments, the flow cell 112 may be configured to transition from a locked configuration to an unlocked configuration and move to the imager 116. In some embodiments, the flow cell 112 may transition from an unlocked configuration to a second locked configuration so that the flow cell 112 can be positioned relative to the imager 116 (e.g., without mechanical, fluid, or thermal disturbances). In some embodiments, multiple flow cells 112 may be present in the sequencing system 110 simultaneously. For example, a second flow cell may be used for a sequencing cycle while a first flow cell is used for an imaging cycle. In this way, the sequencing system 110 may have improved sequencing efficiency and high sample throughput.In some embodiments, the imager 116 (e.g., and / or any part of the optical system) may be fluidically isolated from the sequencer 114 to prevent fluid exposure to the imager 116 and / or improve image quality. In some embodiments, the movement mechanism may include at least one of a movable arm and / or an xy stage. In some embodiments, the processors of the sequencing system 110 (e.g., a dedicated processor 118, an FPG(or more) 120, and / or the CPU of the computer system 126) may each be configured to perform different tasks so that simultaneous sequencing and imaging can be performed.

[0087] In one embodiment, images of the flow cell may be captured in groups, with each image within the group matching only one of the fluorescent elements or captured at a wavelength or spectrum that includes it. In another embodiment, the image may be captured as a single image capturing all wavelengths of the fluorescent elements.

[0088] The resolution of the imager 116 controls the level of detail in the flow cell image, including pixel size. In existing systems, this resolution is critical as it controls the accuracy with which the spot search algorithm identifies Polony 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 and subranges in between). In some embodiments, the image resolution of the flow cell image can be from about 10 nm to about 900 nm, from about 10 nm to about 500 nm, from about 10 nm to about 200 nm, from about 20 nm to about 500 nm, from about 20 nm to about 200 nm, or any range or subrange in between. One way to improve the accuracy of spot search is to improve the resolution of the imager 116 or to improve the processing performed on the image captured by the imager 116. This can be used to perform detection of Polony centers in pixels other than those detected by the spot search algorithm. Suitable spot search algorithms will be known to those skilled in the art. These methods can improve the detection accuracy of Polony centers without increasing the resolution of the imager 116. The resolution of the imager 116 may be lower than that of existing systems with comparable performance, which can reduce the cost of the sequencing system 110.

[0089] The image quality of the flow cell image can control the base calling accuracy. The imager 116 disclosed herein can improve the base calling accuracy. Alternatively, processing performed on the image captured by the imager 116 can result in better image quality, thereby improving the base calling accuracy using the system disclosed herein.

[0090] After base calling is performed, a processor (e.g., a dedicated processor 118, an FPGA(or more) 120, a computer system 126, or a combination thereof) may optionally perform additional processing and / or analysis of the base calling results. In some embodiments, after base calling is performed, the (processed and / or raw) sequencing reads(or more) can be output from the system to an external device (e.g., a cloud 130 and / or a computer system 400). The sequencing reads(or more) herein may include forward reads (R1), reverse reads (R2), or both. The sequencing reads(or more) herein may be any ordered sequence of A, T, C, and G bases.

[0091] In some embodiments, the sequence determination read(s) can be transmitted (e.g., directly or indirectly) to a computer system 126 for subsequent analysis, such as adapter trimming or fading.

[0092] These sequencing analysis methods, including primary and / or secondary analysis, can be advantageously executed in parallel on computer system 126 without interference or delay with the existing sequencing workflow of system 100. The results of the sequencing analysis can be made available for generating user sequencing results. Some or all of the operations of the sequencing process can be advantageously executed by FPGAs, and data can be communicated between the CPU and FPGAs to reduce the total operation time from methods that operate without FPGAs.

[0093] The operations or actions disclosed herein may be performed by a dedicated processor 118, an FPGA(or more) 120, a 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, an FPGA(or more) 120, a computing system 126, or a combination thereof. In some embodiments, which operations or actions should be performed by a dedicated processor 118, an FPGA(or more) 120, a computing system 126, or a combination thereof may be determined based on one or more of the computation time for a particular operation(or more), the complexity of the computation in a particular operation(or more), the need for data transmission between hardware devices, or a combination thereof.

[0094] The computing system 126 may include one or more general-purpose computers that provide interfaces for running various programs within an operating system such as Windows® or Linux®. Such operating systems typically offer users greater flexibility.

[0095] In some embodiments, the dedicated processor 118 may be a custom processor that has specific hardware or instructions for executing those steps, rather than a general-purpose processor. The dedicated processor 118 may directly execute specific software without an operating system. The absence of an operating system reduces overhead, at the expense of the flexibility of what the dedicated processor 118 can do. The dedicated processor 118 may utilize a custom programming language, which may be designed to operate more efficiently than software running on a general-purpose computer. This can increase the speed at which steps are executed, enabling real-time processing.

[0096] In some embodiments, FPGAs (or multiple FPGAs) 120 may be configured to perform the operations of the sequencing decision analysis method described herein. FPGAs are programmed as hardware to perform only specific tasks. Software steps can be translated into hardware components using a special programming language. Once programmed, the hardware processes the provided digital data directly without executing software. Instead, FPGAs use logic gates and registers to process the digital data. Because there is no overhead required by an operating system, FPGAs generally process data faster than general-purpose computers. As with dedicated processors 118, this comes at the expense of flexibility.

[0097] Furthermore, the absence of software overhead could allow the FPGA120 to operate faster than the dedicated processor 118, although this may depend on the exact processing being performed, as well as on the specific FPGA120 and dedicated processor 118.

[0098] A group of FPGAs 120 may be configured to execute processing steps in parallel. For example, several FPGAs 120 may be configured to perform processing steps on an image, a set of images, a subtile, or a selected region within one or more images. In some embodiments, each FPGA 120 may execute each step or substep of the processing steps simultaneously, reducing the time required to process the data. This allows the processing steps to be completed in real time or near real time. Further considerations of the use of FPGAs are provided below.

[0099] By executing processing steps in real time, the system 100 may use less memory because data can be processed upon receipt rather than being stored for subsequent analysis. This offers an advantage over conventional systems, which may store data before it is processed, requiring more memory and / or requiring access to and communication with a computer system located in the cloud 130. In some embodiments, the data storage device 122 is used to store information used in or obtained from sequencing analysis. For example, DNA sequences determined after adapter trimming may be stored in the data storage 122. Compressed and / or uncompressed sequencing data may be stored in the data storage. FASTQ files may also be stored in the data storage 122.

[0100] The user interface 124 can be used by the user to operate the sequencing system or to access data stored in the data storage 122 or the computer system 126.

[0101] Computer system 126 may control the general operation of the sequencing system and may be coupled to the user interface 124. In some embodiments, computer system 126 may perform one or more steps in sequencing analysis, such as base calling, adapter trimming, demultiplexing, and fading. In some embodiments, computer system 126 may be structurally and / or functionally similar to computer system 800, as described in more detail in Figure 8. Computer system 126 may include memory configured to store information about the operation of the sequencing system 110, such as configuration information, instructions for operating the sequencing system 110, or user information. Computer system 126 may be configured to pass information between the sequencing system 110 and the cloud 130. For example, computer system 126 may be configured to receive base calling results from a dedicated processor 118 and / or FPGA(s) and send the base calling results to the cloud 130 for storage and / or further analysis.

[0102] As discussed above, the sequencing system 110 may have a dedicated processor 118, an FPGA(or more) 120, or a computer system 126. The sequencing system 110 may use one, two, or all of these elements to achieve the required processing described above. In some embodiments, when these elements are present together, the processing tasks are divided among them. The FPGA(or more) 120 may be used to perform some or all of the sequencing analysis operations, while the computer system 126 may perform other processing functions for the sequencing system 110. Distributing processing across the dedicated processor(or more) 118, FPGA(or more) 120, and / or a general-purpose processor (e.g., in the computer system 126) can enable parallel processing and / or improve the efficiency of processing steps. For example, complex processing steps may be assigned to the dedicated processor(or more) 118 and / or FPGA(or more) 120 while processing for the general operation of the system 110 is performed by the computer system 126.

[0103] Those skilled in the art will understand that various combinations of these elements can enable various system embodiments that balance processing efficiency and speed with the cost of processing elements. The cloud 130 may be a separate network, server, remote storage, or some other remote computing system from the sequencing system 110. Connecting to the cloud 130 may enable access to data stored outside the sequencing system 110 or enable software updates in the sequencing system 110.

[0104] Flow cell device In some embodiments, disclosed herein are flow cell devices and systems that can be used to perform or facilitate DNA sequencing analysis. The flow cell devices herein can be used to immobilize a template nucleic acid molecule derived from a biological sample and then introduce a repeating flow of sequencing reagents (e.g., binding sequencing, synthesis sequencing, and / or avidite sequencing) to attach labeled nucleotides or labeled polyvalent molecules to specific locations on the template sequence. A series of labeled signals are detected and decoded to reveal the nucleotide sequence of the template molecule, e.g., an immobilized and / or amplified nucleic acid template molecule attached to the surface of the flow cell.

[0105] Figure 7 shows an exemplary embodiment of the flow cell device 200. The flow cell device 200 may include one or more substrates placed thereon, a support (e.g., as a surface) having several channels (not shown), an inlet (not shown), and an outlet (not shown). In some embodiments, a liquid (280) is dispensed from a dispenser into the flow cell device 200.

[0106] In some embodiments, the support 210 of the flow cell device 200 disclosed herein may be configured to define or accept one or more channels and / or one or more substrates. In some embodiments, the support 210 may be solid (i.e., rigid and stable in shape). At least a portion of the support 210 may be transparent so that light transmitted from the light source of the imager (116 in Figure 1) can pass through the transparent portion of the support 210 and reach the sample located on the flow cell device 200.

[0107] The support 210 may contain or accept one or more substrates. When the flow cell device 200 is placed in an sequencing system (e.g., sequencing system 110) for imaging, the upper substrate can be closer to the camera of the imager (e.g., imager 116) along the z-direction than the lower substrate. The lower substrate can be closer to the xy stage of the sequencing system 110 for holding and supporting the flow cell 200 during sequencing than the upper substrate.

[0108] In some embodiments, the flow cell device 200 may further include an intermediate substrate between the upper substrate and the lower substrate.

[0109] Each substrate may have a predetermined thickness. In some embodiments, some or all of the 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 uniform thickness may encompass the channel(s) or imaging region of a flow cell device.

[0110] In some embodiments, the upper and / or lower substrate may have a first thickness, and the intermediate substrate may have a second thickness less than the first thickness. In some embodiments, the upper and / or lower substrate may have a thickness of about 0.2 mm to about 5 mm (including all ranges and subranges in between). In some embodiments, the upper and / or lower substrate may have a thickness of about 0.6 mm to about 3 mm (including all ranges and subranges in between). In some embodiments, the upper and / or lower substrate may have a thickness of about 0.8 mm to about 2 mm (including all ranges and subranges in between). In some embodiments, the upper and / or lower substrate may have a thickness of about 0.8 mm to about 1.5 mm (including all ranges and subranges in between).

[0111] In some embodiments, the intermediate substrate may have a thickness of about 40 μm to 200 μm (including all ranges and subranges within that range). In some embodiments, the intermediate substrate may have a thickness of about 40 μm to 150 μm (including all ranges and subranges within that range). In some embodiments, the intermediate substrate may have a thickness of about 40 μm to 70 μm (including all ranges and subranges within that range).

[0112] In some embodiments, the intermediate substrate can have a thickness of about 80 μm to 120 μm (including the entire range and sub-ranges therein).

[0113] In some embodiments, the substrate(s) can form an elongated shape extending along the y-axis on the surface of the support 210 on which it is placed. In some embodiments, the substrate(s) can have various shapes such as rectangles, squares, or ellipses.

[0114] In some embodiments, one or more substrates may be planar or substantially planar. In some embodiments, one or more substrates may not have any curvature perceptible to the naked eye, and therefore one or more substrates may be planar. However, in certain embodiments, the substrates do not need to be planar. Alternatively, part or all of one or more substrates may be curved.

[0115] In some embodiments, the support 210 or one or more substrates may include 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 include tape, such as pressure-sensitive adhesive (PSA) tape.

[0116] The substrate(s) may define one or more channels 250 of the flow cell device 200 (for example, extending longitudinally along the top surface of the flow cell device 200). The channels 250 may allow a fluid (for example, a liquid or gas) to flow through them.

[0117] The gases described herein may include one type of gas or a combination of different types of gases. In some embodiments, the gas includes air. The gas may include dry air. In some embodiments, the gas includes one or more inert gases (e.g., argon or nitrogen). In some embodiments, the gas includes one or more active gases.

[0118] The sequencing reagents described herein may include liquids. In some embodiments, the reagents can remove bubbles larger than a given size (for example, to improve the accuracy and / or reproducibility of reactions performed in a flow cell device, to enhance the clarity of images captured by the optical systems of this disclosure, and / or to 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 re-wet the surface of one or more channels 250 after the surface has been at least partially dried by a gas gap.

[0119] In some embodiments, the channel 250 may include a microfluidic channel. In some embodiments, the gap or height between the upper and lower inner surfaces of the substrate defining the channel 250 along the z-direction is approximately 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm, or any range in between. In some embodiments, the gap or height of the channel 250 is approximately 100 μm or less. In some embodiments, the gap or height of the channel 250 is approximately 60 μm, 50 μm, or 40 μm or less.

[0120] In some embodiments, the length of channel 250 is approximately 120 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, or 30 mm along the y-direction, or any range in between. In some embodiments, the length of channel 250 is approximately 100 μm or less. In some embodiments, the length of channel 250 is approximately 80 mm, 75 mm, 70 mm, 65 mm, 60 mm, 55 mm, or 50 mm or less.

[0121] In some embodiments, the width of the channel 250 is approximately 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 10 mm, 15 mm, 8 mm, or 5 mm along the x-direction, or any range in between. In some embodiments, the length of the channel 250 is approximately 10 mm or approximately 7 mm or less. In some embodiments, the width of the channel 250 is approximately 40 mm, 35 mm, 30 mm, 25 mm, 20 mm, or 15 mm or less.

[0122] In some embodiments, the distance between two adjacent channels 250, or the distance from the end of a channel 250 along the x-axis to the end of a flow cell device 200, is approximately 0.5 mm to approximately 15 mm. In some embodiments, the distance between two adjacent channels 250, or the distance from the end of a channel 250 along the x-axis to the end of a flow cell device 200, is approximately 1 mm to approximately 5 mm.

[0123] In some embodiments, the flow cell device 200 may have two or more channels 250, and all channels 250 may have unformed sizes and shapes. In some embodiments, the flow cell device 200 may have channels 250 of different sizes and / or shapes.

[0124] The flow cell device 200 may include one or more inlets and one or more outlets. Channels 250 may run or extend from their corresponding inlets to their corresponding outlets, thereby enabling fluid communication from the corresponding inlets to the corresponding outlets. Sequencing reagents are introduced into the flow cell device 200 through the inlets, flow through each of the channels 250, interact with the sample located therein, and then exit through the outlets.

[0125] The size and shape of the inlet and outlet can be customized to suit various sequencing applications.

[0126] The flow cell device, fluid control device, and system may include an open landing area through which reagent 280 can flow. Figure 7 shows a flow device with an open landing area.

[0127] In some embodiments, the open landing area can be at least partially formed by a corresponding inlet. The open landing area can be located on the lower substrate. For example, the open landing area can be formed by a void in corresponding areas of the intermediate and upper substrates, or by their extensions or openings. In some embodiments, each channel 250 can be coupled to a corresponding open landing area. The open landing area can be fluidly connected to its corresponding channel(s). 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 surrounding the system and apparatus.

[0128] Nest bank and movable arm In some embodiments, the sequencing system 2000 disclosed herein may include an optical system 2020 having an objective lens, and an xy stage 2010, an xy stage configured to hold a sample to be imaged on thereon and to move the sample in the xy plane relative to the objective lens, on which the sample is fixed to one or more flow cell devices as shown in Figure 2. The sequencing system 2000 may further include a nest bank 2050 configured to provide fluid and thermal communication to a sample when one or more flow cell devices are coupled to the nest bank 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 nest bank 2050 during sequencing execution. The nest bank 2050 may include one or more identical nest modules. As used herein, “nest module” means one or more regions of a nest bank configured to receive the carrier 2051 and to provide fluid and thermal-fluid communication to the carrier 2051 and / or a flow cell device located within it.

[0129] In some embodiments, the sequencing system 2000 comprises a housing (not shown) configured to hold one or more of the following: an optical system, an xy stage 2010, a nesting bank 2050, a carrier 2051, and a moving mechanism 2040 including a movable arm. In other words, the nesting bank 2050, the moving mechanism 2040, and the carrier(s) 2051 can be positioned within an integrated console box of the sequencing system. Alternatively, one or more of the structural elements (e.g., the nesting bank and the movable arm) can be positioned at least partially outside the housing of the sequencing system 2000.

[0130] The xy stage described herein may be equivalent to the moving stage and / or sample stage of an sequencing system. The xy stage may be automatically driven by an actuator (e.g., the first actuator) with first spatial accuracy. Various actuators (e.g., motors, cams, or gear systems) may be used herein. In some embodiments, the xy stage may be manually driven (e.g., by a user). In some embodiments, the xy stage 2010 (e.g., in Figures 2 and 27A) includes a base and a movable stage coupled thereon. In some embodiments, the xy stage 2010 may be driven to move in three-dimensional space (3D). In some embodiments, the xy stage 2010 may be movable. In some embodiments, the xy stage 2010 is driven to move by a predetermined distance, for example, to focus a sample relative to an objective lens. The predetermined distance may be 3D. In some embodiments, the predetermined distance may be along the xy plane. In some embodiments, the predetermined distance is based on the distance between two adjacent microfluidic channels of a flow cell device. In some embodiments, the xy stage 2010 is driven to move from a first position (e.g., a predetermined starting position) to a second position (e.g., a predetermined stopping position). For example, the xy stage 2010 may be driven to move between two different spatial positions (e.g., a starting position and a stopping position) so that each of the two microfluidic channels of the same flow cell device can be positioned relative to the objective lens for imaging. In some embodiments, the xy stage 2010 is driven to move the sample relative to the objective lens for imaging. The xy stage 2010 can move in 3D from a predetermined starting position and stopping position at various speeds and / or with spatial accuracy (e.g., ±0.1 mm, ±0.100 μm). The time required to move the sample to a predetermined position relative to the objective lens can be between 0.5 seconds and 10 seconds (including all ranges and sub-ranges within that range).The time required to move the sample to a predetermined position relative to the objective lens can be between 0.5 and 3 seconds (including the entire range and sub-ranges within that time). Various speeds can be used to optimize the sample movement time and accuracy. For example, the speed can range from 0.001 mm / second to 2 meters / second, e.g., 0.001 mm / second to 100 cm / second, 0.01 mm / second to 50 cm / second, or 0.1 mm / second to 50 mm / second.

[0131] In some embodiments, the xy stage 2010 in the sequencing system 2000 differs from the sample stage and / or transfer stage in existing sequencing systems in that the xy stage 2010 (or near the xy stage 2010) is not in fluid or thermal communication with one or more flow cell devices when a flow cell device is immobilized on the xy stage 2010. In existing systems, fluid and thermal communication (e.g., fluid or heat exchange) or connection (fluid pathways and physical contact) may exist while the sample is immobilized on the xy stage (even while the sample is being imaged). In contrast, the sequencing system 2000 disclosed herein lacks fluid and thermal communication (e.g., fluid or heat exchange) or connection (fluid pathways and physical contact), and the sample has no fluid and / or thermal communication or connection when the sample is on the xy stage.

[0132] In some embodiments, the xy stage 2010 can be coupled with a kinematic mount for precise positioning. For example, the magnetic contacts of the kinematic mount can be engaged via inductive magnetism to load a flow cell device and disengaged to release a flow cell device to load the next flow cell device. The array determination system 2000, with the moving mechanism 2040, nest bank 2050, and xy stage 2010, can produce higher imaging reliability, manufacturing robustness, and flexibility compared to existing systems because imaging is completely isolated from fluid and thermal communication. The movement of the xy stage 2010 (e.g., along the x-axis) can be enabled by a cam system, which reduces costs and increases speed and flexibility.

[0133] In some embodiments, the xy stage 2010 may be coupled with one or more sensors to provide feedback on the alignment of the flow cell device to the xy stage 2010 and / or the optical system. Various sensors can be used, such as a visible light sensor, an audio sensor, other optical sensors such as infrared, pressure sensors, and / or force sensors. Feedback from one or more sensors may be transmitted to a processor in the array determination system 2000 or to an external processor, and the feedback can be used to fine-tune the positioning of the flow cell device to the xy stage 2010 and / or the optical system so that it aligns with the objective lens for imaging. For example, an audio sensor may be used to detect sound waves reflected from the carrier 2051 and determine the distance between the carrier 2051 and the aligned imaging position (e.g., the difference between the actual position of the carrier 2051 and the desired position of the carrier 2051). In another example, a camera(s) may be used to detect whether the carrier is positioned at the aligned imaging position.

[0134] In some embodiments, the xy stage 2010 may include an external hardware processor, separate from the processor of the sequencing system 2000. The hardware processor of the xy stage 2010 may communicate with one or more sensors to process feedback from one or more sensors and generate instructions for actuators (multiple) (e.g., chip tilt devices, motors) to move the xy stage 2010 to a desired 3D position, without communicating with the sequencing system's processor(s) (e.g., one or more dedicated processors 118, multiple FPGAs 120, or processors of the computer system 126). The movement can be performed with a predetermined precision (i.e., ±0.1 mm, ±0.100 μm). In some embodiments, the xy stage 2010 may include an external hardware processor, separate from the sequencing system's processor, to enable autonomous or semi-autonomous movement of the xy stage 2010 based on feedback from one or more sensors.

[0135] In some embodiments, the xy stage does not include any hardware processors outside of the processor of the sequencing system. One or more sensors may communicate with one or more processors of the sequencing system 2000 (e.g., one or more dedicated processors 118, FPGAs 120, or processors of the computer system 126) to process feedback from the one or more sensors and generate commands for actuators 1 or more (e.g., chip tilt devices, motors) to move the xy stage 2010 to a desired 3D position.

[0136] In some embodiments, the movement of the xy stage can be linear. For example, the xy stage 2010 may move along the x, y, or any linear axis in 3D. In some embodiments, the movement of the xy stage is nonlinear (e.g., rotation in 3D).

[0137] In some embodiments, the optical system 2020 may be movable in one or more directions in 3D. In some embodiments, the optical system 2020 may be coupled with a kinematic mount for precise positioning in 3D. In some embodiments, the optical system 2020 may be movable with a predetermined spatial accuracy and within a predetermined speed range. For example, the xy stage 2010 may move linearly along the x-axis, and the optical system (e.g., at least the objective lens) may move linearly along the y-axis, and the combination of movement of the optical system and the xy stage 2010 may enable positioning of the sample relative to the objective lens for imaging. In another example, either the xy stage 2010 or the optical system may move nonlinearly (e.g., rotate around a predetermined origin), and the other of the xy stage 2010 or the optical system may move linearly, and the combination of movement of the optical system and the xy stage 2010 may enable positioning of the sample relative to the objective lens for imaging. The xy stage 2010 and optical system may be movable in multiple directions relative to each other in order to precisely position the sample relative to the objective lens.

[0138] In some embodiments, the movable arm may be automatically driven by an actuator (e.g., a second actuator) with a second spatial precision. The first actuator, the second actuator, or both may be controlled by one or more hardware processors of the sequencing system 2000 (e.g., one or more dedicated processors 118, FPGAs 120, and processors of the computer system 126). The movable arm may be automatically driven to move in three dimensions (3D). The movement of the movable arm in each of the three dimensions may have the same or different spatial precisions. 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 the sample from the nest bank 2050 to the xy stage 2010 may be between 0.5 seconds and 10 seconds (including all ranges and subranges within that range). The time required to move the sample from the nest bank 2050 to the xy stage 2010 may be between 0.5 seconds and 3 seconds (including all ranges and subranges within that range). The movable arm of the moving mechanism 2040 can move at various speeds, for example, the speed can be in the range of 1 mm / sec to 2 m / sec (including all ranges and sub-ranges in between). As further examples, the speed can be in the range of 1 mm / sec to 500 centimeters (cm) / sec, 10 mm / sec to 50 cm / sec, or 1 cm / sec to 100 cm / sec.

[0139] In some embodiments, the movable arm of the moving mechanism 2040 is configured to move one or more flow cell devices between two spatial positions in three dimensions (e.g., between the xy stage 2010 and the nest bank 2050) with a first spatial accuracy. For example, the movable arm may be configured to move a flow cell device from the nest bank 2050 to the xy stage 2010 to image a portion of the flow cell device, and / or to move it from the xy stage 2010 to the nest bank 2050 after imaging.

[0140] Figures 27A–27C illustrate different exemplary embodiments of a sequencing system including a movable arm disclosed herein. As shown in Figures 27A–27C, the sequencing system may comprise one or more flow cell devices, each flow cell carrier device 2051, located in a nest bank 2050 positioned in a first position relative to an optical system 2020 including an objective lens. The nest bank 2050 may be positioned at a certain distance from the objective lens so that there is separation between the optical system and the fluid and / or temperature changes as the reagent flows over the flow cell devices. In some embodiments, each flow cell carrier 2051 may be coupled to a respective xy stage 2010, and a moving mechanism 2040 may pick up the carrier 2051 (e.g., using the respective xy stage 2010) and move the carrier 2051 to a second position near the objective lens, thereby positioning the carrier 2051 in the second position. In some embodiments, the array determination system may include one xy stage 2010 positioned near the objective lens, and a moving mechanism 2040 may be configured to move a flow cell carrier 2051 onto the xy stage 2010 for imaging.

[0141] The movable arm may include a gripping portion (e.g., 2043 in Figure 27A) configured to grasp or otherwise hold the carrier 2051. In some embodiments, the movable arm and the carrier 2051 may be configured to transition between a coupled state and a discoupled state (further described in Figures 30-31). In the coupled state, the movable arm may be securely coupled to the flow cell device and its carrier 2051. When in the coupled state, the movable arm may reliably move the carrier, for example, between the xy stage 2010 for imaging and the nest bank 2050. The movable arm may be configured to transition to a discoupled state, in which case the carrier 2051 is detached from the movable arm when the flow cell device and its carrier 2051 are in a target position. The gripping portion 2043 of the movable arm may be configured to receive and / or hold the carrier 2051 when the carrier 2051 is in a discoupled position relative to the nest bank, or when the carrier is in a discoupled position relative to the xy stage. In some embodiments, the movable arm comprises a horizontal arm 2041 (e.g., mechanically supported) coupled to a vertical arm 2042 (e.g., a bar, support, track, extension, etc.) as shown in Figure 27A. The horizontal arm 2041, the vertical arm 2042, and the gripping section 2043 can move in 3D relative to the housing of the sequencing system or any other reference point of the sequencing system, so that the gripping section 2043 can grip the flow cell carrier 2051 and move between the nest bank 2050 and the xy stage 2010. In some embodiments, the horizontal arm 2041 and / or the vertical arm 2042 may be positioned above the nest bank 2050, and the gripping section 2043 may be facing downward. As shown in Figure 27B, the movable arm of the moving mechanism 2040 may be coupled to the surface of the sequencing system on which the nest bank 2050 and the xy stage 2010 are located. In some embodiments, the base of the movable arm may be fastened to the surface of the array determination system, and the movable arm 2040 may include one or more joints from which the movable arm can bend and / or rotate.In some embodiments, the movable arm may include, for example, one or more of the following: an upper arm (e.g., a first part), a joint, a forearm (e.g., a second part), a wrist, and a gripping part attached to the forearm, as shown in Figure 27B. The upper arm and forearm may be connected to each other via a first joint, and the gripping part may be connected to the forearm via a second joint. Some or all of the components of the movable arm may be individually movable relative to the housing of the array determination system or any other reference point of the array determination system. In such embodiments, the movable arm may move in 3D with 6 degrees of freedom by combining the movements of one or more of the components of the movable arm.

[0142] Figures 30-31 show exemplary embodiments of the movable arm disclosed herein. The movable arm may include a coupled state as shown in Figure 30, in which the movable arm is securely coupled to the flow cell device 200 and its carrier 2051. When the carrier 2051 is coupled, the movable arm can reliably move the carrier 2051, for example, between the xy stage 2010 and the nest bank 2050 for imaging. The movable arm may include an uncoupled state in which the flow cell device 200 and its carrier 2051 are detached from the movable arm, as shown in Figure 31. In some embodiments, the gripping portion of the movable arm in the coupled state may have a greater width than the gripping portion in the uncoupled state. For example, the unconnected gripping portion may have a neutral position having a width smaller than the width of the carrier 2051, and may be configured to extend to match the width of the carrier 2051 so that the gripping portion applies a clamping force to the carrier 2051 and securely holds the carrier 2051 between one or more extensions of the gripping portion (e.g., fingers, nails).

[0143] In some embodiments, the movable arm comprises one or more arm elements that are movable relative to each other. As shown in Figures 30-31, the horizontal arm 2041 can support two different forearms 2041 to move along at least a horizontal plane (e.g., the xy-plane). In some embodiments, the two different forearms 2041 can also move along the z-axis, which is perpendicular to the horizontal plane.

[0144] In some embodiments, the gripping portion 2043 may use various mechanisms for gripping or otherwise holding the carrier 2051 and for moving the carrier together with the gripping portion. For example, the gripping portion 2043 may include fingers as shown in Figures 27A and 27B. In another example, the gripping portion 2043 may lack any finger-like structures. In some embodiments, the gripping portion may use frictional, magnetic, or electromagnetic forces to grip or otherwise hold the carrier 2051 so that the carrier 2051 moves together with the gripping portion.

[0145] In some embodiments, the gripping portion is movably mounted on a horizontal or vertical arm. In some embodiments, the gripping portion is configured to move in 3D relative to the housing of the sequencing system or any other reference point of the sequencing system.

[0146] In some embodiments, the moving mechanism 2040 comprises multiple tracks, as shown, for example, in Figure 27C. Each track may connect a carrier 2051 coupled to a nest bank 2050 to the xy stage 2010. In some embodiments, the carrier 2051 may be driven by a motor or, otherwise, an actuator to move along the track between the xy stage 2010 and the nest bank 2050. Compared to the embodiments in Figures 27A-27B, the tracks reduce the flexibility of the flow cell device as it moves in 3D, but because the travel tracks are in a predetermined layout, they can be simpler, less prone to variation in movement, or more compact.

[0147] In some embodiments, the moving mechanism 2040 comprises one or more belt conveyors, which function similarly to trucks to move carriers between the xy stage 2010 and the nest bank 2050.

[0148] Figure 25 shows an exemplary layout of the transport mechanism 2040, nest bank 2050, and optical system (including objective lens) relative to the carrier(s) 2051. In some embodiments, the carrier 2051 may have at least two docking positions (fluid control position 2503 and imaging position 2502). Multiple flow cell carriers may be arranged in a single sequencing system, and multiple carriers may share a common optical system. At the fluid control position 2503, the dispenser 2070 may dispense fluids such as library solution, sample, reagents, and wash buffers into the open landing area(s) of the flow cell device located on the carrier 2051. The dispenser 2070 may include various embodiments of dispensing tips, e.g., pipette tips and removable cartridges (containing different reagents and communicating fluidly with the dispensing tip).

[0149] In some embodiments, the movement of the movable arm (e.g., movement at the gripping or distal end of the movable arm) can be linear, thereby causing the carrier 2051, which is moved by the moving mechanism 2040, to also move linearly. For example, the movable arm can move the carrier 2051 and the sample along the x-axis or y-axis in the xy-plane. In some embodiments, the movement of the movable arm can be nonlinear (e.g., rotation in 3D), and the carrier 2051, which is carried by the gripping portion of the movable arm, also moves nonlinearly accordingly.

[0150] The samples disclosed herein may include a variety of samples sequenced on a sequencing system. The samples herein may be 2D or 3D samples, including in-situ samples such as cells and / or tissues. Fluids may be extracted from a flow cell device using an extraction pump, which may be connected to a carrier 2051 and a flow cell via connections 1006 in Figures 24A and 25. Connection 1006 may be a quick-connect connection with a gasket(s) or other sealing component to prevent leakage and allow for sealed fluid communication. In some embodiments, the extraction pump is coupled to a nest bank. In some embodiments, the extraction pump is coupled to a flow cell.

[0151] At imaging position 2502, the optical system can perform imaging and, if necessary, be in optional fluid communication with a flow cell device.

[0152] The flow cell device carrier can be moved between docking positions using a moving mechanism 2040. For example, the moving mechanism 2040 may include a movable arm, a belt conveyor, a roller conveyor, a rail system, or a pick-and-place robot.

[0153] In some embodiments, the arrangement determination system may include a plurality of flow cell carriers, each having its own fluid control line 1001 connected to one or more pumps. The moving mechanism 2040 may move the plurality of carriers 2051 and manage their operation using an algorithm that prevents the plurality of fluid control lines 1001 from becoming entangled with each other or otherwise interfering with each other.

[0154] In some embodiments, the xy stage 2010 is configured to be driven to move to a 3D position with a predetermined spatial accuracy (e.g., a second spatial accuracy) different 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 accurate than the first spatial accuracy. For example, the first spatial accuracy may be 1 mm and the second spatial accuracy may be 0.05 mm. Having different spatial accuracys can be advantageous in that it may allow for coarser movement of the moving mechanism 2040 to move the sample to the stage, and allow for finer adjustment of the spatial position of the xy stage 2010 relative to the objective lens for focusing and imaging.

[0155] In some embodiments, the xy stage 2010 may include one or more fasteners configured to detachably secure a flow cell device thereto. Various fasteners can be used herein. In non-limiting examples, the fastener(s) may include one or more clamps using mechanical (e.g., snap-fit, friction-fit), magnetic, or electromagnetic force(s).

[0156] In some embodiments, each carrier 2051 includes a coupled position in which the carrier 2051 is removably mounted and secured to the xy stage 2010 by fasteners. In the coupled position, the carrier 2051 is in sealed fluid communication with the xy stage 2010. In some embodiments, each carrier 2051 includes a detached position in which the carrier 2051 is removable from the xy stage 2010 and is not secured to the xy stage 2010. In the detached position, the carrier 2051 is not in fluid communication with the xy stage 2010. Furthermore, the carrier 2051 may include one or more valves or other fluid control stoppers to prevent fluid within the carrier 2051 from leaking out of the carrier 2051.

[0157] In some embodiments, the xy stage 2010 does not include a pump or fluid path that can connect to a flow cell device coupled thereon and enable fluid communication. The absence of a pump or fluid path on the xy stage 2010 may simplify the sequencing system and make it more cost-effective. Furthermore, separating the fluid system from the xy stage 2010 and the optical system may advantageously eliminate or minimize contamination or leakage during imaging.

[0158] In some embodiments, the xy stage 2010 may include one or more pumps configured to deliver 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 stoppers that can eliminate or reduce the possibility of leakage from the flow cell device while coupled on the xy stage 2010.

[0159] 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 within a predetermined temperature and / or temperature range. In some embodiments, the xy stage 2010 may include one or more temperature sensors. 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 regulate the temperature (e.g., the heating device and / or cooling device) to achieve the predetermined temperature. Various thermal devices and sensors can be used herein.

[0160] 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 include, among other things, air tables.

[0161] Figure 32A shows an exemplary embodiment of the xy stage 2010 coupled to a mechanical decoupler or mechanical isolator 2014. The mechanical isolator 2014 can prevent or otherwise minimize external mechanical disturbances (e.g., vibrations reaching the flow cell device 200 and / or optical system 2020) so that external disturbances to the flow cell device 200 do not interfere with imaging and / or cause undesirable movement of the sample(s) immobilized on the flow cell device 200. Various mechanical isolators 2014 can be used to isolate external mechanical disturbances.

[0162] 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 dump devices (e.g., 2503 in Figures 32A-32B) configured to capture energy from the light source of the optical system and prevent the 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 dump device(s) capture the excitation light traveling from the optical system through the flow cell device 200 and then to the beam dump device(s). In some embodiments, the beam dump device(s) capture the emitted light emitted from the sample and traveling to the beam dump device(s). In some embodiments, the beam dump device(s) may include a laser beam dump device. In some embodiments, the beam dump device may include a variety of components configured to absorb photon energy, such as graphite, tungsten, concrete, or marble.

[0163] In some embodiments, the optical system 2020 or some structural elements of the optical system (e.g., objective lenses) may be positioned on top of the flow cell device 200, as shown in Figures 32A to 32B. In some embodiments, the optical system 2020 may be positioned in various different positions relative to the flow cell device 200 (and should not be limited to the embodiments shown in Figures 32A to 32B). For example, the optical system 2020 or some structural elements of the optical system (e.g., objective lenses) 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.

[0164] 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 shows a schematic diagram of an exemplary embodiment of the xy stage 2010 and the carrier 2051. In some embodiments, the mounting element(s) 2509 may include precision kinematic mounts. In some embodiments, the mounting element(s) 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(s) 2509 may be secured to the xy stage 2010 via various fastening elements, such as magnetic latches.

[0165] In some embodiments, the mounting element(s) 2509 may include Maxwell or Kelvin couplings. In some embodiments, the mounting element(s) 2509 may include precision kinematic mounting elements. In some embodiments, the mounting element(s) 2509 has a total of fewer than 10, 9, 8, 7, 6, or 5 contacts with the xy stage 2010. In some embodiments, the mounting element has a total of fewer than 6, 5, 4, or 3 contacts with the xy stage 2010. In some embodiments, the mounting element(s) 2509 includes various geometric shapes that satisfy the principle of exact constraint design. For example, in some embodiments, the number of constraint points of the mounting element(s) 2509 is equal to the number of degrees of freedom of the constrained flow cell device carrier 2051. As an example, the mounting element(s) 2509 may consist of three elements, each having a spherical surface, and each having a concave tetrahedron, a V-groove directed toward the tetrahedron, and a plane that rests on the xy stage 2010. The tetrahedron may provide three contact points, while the V-groove may provide two, and the plane may provide one, resulting in a total of six contact points with the xy stage 2010. As another example, the xy stage 2010 may include three V-grooves, while the mounting element(s) 2509 consists of three elements, each having a curved surface configured to be positioned on the corresponding groove. Each of the three V-grooves provides two contact points with the corresponding mounting element(s) 2509, for a total of six contact points. In some embodiments, the mounting element(s) 2509 is configured to control the positional accuracy of the carrier 2051 within a range of 0.1 nm to 0.1 mm (including all ranges and sub-ranges in between). In some embodiments, the mounting element(s) 2509 is configured to control the positional accuracy of the carrier within a range of 1 nm to 1 μm (including all ranges and subranges in between). In some embodiments, the mounting element(s) 2509 is configured to control the positional accuracy of the carrier(s) 2051 within a range of 0.1 μm to 0.1 mm (including all ranges and subranges in between).In some embodiments, the mounting element(s) 2509 is configured to control the positional accuracy of the carrier 2051 within a range of 1 μm to 0.1 mm (including all ranges and sub-ranges in between).

[0166] In some embodiments, when the flow cell device 200 and its carrier 2051 are in the imaging position, there may be a gap zone 2508 between the flow cell device 200 and the xy stage 2010. The gap zone 2508 lacks any heater / cooler or fluid connections to the flow cell device 200 or its carrier 2051. The gap zone 2508 is maintained to reduce mechanical and thermal stresses that may be applied to the flow cell device 200 from heater / cooler or fluid paths, so that flow cell images can be acquired with less thermal or mechanical disturbance when the flow cell device 200 is in the imaging position. Thus, the gap zone 2508 can improve the image quality of flow cell images by reducing mechanical disturbances (e.g., vibration, shaking, strain of the flow cell device 200, etc.) and 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 determined in advance. The height of the air gap may be adjustable by adjusting the relative position of the flow cell device 200 with respect to the xy stage 2010. The air gap can have a height of 0.01 mm to 1 cm (including all ranges and subranges within that range). The air gap can have a height of 0.1 mm to 5 cm (including all ranges and subranges within that range). The air gap can have a height of 1 mm to 50 cm (including all ranges and subranges within that range).

[0167] Figure 32B shows another embodiment of the xy stage 2010, also referred to as the imaging dock 2010. In this embodiment, the xy stage 2010 includes a beam dump 2503. This beam dump 2503 may be in direct contact with the flow cell device 200, the carrier 2051, or both. In some embodiments, the beam dump 2053 may be in contact with the flow cell device 200 and / or carrier 2051 with a minimum contact force or any force within a given force range. While we do not wish to be constrained by theory, it is believed that a minimum contact force with the beam dump reduces mechanical stress and the possibility of heat or vibration transmission. Contact with a given force may be maintained by balancing a locking force 2504b that is slightly greater than the damping force 2504c, and both the locking force 2504b and the damping force 2504c may be applied to the beam dump 2503 through various mechanisms. For example, the locking force 2504b may be applied through one or more magnetic latches located on the flow cell device 200, the carrier 2051, and / or the xy stage 2010. The magnetic latches may attract the carrier 2051 and thus attract the flow cell device 200 toward the xy stage 2010, and / or attract the xy stage 2010 toward the carrier 2051.

[0168] In some embodiments, the beam dump 2503 may generate a damping force 2504c that pulls the beam dump away from the flow cell device 200 and the carrier 2051. In some embodiments, the damping force 2504c may be generated by using a biasing mechanism (e.g., a spring-deformable member, a flexible material, etc.). In some embodiments, the damping force 2504c can offset the locking force 2504b. The net effect of the damping force 2504c and the locking force 2504b may be used to minimize the amount of force applied to the flow cell 200. The total force by combining the locking force 2504b and the damping force 2504c can be adjusted, for example, by selecting various combinations of magnets and springs.

[0169] In some embodiments, the locking force 2504b and the damping force 2504c may be within a predetermined range. In some embodiments, the net force resulting from the combination of the locking force 2504b and the damping force 2504c may be within a predetermined range. In some embodiments, the net force may be in the range of -0.001 Newtons (N) to 0.001 N (for example, a positive force is an upward force toward the flow cell device along z). In some embodiments, the net force may be in the range of -0.01 N to 0.01 N (for example, a positive force is an upward force toward the flow cell device along z). In some embodiments, the net force may be in the range of -0.05 N to 0.05 N (for example, a positive force is an upward force toward the flow cell device along z). In some embodiments, the net force may be in the range of -0.5 N to 0.5 N (for example, a positive force is an upward force toward the flow cell device along z). In some embodiments, the net force may be in the range of -10N to 10N (for example, a positive force is an upward force toward the flow cell device along z).

[0170] Figure 26A shows a schematic diagram of an example of an xy stage 2010. The xy stage 2010 may secure the carrier 2051 using a coupling mechanism 2012, such as a clamp, fastener, bolt, anchor, or rivet. The xy stage 2010 may include a temperature controller (e.g., a heating device and / or cooling device) 2013 optimized to maintain a predetermined temperature of the sample for incubation, imaging, etc., during library preparation. The xy stage 2010 may include a positioning device 2014 (e.g., a tip tilt device) that positions the carrier 2051 to a 3D position with a predetermined precision (e.g., a first precision) for imaging. In some embodiments, the xy stage 2010 is motor-driven or otherwise driven by actuators 2015 for movement and / or rotation to position the carrier 2051 in 3D for imaging. In some embodiments, the xy stage 2010 is driven via an actuator 2015 to move to a position that is relatively low in precision but accurate enough to bring the sample near a predetermined position (e.g., the imaging position). The position adjustment device 2014 then fine-tunes the sample position to the predetermined position with higher precision. 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, enables the sample to be accurately and reliably positioned at a predetermined 3D position for imaging.

[0171] In some embodiments, the xy stage 2010, a moving mechanism (e.g., moving mechanism 2040, not shown), or both are driven to move with a relatively low precision, but with a precision that allows the sample to be moved to a vicinity of a predetermined position (e.g., within a few millimeters of the predetermined position). A position adjustment device 2014 located on the xy stage 2010 then fine-tunes the position of the sample to the predetermined position with higher precision. In some embodiments, a combination of moving the stage (and sample) relative to the housing or any other reference point and moving the sample relative to the stage allows the sample to be precisely and reliably positioned at a predetermined 3D position for imaging.

[0172] As a non-limiting example, while carrier 2051 is coupled to nest bank 2050 for fluid dosing into the corresponding flow cell, the moving mechanism 2040 may be prevented from moving carrier 2051 away from nest bank 2050, and xy stage 2010 may move to a position between optical system 2020 and nest bank 2050 to await the sample, saving travel distance and time for the moving mechanism to transport the sample. After fluid dosing is complete, the moving mechanism 2040 may be allowed to move carrier 2051 to xy stage 2010, which can then return to optical system 2020 in a position for imaging the sample. In this particular embodiment, position adjustment device 2014 may then fine-tune the position of the sample relative to the objective lens for imaging, while xy stage 2010 and moving mechanism 2040 may move the sample with lower spatial accuracy, optionally, if necessary, at a higher speed possible to save time. Arranging such movements with different spatial accuracies can, to some advantage, reduce system complexity, save manufacturing costs, improve the robustness of the sequencing system, and reduce the time it takes for users to perform sequencing.

[0173] In some embodiments, at least some components within the optical system 2020, the xy stage 2010, or both are mounted on a vibration isolator(s) that mechanically detaches the rest of the instrument for the purpose of improving image quality. Such isolation may advantageously allow imaging with minimal operational disturbance and facilitate fluid dispensing and chemical processing without external operational disturbance. In some embodiments, the flow cell device is coupled to a carrier, and a movable arm is configured to move the carrier and flow cell device together. The carrier 2051 may remain fixedly coupled to the flow cell device when the flow cell device is on the xy stage 2010. In some embodiments, the optical system 2020 may move linearly or nonlinearly in 3D. For example, the optical system 2020, or at least some part of the optical system, may move along the x-axis, y-axis, or any other linear axis in 3D. As another example, at least some part of the optical system may rotate around the z-axis in 3D or another axis in 3D. For example, the xy stage 2010 may move linearly along the x-axis, and the optical system (e.g., at least the objective lens) may move linearly along the y-axis, and the combination of movement of the optical system and the xy stage 2010 makes it possible to position the sample in a predetermined position relative to the objective lens for imaging. In another example, either the xy stage 2010 or the optical system 2020 may move nonlinearly (e.g., rotate around a given origin), and the other of the xy stage 2010 or the optical system 2020 may move linearly, and the combination of movement of the optical system and the xy stage makes it possible to position the sample in a predetermined position relative to the objective lens for imaging.

[0174] In some embodiments, the nest bank 2050 of the sequencing system can hold a flow cell device on it, and the flow cell device and the sample on it can be prepared for imaging during sequencing execution.

[0175] Nest banks can be fluid-connected to various reagents and buffers (e.g., washing buffers and / or library loading buffers). Fluid communication between the nest and the reagent or solution container may be via a closed fluid pathway or an open fluid connection that allows for open dispensing of fluids.

[0176] In some embodiments, each nest module of the Nest Bank 2050 is provided with thermal and fluid interfaces to a flow cell device. The nest modules can be linearly distributed in an array, as shown in Figure 27A, or in various other spatial distributions. Thermal incubation can be carried out in a chamber corresponding to an individual flow cell device carrier. One or more heat sinks can be positioned beneath each nest module, as shown in Figures 3 and 4A. The fluid interface can include a dispensing tip that dispenses into the open wells of the flow cell device, or a plug-in that connects to the respective microfluidic channels of the flow cell device.

[0177] Each flow cell device may include an open landing area configured to openly receive fluid from a nest bank. The flow cell device comprises multiple microfluidic channels, and the nest bank is configured to allow fluid communication to each of the multiple microfluidic channels. Fluid communication from the nest bank to each channel may be independent to avoid cross-contamination. For example, different pipette tips can be used to dispense different reagents into different channels through the corresponding openings of the nest bank. In some embodiments, fluid communication from the nest bank to multiple channels may be performed simultaneously to reduce the time required for fluid communication during sequencing execution. In some embodiments, fluid communication from the nest bank to multiple channels may be performed sequentially to simplify the communication process and reduce the complexity and cost of the nest bank.

[0178] Figures 24A and 24B show exemplary embodiments of the carrier 2051 described herein. In some embodiments, for example in Figure 24A, the carrier 2051 may include an opening 1004 on the surface of the carrier 2051 configured to receive a flow cell device (not shown) therein. The carrier 2051 may further include one or more fluid paths 1001 that are in sealed fluid communication with the flow cell device when the flow cell device is removably mounted to the carrier 2051. 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 having a connector, configured to allow sealed fluid communication between the carrier 2051 and the corresponding nest module when the connector is in the connection position. The carrier 2051 may include a valve 1005 positioned between a fluid path connecting to a flow cell device and a port opening 1006 of the carrier, the valve being in an open position when the flow cell device is coupled to the carrier (e.g., not imaging) and in a closed position when the flow cell device is uncoupled (e.g., imaging). The carrier 2051 may include electrical wiring 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 that electrical communicates with one or more of its elements (e.g., a pump 1003, a port 1006, and a fastener (e.g., fastener 2012)). One or more of the battery, sensor, pump, and fastener(s) may be connected to the electrical connector 1008 via electrical wiring to an external power source.

[0179] Figures 5A–5B and 29 show exemplary embodiments of the carrier 2051. In the embodiment shown in Figure 29, the carrier 2051 may not include a valve or a similar structure that functions similarly. In this particular embodiment, the open landing area for suction and / or the outlet for fluid extraction may be oriented upward away from the direction of gravity. Furthermore, the fluid port (e.g., similar to port 1006) may also face upward. Such a structural arrangement may help prevent fluid loss without requiring a valve on the carrier 2051. The upward-facing port(s) may function equivalently to the fluid port 1006, which is the fluid extraction port. In this embodiment, a gripping coupler 2056 may be positioned on the carrier to assist the gripping part (e.g., of a movable arm) in securely gripping the carrier 2051 while moving from the fluid station to the imaging station. The gripping coupler 2056 may be mechanical, electromechanical, or magnetic.

[0180] In some embodiments, one or more fasteners may include one or more positioning criteria. Figure 29 shows that one or more lateral and / or central positioning criteria can be used to reliably position a flow cell relative to an xy stage (not shown) or a nesting bank (not shown).

[0181] In some embodiments, the carrier 2051 may include an electronic chip 2055 embedded in the carrier to identify flow cells, e.g., serial numbers or any other unique IDs, as well as any relevant sequencing information such as identification for sequencing cycles and assays. The electronic chip 2055 may communicate electronically with the processor(s) of the sequencing system. The electronic chip 2055 may communicate electrically with a power source (e.g., a battery on the carrier 2051) or an external power outlet.

[0182] In some embodiments, the carrier 2051 may be equipped with one or more sensors to provide feedback on the alignment of the carrier 2051 to the xy stage and / or optical system. Various sensors can be used, such as cameras, sound sensors, light sensors, thermal sensors, radio frequency sensors, pressure sensors, and / or force sensors. Feedback from one or more sensors may be transmitted to the processor of the sequencing system, and the feedback can be used to fine-tune the positioning of the carrier 2051 and the flow cell device within it to the xy stage and / or optical system so that the sample(s) are aligned with the objective lens for imaging. For example, a sound sensor may be used to detect sound waves reflected from the carrier to determine how far the carrier is from the aligned imaging position. As another example, a camera(s) may be used to detect whether the carrier is positioned at the aligned imaging position.

[0183] In some embodiments, the carrier 2051 may include an external hardware processor, separate from the processor of the sequencing system. The carrier 2051's hardware processor may communicate with one or more sensors to generate commands to actuators (e.g., chip tilt devices, motors) to move the carrier to a desired 3D position relative to a nest bank, for example, without requiring the carrier 2051 to communicate with the sequencing system's processor to process feedback from one or more sensors. The movement can be performed with a predetermined precision. In some embodiments, the carrier 2051 may include an external hardware processor, separate from the sequencing system's processor, to enable autonomous or semi-autonomous movement of the carrier 2051 based on feedback from one or more sensors. A separate processor for the carrier 2051 may also help distribute computing power and increase the operating speed of the sequencing system.

[0184] In some embodiments, the carrier 2051 may not include an external hardware processor to the sequencing system's processor. One or more sensors communicate with the sequencing system's processor to process feedback from one or more sensors and may generate commands to actuators (or multiple actuators) (e.g., chip tilt devices, motors) to move the flow cell device carrier to a desired 3D position.

[0185] In some embodiments, for example in Figure 24B, the pump 1003 is located outside the carrier 2051 and may or may not move with the carrier 2051. Having an external pump 1003 can advantageously allow the carrier 2051 to be simpler, more compact in size, and lighter, thereby facilitating movement by the moving mechanism. In some embodiments, other elements in Figure 24A (e.g., a battery) may be positioned outside the carrier 2051. In some embodiments, the fluid path may include (e.g., flexible or semi-flexible) tubing, allowing for a permanent connection to the pump 1003.

[0186] In some embodiments, the nest bank 2050 is configured to enable fluid and thermal communication with one or more flow cell devices. In some embodiments, the nest bank is configured to enable fluid and thermal communication with various numbers of flow cell devices, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 flow cell devices. For example, Figure 2 shows a nest bank configured to hold three flow cell devices.

[0187] The Nest Bank 2050 is configured to hold each flow cell device in an unlocked position, where the flow cell device is removable from the Nest Bank. The Nest Bank is configured to hold the flow cell device in a locked position, where the flow cell device is spatially aligned with the Nest Bank, fixedly coupled to the Nest Bank, and allows for sealed fluid and thermal communication between the Nest Bank and the flow cell device. The Nest Bank 2050 is configured to transition between the unlocked and locked positions when a flow cell device is placed on a portion of the Nest Bank (e.g., by a transfer mechanism). In some embodiments, the Nest Bank 2050 may automatically transition from the unlocked to the locked position when a flow cell device is placed on or near a portion of the Nest Bank. In some embodiments, sequencing (e.g., reagent flow) may be blocked until the flow cell device transitions to the locked position in the Nest Bank 2050, as described below.

[0188] The carrier 2051 is configured to be spatially aligned with the nest bank in the locked position, thereby aligning the corresponding flow cell device within it with the nest bank 2050. Figures 5A and 5B show top and exploded views of the flow cell device coupled to the corresponding carrier 2051. In this embodiment, pins embedded in the frame of the carrier 2051 are configured to clamp the carrier 2051 and / or the flow cell device piece together. Ribs in the frame of the carrier 2051 can push the flow cell device in a position aligned with the carrier 2051, and therefore aligned with the nest bank 2050.

[0189] The nest bank 2050 may comprise one or more fasteners. The one or more fasteners may use various mechanisms to fasten carriers and corresponding flow cell devices to the nest bank 2050. The one or more fasteners may utilize magnetism. Figures 3 and 4A-4B show exemplary embodiments of the nest bank where one or more fasteners 2052 (also referenced in Figure 26B) may include multiple magnets. Each magnet may be, for example, a rare-earth magnet, an electromagnetic coil, or both. In some embodiments, one or more fasteners may be switchable on or off. For example, one or more fasteners may be controlled by one or more processors to switch between on and off states. In some embodiments, one or more fasteners may lack mechanical fasteners that can be driven by physical actuators such as motors, clamps, or springs. One or more fasteners 2052 may be pressed by a pin 2057 to ensure it is in the locked position, as shown in Figure 4B. One or more fasteners, either alone or in combination with push pins, are spatially aligned with respect to the nest bank 2050, ensuring that the flow cell device and its corresponding carrier 2051 are in a locked position relative to the nest bank 2050, so that sealed fluid and thermal communication (e.g., physical contact with a heat sink) is possible in such a locked position.

[0190] In some embodiments, the nest bank 2050 may comprise one or more fasteners, each configured to removably fasten and secure a corresponding carrier to the nest bank. Various fasteners may be used herein. For example, each fastener may comprise one or more clamps. One or more clamps may be driven by different forces, such as magnetic or electromagnetic forces.

[0191] In some embodiments, the nest bank 2050 may include one or more pumps configured to enable fluid communication with the flow cell device when the corresponding carrier 2051 is coupled to the nest bank 2050. Each flow cell device carrier may include a coupled position in which the carrier 2051 is detachably attached to the nest bank 2050 via fasteners, fixed, and in sealed fluid communication with the nest bank. Each carrier 2051 may include a detached position in which the flow cell device carrier is detachable from the nest bank 2050. In the detached position, the flow cell device carrier is not in fluid communication with the nest bank 2050, and the fluid in the carrier 2051 is sealed from leakage. Sealing may be enabled by one or more valves (e.g., 1005 in Figure 24A).

[0192] The nest bank 2050 may include a 3D moving device configured to position the carrier 2051 relative to the rest of the nest bank 2050 with a third spatial precision while the carrier 2051 remains coupled to the nest bank 2050. The third spatial position may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more times greater than the first spatial precision of the movable arm.

[0193] In some embodiments, the nest bank 2050 may include a 3D moving device configured to position the carrier 2051 relative to the dispensing tip with a fourth spatial precision, while remaining coupled to the nest bank. The movement can advantageously fine-tune the position of the open landing area(s) of the flow cell device relative to the dispensing tip(s) to ensure safe and accurate fluid administration to the flow cell device. The fourth spatial position may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more times greater than the first spatial precision of the movable arm. In some embodiments, the 3D moving device can drive the carrier 2051 to move in 3D or rotate around an axis in 3D. For example, the 3D moving device may be a piezoelectric stage or actuator that moves along z and can tip or tilt within a given angular range.

[0194] In some embodiments, the nest bank 2050 may be equipped with one or more sensors to provide feedback on the alignment of the carrier 2051 relative to the nest bank 2050. Various sensors, such as cameras, sound sensors, light sensors, thermal sensors, radio frequency sensors, pressure sensors, and / or force sensors, can be used. Feedback from one or more sensors may be transmitted to the processor of the sequencing system, and the feedback can be used to fine-tune the positioning of the flow cell device carrier and the flow cell device within it relative to the nest bank 2050 so that the sample(s) are aligned so as to be securely coupled to the nest bank 2050. For example, a sound sensor may be used to detect sound waves reflected from the carrier to determine how far the carrier 2051 is from its aligned position relative to the nest bank 2050. As another example, a camera(s) may be used to detect whether the carrier 2051 is positioned in an aligned position relative to the nest bank 2050 (and / or dispenser 2070).

[0195] It should be noted that the sensor may be equipped with one or more structural elements of the array determination system (e.g., xy stage 2010, movement mechanism, carrier 2051, and nest bank 2050) to facilitate the efficient and accurate positioning of carrier 2050 relative to xy stage 2010 and / or optical system 2020, and to facilitate the efficient and accurate positioning of carrier 2051 relative to nest bank 2050.

[0196] Alternatively, the nest bank 2050 may not include such a 3D moving device that moves the carrier 2051 relative to the rest of the nest bank 2050 or the dispensing tip. Instead, the dispenser 2070 may include a 3D moving device configured to move the dispensing tip in 3D while the nest bank 2050 and carrier 2051 remain fixed relative to a reference point. As a result, the dispensing tip and the open landing area are aligned with each other for accurate and safe fluid administration.

[0197] Figure 26B shows a schematic diagram of a nest bank 2050 having one nest bank module 2051. The nest bank modules disclosed herein are equivalent to a flow cell device carrier (e.g., carrier 2051) docked to the nest bank. Although not shown in this embodiment, two or more nest bank modules, or equivalent carriers 2051, can be included in the same nest bank.

[0198] The nest bank may independently secure each carrier 2051 using several clamping mechanisms 2052. In some embodiments, the flow cell device may include an open landing area that receives open dispensing by a dispenser 2070, as shown in Figure 27A. In such embodiments, the nest bank may include a pump and an optional valve for sealing and mating with the flow cell carrier while the carrier is in a coupled position relative to the nest bank, and for extracting fluid (e.g., wastewater) from the flow cell.

[0199] Figure 31 shows a schematic diagram of a nest bank 2050 having one nest bank module 2051. In this particular embodiment, the nest bank module, or equivalent herein, the carrier 2051, is in a locked position together with the nest bank 2050. As shown in Figure 31, in some embodiments, the flow cell device 200 may include a flow cell frame 2092 that encloses at least a portion of the flow cell device 200 (e.g., one or more ends of the flow cell device 200, and several areas of the flow cell device 200 along the xy plane). The flow cell frame 2092 may include a variety of materials, including but not limited to metal, plastic, silicone, and rubber. In some embodiments, the flow cell frame 2092 is configured to hold one or more gaskets 2093 in place relative to the flow cell device 200. The gaskets 2093 may facilitate a sealed fluid communication between the flow cell device 200 and the nest bank 2050. In some embodiments, the gasket(s) 2093 may be connected to the fluid manifold 2095 of the nest bank 2050. The fluid manifold 2095 may be in fluid communication with one or more fluid lines 1001 connected to one or more pumps.

[0200] In some embodiments, the carrier 2051 may include one or more fasteners or docking features 2094 that can be coupled to features matching the nest bank 2050 in order to securely couple the carrier to the nest bank 2050. Various mechanical elements can be used as fasteners or docking features (e.g., clamps, clips, bolts, magnets, snap-fit ​​components, latches, adhesives, etc.). An exemplary docking feature 2094 may include an alignment retainer and a magnetic latch. The docking feature may be configured to maintain a reliable connection (e.g., sealing and alignment) from the flow cell gasket 2093 to the fluid manifold 2095. The docking feature may be configured to maintain a reliable connection (e.g., sealing and alignment) from the flow cell device 200 to the heater and / or cooler 2053 of the nest bank 2050.

[0201] Similar to the xy stage (not shown), the nest bank 2050 may include a heating and / or cooling device 2053 optimized for developing fluids and assays for chemical processing. In some embodiments, the nest bank 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 immobilized on 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 configured to blow cold or hot air, a microwave, an infrared light source, and / or an electromagnetic wave source. In some embodiments, the temperature controller of the nest bank may be structurally and / or functionally similar to the temperature controller of the xy stage.

[0202] As shown in Figure 26B, similar to the xy stage, the nest bank 2050 may include a positioning device 2054 (e.g., a tip-tilt device) that positions the carrier 2051 to a 3D position with a predetermined precision (e.g., a first precision) for imaging. The positioning device 2054 specified herein is configured to move along the z-axis and tip and / or tilt within a predetermined angular range for high-speed and accurate multi-axis positioning. The tip or tilt may be performed around an axis in 3D (e.g., the x-axis, y-axis, or other axis in the xy-plane). The tip angle or tiling angle can be in a range of values. For example, the tip angle or tilt angle may 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.

[0203] In some embodiments, for example, in embodiments having an in-situ sample, the fluid station or at least a portion of the fluid station may be outside the housing of the sequencing system to facilitate sample preparation. In some embodiments, the dispenser (e.g., dispenser 2070) and nesting module may be outside (at least partially) the housing of the sequencing system so that sample preparation can be monitored as it progresses.

[0204] In some embodiments, the nest bank 2050 includes one or more reagent containers. In some embodiments, one or more reagent containers may be disposable. In some embodiments, a transport mechanism (e.g., a movable arm) is configured to immerse a flow cell device in at least some of the one or more reagent containers. For example, as shown in Figure 6, the nest bank may include three different reagent containers for each flow cell device, and the transport mechanism may transport the flow cell device and immerse it in each container for a predetermined duration and temperature. Some of the reagent containers may hold wash buffers in order to reduce cross-contamination between buffers. For example, a first flow cell device may be immersed in reagent container A, then moved to an xy stage for imaging, then immersed in reagent container B for washing, and then immersed in reagent container C to flow a second type of reagent into a microfluidic channel.

[0205] In some embodiments, the sequencing system may not include a moving mechanism that includes a movable arm for moving the flow cell device carrier between a nest bank (e.g., nest bank 2050) and an optical system (e.g., optical system 2020). In such alternative embodiments, the optical system may move relative to the nest bank to position the flow cell in a predetermined position relative to the objective lens for imaging. For example, the optical system may move linearly along the y-axis, and the nest bank or flow cell carrier detached from the nest bank may move linearly along the x-axis, and their combined movement may allow the sample to be positioned below the objective lens for imaging. In another example, the optical system may be fixed relative to a housing or another reference point of the sequencing system, and the xy stage and / or flow cell device carrier may move linearly in the xy plane to allow the sample to be positioned below the objective lens for imaging. As yet another example, the optical system may be fixed to another reference point of the housing or array determination system, and the xy stage and / or flow cell device carrier may move nonlinearly in the xy plane (e.g., rotate around the z axis) to position the flow cell device carrier relative to the fixed optical system for imaging.

[0206] It should be noted that the movable arms, xy stages, nest banks, and flow cell device carriers described herein may be driven independently to linear or nonlinear motions, or multiple motions, so that a 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 certain volume of reagent(s) and dispenses it into the flow cell device moves with the nest bank and therefore remains fixed relative to the nest bank to ensure proper fluid administration to the flow cell when the flow cell carrier is coupled to the nest bank.

[0207] Disclosed herein are sequencing methods using the sequencing systems disclosed herein. A sequencing method may include one or more operations disclosed herein. The method may include some or all of the operations disclosed herein. The operations may, but are not limited to, the order described herein.

[0208] The method can be performed by one or more hardware processors disclosed herein. In some embodiments, the processor may include one or more processing units, integrated circuits, or combinations 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.

[0209] In some embodiments, some or all operations in the method can be performed by an FPGA(multiple) (e.g., FPGA(multiple) 120). In embodiments where some operations are performed by an FPGA(multiple), data following the operations performed by the FPGA(multiple) can be communicated by the FPGA(multiple) to a CPU(multiple) (e.g., the CPU(multiple) of computer system 126), so that the CPU(multiple) can use such data to perform subsequent operations(multiple) in method 500. Similarly, data can also be communicated from the CPU(multiple) to the FPGA(multiple) for processing by the FPGA(multiple). In some embodiments, all operations in method 500 can be performed by a CPU(multiple). Alternatively, operations performed by a CPU(multiple) can be performed by a dedicated processor or another processor such as a GPU(multiple). In some embodiments, all operations in method 500 can be performed by an FPGA(multiple).

[0210] In some embodiments, the method herein may include the following operations: (a) moving a first flow cell device from a nest bank to an xy stage, wherein the first flow cell device includes a first sample immobilized thereon; (b) moving the xy stage and the first sample thereon relative to the objective lens of the optical system of the sequencing system; (c) imaging the first sample immobilized on the first flow cell device on the xy stage using the optical system; (d) moving the first flow device from the xy stage to a nest bank; and (e) simultaneously enabling fluid and thermal communication between the nest bank and a second flow cell device between one or more of (a) to (d).

[0211] In some embodiments, actions (a) to (d) occur during the same flow cycle of the sequencing run. In some embodiments, action (e) occurs within the same flow cycle as actions (a) to (d). Actions can be repeated in each individual cycle of the sequencing run. For example, an action can be repeated at least once within each cycle. As another example, some of the actions can be repeated multiple times within a single cycle. For example, moving the flow cell device to the xy stage can occur within the same cycle, after each reagent administration to the flow cell device.

[0212] In some embodiments, some of the operations may be performed immediately after the completion of a preceding operation in order to avoid wasting time when performing sequencing. For example, even if the second sample is being imaged immediately after the first reagent has been administered to the first sample, the movable arm may be moved to a position close to the xy stage and objective lens so that the first sample can be quickly moved to the imaging position after the second sample has been imaged.

[0213] While one movable arm is shown in the exemplary embodiment of Figure 2, two or more movable arms may be included, each arm capable of moving one or more corresponding flow cell devices to improve system efficiency and throughput and reduce the idling time of the imaging system.

[0214] In some embodiments, the method further includes (f) moving a second flow cell device from a nest bank to an xy stage, wherein the second flow cell device includes a second sample immobilized thereon; (g) moving the xy stage and the second sample thereon with respect to the objective lens of the optical system of the sequencing system; (h) imaging the second sample immobilized on the second flow cell device on the xy stage using the optical system; (i) moving the first flow device from the xy stage to a nest bank; and (j) enabling fluid and thermal communication between the nest bank and the first flow cell device (for example, simultaneously) between one or more of (f) to (i).

[0215] In some embodiments, the sequencing method further includes repeating operations (a) to (e). Each iteration of operations (a) to (e) can be performed within a single cycle or across different flow cycles of sequencing execution. In some embodiments, the sequencing method further includes repeating operations (f) to (j). Each iteration of operations (f) to (j) can be performed within a single cycle or across different flow cycles of sequencing execution. Each iteration of operations (f) to (j) can be performed after operations (a) to (e).

[0216] In some embodiments, the sequencing method further includes repeating operations (a) to (j) a certain number of times for a nested bank holding two different flow cell devices. A similar operation can also be repeated a certain number of times for any additional flow cell devices configured to be held by the nested bank. In some embodiments, the number of iterations is in the range of 1 to 500 (including all ranges and subranges in between). In some embodiments, the number of iterations corresponds to the number of cycles in the sequencing run.

[0217] In some embodiments, enabling fluid communication in operation (f) between the nest bank and the first flow cell device 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, reversibly fastening the carrier to the nest bank via one or more fasteners to enable sealed fluid communication between the nest bank and the carrier, and enabling physical contact with a heat dissipation element. The one or more fasteners between the carrier and the flow cell device may include screws, pins, mechanical clamps, or other structures involving magnetism.

[0218] In some embodiments, the operation (a) of moving the first flow cell device from the nest bank to the xy stage is performed within a first flow cycle of the sequence determination run, and the operation (f) of moving the first flow cell device from the nest bank to the xy stage is performed within a second flow cycle different from the first flow cycle. In some embodiments, during one or more of (a) to (e), the operation (f) of simultaneously enabling fluid and thermal communication between the nest bank and the first flow cell device includes switching one or more fasteners to the ON state to enable physical contact for sealed fluid and thermal communication. For example, switching on the power supply to the electromagnetic coil.

[0219] In some embodiments, enabling simultaneous fluid and thermal communication between the nest bank and the first flow cell device during one or more of operations (f)(a) to (e) may involve immersing the flow cell device in at least some of one or more reagent containers in a predetermined order.

[0220] In some embodiments, each of operations (a) to (b) and (d) to (g) is completed within 0.5 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or less than 10 seconds. In some embodiments, each of operations (a) to (b) and (d) to (g) is completed within 0.5 seconds, 1 second, 2 seconds, or less than 3 seconds.

[0221] Computer system Various embodiments of this method can be implemented using one or more computer systems (for example, computer system 800 shown in Figure 8). One or more computer systems 800 may be used, for example, to implement any of the embodiments considered herein, as well as combinations and subcombinations thereof.

[0222] The computer system 800 may include one or more hardware processors 404. The hardware processors 804 may be a central processing unit (CPU), a graphics processing unit (GPU), or a combination thereof. The processors 804 may be connected to a bus or communication infrastructure 806.

[0223] The computer system 800 may also include user input / output devices 803, such as monitors, keyboards, and pointing devices, which can communicate with the communication infrastructure 406 via user input / output interfaces 802. The user input / output devices 803 may be coupled to the user interface 124 in Figure 1.

[0224] One or more of the processors 804 may be graphics processing units (GPUs). In one embodiment, a GPU may be a processor that is a dedicated electronic circuit designed to handle mathematically intensive applications. A GPU may have parallel architectures that are efficient for parallel processing of large data blocks, such as mathematically intensive data common to computer graphics applications, images, videos, vector processing, array processing, etc., as well as encryption (including brute-force cracking), generation of cryptographic hashes or hash arrays, solving partial hash reversal problems, and / or generating the results of other proof-of-work calculations for some blockchain-based applications. Due to the general-purpose computing (GPGPU) capabilities on the graphics processing unit, a GPU may be particularly useful in at least the image recognition and machine learning embodiments described herein.

[0225] Additionally, one or more of the processors 404 may include a hardware-accelerated cryptographic coprocessor, a logic coprocessor or other implementation for accelerating cryptographic computations, or other specialized mathematical functions. Such an accelerated processor may further include an instruction set(s) for accelerating using the coprocessor and / or other logic to facilitate such acceleration.

[0226] The computer system 800 may also include main memory or primary memory 808, and a data storage device such as random access memory (RAM). The main memory 808 may include one or more levels of cache. The main memory 808 may store control logic (i.e., computer software) and / or data within it.

[0227] The computer system 800 may also include one or more secondary data storage devices or secondary memory 810. The secondary memory 810 may include, for example, a main storage drive 812 and / or a removable storage device or drive 814. The main 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, a compact disk drive, an optical storage device, a tape backup device, and / or any other storage device / drive.

[0228] The removable storage drive 814 may interact with the removable storage unit 818.

[0229] The removable storage unit 818 may include a computer-accessible or readable storage device that stores computer software and / or data. The software may include control logic. The software may include instructions that can be executed by the hardware processor(s) 804. The removable storage unit 818 may be a floppy disk, magnetic tape, compact disk, DVD, optical storage disk, and / or any other computer data storage device. The removable storage drive 814 may read from and / or write to the removable storage unit 818.

[0230] The secondary memory 810 may include other means, devices, components, equipment or other techniques that enable the computer system 800 to access computer programs and / or other instructions and / or data. Such means, devices, components, equipment or other techniques may include, for example, a removable storage unit 822 and an interface 820. Examples of removable storage units 822 and interfaces 820 may include program cartridges and cartridge interfaces (such as those found in video game devices), removable memory chips (such as EPROMs or PROMs) and associated sockets, memory sticks and USB ports, memory cards and associated memory card slots, and / or any other removable storage units and associated interfaces.

[0231] The computer system 800 may further include a communication interface or network interface 824. The communication interface 824 may enable the computer system 800 to communicate and interact with any combination of external devices, external networks, external entities, etc. (referenced individually and collectively by reference number 828). For example, the communication interface 824 may enable the computer system 800 to communicate with an external or remote device 828 via a communication path 826, which may be wired and / or wireless (or a combination thereof) and may include any combination of LAN, WAN, internet, etc. Control logic and / or data may be transmitted to and from the computer system 800 via the communication path 826. In some embodiments, the communication path 826 is a connection to the cloud 130, as shown in Figure 1. External devices, etc., referenced by reference number 828 may be devices, networks, entities, etc., within the cloud 130.

[0232] The computer system 800 can also be any of a number of non-limiting examples, or any combination thereof, such as a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet, a smartphone, a smartwatch, or other wearable, an appliance, part of the Internet of Things (IoT), and / or an embedded system.

[0233] It should be understood that the framework described herein can be implemented as a method, process, apparatus, system, or product, such as a non-transitory computer-readable medium or device. For purposes of illustration, this framework can be described in the context where a distributed ledger is publicly available or at least accessible by untrusted third parties. As a current use case, blockchain-based systems are mentioned. However, it should be understood that this framework can also be applied to settings where confidential information or other confidential information may need to pass through the hands of untrusted third parties, and this technology is not limited to the use of distributed ledgers or blockchains.

[0234] The computer system 800 can be a client or server that accesses or hosts any application and / or data through any delivery paradigm, including but not limited to a remote or distributed cloud computing solution, local or on-premises software (e.g., an "on-premises" cloud-based solution), a "service as" model (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 a hybrid model that includes any combination of the above examples or other services or delivery paradigms).

[0235] Any applicable data structures, file formats, and schemas can be derived from standards including, but not limited to, JavaScript® Object Notation (JSON), Extensible Markup Language (XML), yet another markup language (YAML), Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), MessagePack, XML User Interface Language (XUL), or other functionally similar representations alone or in combination. Alternatively, unique data structures, formats, or schemas can be used exclusively or in combination with known standards or open standards.

[0236] Any related data, files, and / or databases may be stored, retrieved, accessed, and / or transmitted in human-readable formats such as numerical, text, graphic, or multimedia formats, further including various types of markup languages, among other possible formats. Alternatively, or in combination with the above formats, 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.

[0237] Interfaces or interconnections between various systems and layers may use any number of mechanisms, such as any number of protocols, programmatic frameworks, floor plans, or application programming interfaces (APIs), including, but not limited to, the Document Object Model (DOM), Discovery Services (DS), NSUserDefaults, Web Services Description Language (WSDL), Message Exchange Pattern (MEP), Web Distributed Data Exchange (WDDX), Web Hypertext Application Technologies Working Group (WHATWG), HTML5 Web Messaging, Representation 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 mechanisms capable of achieving similar functionality and results.

[0238] Such interfaces or interconnections may also utilize uniform resource identifiers (URIs), which may further include uniform resource locators (URLs) or uniform resource names (URNs). Other forms of unified and / or unique identifiers, locators, or names may be used exclusively or in combination with forms such as those described above.

[0239] Any of the protocols or APIs described above may interface with, implement, compile, or interpret in any programming language, procedural language, functional language, or object-oriented language. Non-exclusive examples include virtually any other language by any kind of framework, runtime environment, virtual machine, interpreter, stack, engine, or similar mechanism in C, C++, C#, Objective-C, Java®, Scala, Clojure, Elixir, Swift, Go, Perl, PHP, Python®, Ruby, JavaScript®, WebAssembly, or any other library or schema (including, but not limited to, Node.js, V8, Knockout, jQuery, Dojo, Dijit, OpenUI5, AngularJS, ExpressJS, Backbone.js, Ember.js, DHTMLX, Vue, React, Electron, etc.).

[0240] In some embodiments, a tangible non-temporary device or product including a tangible non-temporary computer-usable or readable medium storing control logic (software) may be referred to herein as a computer program product or program storage device. This includes, but is not limited to, a tangible product embodying a computer system 800, main memory 808, secondary memory 810, and removable storage units 818 and 822, as well as any combination thereof. When such control logic is executed by one or more data processing devices (such as computer system 800), such data processing devices can be made to operate as described herein.

[0241] Based on the teachings contained herein, methods for creating and using embodiments of the disclosure using data processing devices, computer systems, and / or computer architectures other than those shown in Figure 8 will be apparent to those skilled in the art in the relevant field. In particular, embodiments may operate with software, hardware, and / or operating system implementations other than those described herein.

[0242] Imaging system The imager 116 in Figure 1 may include one or more optical systems 2020. Further disclosed herein are optical system design guidelines, as well as 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 provide a larger field of view, increased spatial resolution, improved modulation transfer, contrast-to-noise ratio, and image quality, a higher spatial sampling frequency, faster transitions between image captures when repositioning the sample surface to capture a series of images (e.g., of different fields of view), and an improved imaging system duty cycle, thus enabling higher throughput image acquisition and analysis.

[0243] In some cases, for example, improved imaging performance for dual-side (flow cell) imaging applications can be achieved by using an electro-optical phase plate in combination with the objective lens to compensate for optical aberrations caused by the fluid layer separating the upper (near) and lower (far) inner surfaces of the flow cell. In some cases, this design technique can also compensate for vibrations introduced by a motion-driven compensator that moves in or out of the optical path depending on which surface of the flow cell is being imaged.

[0244] In some cases, for example, the improvement of imaging performance for dual-sided (flow cell) imaging applications, including the use of thick flow cell walls (e.g., the thickness of the wall (or cover slip) exceeds 700 μm) and fluid channels (e.g., the height or thickness of the fluid channel is 50 - 200 μm), can be achieved even when using commercially available off-the-shelf objective lenses by using a tube lens design that corrects the optical aberrations caused by the thick flow cell wall and / or the intermediate fluid layer in combination with the objective lens.

[0245] In some cases, for example, the improvement of imaging performance for multi-channel (e.g., two-color or four-color) imaging applications can be achieved by using multiple tube lenses (one for each imaging channel), and each tube lens design is optimized for the specific wavelength range used in that imaging channel.

[0246] Exemplary embodiments disclosed herein may include a fluorescence imaging system, the 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 within a specified field of view of a sample surface when the sample surface is exposed to the excitation light, the numerical aperture of the objective lens being a numerical aperture that falls within a range defined by 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, at least 0.9, or any two of the foregoing, the working distance of the objective lens being a working distance that falls within a range defined by at least 400 microns (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 any two of the foregoing, and the field of view being at least 0.1 mm 2 , at least 0.2 mm 2 , at least 0.5 mm 2 , at least 0.7 mm 2 , at least 1 mm 2 , at least 2 mm2 at least 3mm 2 at least 5mm 2 , or at least 10 mm 2 an objective lens having a field of view that falls within the range defined by, or any two of the above, and c) at least one image sensor, wherein the pixel dimensions of the image sensor are selected such that fluorescence collected by the objective lens is imaged onto the image sensor and the spatial sampling frequency of the fluorescence imaging system is at least twice the optical resolution of the fluorescence imaging system.

[0247] 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 500 μm to 7,000 μm, 100 μm to 5,000 μm, or 500 μm to 2,000 μm. In some embodiments, the field of view is at least 2.5 mm. 2 It may have an area of ​​at least 3 mm. In some embodiments, the field of view is at least 3 mm 2 It may have an area of ​​0.5 mm. In some embodiments, the field of view is 0.5 mm. 2 ~10mm 2 , 1mm 2 ~10mm 2 , 1mm 2 ~5mm 2 , 1.5mm 2 ~5mm 2 , 2mm 2 ~5mm 2 , or 2.5mm 2 ~3mm 2It may have an area of ​​. In 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 2 to 10 times the optical resolution, 2 to 5 times the optical resolution, or 2.5 to 3 times the optical resolution. In some embodiments, the system may further include an XYZ translation stage so that the system is configured to acquire a series of two or more fluorescence images in an automated manner, and each image in the series of images may or may not 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 lens focal plane while acquiring images for 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 within the range defined by any two of the above. In some embodiments, the system further includes an autofocusing mechanism configured to adjust the position of the focal plane before acquiring images of different fields of view if an error signal indicates that the difference in position 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 comprises 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 difference in position between the focal plane of each of the three or more image sensors and the sample plane is less than 100 nm. In some embodiments, the difference in position between the focal plane of each of the three or more image sensors and the sample plane is less than 50 nm. In some embodiments, the total time required to reposition the sample plane, adjust the focus as necessary, and acquire images is less than 0.4 seconds per field of view.In some embodiments, the total time required to reposition the sample surface, adjust the focus as needed, and acquire an image is less than 0.3 seconds per field of view.

[0248] Furthermore, this specification provides a fluorescence imaging system for two-plane imaging of a flow cell, comprising: a) an objective lens configured to collect fluorescence emanating from a specified field of view of a sample plane within a flow cell; and b) at least one tube lens positioned between the objective lens and at least one image sensor, wherein at least one tube lens is configured to correct the imaging performance index of the combination of the objective lens, at least one tube lens, and at least one image sensor when imaging the inner surface of the flow cell, and the flow cell has a minimum thickness of 700 μm (e.g., 500 μm to 3,000 μm, 700 μm to 2,000 μm, 700 to 1,500 μm, 1,000 μm to 2,000 μm, 2,000 μm to 3,000 μm). A fluorescence imaging system is disclosed, having a wall thickness of 00 μm (or any range between them) and a gap of at least 50 μm (e.g., 50 μm to 1,000 μm, 50 to 200 μm, 100 μm to 500 μm, 100 μm to 200 μm, or any range between them) between the upper inner surface and the lower inner surface, wherein the imaging performance metric is substantially the same when imaging the upper inner surface or the lower inner surface of the flow cell, without moving the optical compensator in or out of the optical path, 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 in or out of the optical path.

[0249] 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, a commercially available microscope objective lens may have a numerical aperture of at least 0.3. In some embodiments, the objective lens may have a working distance of at least 700 μm. In some embodiments, the objective lens may be corrected to compensate for a coverslip thickness (or flow cell wall thickness) of 0.17 mm, or a coverslip thickness (or flow cell wall thickness) greater than or less than 0.17 mm. In some embodiments, the optical system may be corrected to compensate for the coverslip thickness, flow cell thickness, or the distance between desired focal planes. In some embodiments, such correction may be made by inserting corrective optical equipment, such as a lens or optical assembly, into the optical path of the optical system. In some embodiments, such correction may be made without inserting corrective optical equipment, 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, the electro-optic phase plate may provide correction for optical aberrations caused by the fluid filling the gap between the upper and lower inner surfaces of the flow cell. In some embodiments, at least one tube lens may be a composite lens comprising three or more optical components. In some embodiments, at least one tube lens may be a composite lens comprising four optical components, comprising one or more of 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 above order or in any alternative order. In some embodiments, at least one tube lens is configured to correct the imaging performance metric for a combination of the objective lens, at least one tube lens, and at least one image sensor when imaging the inner surface of a flow cell having a wall thickness of at least 1 mm.In some embodiments, at least one tube lens is configured to compensate for the imaging performance metric of the combination of the objective lens, at least one tube 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, at least one tube lens is configured to compensate for the imaging performance metric of the combination of the objective lens, at least one tube lens, and at least one image sensor when imaging the inner surface of a flow cell having a gap of at least 200 μm. In some embodiments, the system includes a single objective lens, two tube lenses, and two image sensors, each of the two tube lenses designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the system includes a single objective lens, three tube lenses, and three image sensors, each of the three tube lenses designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the system includes a single objective lens, four tube lenses, and four image sensors, each of the four tube lenses designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the design of the objective lens or at least one tube lens is configured to optimize the modulation transfer function in the medium to high spatial frequency range. In some embodiments, the imaging performance metrics include measurements of the modulation transfer function (MTF) at one or more specified spatial frequencies, focus blur, spherical aberration, chromatic aberration, coma aberration, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the difference in imaging performance metrics for imaging the upper inner surface and the lower inner surface of the flow cell is less than 10%. In some embodiments, the difference in imaging performance metrics for imaging the upper inner surface and the lower inner surface of the flow cell is less than 5%.In some embodiments, using at least one tube lens improves the imaging performance metric for dual-side imaging to at least the same level or better compared to a conventional system comprising an objective lens, a motion-driven compensator, and an image sensor. In some embodiments, using at least one tube lens improves the imaging performance metric for dual-side imaging to at least 10% compared to a conventional system comprising an objective lens, a motion-driven compensator, and an image sensor.

[0250] Disclosed herein is an illumination system for use in imaging-based solid-phase genotyping and sequencing applications, the illumination system comprising: a) a light source; and b) a liquid light guide configured to collect the light emitted by the light source and deliver it to a designated illumination field on the surface of a support containing a tethered biomacromolecule.

[0251] In some embodiments, the illumination system further comprises a focusing lens. In some embodiments, the designated illumination field is at least 2 mm 2 It has an area of ​​. In some embodiments, the light delivered to the designated illumination field is of uniform intensity across the designated field of view to the imaging system used to acquire an image of the support surface. In some embodiments, the designated field of view is at least 2 mm 2 It has an area of ​​. In some embodiments, the light delivered to a designated illumination field has a uniform intensity across the designated field of view when the coefficient of variation (CV) of light intensity is less than 10%. In some embodiments, the light delivered to a designated illumination field has a uniform intensity across the designated field of view when the coefficient of variation (CV) of light intensity is less than 5%. In some embodiments, the light delivered to a designated illumination field has a speckle contrast value of less than 0.1. In some embodiments, the light delivered to a designated illumination field has a speckle contrast value of less than 0.05.

[0252] It will be understood by those skilled in the art that the disclosed optical systems, imaging systems, or modules may, in some cases, be standalone optical systems designed to image a sample or substrate surface. In some cases, they may comprise one or more processors or computers. In some cases, they may comprise one or more software packages that provide 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, tungsten halogen 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 comprise mechanical and / or opticmechanical components such as XY translation stages, XYZ translation stages, and piezoelectric focusing mechanisms. In some cases, they may function as modules, components, subassemblies, or subsystems of larger systems designed for genomics applications (e.g., genetic testing and / or nucleic acid sequencing applications). For example, in some cases, they may function as modules, components, subassemblies, or subsystems of a larger system further comprising light-shielding and / or other environmental control housings, temperature control modules, fluid control modules, fluid distribution robotics, pick-and-place robotics, one or more processors or computers, one or more local and / or cloud-based software packages (e.g., equipment / 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 associated software), display modules, or any combination thereof.

[0253] Method for sequencing This disclosure provides a method for sequencing immobilized or unimmobilized nucleic acid template molecules. The method can operate in a sequencing system 110, for example, in a sequencer 114. In some embodiments, the immobilized nucleic acid template molecule comprises a plurality of nucleic acid template molecules, each having one copy of the target sequence of interest. In some embodiments, the nucleic acid template molecule having one copy of the target sequence of interest can be generated by performing bridge amplification using a linear library molecule. In some embodiments, the immobilized nucleic acid template molecule comprises a plurality of nucleic acid template molecules (e.g., concatemer template molecules), each having two or more tandem copies of the target sequence of interest. In some embodiments, the nucleic acid template molecule, including concatemer template molecules, can be generated by performing rolling circle amplification of a cyclic library molecule. In some embodiments, the unimmobilized nucleic acid template molecule comprises a cyclic molecule. The sequencing method can use a soluble (e.g., unimmobilized) sequencing polymerase or a sequencing polymerase immobilized on a support. Those skilled in the art will be able to select polymerases suitable for the various sequencing methods disclosed herein.

[0254] In some embodiments, the sequencing reaction uses detectably labeled nucleotide analogs. In some embodiments, the sequencing reaction uses a two-step sequencing reaction comprising a first step of binding to a detectably labeled polyvalent molecule (see Figures 11-15) and a second step of incorporating the nucleotide analog (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.

[0255] In some embodiments, the immobilized concatemer template molecules include tandem repeat units of the target sequence (e.g., also referred to as the insertion region) and any adapter sequences. For example, the tandem repeat units can include (i) a left universal adapter sequence having a binding sequence for a first surface primer (920) (e.g., a surface pinning primer), (ii) a left universal adapter sequence having a binding sequence for a first sequencing primer (940) (e.g., a forward sequencing primer), (iii) the target sequence (910), (iv) a right universal adapter sequence having a binding sequence for a second sequencing primer (950) (e.g., a reverse sequencing primer), (v) a right universal adapter sequence having a binding sequence for a second surface primer (930) (e.g., a surface capture primer), and (vii) a left index sequence (960) and / or a right index sequence (070) (which can be sample index sequences). In some embodiments, the tandem repeat unit further includes a left unique identifier sequence (980) and / or a right unique identifier sequence (990). In some embodiments, the tandem repeat unit further includes at least one binding sequence for a compaction oligonucleotide. FIGS. 9 and 10 show exemplary embodiments of a single unit of a linear library molecule or a concatemer template molecule.

[0256] The immobilized concatemer template molecule can self-destruct into compact nucleic acid nanoballs. During the RCA reaction, the size and / or shape of the nanoballs can be further compacted by including one or more compaction oligonucleotides with binding sites on the concatemer template molecule. Increasing the number of tandem repeat units in each concatemer template molecule increases the number of sites along the concatemer template molecule for hybridizing to multiple sequencing primers (e.g., sequencing primers with universal sequences) that function as multiple start sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction uses detectably labeled nucleotides and / or detectably labeled polyvalent molecules (e.g., polyvalent molecules with nucleotide units), the signals emitted by the nucleotides or nucleotide units involved in parallel sequencing reactions along the concatemer template molecule result in increased signal intensity for each concatemer template molecule. Multiple parts of a given concatemer template molecule can be sequenced simultaneously. Furthermore, multiple binding complexes can form along specific concatemer template molecules, each containing a sequencing polymerase bound to the template / primer double-chain and polyvalent molecule. These multiple binding complexes remain stable without dissociation, resulting in increased duration, which in turn increases signal intensity and reduces imaging time.

[0257] Method for sequencing using nucleotide analogues This disclosure provides a method for sequencing any of the immobilized template molecules described herein, comprising the steps of (a) contacting a sequencing polymerase with (i) a nucleic acid template molecule and (ii) a nucleic acid sequencing primer, wherein the contact is carried out under conditions suitable for binding the sequencing polymerase to the nucleic acid template molecule that hybridizes to the nucleic acid primer, and the nucleic acid template molecule that hybridizes to the nucleic acid primer forms a nucleic acid double helix. In some embodiments, the sequencing polymerase includes recombinant mutant sequencing polymerases that can bind to and incorporate 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).

[0258] In some embodiments of the sequencing method, the sequencing primers (e.g., first and / or second sequencing primers) include a 3'-extendable or 3'-non-extendable end. In some embodiments, the plurality of nucleic acid template molecules include amplified template molecules (e.g., clone-amplified template molecules). In some embodiments, the plurality of nucleic acid template molecules include one copy of the target sequence of interest. In some embodiments, the plurality of nucleic acid molecules include two or more tandem copies of the target sequence of interest (e.g., concatemer template molecules). In some embodiments, the plurality of nucleic acid template molecules include the same target sequence. In some embodiments, the individual nucleic acid template molecules in the plurality of nucleic acid template molecules include different target sequences. In some embodiments, the plurality of nucleic acid primers are in solution or immobilized on a support (e.g., support 210 of the flow cell device disclosed herein). In some embodiments, when the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are immobilized on a support, binding with the first sequencing polymerase produces a plurality of immobilized first complex polymerases. In some embodiments, multiple nucleic acid template molecules and / or nucleic acid primers are arranged on a support. 2 ~10 15It is immobilized on several different sites. In some embodiments, the binding of multiple template molecules and nucleic acid primers to multiple first sequencing polymerases is performed on the support. 2 ~10 15 Multiple immobilized first complex polymerases are generated at different sites on the support. In some embodiments, the multiple immobilized first complex polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the multiple immobilized first complex polymerases are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes containing sequencing polymerases, polyvalent molecules, nucleotides, and / or divalent cations) to flow onto the support using the sequencing system 110 described herein, thereby allowing the multiple immobilized complex polymerases on the support to react with the solution of reagents in a massively parallel manner.

[0259] In some embodiments, the sequencing method further includes step (b): contacting a sequencing polymerase with a plurality of nucleotides, and at least one nucleotide, under conditions suitable for polymerase catalytic nucleotide incorporation, which is suitable for conjugating the sequencing polymerase bound to the nucleic acid double helix and for extending the sequencing primer by the one nucleotide. In some embodiments, the sequencing polymerase contacts 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 termination moiety at the sugar 2' or 3' position. In some embodiments, the chain termination moiety is removable from the sugar 2' or 3' position to convert the chain termination moiety to an OH or H group. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain termination moiety. In some embodiments, at least one nucleotide in the plurality of nucleotides is labeled with a detectable reporter moiety (e.g., a fluorophore) that emits a detectable signal. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to the nucleic acid base. In some embodiments, the fluorophore is attached to the nucleic acid base using a linker, which is cleavable / removable from the nucleic acid base. In some embodiments, at least one of the nucleotides in a plurality of nucleotides is not labeled with a detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to the nucleotide corresponds to the identity of the nucleic acid base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), and thus can enable the detection and identification of the nucleic acid base in the nucleic acid template molecule, corresponding to the corresponding complementary nucleic acid base in the nucleic acid template molecule. When the incorporated chain termination nucleotide is detectably labeled, step (b) may further include detecting the signal released from the incorporated chain termination nucleotide.In some embodiments, step (b) further includes identifying the nucleic acid base of the incorporated chain termination nucleotide.

[0260] In some embodiments, the sequencing method further includes step (c): removing the chain termination portion from the incorporated chain termination nucleotide to generate an extendable 3'OH group. In some embodiments, step (c) further includes removing a detectable label from the incorporated chain termination nucleotide. In some embodiments, the sequencing polymerase remains bound to a template molecule that hybridizes to a sequencing primer extended by a single nucleic acid base.

[0261] In some embodiments, the method for sequencing further includes step (d): repeating steps (b) to (c) at least once.

[0262] A two-step method for nucleic acid sequencing This disclosure provides a two-step method for sequencing any of the immobilized template molecules described herein. In some embodiments, the first step generally includes conjugating a polyvalent molecule to a complex polymerase to form a polyvalent complex polymerase, and detecting the polyvalent complex polymerase.

[0263] In some embodiments, the first step includes (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 carried out under conditions suitable for binding the plurality of first sequencing polymerases to the plurality of nucleic acid template molecules and the plurality of nucleic acid primers, thereby forming a plurality of first complex polymerases, each comprising a first sequencing polymerase that binds to a nucleic acid duplex, the nucleic acid duplex comprising a nucleic acid template molecule that hybridizes to the nucleic acid primers. In some embodiments, the first polymerase includes recombinant mutant sequencing polymerase.

[0264] In some embodiments, the sequencing primers include oligonucleotides having a 3'-extendable or 3'-non-extendable end. In some embodiments, the plurality of nucleic acid template molecules include amplified template molecules (e.g., clone-amplified template molecules). In some embodiments, the plurality of nucleic acid template molecules include one copy of the sequence of interest. In some embodiments, the plurality of nucleic acid molecules include two or more tandem copies (e.g., concatemers) of the sequence of interest. In some embodiments, the nucleic acid template molecules in the plurality of nucleic acid template molecules include the same sequence of interest. In some embodiments, the individual nucleic acid template molecules in the plurality of nucleic acid template molecules include different sequences of interest. In some embodiments, the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are in solution or immobilized on a support (e.g., support 210 of a flow cell device disclosed herein). In some embodiments, when the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are immobilized on a support, binding with a first sequencing polymerase produces a plurality of immobilized first complex polymerases. In some embodiments, multiple nucleic acid template molecules and / or nucleic acid primers are arranged on a support. 2 ~10 15 It is immobilized on several different sites. In some embodiments, the binding of multiple template molecules and nucleic acid primers to multiple first sequencing polymerases is performed on the support. 2 ~10 15 Multiple immobilized first complex polymerases are generated at different sites on the support. In some embodiments, the multiple immobilized first complex polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the multiple immobilized first complex polymerases are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes containing sequencing polymerases, polyvalent molecules, nucleotides, and / or divalent cations) to flow onto the support using the sequencing system 110 described herein, thereby allowing the multiple immobilized complex polymerases on the support to react with the solution of reagents in a massively parallel manner.

[0265] In some embodiments, the sequencing method further includes step (b): contacting a plurality of first complex polymerases with a plurality of polyvalent molecules to form a plurality of polyvalent complex polymerases (e.g., binding complexes). In some embodiments, the individual polyvalent molecules in the plurality of polyvalent molecules include a core 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 11-15). In some embodiments, the contact in step (b) is carried out under conditions suitable for binding complementary nucleotide units of the polyvalent molecules to at least two of the plurality of first complex polymerases, thereby forming a plurality of polyvalent complex polymerases. In some embodiments, the conditions are suitable for inhibiting the polymerase-catalyzed incorporation of complementary nucleotide units into the primers of the plurality of polyvalent complex polymerases. In some embodiments, the multiple polyvalent molecules include at least one polyvalent molecule having multiple nucleotide arms (e.g., Figures 9–14), each of which nucleotide arms is attached to a nucleotide analog (e.g., a nucleotide analog unit), the nucleotide analog having a chain termination moiety at the sugar 2' and / or 3' positions. In some embodiments, the multiple polyvalent molecules include at least one polyvalent molecule containing multiple nucleotide arms, each of which nucleotide arms is attached to a nucleotide unit lacking a chain termination moiety. In some embodiments, at least one of the polyvalent molecules in the multiple polyvalent molecules is labeled with a detectable reporter moiety that emits a signal. In some embodiments, the detectable reporter moiety includes a fluorophore. In some embodiments, the contacting in step (b) is carried out in the presence of at least one non-catalyzable cation, including strontium, barium, and / or calcium.

[0266] In some embodiments, the sequencing method further includes step (c): detecting multiple polyvalent complex polymerases. In some embodiments, detection involves detecting a signal released by a polyvalent molecule bound to the complex polymerase, where the complementary nucleotide units of the polyvalent molecule bind to a primer, but the incorporation of the complementary nucleotide units is inhibited. In some embodiments, the polyvalent molecule is labeled with a detectable reporter portion to enable detection. In some embodiments, the labeled polyvalent molecule includes a fluorophore attached to the core, linker, and / or nucleotide units of the polyvalent molecule.

[0267] In some embodiments, the sequencing method further includes step (d): identifying the nucleic acid bases of complementary nucleotide units bound to a plurality of first complex polymerases, thereby determining the identity of the corresponding nucleic acid bases in the nucleic acid template molecule, and thus determining the sequence of the nucleic acid template molecule. In some embodiments, the polyvalent molecule is labeled with a detectable reporter portion corresponding to a specific nucleotide unit attached to a nucleotide arm, enabling the identification of complementary nucleotides (e.g., adenine, guanine, cytosine, thymine, or uracil, which are nucleotide bases) in the nucleic acid molecule bound to a plurality of first complex polymerases.

[0268] In some embodiments, the sequencing method further includes step (e): dissociating multiple polyvalent complex polymerases, removing multiple first sequencing polymerases and their bound polyvalent molecules, and retaining multiple nucleic acid double helixes.

[0269] The second step of a two-step sequencing method generally involves nucleotide incorporation. In some embodiments, the sequencing method further comprises: step (f): contacting a plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contact is carried out 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 of which comprises a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerase includes recombinant mutant sequencing polymerases.

[0270] In some embodiments, the plurality of first sequencing polymerases in step (a) have amino acid sequences that are 100% identical to the amino acid sequences of the plurality of second sequencing polymerases in step (f). In some embodiments, the plurality of first sequencing polymerases in step (a) have amino acid sequences that are different from the amino acid sequences of the plurality of second sequencing polymerases in step (f).

[0271] In some embodiments, the sequencing method further comprises: step (g): contacting a plurality of second complex polymerases with a plurality of nucleotides, wherein the contact is carried out under conditions suitable for conjugating 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 carried out under conditions suitable for promoting the polymerase catalyst incorporation of the conjugated complementary nucleotides into the primer of the nucleotide complex polymerase, thereby extending the sequencing primer by one nucleotide. In some embodiments, the incorporation of the nucleotides 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 carried out in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides include native nucleotides (e.g., dissimilar nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides include removable 2' and / or 3' chain termination portions. In some embodiments, the plurality of nucleotides include non-removable 2' and / or 3' chain termination portions. In some embodiments, at least one of the nucleotides in the plurality of nucleotides is not labeled with a detectable reporter portion. In some embodiments, the plurality of nucleotides are not labeled with a detectable reporter portion. In some embodiments, the plurality of nucleotides include a plurality of nucleotides labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker, and the linker is either cleavable / removable from the base or not removable from the base.In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to a nucleotide can correspond to a nucleic acid base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to enable the detection and identification of the nucleic acid base.

[0272] In some embodiments, when multiple nucleotides in step (g) are detectably labeled, the sequencing method further includes step (h): detecting the labeled nucleotides incorporated into the primers of a nucleotide complex polymerase. In some embodiments, the multiple nucleotides are labeled with a detectable reporter portion to enable detection. In some embodiments, when the multiple nucleotides in step (g) are unlabeled, the detection in step (h) is omitted.

[0273] In some embodiments, when multiple nucleotides in step (g) are detectably labeled, the sequencing method further includes step (i): identifying the bases of the nucleotides to be incorporated into the primers of the nucleotide complex polymerase based on the detection of the labels as described above. In some embodiments, the identification of the incorporated nucleotides in step (i) can be used to confirm the identity of the complementary nucleotides of the polyvalent molecule 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, if the multiple nucleotides in step (g) are unlabeled, the identification in step (i) is omitted.

[0274] In some embodiments, the sequencing method further includes removing the chain termination portion from the incorporated nucleotide when step (j): step (g) is performed by contacting a plurality of second complex polymerases with a plurality of nucleotides, each containing at least one nucleotide having a 2' and / or 3' chain termination portion.

[0275] In some embodiments, the sequencing method further includes step (k): repeating steps (a) to (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 in steps (c) and (d) by detecting and identifying a polyvalent molecule that binds to the sequencing polymerase but is not incorporated into the 3' end of the primer. In some embodiments, the sequence of the nucleic acid template molecule can be determined (or confirmed) in steps (h) and (i) by detecting and identifying a nucleotide incorporated into the 3' end of the primer.

[0276] In some embodiments of the sequencing method, the binding of a plurality of first complex polymerases to a plurality of polyvalent molecules forms at least one avidity complex, and the method includes (a) forming a first binding complex by binding a first nucleic acid primer, a first sequencing polymerase, and a first polyvalent molecule to a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein the first nucleotide unit of the first polyvalent molecule binds to the first sequencing polymerase; and (b) forming a second binding complex by binding a second nucleic acid primer, a second sequencing polymerase, and a first polyvalent molecule to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first polyvalent molecule binds to the second sequencing polymerase, and the first and second binding complexes, containing the same polyvalent molecule, form an avidity complex. In some embodiments, the first sequencing polymerase includes 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 concatemer template molecule comprises a tandem repeat sequence of the sequence of interest and at least one universal sequencing primer binding site. The first and second nucleic acid primers can bind to the sequencing primer binding sites along the concatemer template molecule. Exemplary polyvalent molecules are shown in Figures 11–14, and an exemplary single unit of the concatemer template molecule is shown in Figures 9–10.

[0277] In some embodiments of the sequencing method, the method comprises conjugating a plurality of first complex polymerases with a plurality of polyvalent molecules to form at least one avidity complex, the method comprising: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different parts of a concatemer template molecule to form at least first and second complex polymerases on the same concatemer template molecule; and (b) contacting a plurality of polyvalent molecules with at least first and second complex polymerases on the same concatemer template molecule under conditions suitable for conjugating a single polyvalent molecule from the plurality of polyvalent molecules to the first and second complex polymerases, wherein at least a first nucleotide unit of the single polyvalent molecule conjugates to a first complex polymerase containing a first primer hybridized to a first part of the concatemer template molecule, thereby forming a first binding complex (e.g., a first ternary complex). The contact is carried out under conditions suitable for inhibiting polymerase-catalyzed integration of the bound first and second nucleotide units in the first and second binding complexes, which bind to the same polyvalent molecule, and the first and second binding complexes bind to the same polyvalent molecule, and the contact is carried out under conditions suitable for inhibiting polymerase-catalyzed integration of the bound first and second nucleotide units in the first and second binding complexes, which bind to the same polyvalent molecule, and the first and second binding complexes form an avidity complex; (c) detecting the first and second binding complexes on the same concatemer template molecule; and (d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule. In some embodiments, the multiple sequencing polymerases include any wild-type or mutant sequencing polymerase described herein or known in the art (e.g., U.S. Patent No. 11,859,241). In some embodiments, the concatemer template molecule includes a tandem repeat sequence of the sequence of interest and at least one universal sequencing primer binding site.Multiple nucleic acid primers can bind to sequencing primer binding sites along concatemer template molecules. Exemplary polyvalent molecules are shown in Figures 9–12.

[0278] Sequence determination by concatenation 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 binding-by-binding (SBB) procedure using unlabeled chain termination nucleotides. In some embodiments, the binding-by-binding (SBB) method includes (a) sequentially contacting a primed template nucleic acid molecule (e.g., including one or more copies of the sequence of interest as described above) with at least two separate mixtures under ternary complex stabilization conditions, wherein at least two separate mixtures each contain a polymerase and a nucleotide, thereby obtaining a primed template nucleic acid that has been contacted with nucleotide congeners for the base types of the first, second, and third base types in the template under ternary complex stabilization conditions; (b) examining the at least two separate mixtures to determine whether a ternary complex has been formed; and (c) A method for identifying the next correct nucleotide for a primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a nucleotide homogeneity of the first, second, and third base types, and based on the absence of a ternary complex in step (b), the next correct nucleotide is presumed to be a nucleotide homogeneity of the fourth base type; (d) adding the next correct nucleotide to a primer on the primed template nucleic acid molecule after step (b), thereby generating an extended primer; and (e) repeating steps (a) to (d) at least once on the primed template nucleic acid containing the extended primer. Exemplary sequencing methods by binding are described in U.S. Patents No. 10,246,744 and No. 10,731,141 (the contents of both patents are incorporated herein by reference in their entirety).

[0279] Method for sequencing using phosphate-labeled nucleotides This disclosure provides a method for sequencing using a sequencing system, wherein the method uses an immobilized sequencing polymerase to which an unimmobilized template molecule is bound, and the sequencing reaction is carried out using a phosphate-labeled nucleotide. In some embodiments, the sequencing method includes step (a): providing a support on which a plurality of sequencing polymerases are immobilized. In some embodiments, the sequencing polymerase includes a processive DNA polymerase. In some embodiments, the sequencing polymerase includes a wild-type or mutant DNA polymerase, for example, Phi29 DNA polymerase. In some embodiments, the support includes a plurality of separate compartments, and the sequencing polymerases are immobilized at the bottom of the compartments. In some embodiments, the separate compartments include a silica bottom through which light can pass. In some embodiments, the separate compartments include a silica bottom composed of a nanophotonic constraint structure including holes in a metal cladding film (e.g., an aluminum cladding film). In some embodiments, the holes in the metal cladding have small openings, for example, about 70 nm. In some embodiments, the height of the nanophotonic confinement structure is approximately 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.

[0280] In some embodiments, the sequencing method further includes step (b): contacting a plurality of immobilized sequencing polymerases with a plurality of single-stranded cyclic nucleic acid template molecules and a plurality of oligonucleotide sequencing primers under conditions suitable for each immobilized sequencing polymerase to bind to the single-stranded cyclic template molecule and for each sequencing primer to hybridize to each single-stranded cyclic template molecule, thereby generating a plurality of polymerase / template / primer complexes. In some embodiments, each sequencing primer hybridizes to a universal sequencing primer binding site on the single-stranded cyclic template molecule.

[0281] In some embodiments, the sequencing method further comprises step (c): contacting multiple polymerase / template / primer complexes with multiple phosphate-labeled nucleotides, each containing an aromatic base, a 5-carbon sugar (e.g., ribose or deoxyribose), and a phosphate chain containing 3 to 20 phosphate groups, wherein the terminal phosphate groups are bound to a detectable reporter moiety (e.g., a fluorophore). The first, second, and third phosphate groups may be called alpha, beta, and gamma phosphate groups. In some embodiments, the specific detectable reporter moiety attached to the terminal phosphate groups corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to enable the detection and identification of nucleic acid bases. In some embodiments, the multiple polymerase / template / primer complexes are contacted with multiple phosphate-labeled nucleotides under conditions suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase is capable of binding complementary phosphate-labeled nucleotides and incorporating complementary nucleotides on the opposite side of the nucleotide in the template molecule. In some embodiments, polymerase-catalyzed nucleotide incorporation reactions involve cleavage between alpha-phosphate and beta-phosphate groups, thereby releasing polyphosphate chains bonded to fluorophores.

[0282] In some embodiments, the sequencing method further includes step (d): detecting a fluorescent signal emitted by a phosphate-labeled nucleotide that is bound by a sequencing polymerase and incorporated into the terminals of a sequencing primer. In some embodiments, step (d) further includes identifying the phosphate-labeled nucleotide that is bound by a sequencing polymerase and incorporated into the terminals of a sequencing primer.

[0283] In some embodiments, the sequencing method further includes step (d): repeating steps (c) to (d) at least once. In some embodiments, the sequencing method using phosphate-labeled nucleotides can be carried out according to the methods described in U.S. Patent No. 7,170,050, No. 7,302,146, and / or No. 7,405,281.

[0284] Sequence determination polymerase This disclosure provides methods for sequencing nucleic acid template molecules, wherein any of the sequencing methods described herein utilize at least one type of sequencing polymerase and a plurality of nucleotides, or at least one type of sequencing polymerase and a plurality of nucleotides and a plurality of polyvalent molecules. In some embodiments, the sequencing polymerase(s) can incorporate complementary nucleotides opposite to the nucleotides in the template molecule. In some embodiments, the sequencing polymerase(s) can bind to complementary nucleotide units of the polyvalent molecules opposite to the nucleotides in the template molecule. In some embodiments, the plurality of sequencing polymerases include recombinant mutant polymerases.

[0285] Examples of polymerases suitable for use in sequencing of nucleotides and / or polyvalent molecules include: Klen's DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaeon; Candidatus Hadarchaeum Yellowstonense; Hadesarchaea archaeon; Euryarchaeota archaeon; Thermoplasmata archaeon; Thermococcus polymerase, e.g., Thermococcus litoralis; bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; bacteriophage polymerases, e.g., T4, RB69, and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alpha and epsilon; 9 degree Examples of DNA polymerases include, but are not limited to, N polymerase; reverse transcriptases, e.g., HIV-type M or O reverse transcriptase; avian myeloblastosis virus reverse transcriptase; Moloney's mouse leukemia virus (MMLV) reverse transcriptase; or telomerase. Further non-limiting examples of DNA polymerases include those from various archaeal genera, e.g., Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, which include such polymerases known in the art, e.g., 9 degrees N, VENT®, DEEP VENT®, THERMINATOR®, Pfu, KOD, Pfx, Tgo, and RB69 polymerase.Additional 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).

[0286] nucleotide This disclosure provides methods for sequencing nucleic acid template molecules, wherein any of the sequencing methods described herein utilize at least one nucleotide or at least one or more nucleotides. A nucleotide comprises a base, a sugar, and at least one phosphate group. In some embodiments, at least one nucleotide in a plurality of nucleotides comprises an aromatic base, a pentacosate (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). A plurality of nucleotides may include at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. A plurality of nucleotides may include 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 in a plurality of nucleotides is not a nucleotide analog. In some embodiments, at least one nucleotide in a plurality of nucleotides includes a nucleotide analog.

[0287] In some embodiments, in any of the methods for sequencing nucleic acid molecules described herein, at least one nucleotide in a plurality of nucleotides comprises a chain of 1, 2, or 3 phosphorus atoms, the chain typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide in a plurality of nucleotides is an analog having a phosphorus chain, in which the phosphorus atoms together are linked to intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise a substituted side chain group comprising O, S, or BH3. In some embodiments, the chain comprises a phosphate group substituted with analogs comprising phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoramidite groups.

[0288] In some embodiments, in any of the methods for sequencing nucleic acid molecules described herein, at least one nucleotide in a plurality of nucleotides comprises a terminator nucleotide analog, the terminator nucleotide analog having a chain termination portion (e.g., a blocking portion) at the 2' sugar position, the 3' sugar position, or both the 2' and 3' sugar positions. In some embodiments, the chain termination portion can inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides containing the chain termination portion in the nascent chain during a primer extension reaction. In some embodiments, the chain termination portion is attached to the 3' sugar position, where the sugar comprises a ribose or deoxyribose sugar portion. In some embodiments, the chain termination portion is removable / cleavable from the 3' sugar position to produce a nucleotide having a 3'OH sugar group that can be extended by a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion includes alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, keto groups, isocyanate groups, phosphate groups, thio groups, disulfide groups, carbonate groups, urea groups, silyl groups, or acetal groups. In some embodiments, the chain termination portion can be cleaved / removed from the nucleotide, for example, by reacting the chain termination portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl chain termination portions can be cleaved with tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4) having piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the aryl and benzyl chain termination portions can be cleaved using H2Pd / C. In some embodiments, the chain termination portions, which are amines, amides, ketos, isocyanates, phosphates, thios, and disulfides, can be cleaved using phosphines or thiol groups containing beta-mercaptoethanol or dithiotitol (DTT).In some embodiments, the chain-ending carbonate can be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-ending urea and silyl can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the chain-ending portion may be cleaved / removed using nitrite. In some embodiments, the chain-ending portion may be cleaved / removed using a solution containing nitrite, for example, a combination of nitrite and an acid, for example, acetic acid, sulfuric acid, or nitric acid. In some further embodiments, the solution may contain an organic acid.

[0289] In some embodiments, at least one nucleotide in a plurality of nucleotides comprises a terminator nucleotide analog having a chain termination moiety (e.g., a blocking moiety) at the 2', 3', or 2' and 3' sugar positions. In some embodiments, the chain termination moiety comprises an azide, azido, or azidomethyl group. In some embodiments, the chain termination moiety comprises a 3'-O-azide or 3'-O-azidomethyl group. In some embodiments, the azide, azido, and azidomethyl groups that constitute the chain termination moiety are cleavable / removable using a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. 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 cleavage agent comprises 4-dimethylaminopyridine (4-DMAP). In some embodiments, a chain termination portion comprising one or more of a 3'-O-amino group, a 3'-O-aminomethyl group, a 3'-O-methylamino group, or derivatives thereof can be cleaved using nitrite via a nitrite-utilizing mechanism or by using a nitrite-containing solution. In some embodiments, a chain termination portion comprising one or more of a 3'-O-amino group, a 3'-O-aminomethyl group, a 3'-O-methylamino group, or derivatives thereof can be cleaved using a nitrite-containing solution. In some embodiments, the nitrite can be combined with or in contact with an acid such as acetic acid, sulfuric acid, or nitric acid. In some further embodiments, the nitrite can be combined with or in contact with an organic acid such as formic acid, acetic acid, propionic acid, butyric acid, or isobutyric acid. In some embodiments, the chain termination portion comprises a 3'-acetal moiety that can be cleaved with a palladium deblocking reagent (e.g., Pd(0)).

[0290] In some embodiments, the nucleotide includes a chain termination moiety selected from the group consisting of 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azide, 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'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tertbutyl, 3'-fluorenylmethyloxycarbonyl, 3'tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino group, 3'-phosphorothioate, 3-O-benzyl, and 3'-O-benzyl, 3-acetal moiety, or derivatives thereof.

[0291] In some embodiments, a plurality of nucleotides includes at least one nucleotide labeled with a detectable reporter moiety. In some embodiments, a plurality of nucleotides includes a plurality of nucleotides labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety includes a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base by a linker, and the linker is cleavable / removable from the base. In some embodiments, at least one of the nucleotides in the plurality of nucleotides is not labeled with a detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to enable detection and identification of the nucleotide base.

[0292] In some embodiments, a cleavable linker on a nucleotide base (e.g., a linker attached to a nucleotide-detectable reporter portion) includes a cleavable moiety comprising an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the cleavable linker is cleavable / removable from the nucleic acid base by reacting the cleavable moiety with a chemical agent, pH change, light, or heat. In some embodiments, the cleavable moieties alkyl, alkenyl, alkynyl, and allyl are cleavable using tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4) having piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the cleavable moieties aryl and benzyl are cleavable with H2Pd / C. In some embodiments, the cleavable moieties—amines, amides, ketos, isocyanates, phosphates, thios, and disulfides—can be cleaved with phosphines or thiol groups containing beta-mercaptoethanol or dithiotitol (DTT). In some embodiments, the cleavable moieties—carbonates—can be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the cleavable moieties—ureas and silyls—can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0293] In some embodiments, the cleavable linker on the nucleotide base includes a cleavable moiety comprising an azide, azido, or azidomethyl group. In some embodiments, the cleavable moiety, which is the azide, azido, or azidomethyl group, is cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound includes a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent includes 4-dimethylaminopyridine (4-DMAP).

[0294] In some embodiments, in any of the methods for sequencing nucleic acid molecules described herein, the chain termination portion (e.g., at the 2' and / or 3' sugar positions) and the cleavable linker on the nucleotide base have the same or different cleavable portions. In some embodiments, the chain termination portion (e.g., at the 2' and / or 3' sugar positions) and the detectable reporter portion linked to the base are chemically cleavable / removable with the same chemical agent. In some embodiments, the chain termination portion (e.g., at the 2' and / or 3' sugar positions) and the detectable reporter portion linked to the base are chemically cleavable / removable with different chemical agents.

[0295] polyvalent molecules This disclosure provides a method for sequencing a nucleic acid template molecule, wherein any of the sequencing methods described herein utilizes at least one polyvalent molecule. In some embodiments, the polyvalent molecule comprises a plurality of nucleotide arms, which are attached to a core and have one of the following configurations: a starburst, a helter skelter, or a bottlebrush configuration (e.g., Figure 11). In some embodiments, the polyvalent molecule comprises (1) a core and (2) a plurality of nucleotide arms, which comprises (i) a core attachment portion, (ii) a spacer containing a PEG portion, (iii) a linker, and (iv) a nucleotide unit, where the core 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 group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, and both linker chains have 2 to 6 subunits. In some embodiments, the linker also comprises an aromatic portion. An exemplary nucleotide arm is shown in Figure 15. Exemplary polyvalent molecules are shown in Figures 11 to 14. An exemplary spacer is shown in Figure 16 (top), and an exemplary linker is shown in Figure 16 (bottom) and Figure 17. Exemplary nucleotides attached to the linker are shown in Figures 18 to 21. An exemplary biotinylated nucleotide arm is shown in Figure 22.

[0296] In some embodiments, the polyvalent molecule comprises a core attached to multiple nucleotide arms, each having the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0297] In some embodiments, a polyvalent molecule comprises a core attached to a plurality of nucleotide arms, each arm comprising a nucleotide unit. A nucleotide unit comprises an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). A plurality of polyvalent molecules may include one type of polyvalent molecule having one type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. A plurality of polyvalent molecules may also be included in a mixture of any combination of two or more types of polyvalent molecules, where each individual polyvalent molecule in the mixture comprises a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0298] In some embodiments, the nucleotide unit comprises a chain of one, two, or three phosphorus atoms, the chain typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain, within the phosphorus chain, the phosphorus atoms together are bonded to intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise a substituted side chain group comprising O, S, or BH3. In some embodiments, the chain comprises a phosphate group substituted with analogs comprising phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoramidite groups.

[0299] In some embodiments, the polyvalent molecule comprises a core attached to multiple nucleotide arms, each nucleotide arm comprising a nucleotide unit, the nucleotide unit being a nucleotide analog having a chain termination portion (e.g., a blocking portion) at the 2', 3', or both 2' and 3' sugar positions. In some embodiments, the nucleotide unit contains the chain termination portion (e.g., a blocking portion) at the 2', 3', or both 2' and 3' sugar positions. In some embodiments, the chain termination portion can inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent chain during a primer extension reaction. In some embodiments, the chain termination portion is attached at the 3' sugar position, where the sugar comprises a ribose or deoxyribose sugar portion. In some embodiments, the chain termination portion is removable / cleavable from the 3' sugar position to produce a nucleotide having a 3'OH sugar group that can be extended by a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion includes an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the chain termination portion can be cleaved / removed from the nucleotide unit, for example, by reacting the chain termination portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl chain termination portions can be cleaved with tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4) having piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the aryl and benzyl chain termination portions can be cleaved using H2Pd / C. In some embodiments, the chain termination portions, which are amines, amides, ketos, isocyanates, phosphates, thios, and disulfides, can be cleaved using phosphines or thiol groups containing beta-mercaptoethanol or dithiotitol (DTT).In some embodiments, the chain-ending portion carbonate can be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-ending portions urea and silyl can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0300] In some embodiments, the nucleotide unit contains a chain termination moiety (e.g., a blocking moiety) at the 2', 3', or both 2' and 3' positions. In some embodiments, the chain termination moiety contains an azide, azido, or azidomethyl group. In some embodiments, the chain termination moiety contains a 3'-O-azide or 3'-O-azidomethyl group. In some embodiments, the azide, azido, and azidomethyl groups that constitute the chain termination moiety are cleavable / removable using a phosphine compound. In some embodiments, the phosphine compound contains a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound contains tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent contains 4-dimethylaminopyridine (4-DMAP).

[0301] In some embodiments, a nucleotide unit comprising a chain termination portion selected from the group consisting of 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azide, 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'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tertbutyl, 3'-fluorenylmethyloxycarbonyl, 3'tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino group, 3'-phosphorothioate, and 3'-O-benzyl, or derivatives thereof.

[0302] In some embodiments, the polyvalent molecule comprises a core attached to multiple nucleotide arms, the nucleotide arms comprising spacers, linkers, and nucleotide units, and the core, linkers, and / or nucleotide units are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, all fluorophores are present on and identical to those on individual polyvalent molecules (i.e., have the same excitation and emission spectra). In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) bound to the polyvalent molecule can correspond to a base of a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to enable the detection and identification of nucleotide bases.

[0303] In some embodiments, at least one nucleotide arm of the polyvalent molecule has a nucleotide unit attached to a detectable reporter moiety. In some embodiments, the detectable reporter moiety is attached to a nucleotide base. In some embodiments, the detectable reporter moiety includes a fluorophore. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to the polyvalent molecule can correspond to the identity of a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) of a nucleotide unit(s) to enable the detection and identification of the nucleotide base.

[0304] In some embodiments, the core of the polyvalent molecule comprises an avidin-like or streptavidin-like moiety, and the core-attached moiety comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type moiety, and the streptavidin-type or avidin-type moiety comprises an avidin protein, as well as any derivatives, analogs, and other non-natural forms of avidin, which can bind to at least one biotin moiety. Other forms of the avidin moiety include natural and recombinant avidin and streptavidin, as well as derivatized molecules, such as non-glycosylated avidin and cleaved streptavidin. For example, the avidin portion includes deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinii), and derivatized forms, such as N-acylavidins, such as N-acetyl, N-phthalyl, and N-succinylavidin, as well as commercially available products such as EXTRAVIDIN®, CAPTAVIDIN®, NEUTRAVIDIN, and NEUTRALITE AVIDIN.

[0305] In some embodiments of the sequencing methods described herein, the method may include forming a binding complex, the binding complex comprising (i) a polymerase, a primer and a double-stranded nucleic acid template molecule, and a nucleotide, or the binding complex comprising (ii) a polymerase, a primer and a double-stranded nucleic acid template molecule, and a nucleotide unit of a polyvalent molecule. In some embodiments, the binding complex has a duration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more than 1 second. In some embodiments, the binding complex has a duration of approximately 0.1–0.25 seconds, or approximately 0.25–0.5 seconds, or approximately 0.5–0.75 seconds, or approximately 0.75–1 second, or approximately 1–2 seconds, or approximately 2–3 seconds, or approximately 3–4 seconds, or approximately 4–5 seconds, and / or the method is performed or may be performed at a temperature 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, or 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) can remain stable until it is subjected to conditions that cause dissociation of the interaction between the polymerase, template molecule, primer, and / or nucleotide units or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with one of a washing agent, EDTA, and / or water, or any combination thereof. In some embodiments, the Disclosure provides a method wherein the binding complex is deposited on, attached to, or hybridized to a surface exhibiting a contrast-to-noise ratio greater than 20 in the detection step. In some embodiments, the Disclosure provides a method wherein the contact is performed under conditions that stabilize the binding complex when a nucleotide or nucleotide unit is complementary to the next base of the template nucleic acid, and destabilize the binding complex when a nucleotide or nucleotide unit is not complementary to the next base of the template nucleic acid.

[0306] Compaction oligonucleotides This disclosure provides a method for preparing nucleic acid template molecules for sequencing (e.g., compaction oligonucleotides, as described herein). Suitable compaction oligonucleotides are known in the art and are described, for example, in WO2024 / 040058A1 (the contents of which are incorporated herein by reference in their entirety).

[0307] Exemplary compaction oligonucleotides include single-stranded linear oligonucleotides having a 5' region that can hybridize to a first portion of a concatemer template molecule, and compaction oligonucleotides having a 3' region that can hybridize to a second portion of a concatemer template molecule (e.g., the same concatemer template molecule). In some embodiments, hybridization of compaction oligonucleotides to individual concatemer template molecules causes the concatemer molecule to collapse or fold into DNA nanoballs, which are more compact in shape and size compared to the non-collapsed DNA molecule. Spot images of DNA nanoballs can be represented as Gaussian spots, and their size can be measured as full width half-value (FWHM). Smaller spot sizes, indicated by smaller FWHMs, typically correlate with improved spot imaging. In some embodiments, the FWHM of DNA nanoball spots can be about 10 μm or less. DNA nanoballs can be compact nucleic acid structures with smaller full width half-values ​​(FWHMs) compared to concatemers that have not collapsed / folded into DNA nanoballs.

[0308] In some embodiments, the compaction oligonucleotide comprises a single-stranded oligonucleotide containing DNA, RNA, or a combination of DNA and RNA. The compaction oligonucleotide can be any of the following lengths: 20 to 150 nucleotides, 30 to 100 nucleotides, or 40 to 80 nucleotides.

[0309] In some embodiments, the compaction oligonucleotide includes a 5' region and a 3' region, and optionally, an intervening region between the 5' and 3' regions. The intervening region can be of any length, for example, about 2 to 20 nucleotides. The intervening region contains a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intervening region contains a non-homopolymer sequence.

[0310] The 5' region of the compaction oligonucleotide can be fully complementary or partially complementary to the first portion of the concatemer template molecule along its length. The 3' region of the compaction oligonucleotide can be fully complementary or partially complementary to the second portion of the concatemer template molecule along its length. The 5' region of the compaction oligonucleotide can hybridize to the first universal sequence portion of the concatemer template molecule. The 3' region of the compaction oligonucleotide can hybridize to the second universal sequence portion of the concatemer molecule. The 5' and 3' regions of the compaction oligonucleotide can hybridize to the concatemer, bringing together the distal portions of the concatemer, causing concatemer compaction, and forming DNA nanoballs.

[0311] The 5' region of a compaction oligonucleotide can have the same sequence as the 3' region. The 5' region of a compaction oligonucleotide can have a different sequence from the 3' region. The 3' region of a compaction oligonucleotide can have a sequence that is the reverse sequence of the 5' region.

[0312] Support and low nonspecific coating In some embodiments, the flow cell device herein may include a support 210 (e.g., a solid support disclosed herein). This disclosure provides sequencing compositions and methods using a support on which a plurality of oligonucleotide surface primers are immobilized. 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 typically used for nucleic acid capture, amplification, and sequencing workflows, such as dyes, nucleotides, enzymes, and nucleic acid primers. The surface coatings exhibit a lower background fluorescence signal or a higher contrast-to-noise (CNR) ratio compared to conventional surface coatings.

[0313] Low nonspecific binding coatings comprise one or more layers (Figure 23). In some embodiments, multiple surface primers are immobilized on the low nonspecific binding coating. In some embodiments, at least one surface primer is embedded within the low nonspecific binding coating. Low nonspecific binding coatings enable improved nucleic acid hybridization and amplification performance. Generally, a support comprises a substrate (or support structure) and one or more layers of low-binding chemically modified layers attached covalently or acovalently, e.g., silane layers, polymer films, and one or more covalently or acovalently attached surface primers that can be used to tether 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 binding chemistry used to crosslink one or more layers to and / or to the support, and the total number of layers, can be varied so that nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the coating is minimized or reduced compared to an equivalent monolayer. The coating formulations described herein may be varied so that nonspecific hybridization on the coating is minimized or reduced compared to an equivalent monolayer. The coating formulations may be varied so that nonspecific amplification on the coating is minimized or reduced compared to an equivalent monolayer. The coating formulations may be varied so that the specific amplification rate and / or yield on the coating is maximized. Amplification levels suitable for detection are achieved in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or fewer, or more than 30 amplification cycles, in some cases disclosed herein.

[0314] A support structure comprising one or more chemically modified layers, such as a layer of low nonspecific binding polymer, may be independent or integrated within another structure or assembly. For example, in some embodiments, the support structure may comprise one or more surfaces within an integrated or assembled flow cell device as described herein. The support structure may comprise one or more surfaces within a microplate format, such as the bottom surfaces of wells in a microplate. In some embodiments, the support structure comprises the inner surface of a capillary (e.g., the lumen surface). In some embodiments, the support structure comprises the inner surface of a capillary etched within a planar chip (e.g., the lumen surface).

[0315] The bonding chemistry used to graft the first chemically modified layer onto the surface of the support generally depends on both the material from which the surface is manufactured and the chemical properties of the layer. In some embodiments, the first layer may adhere covalently to the surface. In some embodiments, the first layer may adhere to the support non-covalently, e.g., by adsorption, via non-covalent interactions between the support and the molecular components of the first layer, e.g., electrostatic interactions, hydrogen bonds, or van der Waals interactions. In any case, the support may be treated before the adhesion or deposition of the first layer. Any of the various surface preparation techniques known to those skilled in the art may be used to clean or treat the surface. For example, a glass or silicon surface may be acid-washed using a piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)), a base treatment in KOH and NaOH, and / or cleaned using an oxygen plasma treatment method.

[0316] Silane chemistry has developed non-limiting methods for covalently modifying silanol groups on glass or silicon surfaces to bond more reactive functional groups (e.g., amine or carboxyl groups), which can then be used in bonding linker molecules (e.g., linear hydrocarbon molecules of various lengths, e.g., 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 in the preparation of any of the disclosed low-bonding coatings include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), any of the various PEG silanes (e.g., those with molecular weights such as 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., those containing free amino functional groups), maleimide-PEG silanes, and biotin-PEG silanes.

[0317] Any of the various 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 in the preparation of one or more chemically modified layers on a support, and the selection of components used may vary to modify 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 prepare one or more layers of low nonspecific binding material in any of the disclosed coatings include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyester, dextran, poly-lysine, and poly-lysine copolymers, or any combination thereof. Examples of conjugation chemistry that can be used to graft one or more layers of a material (e.g., polymer layers) onto a surface and / or to crosslink the layers with each other include, but are not limited to, biotin-streptavidin interactions (or their variants), his-tag-Ni / NTA conjugation chemistry, methoxyether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxys, azides, hydrazides, alkynes, isocyanates, and silanes.

[0318] A low nonspecific bonding surface coating can be applied uniformly across the entire support. Alternatively, the surface coating can be patterned such that the chemically modified layer is limited to one or more distinct regions of the support. For example, the coating can be patterned using photolithography techniques to create a regular array or random pattern of chemically modified regions on the support. Alternatively or in combination, the coating can be patterned using, for example, contact printing and / or inkjet printing techniques. In some embodiments, the regular 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 distinct regions.

[0319] In some embodiments, low nonspecific binding coatings include hydrophilic polymers that are nonspecifically adsorbed to or covalently grafted onto a support. Typically, passivation is performed using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene), or other hydrophilic polymers having different molecular weights and end groups bound to the support, for example, using silane chemistry. End groups distal to the surface may include, but are not limited to, biotin, methoxyether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In some embodiments, two or more layers of hydrophilic polymers, e.g., linear polymers, branched polymers, or highly branched polymers, may be deposited on the surface. In some embodiments, the two or more layers may be covalently bound to each other or internally crosslinked to improve the stability of the resulting coating. In some embodiments, surface primers described herein (e.g., surface capture primers and surface pinning primers) having different nucleotide sequences and / or base modifications (or other biomolecules, e.g., enzymes or antibodies) may be tethered to the resulting layer at varying surface densities. In some embodiments, for example, both the surface functional group density and the surface primer concentration may be varied to achieve a desired surface primer density range. Additionally, the surface primer density can be controlled by diluting the surface primer with other molecules possessing the same functional groups. For example, to reduce the final primer density, an amine-labeled surface primer can be diluted with amine-labeled polyethylene glycol in reaction with an NHS ester-coated surface. Surface primers having linkers of different lengths between the hybridization region and the surface-bonded functional groups can also be applied to control the surface density. Suitable linker examples include poly-T and poly-A chains at the 5' end of the primer (e.g., 0 to 20 bases), 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 may be tethered to a surface, and then fluorescent leads may be compared to those for a dye solution of known concentration.

[0320] In some embodiments, the low nonspecific bonding coating comprises a functionalized polymer coating layer covalently bonded to at least a portion of a support via chemical groups on the support, a primer grafted onto 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-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM).

[0321] Supports containing multilayer coatings of PEG and other hydrophilic polymers have been developed to adjust primer surface density and add additional dimensionality to hydrophilic or amphoteric coatings. By using hydrophilic and amphoteric surface layering techniques, including but not limited to the polymer / copolymer materials described below, it is possible to significantly increase the primer load density on the support. Conventional PEG coating techniques use monolayer primer deposition, which has generally been reported for single-molecule applications but does not yield high copy numbers for nucleic acid amplification applications. As described herein, “layering” can be achieved with any compatible polymer or monomer subunit so that a surface containing two or more highly crosslinked layers can be sequentially constructed using conventional crosslinking techniques. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some embodiments, different layers may adhere to each other via any of a variety of conjugation reactions, including, but not limited to, biotin-streptavidin bonding, 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 may be constructed in solution and then layered onto a surface in a series of steps.

[0322] Examples of materials from which support structures can 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 polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic support structures are intended.

[0323] The support structure may be any of a variety of geometric shapes and dimensions known to those skilled in the art, and may comprise any of a variety of materials known to those skilled in the art. For example, the support structure may be locally planar (e.g., including a microscope slide or the surface of a microscope slide). Generally, the support structure may 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 a non-porous bead or particle of an irregular shape). In some embodiments, the surface of the support structure used for nucleic acid hybridization and amplification may be a solid non-porous surface. In some embodiments, the surface of the support structure used for nucleic acid hybridization and amplification may be porous, so that the coating described herein penetrates the porous surface and the nucleic acid hybridization and amplification reactions performed thereon may occur within the pores. In some embodiments, the support geometry may comprise one or more channels, inlets, and / or outlets as described herein.

[0324] A support structure comprising one or more chemically modified layers, such as a layer of low nonspecific binding polymers, may be independent or integrated within another structure or assembly. For example, the support structure may comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may comprise one or more surfaces within a microplate format, such as the bottom surface of a well in a microplate. In some embodiments, the support structure comprises the inner surface of a capillary (e.g., the lumen surface). In some embodiments, the support structure comprises the inner surface of a capillary etched within a planar chip (e.g., the lumen surface). In some embodiments, the support comprises one or more inner surfaces of the flow cell device described herein.

[0325] As described above, the low nonspecific binding supports of this disclosure exhibit reduced nonspecific binding of 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 by a given support surface can be evaluated either qualitatively or quantitatively. Exposing the surface to, for example, a fluorescent dye (e.g., cyanine, e.g., Cy3 or Cy5, fluorescein, coumarin, rhodamine, or other dyes disclosed herein), a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and / or a fluorescently labeled protein (e.g., polymerase) under a set of standardized conditions, followed by a specific rinsing protocol and fluorescence imaging, can be used as a qualitative tool for comparing nonspecific binding on supports containing different surface formulations. In some embodiments, exposing a surface to, for example, a fluorescent dye, fluorescently labeled nucleotide, fluorescently labeled oligonucleotide, and / or fluorescently labeled protein (e.g., polymerase) under a set of standardized conditions, followed by a specific rinsing protocol and fluorescence imaging, can be used as a quantitative tool for comparing nonspecific binding on supports containing different surface formulations, provided that the fluorescence imaging is performed under conditions where the fluorescence signal is linearly related (or in a predictable manner) to the number of fluorophores on the support (e.g., under conditions where fluorophore signal saturation and / or self-quenching is not a concern), and care is taken to ensure that suitable calibration criteria are used. In some embodiments, other techniques known to those skilled in the art, e.g., radioisotope labeling and counting methods, can be used for quantitative assessment of the degree to which nonspecific binding is exhibited by different support surface formulations of this disclosure.

[0326] Some surfaces disclosed herein exhibit a ratio of specific fluorescence to nonspecific fluorescence of fluorophores such as Cy3, 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 encompassed by the range described herein.

[0327] The degree of nonspecific binding exhibited by the disclosed low-binding supports may be evaluated under a set of standardized incubation and rinsing conditions using a standardized protocol for contacting the surface with labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-strand binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., followed by detection of the amount of label remaining on the surface and comparison of 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. Thus, in some embodiments, the degree of nonspecific binding exhibited by a given support surface formulation may be evaluated in terms of 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 are 1 μm 2 Less than 0.001 molecules per unit, 1 μm 2 Less than 0.01 molecules per unit, 1 μm 2 Less than 0.1 molecules per unit, 1 μm 2 Less than 0.25 molecules per unit, 1 μm 2Less than 0.5 molecules per unit, 1 μm 2 Less than 1 molecule per unit, 1 μm 2 Less than 10 molecules per unit, 1 μm 2 Less than 100 molecules per unit, or 1 μm 2 It may exhibit nonspecific protein binding of less than 1,000 molecules per unit (or nonspecific binding of other specific molecules (e.g., cyanine, e.g., Cy3 or Cy5, fluorescein, coumarin, rhodamine, or other dyes disclosed herein)). A given support surface of this disclosure exhibits nonspecific binding of any value within this range, e.g., 1 μm 2 Those skilled in the art will recognize that nonspecific binding of fewer than 86 molecules per unit may be possible. For example, some modified surfaces disclosed herein were contacted with a 1 μM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate-buffered saline (PBS) buffer for 15 minutes, followed by rinsing three times with deionized water, and then bound to 0.5 molecules / μm. 2 It exhibits nonspecific protein binding of less than 1 μm. Some modified surfaces disclosed herein have a thickness of 1 μm. 2It shows nonspecific binding of less than 0.25 Cy3 dye molecules per molecule. 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-propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were observed. The Biosciences (Biosciences) was incubated on a low-binding coated support at 37°C for 15 minutes in a 384-well plate format. Each well was rinsed 2-3 times with 50 μl of deionized RNase / DNase-free water and 2-3 times with 25 mM ACES buffer (pH 7.4). The 384-well plates were imaged in a GE Typhoon instrument with a PMT gain setting of 800 and a resolution of 50-100 μm, using Cy3, AF555, or Cy5 filter sets as specified by the manufacturer (according to the dye tests performed). For higher resolution imaging, images were taken using a total internal reflection fluorescence (TIRF) objective lens (100×, 1.5NA, Olympus), a CCD camera (e.g., Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera, or Olympus DP80 color and monochrome camera), and an illumination source (e.g., Olympus 100W). The images were collected using an Olympus IX83 microscope (e.g., an inverted fluorescence microscope) with an excitation wavelength of 532 nm or 635 nm (Hg lamp, Olympus 75W Xe lamp, or Olympus U-HGLGPS fluorescence light source) (Olympus Corp., Center Valley, Pa.).The dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, NY) and are, for example, 405, 488, 532, or 633 nm dichroic reflectors / beam splitters, and the bandpass filters were selected as 532LP or 645LP to match the appropriate excitation wavelength. Some of the modified surfaces disclosed herein are 1 μm thick. 2 It exhibits nonspecific binding of less than 0.25 dye molecules per unit. In some embodiments, the coated support was immersed in a buffer (e.g., 25 mM ACES, pH 7.4) while acquiring images.

[0328] In some embodiments, the surfaces disclosed herein exhibit a ratio of specific fluorescence signals to nonspecific fluorescence signals for fluorophores such as Cy3, 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 encompassed by the range described herein.

[0329] Low background surfaces consistent with the disclosures herein may exhibit specific dye attachment (e.g., Cy3 attachment) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) ratios 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, and 50:1, or more than 50 attached specific dye molecules per nonspecifically adsorbed molecule. Similarly, low background surfaces consistent with the disclosures herein, with fluorophores, e.g., Cy3, attached when subjected to excitation energy, may exhibit specific fluorescence signal to nonspecific adsorbed dye fluorescence signal ratios 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 greater than 50:1 (e.g., resulting from Cy3-labeled oligonucleotides attached to the surface).

[0330] In some embodiments, the degree of hydrophilicity (or "wettability" with aqueous solutions) of the disclosed support surface may be evaluated, for example, by measuring the water contact angle, in which a small water droplet is placed on the surface and its contact angle with the surface is measured, for example, using an optical tensiometer. In some embodiments, a static contact angle may be determined. In some embodiments, an advancing or receding contact angle may be determined. In some embodiments, the water contact angle for the surface-treated hydrophilic low-bonding support disclosed herein may be in the range of about 0 to about 30 degrees. In some embodiments, the water contact angle for the surface-treated hydrophilic low-bonding support disclosed herein may be 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 or less. In many cases, the contact angle is 40 degrees or less. It will be recognized by those skilled in the art that a given hydrophilic low-bonding support surface of this disclosure may exhibit a water contact angle having a value within this range.

[0331] In some embodiments, the hydrophilic surfaces disclosed herein often facilitate a reduction in washing time in bioassays by reducing the nonspecific binding of biomolecules to low-binding surfaces. In some embodiments, a suitable washing step may be performed in less than 60, 50, 40, 30, 20, 15, or 10 seconds. For example, a suitable washing step may be performed in less than 30 seconds.

[0332] Some of the low-binding surfaces of this disclosure exhibit significant improvements in stability or durability against prolonged exposure to solvents and high temperatures, or against repeated cycles of solvent exposure or temperature changes. For example, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups on the surface or tethered biomolecules (e.g., oligonucleotide primers) on the surface and monitoring the fluorescence signal before, during, and after prolonged exposure to solvents and high temperatures, or to repeated cycles of solvent exposure or temperature changes. 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 minutes, 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 when measured over these periods). In some embodiments, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages when measured over this range of cycles) over repeated exposure to solvent changes and / or temperature changes over 5, 10, 20, 30, 400, 500, 600, 700, 800, 900, or 1,000 cycles.

[0333] In some embodiments, the surfaces disclosed herein may exhibit a high ratio of specific signals to non-specific signals or other backgrounds. For example, when used for nucleic acid amplification, some surfaces may exhibit amplification signals at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than the signals of adjacent non-aggregated regions of the surface. Similarly, some surfaces may exhibit amplification signals at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than the signals of adjacent amplified nucleic acid aggregated regions of the surface.

[0334] In some embodiments, the disclosed low-background surface fluorescence images, when used in nucleic acid hybridization or amplification applications to produce polony of hybridized or clone-amplified nucleic acid molecules (e.g., directly or indirectly labeled with fluorophores), exhibit contrast-to-noise ratios (CNRs) 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.

[0335] One or more types of primers may adhere to or tether to the surface of the support. In some embodiments, one or more types of adapters or primers may include spacer sequences, adapter sequences for hybridization to adapter-ligated target library nucleic acid sequences, forward amplification primers, reverse amplification primers, sequencing primers, surface capture primers, surface pinning primers, and / or molecular barcode sequences, or any combination thereof. In some embodiments, one primer sequence or adapter 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 adapter sequences may be tethered to at least one layer of the surface. In some embodiments, the support includes a plurality of primers tethered to the support, and all primers include the same sequence (e.g., the same surface capture sequence). In some embodiments, the support includes two or more primers tethered to the support, and each plurality of primers includes a primer that includes the same sequence, but different from the corresponding sequence in the other plurality of primers. As a non-limiting example, the first set of primers may include a first surface capture array, and the second set of primers may include a second surface capture array, where the first and second capture arrays are not identical.

[0336] In some embodiments, the tethered adapter sequence and / or primer sequence may be in the range of about 10 to about 100 nucleotides in length. In some embodiments, the tethered adapter sequence and / or primer sequence may 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 length. In some embodiments, the tethered adapter sequence and / or primer sequence may be up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 nucleotides in length. Any of the lower and upper limits described in this paragraph may be combined to form a range included in the disclosure; for example, in some embodiments, the length of the tethered adapter sequence and / or primer sequence may be in the range of about 20 to about 80 nucleotides. It will be recognized by those skilled in the art that the length of the tethered adapter sequence and / or primer sequence may have any value within this range, for example, about 24 nucleotides.

[0337] In some embodiments, the surface density obtained of the primer (e.g., capture primer) on the surface of the low-binding support of this disclosure is 1 μm 2 Approximately 100 primer molecules per unit, ~1 μm in size. 2 The number of primer molecules per unit area can range from approximately 100,000. In some embodiments, the resulting surface density of the primers on the low-binding support surface of this disclosure is 1 μm 2 Approximately 1,000 primer molecules per unit, ~1 μm in size. 2 The number of primer molecules per unit area can range from approximately 1,000,000. In some embodiments, the surface density of the primers is 1 μm 2 The number of molecules per unit may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000. In some embodiments, the surface density of the primer is 1 μm 2The number of molecules per unit may be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000. Any combination of the lower and upper limits described in this paragraph may form the range included in this disclosure, for example, in some embodiments the surface density of the primer is 1 μm 2 Approximately 10,000 molecules per unit ~ 1 μm 2 The number of molecules per surface can range from approximately 100,000. The surface density of primer molecules is any value within this range, for example, 1 μm. 2 Those skilled in the art will recognize that it may contain approximately 455,000 molecules per unit. In some embodiments, the surface density of the target library nucleic acid sequence initially hybridized to the adapter or primer sequence on the support surface may be less than or equal to that indicated for the surface density of the tethered primer. In some embodiments, the surface density of the cloned amplified target library nucleic acid sequence hybridized to the adapter or primer sequence on the support surface may encompass the same range as that indicated for the surface density of the tethered primer.

[0338] The local densities listed above are for a surface that is, for example, 500,000 / μm 2 The density does not exclude variations across the surface, including regions having a certain oligodendrology density, and also including at least a second region having a substantially different local density.

[0339] In some embodiments, the performance of nucleic acid hybridization and / or amplification reactions using the disclosed reaction formulations and low-binding supports may be evaluated using fluorescence imaging techniques, and the contrast-to-noise ratio (CNR) of the image provides an important metric in evaluating amplification specificity and nonspecific binding on the support. CNR is generally defined as CNR = (signal - background) / noise. The background term is generally considered to be the signal measured over the interstitial region surrounding a specific feature (diffraction-limited spot, DLS) in a particular region of interest (ROI). While the signal-to-noise ratio (SNR) is often considered a benchmark for overall signal quality, it can be shown that an improved CNR can offer a significant advantage over SNR as a benchmark for signal quality in applications requiring rapid image capture (e.g., sequencing applications where cycle time must be minimized). With a high CNR, the imaging time required to achieve accurate identification (and therefore, accurate base calling in the case of sequencing applications) can be significantly reduced, even with a moderate improvement in CNR. Improved CNR in imaging data regarding imaging integration time provides a method for more accurately detecting features such as clone-amplified nucleic acid colonies on the support surface.

[0340] In most ensemble-based sequencing methods, the background term is typically measured as a signal associated with "interstitial" regions. "Interstitial" background (B inter ) in addition to the "intrastial" background (B intra The background signals are located within the region occupied by the amplified DNA colonies. The combination of these two background signals determines the achievable CNR, which then directly impacts optical instrument requirements, architecture costs, reagent costs, run time, cost / genome, and ultimately, the accuracy and data quality for circular array-based sequencing applications. interBackground signals arise from various sources, some examples of which include autofluorescence from consumable flow cells, nonspecific adsorption of detection molecules resulting in spurious fluorescence signals that can obscure signals from ROIs, and the presence of nonspecific DNA amplification products (e.g., those arising 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 arising from individual DNA colonies (i.e., (signal)-B (interstitial) in the FOV) provide distinguishable features that can be classified. In some embodiments, intrastitial background (B (intrastitial)) can contribute to confounding fluorescence signals that are not specific to the target of interest but are present in the same ROI, thus making averaging and subtraction much more difficult.

[0341] Nucleic acid amplification on the low-binding coated supports described herein may reduce the interstitial (B) background signal by reducing nonspecific binding, resulting in improvements in specific nucleic acid amplification and a reduction in nonspecific amplification that can affect background signals arising from both interstitial and intrastitial regions. In some embodiments, the disclosed low-binding coated supports, used optionally in combination with the disclosed hybridization and / or amplification reaction formulations, may provide improvements in CNR by 2, 5, 10, 100, 250, 500, or 1000 times compared to those achieved using conventional supports and hybridization, amplification, and / or sequencing protocols. 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 supports and nucleic acid hybridization and amplification formulations for other detection modes, including both optical and non-optical detection modes.

[0342] 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 a combination of these. In some embodiments, the support can be cylindrical and include, for example, a capillary or the inner surface of a capillary.

[0343] 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 raised areas, etching, pores, three-dimensional scaffolding, or a combination thereof.

[0344] In some embodiments, the support comprises beads having any of the following shapes, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0345] The support can be manufactured from any of the following materials, which 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 polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are intended.

[0346] In some embodiments, the surface of the support is coated with one or more compounds to create a passivation layer on the support. In some embodiments, the support includes a low nonspecific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. Generally, the support may include a low-binding chemical modification layer that is covalently or non-covalently bonded, e.g., a silane layer, a polymer film, and one or more layers of one or more covalently or non-covalently bonded oligonucleotides that can be used to immobilize multiple nucleic acid template molecules on the support.

[0347] In some embodiments, the degree of hydrophilicity (or "wettability" with aqueous solutions) of a surface coating can be evaluated, for example, by measuring the water contact angle, which is measured, for example, using an optical tensile meter, when a small water droplet is placed on the surface. In some embodiments, a static contact angle can be determined. In some embodiments, an advancing or receding contact angle can be determined. In some embodiments, the water contact angle of a surface-treated hydrophilic low-bonding support disclosed herein may be in the range of about 0 to about 30 degrees. In some embodiments, the water contact angle of a surface-treated hydrophilic low-bonding support disclosed herein may be 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 or less....

Claims

1. A sequencing system, Optical system 2020 equipped with an objective lens, x-y stage 2010, which holds a sample to be imaged on it and is configured to move the sample in the x-y plane relative to the objective lens, and the sample is fixed on one or more flow cell devices, x-y stage 2010, A nest bank 2050, configured to provide fluid and thermal communication to the sample when one or more flow cell devices are coupled to the nest bank, A sequence determination system comprising: a moving mechanism 2040, which optionally includes a movable arm configured to move one or more flow cell devices between the x-y stage 2010 and the nest bank 2050 during sequence determination execution.

2. The array determination system according to claim 1, wherein the x-y stage 2010 is automatically driven by a first actuator with a first spatial accuracy.

3. The arrangement determination system according to claim 1 or 2, wherein the movable arm is automatically driven by a second actuator with a second spatial accuracy.

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

5. The aforementioned sequencing system The array determination system according to any one of the prior claims, further comprising a housing configured to hold therein one or more of the optical system 2020, the x-y stage 2010, the nest bank 2050, and the moving mechanism 2040.

6. The arrangement determination system according to any one of the prior claims, wherein the movable arm is automatically driven to move in three dimensions (3D).

7. The arrangement determination system according to any one of the prior claims, wherein the movement in each of the three dimensions has one or more predetermined spatial accuracies.

8. The sequencing system according to any one of the prior claims, wherein the sequencing system lacks fluid or thermal communication to one or more flow cell devices on or near the x-y stage 2010 when the flow cell devices are fixed on the x-y stage 2010.

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

10. The sequence determination system according to any one of the prior claims, wherein the flow cell device comprises a plurality of microfluidic channels, and the nest bank 2050 is configured to allow fluid communication to each of the plurality of microfluidic channels independently and simultaneously.

11. The sequence determination system according to any one of the prior claims, wherein the flow cell device comprises a plurality of microfluidic channels, and the nest bank 2050 is configured to allow fluid communication to each of the plurality of microfluidic channels independently and sequentially.

12. The sequence determination system according to any one of the prior claims, wherein the flow cell device comprises a plurality of microfluidic channels, and the nest bank 2050 is configured to allow independent fluid communication to each of the plurality of microfluidic channels without cross-contamination.

13. The arrangement determination system according to any one of the prior claims, wherein the x-y stage 2010 is driven to move a predetermined distance in the x-y plane.

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

15. The array determination system according to any one of the prior claims, wherein the nest bank 2050 is configured to enable fluid and thermal communication with one or more flow cell devices.

16. The arrangement determination system according to any one of the prior claims, wherein the nest bank 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 nest bank 2050.

17. The arrangement determination system according to any one of the prior claims, wherein the nest bank 2050 is configured to hold each of the flow cell devices in an unlocked position in which the flow cell devices are removable from the nest bank 2050, and in a locked position in which the flow cell devices are spatially aligned with the nest bank 2050, fixedly coupled to the nest bank 2050, and sealed fluid and thermal communication is possible between the nest bank and the flow cell devices.

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

19. The arrangement determination system according to any one of the prior claims, wherein the movable arm is configured to move the carrier 2051 and the flow cell device together.

20. The array determination system according to any one of the prior claims, wherein the carrier 2051 is configured to be spatially aligned with respect to the nest bank at the locked position.

21. The sequencing system according to any one of the prior claims, wherein the nest bank 2050 comprises one or more fasteners.

22. The arrangement determination system according to claim 21, wherein one or more fasteners use magnetic force.

23. The arrangement determination system according to claim 21 or 22, wherein one or more fasteners include a rare earth magnet, an electromagnetic coil, or both.

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

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

26. The array determination system according to any one of the prior claims, wherein the movable arm is configured to move one or more flow cell devices between the x-y stage 2010 and the nest bank 2050 with a first spatial accuracy.

27. The array determination system according to any one of the prior claims, wherein the movable arm includes a gripping portion configured to grip the carrier 2051 when the carrier 2051 is in a detached position relative to the nest bank 2050, or when the carrier 2051 is in a detached position relative to the x-y stage 2010.

28. The arrangement determination system according to any one of the prior claims, wherein the movable arm includes a horizontal arm that is mechanically supported by a vertical arm.

29. The arrangement determination system according to any one of the prior claims, wherein the movable arm comprises an upper arm, a joint, a forearm, a wrist, and a gripping portion attached to the forearm.

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

31. The arrangement determination system according to any one of claims 27 to 30, wherein the gripping portion is movably attached to the horizontal arm or the vertical arm.

32. The arrangement determination system according to any one of claims 27 to 31, wherein the gripping portion is configured to move in 3D.

33. The arrangement determination system according to any one of the prior claims, wherein the moving mechanism 2040 comprises a plurality of tracks, each track connecting a carrier 2051 coupled to the nest bank to the x-y stage 2010.

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

35. The array determination system according to any one of the prior claims, wherein the carrier 2051 comprises one or more sensors.

36. The array determination system according to any one of the prior claims, wherein the x-y stage 2010 comprises one or more sensors.

37. The array determination system according to any one of the prior claims, wherein the nest bank 2050 comprises one or more sensors.

38. The array determination system according to any one of claims 35 to 37, wherein one or more sensors are configured to provide feedback to a processor that facilitates the positioning of the carrier 2051 relative to the x-y stage 2010, the optical system 2020, or the nest bank 2050.

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

40. The array determination system according to any one of claims 33 to 39, wherein the plurality of tracks comprises one or more actuators configured to drive one or more of the plurality of tracks in order to move the corresponding carrier 2051 to the x-y stage 2010.

41. The array determination system according to any one of the prior claims, wherein the x-y stage 2010 is configured to be driven to move to a 3D position with a second spatial accuracy.

42. The sequencing system according to claim 41, wherein the second spatial accuracy is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the first spatial accuracy.

43. The aforementioned x-y stage 2010 is The sequencing system according to any one of the prior claims, further comprising a fastener configured to detachably secure the flow cell device thereto.

44. The arrangement determination system according to claim 43, wherein the fastener comprises one or more clamps.

45. The arrangement determination system according to any one of the prior claims, wherein each carrier 2051 includes a bonding position in which the carrier 2051 is detachably attached to the x-y stage 2010.

46. The sequencing system according to any one of the prior claims, wherein each carrier 2051 includes a detachment position from which the carrier 2051 is removable from the x-y stage 2010.

47. The array determination system according to any one of the prior claims, wherein the x-y stage 2010 comprises one or more pumps configured to extract fluid from the flow cell device when the corresponding carrier 2051 is coupled to the x-y stage 2010.

48. The sequence determination system according to any one of the prior claims, wherein the x-y stage 2010 includes a heating device, a cooling device, or both.

49. The array determination system according to any one of the prior claims, wherein the x-y stage 2010 is coupled to a mechanical decoupler configured to isolate the x-y stage from external vibrations or mechanical disturbances.

50. The sequence determination system according to any one of the prior claims, wherein the nest bank 2050 comprises one or more fasteners, each configured to fasten a corresponding carrier 2051 to the nest bank 2050.

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

52. The arrangement determination system according to claim 51, wherein one or more clamps are driven by magnetic force, electromagnetic force, or pressure.

53. The array determination system according to any one of the prior claims, wherein the nest bank 2050 comprises one or more pumps configured to extract fluid from the flow cell device when the corresponding carrier 2051 is coupled to the nest bank 2050.

54. The sequencing system according to any one of the prior claims, wherein each carrier 2051 includes a detachment position from which the carrier 2051 can be removed from the nest bank 2050.

55. The arrangement determination system according to any one of the prior claims, wherein each carrier 2051 includes a coupling position in which the flow cell device carrier is detachably attached to the nest bank 2050 and is in sealed fluid communication with the nest bank 2050.

56. The array determination system according to any one of the prior claims, wherein the nest bank 2050 comprises a 3D moving device configured to position the carrier 2051 relative to the nest bank with a third spatial accuracy.

57. The sequence determination system according to claim 56, wherein the third spatial position is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the first spatial accuracy.

58. The carrier 2051 said, The sequencing system according to any one of the prior claims, comprising an opening on the surface of a carrier 2051 configured therein to receive a flow cell device.

59. The carrier 2051 said, The arrangement determination system according to any one of the prior claims, comprising one or more fluid paths in sealed fluid communication with the flow cell device when the flow cell device is detachably attached to the carrier 2051.

60. The carrier 2051 said, The arrangement determination system according to any one of the prior claims, comprising a pump configured to draw in or push out fluid between the flow cell device and the carrier 2051.

61. The carrier 2051 said, The array determination system according to any one of the prior claims, comprising a valve positioned between a fluid path connected to the flow cell device and a port opening of the carrier 2051, the valve being in an open position when the flow cell device is in the coupling position with respect to the carrier 2051, and in a closed position when the flow cell device is in the detachment position.

62. The carrier 2051 said, The arrangement determination system according to any one of the prior claims, comprising a port opening having a connector, configured to allow sealed fluid communication between the carrier 2051 and the corresponding nest module when the connector is in a connection position.

63. The carrier 2051 said, The array determination system according to any one of the prior claims, comprising electrical wiring having an electrical connector configured to enable electrical communication between the carrier 2051 and the power supply.

64. The array determination system according to any one of the prior claims, wherein the carrier 2051 comprises a battery, a sensor, or both, and the battery or sensor is connected to the electrical connector via the electrical wiring.

65. The sequencing system according to any one of the prior claims, wherein the nest bank 2050 includes one or more reagent containers.

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

67. The sequencing system according to claim 65 or 66, wherein the transfer 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 prior claims, wherein the nest bank 2050 further comprises a cooler, a heater, or both.

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

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

71. The array determination system according to any one of the prior claims, further comprising a beam dump configured to absorb at least a portion of the excitation light generated by the optical system.

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

73. The array determination system according to claim 71 or 72, wherein the beam dump is separated from the flow cell device by a gap zone.

74. The array determination system according to any one of claims 71 to 73, wherein the beam dump contacts the flow cell device with a predetermined locking force.

75. The array determination system according to any one of claims 71 to 74, wherein the beam dump contacts the x-y stage with a predetermined damping force.

76. The array determination system according to claim 75, wherein the predetermined damping force is configured to reduce the predetermined locking force so that it can be customized so that the net force acting on the flow cell device is within a predetermined range.

77. A method for determining sequences, (a) Moving the first flow cell device from the nest bank 2050 to the x-y stage 2010, wherein the first flow cell device includes the first sample immobilized thereon. (b) Moving the x-y stage 2010 and the first sample on it with respect to the objective lens of the optical system of the sequencing determination system, (c) Using the optical system 2020, the first sample immobilized on the first flow cell device on the x-y stage is imaged, (d) Moving the first flow device from the x-y stage 2010 to the nest bank 2050, (e) To enable fluid and thermal communication between the nest bank 2050 and the second flow cell device simultaneously during one or more of (a) to (d), (f) Moving the second flow cell device from the nest bank 2050 to the x-y stage 2010, wherein the second flow cell device includes a second sample immobilized thereon. (g) Moving the x-y stage 2010 and the second sample on it with respect to the objective lens of the optical system 2040 of the sequencing determination system, (h) Using the optical system 2040, image the second sample immobilized on the second flow cell device on the x-y stage 2010, (i) Moving the first flow device from the x-y stage 2010 to the nest bank 2050, A method for determining an array, comprising enabling fluid communication and thermal communication between the nest bank 2050 and the first flow cell device simultaneously during one or more of (j), (f) to (i).

78. A method for determining sequences, (a) Moving the first flow cell device from the nest bank 2050 to the x-y stage 2010, wherein the first flow cell device includes the first sample immobilized thereon. (b) Moving the x-y stage 2010 and the first sample on it with respect to the objective lens of the optical system 2020 of the sequencing system, (c) Using the optical system 2020, image the first sample immobilized on the first flow cell device on the x-y stage 2010, (d) Moving the first flow device from the x-y stage 2010 to the nest bank 2050, (e) To enable fluid and thermal communication between the nest bank 2050 and the second flow cell device simultaneously during one or more of (a) to (d), (f) A sequencing method comprising moving the second flow cell device from the nest bank 2050 to the x-y stage 2010, wherein the second flow cell device contains a second sample immobilized thereon.

79. The aforementioned sequence determination method, The method according to claim 77 or 78, further comprising repeating operations (a) to (e).

80. The aforementioned sequence determination method, The method according to claim 78 or 79, further comprising repeating operations (f) to (j).

81. The aforementioned sequence determination method, The method according to any one of claims 78 to 80, further comprising repeating operations (a) to (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 times.

83. To enable fluid communication between the nest bank 2050 and the first flow cell device, The flow cell device is reversibly fastened to the carrier 2051 via one or more fasteners, thereby enabling sealed fluid communication between the flow cell device and the carrier 2051. The method according to any one of claims 77 to 82, comprising reversibly fastening the carrier 2051 to the nest bank 2050 via one or more fasteners to enable sealed fluid communication between the nest bank 2050 and the carrier 2051 and to enable physical contact with a heat dissipation element.

84. The method according to any one of claims 77 to 83, wherein (a) the movement of the first flow cell device from the nest bank 2050 to the x-y stage 2010 is within a first flow cycle of sequencing execution, and (f) the movement of the first flow cell device from the nest bank 2050 to the x-y stage 2010 is within a second flow cycle of sequencing execution, which is different from the first flow cycle.

85. The method according to any one of claims 77 to 84, wherein each of the operations (a) to (b) and (d) to (g) is completed within 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 the operations (a) to (b) and (d) to (g) is completed within 0.5 seconds, 1 second, 2 seconds, or less than 3 seconds.

87. (e) During one or more of (a) to (d), it is possible to simultaneously enable fluid communication and thermal communication between the nest bank 2050 and the first flow cell device. The method according to any one of claims 77 to 86, comprising switching one or more fasteners to the ON state to enable physical contact for sealed fluid and thermal communication.

88. (e) During one or more of (a) to (d), it is possible to simultaneously enable fluid communication and thermal communication between the nest bank and the first flow cell device. The method according to any one of claims 77 to 87, comprising immersing the flow cell device in at least some of the one or more reagent containers in a predetermined order.

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