Methods for cellularly addressable nucleic acid sequencing

Abstract

To provide systems for analyzing nucleic acids in a biological sample in a manner that retains the spatial and / or cellular origin of the nucleic acids within the biological sample.SOLUTION: Provided is a computer system for identifying a target nucleic acid sequence in situ, which causes one or more processors to execute: (a) contacting a cell or tissue obtained from a human subject containing a target nucleic acid sequence with a detectable nucleotide conjugate, the target nucleic acid sequence being primed; (b) detecting a binding complex; and (c) performing the steps (a) and (b) for at least two other nucleotides of the target nucleic acid sequence, thereby identifying the target nucleic acid sequence in situ.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 904,623, filed September 23, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Emerging diagnostic methods for diseases and disorders such as cancer, infectious diseases, and dysbiosis rely on next-generation sequencing (NGS) methods, which provide high-resolution genetic and genomic data to enable robust, personalized diagnoses, treatment plans, and ultimately cures for previously intractable diseases. While powerful, NGS methods remain limited by the methods available for providing nucleic acid samples to the instruments that perform the actual sequencing. For example, identifying the precise nature of mutations present in a particular tumor requires multiple steps in tumor tissue isolation, nucleic acid isolation, and sample preparation for specific sequencing methods before engaging the instrument to obtain the actual sequence data. Furthermore, deconvoluting and processing sequence data in a way that allows correlation of specific sequences with specific cells or tissues is complicated by the nature of NGS techniques, which often require sample pooling, during which spatial and cellular identity information is lost.

[0003] To provide molecular diagnostics with higher spatial or tissue resolution, various methods have been proposed to address the problem of the loss of cell addressability in NGS.For example, some methods rely on cell separation using unique barcodes to identify the sequences associated with individual cells after sequencing is completed, followed by applying unique barcodes to the nucleic acids of individual cells, and then bulk sequencing.This can be achieved, for example, by exposing individual cells to a lysis and hybridization mixture, such as beads or emulsion, in an isolated environment.These methods may further require enrichment or processing of target cell subpopulations, such as by cell sorting for circulating cells, or by tissue collection for solid tumor cells, followed by dissociation and protease treatment.

[0004] Although such methods can obtain cell-addressable information, they face significant limitations, such as the difficulty of processing solid tissues, and throughput is limited by the ability to isolate, tag, and prepare nucleic acids for sequencing.Similarly, limitations exist related to the need to transfer prepared libraries to a separate instrument, system, or location to perform the sequencing process.This practically limits the sequencing throughput to approximately 50,000 cells per sequencing run, severely limiting the sensitivity and usefulness of these assays when diagnostically relevant tissue samples, secretions, excretions, or exudates, or microbiome samples, contain vast numbers of cells.A certain level of addressability can be achieved by simply physically isolating the sample and performing isolation, library preparation, and sequencing reactions on known sequences.However, this process is labor-intensive and time-consuming, and is not practical as a means of screening a large number of patients or deploying a systematic screening method.

[0005] Therefore, there is a need for cell-addressable sequencing methods, as well as compositions and methods that can increase the accuracy and throughput of cell- or spatially-addressable sequencing methods that avoid the aforementioned limitations of existing techniques. Summary of the Invention

[0006] Aspects disclosed herein provide methods for analyzing a biological sample, the methods comprising: (a) detecting a multivalent binding complex formed between a target nucleic acid sequence of a target nucleic acid molecule or a derivative thereof and a detectable polymer-nucleotide conjugate in the presence of the biological sample or a derivative thereof; and (b) determining the origin of the target nucleic acid sequence in the biological sample or a derivative thereof. In some embodiments, the determining step (b) is performed at least in part by analyzing the relative three-dimensional relationship between the target nucleic acid sequence and a reference point in the biological sample or a derivative thereof. In some embodiments, the method further comprises contacting the biological sample or a derivative thereof with the detectable polymer-nucleotide conjugate in the presence of the biological sample. In some embodiments, the method further comprises binding at least a portion of the target nucleic acid sequence to a capture oligonucleotide molecule bound to a surface of a substrate. In some embodiments, the surface has a water contact angle of 45 degrees or less. In some embodiments, the binding step comprises hybridizing in the presence of a hybridization buffer, the hybridization buffer comprising (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9, and (ii) a second polar aprotic solvent having a dielectric constant of 115 or less. In some embodiments, the method further comprises immobilizing the biological sample or a derivative thereof on a surface in a manner sufficient to fix a relative three-dimensional relationship. In some embodiments, the method further comprises amplifying target nucleic acid sequences on the surface of the substrate using rolling circle amplification. In some embodiments, an image of the surface in the presence of the biological sample or a derivative thereof exhibits a contrast-to-noise ratio of about 5 or greater, as measured by (a) contacting the surface with fluorescently labeled nucleotide molecules comprising nucleic acid sequences complementary to at least a portion of capture oligonucleotides immobilized on the surface, and (b) subsequent to (a), imaging the surface using an inverted microscope and camera under non-signal-saturating conditions while the surface is immersed in buffer.In some embodiments, the method further comprises performing a nucleotide coupling reaction between the nucleotide moiety attached to the polymer-nucleotide conjugate and a target nucleic acid molecule or a derivative thereof. In some embodiments, the target nucleic acid molecule or a derivative thereof is a deoxyribonucleic acid (DNA) molecule. In some embodiments, the biological sample or a derivative thereof comprises a fluid biological sample. In some embodiments, the source is cancer tissue.

[0007] Aspects disclosed herein provide methods for in situ identification of at least a portion of an intracellular component within a cell or tissue, the method comprising: (a) detecting a signal from a multivalent binding complex between the intracellular component or a derivative thereof and a detectable polymer-nucleotide conjugate; and (b) processing at least the signal detected in (a) to identify at least a portion of the intracellular component or a derivative thereof. In some embodiments, the intracellular component or a derivative thereof is a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the method further comprises (c) immobilizing the cell or tissue on a substrate surface. In some embodiments, the method further comprises (d) binding at least a portion of the intracellular component to a capture molecule bound to the surface. In some embodiments, the method further comprises (e) permeabilizing the tissue or lysing the cells prior to detection in (a). In some embodiments, the surface has a water contact angle of 45 degrees or less. In some embodiments, the binding step in (d) comprises hybridizing the capture molecule to at least a portion of the intracellular component in the presence of a hybridization buffer, the hybridization buffer comprising (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9, and (ii) a second polar aprotic solvent having a dielectric constant of 115 or less. In some embodiments, the image of the surface exhibits a contrast-to-noise ratio of about 5 or greater when measured by (a) contacting the surface with fluorescently labeled nucleotide molecules comprising a nucleic acid sequence complementary to at least a portion of the capture oligonucleotides immobilized on the surface, and (b) imaging the surface using an inverted microscope and camera under non-signal-saturating conditions while the surface is immersed in buffer following (a). In some embodiments, the detecting step of the signal from the multivalent binding complex in (a) comprises performing a nucleotide binding reaction between a nucleotide moiety bound to the polymer-nucleotide conjugate and the intracellular component or a derivative thereof. In some embodiments, the tissue is derived from a tumor.

[0008] A system for analyzing a biological sample, the biological sample comprising a substrate including a surface to which a polymer layer suitable for immobilizing the biological sample is attached, the biological sample or derivative thereof comprising a target nucleic acid molecule or derivative thereof, the polymer layer configured to bind to (i) the biological sample or derivative thereof or (ii) the target nucleic acid molecule or derivative thereof, the target nucleic acid molecule or derivative thereof configured to bind to a nucleotide moiety comprising a detectable label, and an image of the surface exhibits a contrast-to-noise ratio of about 5 or greater when acquired using an inverted microscope and camera under non-signal-saturating conditions while the surface is immersed in a buffer solution, the detectable label being a fluorescent dye. In some embodiments, the polymer layer is hydrophobic. In some embodiments, the system further comprises a fixative that fixes the biological sample to the surface when contacted while the biological sample is adjacent to the surface. In some embodiments, the fixative comprises formaldehyde or glutaraldehyde. In some embodiments, the target nucleic acid molecule is a concatemer. In some embodiments, the target nucleic acid molecule comprises a universal sequence region comprising a spatial barcode sequence or a sample barcode sequence configured to preserve the origin of the target nucleic acid molecule in the biological sample. In some embodiments, the image of the surface, when acquired, exhibits a contrast-to-noise ratio of about 10 or greater. In some embodiments, the substrate is a flow cell device comprising a first flow channel and, optionally, a second flow channel. In some embodiments, the substrate is a reflective, transparent, or translucent planar substrate. In some embodiments, the flow cell device is a capillary flow cell device.

[0009] Aspects disclosed herein include a system for analyzing nucleic acid sequence information in a biological sample or a derivative thereof, the system comprising one or more computer processors programmed to: (a) detect a signal from a multivalent binding complex formed between a target nucleic acid sequence of a target nucleic acid molecule or a derivative thereof and a detectable polymer-nucleotide conjugate in the presence of the biological sample or derivative thereof, the signal indicating the identity of a nucleotide in the target nucleic acid sequence; and (b) determine the origin of the target nucleic acid sequence in the biological sample. In some embodiments, the one or more computer processors are programmed to determine the origin of the target nucleic acid sequence in (b) by analyzing the relative three-dimensional relationship between the target nucleic acid molecule or derivative thereof and the biological sample or derivative thereof. In some embodiments, the system further comprises a database configured to store three-dimensional data related to the origin of the target nucleic acid sequence. In some embodiments, the database is further configured to store sequencing data including the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, (b) is performed by correlating the sequencing data and the three-dimensional data. In some embodiments, the one or more computer processors are programmed to identify the target nucleic acid sequence in less than 60 minutes by repeating (a)-(b). In some embodiments, the one or more computer processors are programmed to perform (a)-(b) with a base calling accuracy characterized by a Q-score of greater than 25 for at least 80% of the identified nucleotides. In some embodiments, the detectable polymer-nucleotide conjugate comprises (a) a polymer core and (b) two or more nucleotide moieties attached to the polymer core, wherein the polymer-nucleotide conjugate is configured to form a multivalent binding complex between the two or more nucleotide moieties and the target nucleic acid molecule or derivative thereof.In some embodiments, the one or more nucleotide moieties comprise a nucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog. In some embodiments, the polymer core comprises a polymer having a star, comb, cross, bottlebrush, or dendrimer configuration. In some embodiments, the polymer core comprises branched polyethylene glycol (PEG) molecules. In some embodiments, the system is 1.0 mm. 2 It further includes an optical imaging system that includes a field of view (FOV).

[0010] Aspects disclosed herein provide kits that include (a) a detectable polymer-nucleotide conjugate comprising (i) a polymer core and (ii) two or more nucleotide moieties attached to the polymer core, and (b) instructions for identifying at least a portion of an intracellular component in situ within a cell or tissue by contacting the detectable polymer-nucleotide conjugate with the intracellular component under conditions sufficient to form a multivalent binding complex between the two or more nucleotide moieties and the intracellular component. In some embodiments, the kit includes four of the detectable polymer-nucleotide conjugates, each having a different nucleotide moiety attached.

[0011] Aspects disclosed herein include kits that include (a) a substrate comprising a surface having attached thereto a polymer layer suitable for immobilizing a biological sample or a derivative thereof, and (b) instructions for determining a target nucleic acid sequence and its origin in the biological sample or derivative on the surface. In some embodiments, the kit further includes (a) a hybridization buffer comprising (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9, and (ii) a second polar aprotic solvent having a dielectric constant of 115 or less, and (b) instructions for hybridizing at least a portion of the target nucleic acid sequence to at least a portion of a capture oligonucleotide bound to the surface.

[0012] Citation by reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0013] The novel features of the invention are set forth with particularity in the appended claims. For a better understanding of the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings of which: [Figure 1] 1 is a schematic diagram of one embodiment of a low-binding support comprising a glass substrate and alternating layers of hydrophilic coatings covalently or non-covalently attached to the glass, where the support further comprises chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides and circularization oligonucleotides), in accordance with embodiments of the present disclosure. In alternative embodiments, the support may be made of any substance, such as glass, plastic, or polymeric material. [Figure 2] 1 is a schematic diagram showing a support having immobilized thereon a capture oligonucleotide and a circularization oligonucleotide, in some embodiments, the support has immobilized thereon a plurality of capture oligonucleotides and a plurality of circularization oligonucleotides, according to embodiments of the present disclosure. [Figure 3] 3 is a schematic diagram showing a support having a plurality of capture and circularization oligonucleotides immobilized thereon, and a biological sample (e.g., a tissue sample) disposed on the support (see left side), according to an embodiment of the present disclosure. Figure 3 shows a magnified portion of the support having a series of features (see right side), each of which is circular and labeled for spatial differentiation on the support. Each feature contains a plurality of immobilized capture and circularization oligonucleotides. [Figure 4]1 is a schematic diagram showing a support having immobilized thereon a capture oligonucleotide and a soluble circularized oligonucleotide, in some embodiments, the support has multiple capture oligonucleotides immobilized thereon. [Figure 5A] FIG. 1 is a schematic diagram showing a nucleotide arm of a polymer-nucleotide conjugate according to an embodiment of the present disclosure. [Figure 5B] 1 is a schematic diagram of a polymer-nucleotide conjugate in which a core is attached to multiple nucleotide arms, each nucleotide arm comprising (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, according to an embodiment of the present disclosure. [Figure 5C] 1 is a schematic diagram of a polymer-nucleotide conjugate in the form of a dendrimer comprising branched polymers radiating from a central attachment point or moiety, in which multiple nucleotide arms radiate from the central attachment point, according to an embodiment of the present disclosure. [Figure 5D] FIG. 1 is a diagram of a nucleotide arm of a polymer-nucleotide conjugate comprising a biotin core attachment moiety, a spacer, an aliphatic chain linker, and a nucleotide attached to the linker via a propargyl linkage at the base, according to an embodiment of the present disclosure. [Figure 6A] FIG. 1 shows the structure of the spacer and linker of a polymer-nucleotide conjugate according to an embodiment of the present disclosure. [Figure 6B] FIG. 1 shows the structure of an additional linker of a polymer-nucleotide conjugate according to an embodiment of the present disclosure. [Figure 7] FIG. 1 illustrates a workflow according to an embodiment of the present disclosure. [Figure 8] 8A and 8B are schematic diagrams illustrating non-limiting examples of dual-surface support structures for presenting sample sites for imaging by the imaging system disclosed herein. Figure 8A illustrates imaging of the inner surface before and after the flow cell. Figure 8B illustrates imaging of the outer surface before and after the substrate. [Figure 9]9A and 9B illustrate a non-limiting example of a multi-channel fluorescence imaging module including a dichroic beam splitter for transmitting an excitation beam to a sample and receiving and redirecting the resulting fluorescence emission by reflection to four detection channels configured for detection of fluorescence emission at four different wavelengths or wavelength bands. (A) and (B) are top and bottom isometric views, respectively. [Figure 10] 10A and 10B, which illustrate optical paths within the multi-channel fluorescence imaging module of FIGS. 10A and 10B, including a dichroic beam splitter for transmitting excitation light to a sample and receiving and redirecting the resulting fluorescence emission by reflection to four detection channels configured for detection of fluorescence emission at four different wavelengths or wavelength bands. FIG. 10A is a plan view, and FIG. 10B is a side view. [Figure 11] 11A and 11B illustrate the modulation transfer function (MTF) of an example dual-surface imaging system disclosed herein with a numerical aperture (NA) of 0.3. FIG. 11A is the first surface, and FIG. 11B is the second surface. [Figure 12] 12A and 12B illustrate the MTF of an example dual surface imaging system disclosed herein with an NA of 0.5: Fig. 12A is the first surface; Fig. 12B is the second surface. [Figure 13] 13A and 13B illustrate the MTF of an example dual surface imaging system disclosed herein with an NA of 0.7: Fig. 13A is the first surface; Fig. 13B is the second surface. [Figure 14A] 14A provides plots of the Strehl ratio calculated for imaging a second flow cell surface through a first flow cell surface. Figure 14A is a plot of the Strehl ratio for imaging a second flow cell surface through a first flow cell surface as a function of the thickness of the intervening fluid layer (fluid channel height) for different objective lenses and / or optical system numerical apertures. [Figure 14B]14A and 14B provide plots of the Strehl ratio calculated for imaging a second flow cell surface through a first flow cell surface. Figure 14B is a plot of the Strehl ratio as a function of numerical aperture for imaging the first flow cell surface and the second flow cell surface through an intervening layer of water 0.1 mm thick. [Figure 15] A schematic of the optical ray tracing of an objective lens design designed to image the opposite surface of a 0.17 mm thick cover slip is provided. [Figure 16] FIG. 16 provides a plot of the modulation transfer function for the objective illustrated in FIG. 15 as a function of spatial frequency when used to image the opposite surface of a 0.17 mm thick cover slip. [Figure 17] FIG. 20 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image the opposite surface of a 0.3 mm thick cover slip. [Figure 18] FIG. 16 provides a plot of the modulation transfer function for the objective illustrated in FIG. 15 as a function of spatial frequency when used to image a surface separated from the opposite surface of a 0.3 mm thick cover slip by a 0.1 mm thick layer of aqueous fluid. [Figure 19] FIG. 16 provides a plot of the modulation transfer function for the objective illustrated in FIG. 15 as a function of spatial frequency when used to image the opposite surface of a 1.0 mm thick cover slip. [Figure 20] FIG. 16 provides a plot of the modulation transfer function for the objective illustrated in FIG. 15 as a function of spatial frequency when used to image a surface separated from the opposite surface of a 1.0 mm thick cover slip by a 0.1 mm thick layer of aqueous fluid. [Figure 21] FIG. 16 provides a ray trace of a tube lens design that, when used in conjunction with the objective illustrated in FIG. 15, improves double-sided imaging through a 1 mm thick cover glass. [Figure 22]FIG. 16 provides a plot of the modulation transfer function for the objective and tube lens combination illustrated in FIG. 15 as a function of spatial frequency when used to image the opposite surface of a 1.0 mm thick cover slip. [Figure 23] FIG. 16 provides a plot of the modulation transfer function of the objective and tube lens combination illustrated in FIG. 15 as a function of spatial frequency when used to image a surface separated from the opposite surface of a 1.0 mm thick cover slip by a 0.1 mm thick layer of aqueous fluid. [Figure 24] FIG. 1 illustrates a non-limiting example of a single capillary flow cell with two fluidic adapters. [Figure 25] FIG. 1 illustrates a non-limiting example of a flow cell cartridge designed to hold two capillaries, including a chassis, fluidic adapter, and optionally other components. [Figure 26] FIG. 1 illustrates a non-limiting example of a system comprising a single capillary flow cell connected to various fluid flow regulation components, where the single capillary is adaptable for attachment to a microscope stage or to custom imaging equipment for use in various imaging applications. [Figure 27] FIG. 1 is a schematic diagram illustrating a support having immobilized thereon capture oligonucleotides and circularization oligonucleotides, and an exemplary method for capturing nucleic acids from a cellular biological sample located on the support, according to various embodiments described herein. [Figure 28] 1 is a schematic diagram illustrating a support having immobilized capture oligonucleotides thereon and an exemplary method for capturing nucleic acids from a cellular biological sample located on the support, according to various embodiments described herein, the method including the use of soluble circularized oligonucleotides. DETAILED DESCRIPTION OF THE INVENTION

[0014] Provided herein are spatially and cellularly addressable sequencing methods and compositions, as well as compositions, devices, and kits useful for carrying out the methods and systems described herein. The methods and systems described herein can utilize polymer-nucleotide conjugates in situ in nucleotide conjugation reactions. The nucleotide conjugation reactions can be carried out on hydrophilic surfaces, which provide many of the advantages described herein. Also provided herein are hybridization buffers comprising polar and aprotic solvents in combination with pH buffers. Additionally, optical systems useful for spatially resolving sequencing data are provided. In some embodiments, the optical systems described herein can be configured with a 1.0 mm 2 It has a vision that goes beyond that.

[0015] As shown in FIG. 7 , in some embodiments, the methods described herein include (a) providing a surface (e.g., a low non-specific binding surface) to which a plurality of capture oligonucleotides are attached (701); immobilizing a biological sample containing a target nucleic acid molecule on the surface and optionally permeabilizing the biological sample (702); (c) contacting the plurality of capture oligonucleotides with the target nucleic acid molecule under conditions sufficient to allow hybridization of at least a portion of the plurality of capture oligonucleotides to the target nucleic acid molecule (703); (d) amplifying the target nucleic acid molecule to produce an amplified target nucleic acid molecule or derivative thereof (704); and (e) contacting the amplified target nucleic acid molecule or derivative thereof with one or more polymerases and one or more primer nucleic acid molecules having primer sequences complementary to one or more regions of the amplified target nucleic acid molecule or derivative thereof to produce a primed target nucleic acid molecule or derivative thereof (705). (f) contacting the primed target nucleic acid molecule or derivative thereof with a polymer-nucleotide conjugate (706) comprising two or more nucleotide moieties attached to a polymer (e.g., PEG) core labeled with a detectable label (e.g., a fluorophore); (g) detecting a multivalent binding complex formed between the primed target nucleic acid molecule or derivative thereof and the polymer-nucleotide conjugate (707); (h) washing the surface with a buffer sufficient to remove the polymer-nucleotide conjugate from the primed target nucleic acid molecule or derivative thereof (708); (i) incorporating a nucleotide that does not include a detectable label but optionally includes a protecting group (e.g., azidomethyl) that prevents incorporation of a second nucleotide at the N+1 position on the primed target nucleic acid molecule or derivative thereof (709); and (j) optionally repeating steps (f)-(j) (710).

[0016] Existing methods for spatially addressable sequence identification (also referred to herein as spatial transcriptomics technology) suffer from low sensitivity, non-specificity, and inaccurate spatial location of the transcripts of interest. In contrast, the methods, systems, compositions, and kits described herein overcome these challenges by leveraging, for example, low non-specific binding surfaces, highly efficient hybridization buffers, high copy number nanoball preparation methods, and multivalent molecules.

[0017] The low nonspecific binding and improved signal of the present disclosure significantly improve the contrast-to-noise ratio (CNR) compared to existing methods. The CNR is improved, at least in part, by utilizing highly compact reaction foci (e.g., highly compact nucleic acid clusters with high copy numbers), highly efficient surface hybridization (allowing for precise localization of nucleic acid capture), and very low background, while enabling highly efficient capture, amplification, and clustering of target nucleic acids. Once a biological sample (e.g., tissue, cell suspension) is bound to the substrate, sequencing reactions can be performed in the presence of the biological sample. Analysis of the sequencing reactions can be performed in a manner that provides cellular and / or spatial addressability, allowing sequence data to be associated with tissue, cell type, physiological location, or the spatial location from which it originated.

[0018] The high-efficiency hybridization buffer described herein promotes high stringency (e.g., specificity), speed, and efficacy of nucleic acid hybridization reactions, increasing the efficiency of subsequent amplification and sequencing steps. The high-efficiency hybridization buffer can significantly shorten nucleic acid hybridization time and reduce sample input requirements. The high-efficiency hybridization buffer can be used in nucleic acid annealing workflows under isothermal conditions, eliminating the need for a cooling step for annealing. The high-efficiency hybridization buffer provides precise localization of nucleic acid capture on surfaces for precise spatial localization of nucleic acids (e.g., transcripts) derived from cells or tissues.

[0019] The rolling circle amplification method described herein involves a two-step method that utilizes non-catalytic divalent cations followed by catalytic divalent cations to synchronize rolling circle amplification events on a surface. The rolling circle amplification reaction may be subjected to relaxed conditions and a flexing amplification reaction that generates new concatemers from existing concatemers. Together, these amplification methods generate highly compact nanoballs that encompass high copy numbers of target sequences, improving sequencing signal strength.

[0020] The nucleic acid analysis methods described herein have a higher throughput than existing methods, allowing for analysis of 50,000, 100,000, 150,000, 250,000, 500,000, 750,000, or 1,000,000 or more cells per run, which in principle allows for detection of mutations in as few as one cell per million, thereby achieving a significant degree of diagnostic sensitivity.A further advantage of the nucleic acid methods disclosed herein is that the required reactions can be carried out at a single temperature (e.g., isothermal conditions), such as 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 42°C, 50°C, 60°C, 65°C, 70°C, or 72°C or higher, or within a range defined by any two of the aforementioned temperatures.

[0021] Multivalent molecules used in sequencing reactions offer many advantages not offered by free nucleotides. Multivalent molecules contain a core attached to multiple arms, each tethered to a nucleotide. Multivalent molecules increase the local concentration of nucleotides near the polymerase / template binding site. Multivalent molecules also exhibit increased duration in the formation of a stable ternary complex with the polymerase and nucleic acid template. Thus, labeled multivalent molecules provide shorter imaging times and increased signal intensity during sequencing reactions.

[0022] Cellular and spatial resolution of sequencing data generated using the methods and systems described herein is achieved by the imaging methods and systems described herein, thereby increasing optical resolution and improving image quality in genomics applications.

[0023] Disclosed herein are optical component and system designs for high performance fluorescence imaging methods and systems that can provide any one or more of: a larger field of view; improved optical resolution (including high performance optical resolution); improved contrast; improved image quality; faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., of different fields of view); improved imaging system duty cycle; and higher throughput image acquisition and analysis.

[0024] In some instances, for example, in double-sided (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or cover glass) thickness greater than 700 µm) and fluidic channels (e.g., fluidic channel height or thickness of 50-200 µm), improved imaging performance can be achieved using novel objective lens designs that correct for optical aberrations introduced by imaging the opposite surface of the thick cover glass and / or fluidic channel from the objective.

[0025] In some instances, for example, in double-sided (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or cover slip) thickness greater than 700 μm) and fluidic channels (e.g., fluidic channel height or thickness of 50-200 μm), improved imaging performance can be achieved even when using commercially available, off-the-shelf objectives by using novel objective lens designs that correct for optical aberrations introduced by the thick flow cell walls and / or intervening fluid layers in combination with the object, as opposed to conventional microscope tube lenses that simply form an image at an intermediate image plane.

[0026] In some examples, improved imaging performance, for example in multi-channel (e.g., two-color or four-color) imaging applications, can be achieved by using multiple tube lenses, one for each imaging channel, where each tube lens design is optimized for the particular wavelength band used in the imaging channel.

[0027] In some examples, improved imaging performance, for example in double-sided (flow cell) imaging applications, can be achieved by using an electro-optic phase plate in combination with the objective lens to correct for optical aberrations introduced by the fluid layer separating the top (proximal) and bottom (distal) inner surfaces of the flow cell. In some examples, this design approach can further compensate for vibrations, for example, with a motion-activated compensator that can be moved in and out of the optical path depending on which surface of the flow cell is being imaged.

[0028] Further advantages of the disclosed imaging optical design may include the position and orientation of one or more excitation light sources and one or more detection optical paths relative to the objective lens and the dichroic filter receiving the excitation beam. The excitation beam may be linearly polarized, and the orientation of the linear polarization may be such that s-polarized light is projected onto the dichroic reflective surface of the dichroic filter. Such a feature can potentially improve filtering of the excitation beam and reduce wavefront errors introduced into the emission beam due to, for example, surface deformations of the dichroic filter.

[0029] Although this specification has been primarily discussed in terms of fluorescence imaging (e.g., fluorescence microscopy imaging, fluorescence focusing imaging, two-photon fluorescence, etc.), it will be understood by those skilled in the art that many of the disclosed optical design techniques and features are applicable to other imaging modes, such as bright-field imaging, dark-field imaging, phase-contrast imaging, etc.

[0030] In addition to the optical components and imaging system designs disclosed herein, flow cell devices and systems for performing various genomic analysis methods, including cell-addressable nucleic acid sequencing, are disclosed, which may include various combinations of the disclosed optical, mechanical, fluidic, thermal, electrical, and computing modules or subsystems. Advantages of the disclosed flow cell devices, cartridges, and analysis systems include, but are not limited to, (i) reduced manufacturing complexity and cost of the devices and systems, (ii) significantly reduced consumable costs (e.g., compared to currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components such as syringe pumps and diaphragm valves, and (v) flexible system throughput.

[0031] In some examples, the disclosed capillary flow cell devices and capillary flow cell cartridges can be constructed from commercially available, disposable, single-lumen (e.g., single fluid flow channel) or multi-lumen capillaries that may further include a fluidic adapter, a cartridge chassis, one or more integrated fluidic flow control components, or a combination thereof. In some examples, the disclosed flow cell-based systems may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluidic flow controller module, a temperature regulation module, an imaging module, or any combination thereof. Some of the disclosed design features of capillary flow cell devices, cartridges, and systems include, but are not limited to, (i) a single flow channel configuration; (ii) sealed, reliable, and repeatable switching between reagent streams that can implement a simple load / unload mechanism such that the fluid interface between the system and capillary is reliably sealed, facilitating capillary repositioning and system reuse, allowing precise control of reaction conditions such as reagent concentration, pH, and temperature; (iii) an interchangeable single fluid flow channel device or capillary flow cell cartridge with multiple flow channels that can be interchangeably used to provide flexible system throughput; and (iv) compatibility with a wide variety of detection methods, such as fluorescence imaging.

[0032] Although the disclosed capillary flow cells and microfluidic devices and systems are described primarily in terms of their use in nucleic acid sequencing applications, various aspects of the disclosed devices and systems can be applied not only to nucleic acid sequencing but also to any other type of chemical, biochemical, nucleic acid, cellular, or tissue analysis application. It should be understood that various aspects of the disclosed methods, devices, and systems can be evaluated individually, collectively, or in combination with one another.

[0033] The embodiments described herein provide significant advantages in the diagnosis of cancer, including circulating and solid tumors, the analysis of biopsy samples, e.g., the diagnosis of genetic disorders, the analysis of microbiome samples, e.g., the diagnosis of disorders associated with gut microbiota in the microbial flora, the diagnosis of disorders associated with secretion or exudation, or the assessment of health status or disease risk, which can be assessed with respect to the presence or identity of specific gene sequences in specific cells, tissues, or locations. For example, it may be useful to use high-resolution cell-addressable sequencing techniques to identify the presence of low levels of circulating tumor cells in the diagnosis of hematologic cancers or early metastasis.

[0034] In some embodiments, cells in a tissue or individual cells can be exposed to a surface under conditions optimized for binding (capture) of target nucleic acids, for example, by encapsulating high-density poly-T or poly-dT oligonucleotides for capture of RNA transcripts followed by reverse transcription, or by encapsulating random-sequence capture oligonucleotides for hybridization to genomic, circulating, or organellar DNA. In some embodiments, this capture process can be followed by one or more library preparation steps, such as adding at least one adapter to the captured nucleic acid, where the adapter can include an index sequence, a barcode sequence, and / or a unique molecular identifier (UMI). The adapter addition step can be performed by ligation (e.g., blunt-end ligation) or the use of a "sprint" oligonucleotide. These library preparation steps can result in or further include circularization of the captured nucleic acid. In some embodiments, the circularized nucleic acid molecules can be amplified, such as by rolling circle amplification (RCA), resulting in large, multicopy nucleic acid molecules (e.g., concatemers) containing multiple tandem repeats of the target sequence. In some embodiments, the large, multi-copy nucleic acids can be aggregated by buffer conditions that favor a compact DNA state, by surfaces with a high density of capture oligonucleotides, by the use of bivalent or bispecific oligonucleotides that bridge two or more sites within the large, multi-copy nucleic acid ("clustered oligonucleotides" or "clustered oligos"), or by any combination of the foregoing, or by any method known or that becomes known in the art for producing compact clusters comprising large, multi-copy nucleic acids.

[0035] In some embodiments, surfaces used to capture nucleic acids from cells or tissues can be configured to retain nucleic acids with high activity while simultaneously retaining unwanted proteins, lipids, carbohydrates, or other cellular debris components. Thus, surfaces contemplated herein are capable of binding nucleic acids from cells in tissues or single cells that are lysed in contact with or in proximity to the surface. Furthermore, the surfaces do not retain cellular debris and do not exhibit significant nonspecific binding of additional proteins, such as nucleic acid polymerases, or other molecules, moieties, particles, or items, such as dye molecules or fluorophores.

[0036] In some embodiments, cell lysis (and optionally nucleic acid fragmentation) occurs on or near a surface, such that a substantial amount, such as a representative mass, or substantially all of the DNA, RNA, or other target nucleic acid released from the cell or tissue sample is captured by the surface. The surface can be configured to allow cells to flow over the surface to reach capture sites thereon. Alternatively, the capture surface can be configured such that tissue (e.g., a tissue section) is placed on or in fluid communication with the surface, where a reagent can subsequently flow over the tissue in a manner that facilitates in situ capture of nucleic acids from the tissue, such that the nucleic acids are oriented or positioned within the intact tissue, such that nucleic acids from one cell or region of the tissue are captured in the same position and orientation relative to nucleic acids from other cells or regions of the tissue.

[0037] In some embodiments, nucleic acid capture, adaptoring, circularization, amplification, and clustering can be performed while the nucleic acid is attached to or in close proximity to a surface. Alternatively, one or more of the aforementioned preparation steps can be performed in free solution or while attached to beads.

[0038] Spatially resolved binding of cell-specific nucleic acid complements, such as cell genomes or cell transcriptomes, followed by adapter addition, circularization, amplification and clustering allows the use of sequencing techniques, such as the avidity-based sequencing methods described in US Patent Application No. 62 / 897,172 and US Patent Application No. 16 / 579,794 and those described elsewhere herein, and the foregoing documents are incorporated herein by reference in their entirety.The feasibility of cell or tissue addressable sequencing is further provided by advances in low-binding surfaces, such as those disclosed in US Patent Application No. 16 / 363,842, hybridization methods, such as those disclosed in US Patent Application No. 16 / 543,351, and library preparation methods, such as those disclosed in US Patent Application No. 62 / 767,943 and related published international application WO2020 / 102766, and the contents of the foregoing documents are expressly incorporated herein by reference for all purposes. Thus, in some embodiments, sequence data can be obtained that spatially maps to the cell or tissue from which the genomic or transcriptomic nucleic acid was obtained. In some embodiments, sequence data can be obtained in near one-to-one correspondence with the cellular location of the sample's origin. In some embodiments, sequence data can be obtained in other than one-to-one spatial correspondence with the cellular location within the original sample, but at near-coincident locations relative to other cells or sources of genetic, genomic, or transcriptomic samples within the tissue.

[0039] Solid Support Surface. Provided herein are solid supports comprising a surface (e.g., low non-specific binding). In some examples, the solid support comprises a non-hydrophilic surface. In some examples, the solid carrier comprises a hydrophilic surface. Generally, the disclosed supports can comprise a substrate (i.e., a support structure), one or more layers of covalently or non-covalently attached low-binding chemically modified layers, such as a silane layer or a polymer film, and one or more covalently or non-covalently attached primer sequences that can be used to tether single-stranded template oligonucleotides to the support surface (Figure 1). In some examples, the formulation of the surface, e.g., the chemical composition of one or more layers, the coupling chemistry used to crosslink one or more layers to the support surface and / or to each other, and all layers, can be varied to minimize or reduce non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction complements to the support surface relative to a comparable monolayer. In many cases, the formulation of the surface can be varied to minimize or reduce non-specific hybridization on the support surface relative to a comparable monolayer. The formulation of the surface may be varied to minimize or reduce non-specific amplification on the support surface relative to a comparable monolayer. The formulation of the surface may be varied to maximize the speed and / or yield of specific amplification on the support surface. In some cases disclosed herein, an amplification level suitable for detection is achieved within 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or less or more than 30 amplification cycles.

[0040] Examples of materials from which the substrate or support structure can be fabricated 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. A variety of compositions of glass and plastic substrates are contemplated.

[0041] The substrate or support structure may be provided in any of a variety of geometric shapes and dimensions known to those skilled in the art, or may comprise any of a variety of materials known to those skilled in the art. For example, in some instances, the substrate or support structure may be locally planar (e.g., a glass slide or glass slide surface). Overall, the substrate or support structure may be cylindrical (e.g., a capillary or capillary interior surface), spherical (e.g., the exterior surface of a non-porous bead), or irregular (e.g., the exterior surface of an irregularly shaped non-porous bead or particle). In some instances, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be a solid, non-porous surface. In some instances, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be porous, such that the coatings described herein penetrate the porous surface and nucleic acid hybridization and amplification reactions performed thereon may occur within the pores.

[0042] The substrate or support structure comprising one or more chemically modified layers, e.g., layers of low nonspecific binding polymers, may be freestanding or integrated into other structures or assemblies. For example, in some instances, the substrate or support structure may comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure may comprise one or more surfaces within a microplate format, e.g., the bottom surface of a well in a microplate. As noted above, in some preferred embodiments, the substrate or support structure comprises the interior surface (e.g., luminal surface) of a capillary. In alternative preferred embodiments, the substrate or support structure comprises the interior surface (e.g., luminal surface) of a capillary etched into a planar chip.

[0043] The chemically modified layer can be applied uniformly to the surface of the substrate or supporting structure. Alternatively, the surface of the substrate or supporting structure can be non-uniformly distributed or patterned so that the chemically modified layer is restricted to one or more individual regions of the substrate. For example, the substrate surface can be patterned using photolithography techniques to create an ordered arrangement or a random pattern of chemically modified regions on the surface. Alternatively or in combination, the substrate surface can be patterned using techniques such as contact printing and / or inkjet printing. In some examples, the ordered arrangement or random pattern of chemically modified distinct 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, or any intermediate number ranging therein.

[0044] To achieve a low non-specific binding surface (also referred to herein as a "low binding" or "passivated" surface), a hydrophilic polymer may be non-specifically adsorbed or covalently grafted to a substrate or support surface. Typically, passivation is performed using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilic polymers of different molecular weights with end groups that are coupled to the surface using, for example, silane chemistry, including, but not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide, and bis-silane as end groups distal from the surface. In some examples, two or more layers of hydrophilic polymers, such as linear, branched, or hyperbranched polymers, may be deposited on a surface. In some examples, two or more layers may be covalently bonded to each other or internally crosslinked to improve the stability of the resulting surface. In some examples, oligonucleotide primers with different base sequences and base modifications (or other biomolecules, such as enzymes or antibodies) may be tethered to the resulting surface at various surface densities. In some examples, for example, both the surface functional group density and the oligonucleotide concentration may be varied to target a specific primer density range. In addition, primer density can be adjusted by diluting the oligonucleotides with other molecules carrying the same functional groups. For example, amine-labeled oligonucleotides can be diluted with amine-labeled polyethylene glycol in reaction with an NHS ester-coated surface to reduce the final primer density.Primers with linkers of various lengths between the hybridization region and the surface-attaching functional group can also be applied to adjust surface density. Examples of suitable linkers include poly-T and poly-A chains at the 5' end of the primer (e.g., 0-20 bases), PEG linkers (e.g., 3-20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be tethered to the surface, and the fluorescence readings can then be compared to those of a dye solution of known concentration.

[0045] In some embodiments, the hydrophilic polymer can be a cross-linked polymer. In some embodiments, a cross-linked polymer can include one type of polymer cross-linked with another type of polymer. Examples of cross-linked polymers include polyethylene oxide (PEO) or poly(ethylene glycol) cross-linked with another polymer selected from polyoxyethylene, poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilic polymers. In some embodiments, the cross-linked polymer can be poly(ethylene glycol) cross-linked with polyacrylamide.

[0046] As a result of the surface passivation techniques disclosed herein, proteins, nucleic acids, and other biomolecules do not "stick" to the substrate, i.e., exhibit low nonspecific binding (NSB). Examples using standard monolayer surface preparation with various glass preparation conditions are described below. To achieve extremely low NSB for proteins and nucleic acids, passivated hydrophilic surfaces require novel reaction conditions to improve primer deposition reaction efficiency, hybridization performance, and induce effective amplification. All of these processes require the attachment of oligonucleotides and subsequent protein binding and delivery to the low-binding surface. As described below, when combined with a new primer-surface conjugation formulation (Cy3 oligonucleotide graft titration) and the resulting extremely low nonspecific background (NSB functional testing performed using red and green fluorescent dyes), results are obtained that demonstrate the feasibility of the disclosed approach. In some surfaces disclosed herein, the ratio of specific binding (e.g., hybridization to a tethered primer or probe) to non-specific binding (e.g., Binter) of a fluorophore, such as Cy3, is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value within the ranges herein. In some surfaces disclosed herein, the ratio of specific fluorescent signal to non-specific fluorescent signal of a fluorophore, such as Cy3 (e.g., specific hybridization to a non-specifically bound and labeled oligonucleotide, or non-specific binding (Binter)) is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value within the ranges herein. inter ) or nonspecific amplification (B intra ) labeled oligonucleotides, or combinations thereof (B inter +B intra) in the non-specific amplification of the target gene is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value within the ranges herein.

[0047] Substrates containing multiple coatings of PEG and other hydrophilic polymers have been developed to scale primer surface density and add additional dimensionality to hydrophilic or amphoteric surfaces. Using hydrophilic and amphoteric surface layering techniques, including but not limited to the polymer / copolymer materials described below, the primer loading density on a surface can be significantly increased. Traditional PEG coating techniques use monolayer primer deposition, which has generally been reported in single-molecule applications but does not result in high copy numbers for nucleic acid amplification applications. As described herein, "layering" can be achieved using conventional crosslinking techniques with any compatible polymer or monomer subunit to sequentially build a surface containing two or more highly crosslinked layers. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some examples, the various layers may be attached to one another via any of a variety of conjugation reactions, including, but not limited to, biotin-streptavidin binding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-mamilide reactions, and ionic interactions between positively and negatively charged polymers. In some examples, primer-dense materials may be constructed in solution and then layered onto a surface in multiple steps.

[0048] The attachment chemistry used to graft the first chemically modified layer to the support surface will depend on both the material from which the support is fabricated and the chemical nature of the layer as a whole. In some instances, the first layer may be covalently attached to the support surface. In some instances, the first layer may be non-covalently attached, adsorbed to the surface via non-covalent interactions such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and molecular components of the first layer. In either case, the substrate surface may be treated prior to attachment or deposition of the first layer. The support surface can be cleaned or treated using any of a variety of surface treatment techniques known to those skilled in the art. For example, glass or silicon surfaces can be cleaned using an acid wash using piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) and / or an oxygen plasma treatment method.

[0049] Silane chemistry constitutes one non-limiting approach for covalently modifying silanol groups on glass or silicon surfaces and attaching more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to attach linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, C12, or C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to create any of the disclosed low-binding support surfaces include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), any of the various PEG silanes (e.g., 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silane (i.e., containing a free amino functional group), maleimide-PEG silane, biotin-PEG silane, and the like.

[0050] Any of a variety of molecules known to those of skill in the art, including, but not limited to, amino acids, peptides, nucleic acids, oligonucleotides, other monomers or polymers, or combinations thereof, can be used to create one or more chemically modified layers on a support surface, where the selection of components used can be varied to modify one or more properties of the support surface, such as the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the support surface, or the three-dimensionality (i.e., "thickness") of the support surface. Examples of preferred polymers that can be used to create one or more layers of low nonspecific binding material on any of the disclosed support surfaces include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine, or any combination thereof. Examples of conjugation chemistries that can be used to graft one or more layers of material (e.g., polymer layers) onto a support surface and / or cross-link layers to one another include, but are not limited to, biotin-streptavidin interactions (or variations thereof), his-tag-Ni / NTA conjugation chemistry, methoxy-ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS ester, maleimide, thiol, epoxy, azide, hydrazide, alkyne, isocyanate, and silane.

[0051] One or more layers of the multilayer surface may comprise a branched polymer or may be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl pyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropyl acrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucoside, and dextran.

[0052] In some examples, branched polymers used to fabricate one or more layers of any of the multilayer surfaces disclosed herein may contain at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches. The molecules often exhibit a "power of 2" number of branches, such as 2, 4, 8, 16, 32, 64, or 128 branches.

[0053] An exemplary PEG multilayer includes PEG(8,16,8) (8-arm, 16-arm, 8-arm) on PEGamine-APTES. Similar concentrations are observed for three-layer multi-arm PEG (8-arm, 16-arm, 8-arm) and (8-arm, 64-arm, 8-arm) on PEG-amine-APTES exposed to 8 μM primer, and three-layer multi-arm PEG (8-arm, 8-arm, 8-arm) using star-shaped PEG-amine to replace 16- and 64-arm PEG multilayers with comparable first, second, and third PEG layers is also contemplated.

[0054] The molecular weight of the linear, branched, or hyperbranched polymers used to fabricate one or more layers of any of the multilayer surfaces disclosed herein may be at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 7,500, at least 10,000, at least 12,500, at least 15,000, at least 17,500, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 daltons. In some examples, the molecular weight of the linear, branched, or hyperbranched polymers used to fabricate one or more layers of any of the multilayer surfaces disclosed herein may be up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 17,500, up to 15,000, up to 12,500, up to 10,000, up to 7,500, up to 5,000, up to 4,500, up to 4,000, up to 3,500, up to 3,000, up to 2,500, up to 2,000, up to 1,500, up to 1,000, or up to 500 daltons. Any of the lower and upper limits set forth in this paragraph can be combined to create ranges within the present disclosure, e.g., in some instances, the linear, branched, or hyperbranched polymers used to fabricate one or more layers of any of the multilayer surfaces disclosed herein can range from about 1,500 to about 20,000 daltons. One of skill in the art will recognize that the linear, branched, or hyperbranched polymers used to fabricate one or more layers of any of the multilayer surfaces disclosed herein can be any value within this range, e.g., about 1,260 daltons.

[0055] In some examples, for example, when at least one layer of a multilayer surface comprises a branched polymer, the number of covalent bonds between the branched polymer molecules of the layer being deposited and the molecules of the previous layer can range from about 1 covalent bond per molecule to about 32 covalent bonds per molecule. In some examples, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, or more than 32 covalent bonds per molecule. In some examples, the number of covalent bonds between branched polymer molecules in the new layer and molecules in the previous layer can be up to 32, up to 30, up to 28, up to 26, up to 24, up to 22, up to 20, up to 18, up to 16, up to 14, up to 12, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 per molecule. Any of the lower and upper limits set forth in this paragraph can be combined to create ranges within the present disclosure; for example, in some examples, the number of covalent bonds between branched polymer molecules in the new layer and molecules in the previous layer can range from about 4 to about 16. One of ordinary skill in the art will recognize that the number of covalent bonds between branched polymer molecules in the new layer and molecules in the previous layer can be any value within this range, such as an average number of about 11 in some examples, or about 4.6 in other examples.

[0056] Reactive functional groups remaining after bonding of a layer of material to a support surface can optionally be blocked by attaching small, inert molecules using high-yield coupling chemistries. For example, if amine coupling chemistry is used to attach a new layer of material to a previous layer, the remaining amine groups can be subsequently acetylated or rendered inactive by coupling with a small amino acid such as glycine.

[0057] The number of layers of low nonspecific binding material, e.g., hydrophilic polymeric material, deposited on the surface of the disclosed low-binding supports can range from 1 to about 10. In some examples, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some examples, the number of layers can be up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1. Any of the lower and upper limits described in this paragraph can be combined to form a range within the scope of the present disclosure. For example, in some examples, the number of layers can be from about 2 to about 4. In some examples, all of the layers can comprise the same material. In some examples, each layer can comprise a different material. In some examples, multiple layers can comprise multiple materials. In some examples, at least one layer can comprise a branched polymer. In some examples, all of the layers can comprise a branched polymer.

[0058] One or more layers of low nonspecific binding material may optionally be deposited and / or conjugated to the substrate surface using a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, or any combination thereof. In some examples, the solvent used to deposit and / or conjugate the layer may include alcohol (e.g., methanol, ethanol, propanol, etc.), other organic solvents (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, aqueous buffer solutions (e.g., phosphate buffer, phosphate buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combination thereof. In some examples, the organic components of the solvent mixture used may comprise, in total, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or any percentage ranging or adjacent thereto, with the balance being made up of water or an aqueous buffer. In some examples, the aqueous components of the solvent mixture used may comprise, in total, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or any percentage ranging or adjacent thereto, with the balance being made up of an organic solvent. The pH of the solvent mixture used may be less than 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or greater than 10, or any value ranging or adjacent to the ranges described herein.

[0059] In some examples, one or more layers of low nonspecific binding material may be deposited and / or conjugated to a substrate surface using a mixture of organic solvents, wherein at least one component has a dielectric constant of less than 40 and comprises at least 50% of the total volume of the mixture. In some examples, the dielectric constant of at least one component may be less than 10, less than 20, less than 30, or less than 40. In some examples, at least one component comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, at least 70%, or at least 80% of the total volume of the mixture.

[0060] As noted, the low nonspecific binding supports of the present disclosure exhibit reduced nonspecific binding of proteins, nucleic acids, and other components of hybridization and / or amplification formulations used in solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited by a given support surface can be assessed qualitatively or quantitatively. For example, in some instances, exposure of a surface to fluorescent dyes (e.g., Cy3, Cy5, etc.), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerase) under a set of standard conditions, followed by a specific rinse protocol and fluorescent imaging, can be used as a qualitative tool for comparing nonspecific binding on supports containing various surface formulations. In some instances, exposure of a surface to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerase) under a set of standard conditions, followed by a specific rinse protocol and fluorescent imaging, can be used as a quantitative tool for comparing nonspecific binding on supports containing various surface formulations. provided, however, that care is taken to ensure that fluorescence imaging is performed under conditions in which the fluorescence signal is linearly related (or predictably related) to the number of fluorophores on the support surface (e.g., under conditions in which signal saturation and / or self-quenching of the fluorophores are not an issue) and appropriate calibration standards are used. In some instances, other techniques known to those of skill in the art, such as radioisotope labeling and counting methods, may be used to quantitatively assess the extent to which various support surface formulations of the present disclosure exhibit nonspecific binding.

[0061] Some surfaces disclosed herein have a ratio of specific binding to non-specific binding of a fluorophore, such as Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more than 100, or any intermediate value within the ranges herein. Some surfaces disclosed herein have a ratio of specific fluorescence to non-specific fluorescence of a fluorophore, such as Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more than 100, or any intermediate value within the ranges herein.

[0062] As noted, in some examples, the degree of nonspecific binding exhibited by the disclosed low nonspecific binding supports can be assessed using standard protocols for contacting the surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotide, labeled oligonucleotide, etc., under a series of standardized incubation and washing conditions, followed by detection of the amount of label remaining on the surface and comparison of the resulting signal with appropriate calibration standards. In some examples, the label may comprise a fluorescent label. In some examples, the label may comprise a radioisotope. In some examples, the label may comprise other detectable labels known to those of skill in the art. In some examples, the degree of nonspecific binding exhibited by a given support surface formulation can thus be assessed in terms of the number of nonspecifically bound protein molecules (or other molecules) per unit area. In some examples, a low non-specific binding support of the present disclosure may exhibit non-specific protein binding (or non-specific binding of other specific molecules, e.g., Cy3 dye) of less than 0.001 molecules / μm, less than 0.01 molecules / μm, less than 0.1 molecules / μm, less than 0.25 molecules / μm, less than 0.5 molecules / μm, less than 1 molecule / μm, less than 10 molecules / μm, less than 100 molecules / μm, or less than 1000 molecules / μm. One of skill in the art will recognize that a given support surface of the present disclosure may exhibit non-specific binding within this range, e.g., less than 86 molecules / μm. For example, some modified surfaces disclosed herein exhibit nonspecific protein binding of less than 0.5 molecules / um2 after contact with a 1 uM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS) buffer for 15 minutes, followed by three rinses with deionized water. Some modified surfaces disclosed herein exhibit nonspecific binding of Cy3 dye molecules of less than 0.25 molecules / um2.In an independent nonspecific binding assay, 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-Rho11 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM 7-propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated on low-binding substrate for 15 minutes at 37°C in a 384-well plate format. Each well was rinsed two to three times with 50 μl of RNase / DNase-free deionized water and two to three times with 25 mM ACES buffer, pH 7.4. The 384-well plates were imaged on a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) instrument using a PMT gain setting of 800 and a resolution of 50–100 μm, as specified by the manufacturer, using a Cy3, AF555, or Cy5 filter set (depending on the dye test performed). For higher-resolution imaging, images were collected with an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) equipped with a total internal reflectance fluorescence (TIRF) objective (20x, 0.75 NA or 100x, 1.5 NA, Olympus), an sCMOS Andor camera (Zyla 4.2), and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors, e.g., 405, 488, 532, or 633 nm dichroic reflectors / beamsplitters, were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), and bandpass filters were selected as 532LP or 645LP to match the appropriate excitation wavelength. Some modified surfaces disclosed herein exhibit nonspecific binding of dye molecules of less than 0.25 molecules / μm.

[0063] In some examples, the ratio of specific binding to non-specific binding of a fluorophore, such as Cy3, on a surface disclosed herein is 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 more than 100, or any intermediate value within the ranges herein. In some examples, the ratio of specific fluorescent signal to non-specific fluorescent signal of a fluorophore, such as Cy3, on a surface disclosed herein is 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 more than 100, or any intermediate value within the ranges herein.

[0064] Low background surfaces consistent with the disclosure herein may exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) of at least 3:1, 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 specific dye molecules per nonspecifically adsorbed molecule. Similarly, upon exposure to excitation energy, low background surfaces consistent with the disclosure herein and having attached fluorophores, e.g., Cy3, may exhibit a ratio of specific fluorescent signal (e.g., resulting from Cy3-labeled oligonucleotides attached to the surface) to nonspecifically adsorbed dye fluorescent signal of at least 3:1, 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.

[0065] In some examples, the degree of hydrophilicity (or "wettability" with respect to aqueous solutions) of the disclosed support surfaces can be assessed, for example, via water contact angle measurements, in which a small drop of water is placed on a surface and the contact angle with the surface is measured, for example, using an optical tensiometer. In some examples, a static contact angle can be determined. In some examples, an advancing or receding contact angle can be determined. In some examples, the water contact angle of the hydrophilic, low-binding support surfaces disclosed herein can range from about 0 to about 50 degrees. In some examples, the water contact angle of the hydrophilic, low-binding support surfaces disclosed herein can be 50 degrees, 45 degrees, 40 degrees, 35 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 will be any number within this range or less, for example, 40 degrees or less. Those skilled in the art will recognize that a given hydrophilic, low-binding support surface may exhibit a water contact angle value within this range, for example, a water contact angle of about 27 degrees.

[0066] In some instances, the hydrophilic surfaces disclosed herein facilitate shortening the washing time of bioassays, often due to reduced non-specific binding of biomolecules to the low-binding surface. In some instances, a suitable washing step can be performed in less than 60, 50, 40, 30, 20, 15, or 10 seconds. For example, in some instances, a suitable washing step can be performed in less than 30 seconds.

[0067] Some low-binding surfaces of the present disclosure exhibit significantly improved stability or durability against prolonged exposure to solvents and high temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some instances, 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 fluorescent signal before, during, or after prolonged exposure to solvents and high temperatures, or repeated cycles of solvent exposure or temperature changes. In some examples, 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 percentages measured over these periods) over a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 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 of exposure to solvent and / or elevated temperature. In some examples, 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 measured over this range of cycles) over 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 repeated exposures to solvent and / or temperature changes.

[0068] In some examples, the surfaces disclosed herein may exhibit a high ratio of specific signal to non-specific signal or other background. For example, some surfaces, when used for nucleic acid amplification, may exhibit an amplification signal that is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than the signal of adjacent unpopulated regions of the surface. Similarly, some surfaces exhibit an amplification signal that is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than the signal of adjacent amplified nucleic acid-populated regions of the surface.

[0069] Fluorescence excitation energy varies between specific fluorophores and protocols and may range from less than 400 nm to more than 800 nm in excitation wavelength, consistent with fluorophore selection or other parameters of use of the surfaces disclosed herein.

[0070] Thus, low nonspecific binding surfaces as disclosed herein exhibit a lower background fluorescence signal or a higher contrast-to-noise ratio (CNR) than surfaces known in the art. For example, in some instances, the background fluorescence of a surface at a location spatially separated from or excluded from a labeled feature on the surface (e.g., a labeled spot, cluster, distinct region, subsection, or subset of the surface) comprising a hybridized cluster of nucleic acid molecules or a clonal amplification cluster of nucleic acid molecules produced, for example, by 20 rounds of nucleic acid amplification via thermal cycling, may be 20x, 10x, 5x, 2x, 1x, 0.5x, 0.1x, or less than 0.1x the background fluorescence measured at the same location prior to the previous hybridization or 20 rounds of nucleic acid amplification.

[0071] In some examples, fluorescent images of the disclosed low background surfaces when used in nucleic acid hybridization or amplification applications to generate clusters of hybridized or clonally amplified nucleic acid molecules (e.g., labeled directly or indirectly with a fluorophore) exhibit a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than 250.

[0072] Generally, at least one of the one or more layers of low non-specific binding material includes functional groups for covalently or non-covalently attaching oligonucleotide molecules, e.g., adapter or primer sequences, or may already include covalently or non-covalently attached oligonucleotide adapter or primer sequences when deposited on the support surface. In some examples, the oligonucleotides tethered to the polymer molecules of the at least one third layer may be distributed at multiple depths throughout the layer.

[0073] In some examples, the oligonucleotide adaptor or primer molecule is covalently attached to the polymer in solution, e.g., before being attached to the polymer or deposited on a surface. In some examples, the oligonucleotide adaptor or primer molecule is covalently attached to the polymer after being attached to the polymer or deposited on a surface. In some examples, at least one hydrophilic polymer layer comprises a plurality of covalently attached oligonucleotide adaptor or primer molecules. In some examples, at least two, at least three, at least four, or at least five layers of hydrophilic polymer comprise a plurality of covalently attached adaptor or primer molecules.

[0074] In some instances, oligonucleotide adaptor or primer molecules may be attached to one or more layers of hydrophilic polymers using any of a variety of suitable conjugation chemistries known to those skilled in the art. For example, the oligonucleotide adaptor or primer sequence may contain a moiety reactive with amine groups, carboxyl groups, thiol groups, etc. Examples of suitable amine-reactive conjugation chemistries that can be used include, but are not limited to, reactions involving isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester groups. Examples of suitable carboxyl-reactive conjugation chemistries include, but are not limited to, reactions involving carbodiimide compounds, such as water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl). Examples of suitable sulfydryl-reactive conjugation chemistries include maleimides, haloacetyls, and pyridyl disulfides.

[0075] One or more oligonucleotide molecules may be attached or tethered to the support surface. In some examples, one or more oligonucleotide adapters or primers may include a spacer sequence, an adapter sequence for hybridization to the adapter-ligated template library nucleic acid sequence, a forward amplification primer, a reverse amplification primer, a sequencing primer, and / or a molecular barcoding sequence, or any combination thereof. In some examples, one primer or adapter sequence may be tethered to at least one layer of the surface. In some examples, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adapter sequences may be tethered to at least one layer of the surface.

[0076] The length of the tethered oligonucleotide adaptor and / or primer sequence can range from about 10 nucleotides to about 100 nucleotides. In some examples, the length of the tethered oligonucleotide adaptor and / or primer sequence can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some examples, the length of the tethered oligonucleotide adaptor and / or primer sequence can be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. Any of the lower and upper limits described in this paragraph can be combined to form ranges encompassed within the present disclosure. For example, in some examples, the length of the tethered oligonucleotide adaptor and / or primer sequence can range from about 20 to about 80 nucleotides. One of skill in the art will recognize that the length of the tethered oligonucleotide adaptor and / or primer sequence may have any value within this range, for example, about 24 nucleotides.

[0077] In some instances, the tethered adapter or primer sequence may contain modifications designed to enhance the specificity and efficiency of nucleic acid amplification when performed on a low-binding support. For example, in some instances, the primer may contain a polymerase stop point such that the extension of the primer sequence between the surface conjugation point and the modification site is always in single-stranded form and serves as a loading site for a 5'-3' helicase in some helicase-dependent isothermal amplification methods. Examples of other primer modifications that can be used to create a polymerase stop point include, but are not limited to, the insertion of a PEG chain into the backbone of the primer between two nucleotides toward the 5' end, the insertion of an abasic nucleotide (i.e., a nucleotide containing neither a purine nor a pyrimidine base), or a lesion site that can be bypassed by a helicase.

[0078] As discussed further in the examples below, it may be desirable to alter the surface density of tethered oligonucleotide adapters or primers on the support surface and / or the spacing of tethered adapters or primers away from the support surface (e.g., by changing the length of the linker molecule used to tether the adapters or primers to the surface) to "tune" the support for optimal performance when using a given amplification method. As noted below, adjusting the surface density of tethered oligonucleotide adapters or primers can affect the degree of specific and / or nonspecific amplification observed on the support in a manner that varies depending on the amplification method selected. In some examples, the surface density of tethered oligonucleotide adapters or primers can be altered by adjusting the ratio of molecular components used to create the support surface. For example, when oligonucleotide primer-PEG conjugates are used to create the final layer of a low-binding support, the ratio of oligonucleotide primer-PEG conjugates to unconjugated PEG molecules may be altered. The resulting surface density of tethered primer molecules can then be estimated or measured using any of a variety of techniques known to those skilled in the art. Examples include, but are not limited to, the use of radioisotope labeling and counting methods, the covalent attachment of cleavable molecules containing optically detectable tags (e.g., fluorescent tags) that can be cleaved from a defined area of ​​the support surface, collected in a fixed volume of an appropriate solvent, and then quantified by comparison of the fluorescent signal with that of a calibration solution of known optical tag concentration, or the use of fluorescent imaging techniques in which attention is paid to the labeling reaction conditions and image acquisition environment to ensure that the fluorescent signal is linearly related to the number of fluorophores on the surface (without significant self-quenching for the fluorophores on the surface).

[0079] In some examples, the resulting surface density of oligonucleotide adapters or primers on the low binding support surfaces of the present disclosure may range from about 100 to about 1,000,000 primer molecules per μm 2 . In some examples, the surface density of oligonucleotide adapters or primers is at least 100 per μm, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least At least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 30 The number of molecules may be at least 0,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules.In some examples, the surface density of oligonucleotide adapters or primers is at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 4 50,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 5 5,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, at most 7,000, at most 6,500, at most 6,000 The surface density of the adapter or primer may be 0, at most 5,500, at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, or at most 100 molecules per μm. Any of the lower and upper limits described in this paragraph may be combined to form ranges within the scope of the present disclosure. For example, in some examples, the surface density of the adapter or primer may be from about 10,000 to about 100,000 molecules per μm. One of skill in the art will recognize that the surface density of adapter or primer molecules may have any value within the above range, for example, in some instances, 3,800 molecules per μm2, or in other instances, 455,000 molecules per μm2.In some instances, as discussed further below, the surface density of template library nucleic acid sequences (e.g., sample DNA molecules) that are initially hybridized to adapter or primer sequences on a support surface may be equal to or less than that shown for the surface density of tethered oligonucleotide primers. In some instances, as discussed further below, the surface density of clonal amplified template library nucleic acid sequences that are hybridized to adapter or primer sequences on a support surface may be in the same or a different range than that shown for the surface density of tethered oligonucleotide adapters or primers.

[0080] The local surface densities of adaptor or primer molecules listed above do not exclude variations in density across the surface, so that a surface may include regions having an oligo density of, for example, 500,000 / um2, as well as at least a second region surface having a substantially different local density.

[0081] A solid support for capturing and analyzing DNA. In some embodiments, as shown in FIG. 2, a surface is bound to a plurality of oligonucleotides (e.g., capture oligonucleotides (200)) for capturing target nucleic acids, such as DNA molecules. In some embodiments, the capture oligonucleotides each comprise a single-stranded oligonucleotide. The capture oligonucleotides can be immobilized to the passivated surface by their 5' ends, or an internal portion of the capture oligonucleotide can be immobilized to the passivated surface. Each capture oligonucleotide can comprise an extendable 3' end. As shown in FIG. 2, each capture oligonucleotide can comprise a cleavable region (250) that can be positioned near the end immobilized to the passivated surface. For example, each capture oligonucleotide can comprise a cleavable region near the 5' end. The cleavable region can be cleaved by an enzyme, a chemical compound, light, or heat. In some embodiments, each capture oligonucleotide comprises a target capture region (210) and a universal sequence region (220, 230, 240). In some embodiments, the target capture region of the capture oligonucleotide comprises a sequence that can hybridize to at least a portion of a target nucleic acid. The target capture region may include, for example, a random nucleotide sequence or a target-specific sequence corresponding to the sequence of the captured target nucleic acid. In some embodiments, the universal sequence region includes a sample barcode sequence (220) that can be used to distinguish target nucleic acids from different sample sources in multiplex assays. In some embodiments, the universal sequence region includes a spatial barcode sequence (230) that conveys location information for a cell within a tissue sample or a capture oligonucleotide on a support that conveys location information for a single cell. In some embodiments, the sample barcode sequence (220) may be upstream or downstream of the spatial barcode sequence (230). In some embodiments, the universal sequence region of the capture oligonucleotide includes a circularization anchor region (240) that hybridizes to a portion of another type of oligonucleotide to facilitate circularization of the captured nucleic acid (300).In some embodiments, the universal sequence region of the capture oligonucleotide comprises at least one sequence that binds / hybridizes to a universal primer sequence, such as a sequencing primer sequence and / or an amplification primer sequence. In some embodiments, the circularization anchor region (240) comprises any one of a sequencing primer sequence, an amplification primer sequence, a sample barcode sequence, and / or a spatial barcode sequence, or any combination of two or more thereof. In some embodiments, the circularization anchor region (240) comprises a separate sequence that hybridizes with a portion of another type of oligonucleotide that facilitates circularization of the capture nucleic acid. In some embodiments, the universal sequence region comprises a cleavable region that can be cleaved by an enzyme, a chemical compound, light, or heat.

[0082] Still referring to FIG. 2 , in some embodiments, the surface is bound to a plurality of different types of oligonucleotides (e.g., circularization oligonucleotides (300)) that facilitate circularization of the capture target nucleic acid. In some embodiments, the circularization oligonucleotides each comprise a single-stranded oligonucleotide. The circularization oligonucleotides can be immobilized to the passivated surface by their 5′ ends, or an internal portion of the circularization oligonucleotide can be immobilized to the passivated surface. Each of the circularization oligonucleotides can comprise an extendable 3′ end. Each of the circularization oligonucleotides comprises a homopolymer region (310) and a universal sequence region (320), as shown in FIG. 3 . The homopolymer region may be selected from the group consisting of a poly-T tail, a poly-dT tail, a poly-A tail, a poly-dA tail, a poly-C tail, a poly-dC tail, a poly-G tail, and a poly-dG tail. The homopolymer region can be located at or near the 3′ end of the circularization oligonucleotide. In some embodiments, the universal sequence region of the circularization oligonucleotide hybridizes to the circularization anchor region of the capture oligonucleotide. In some embodiments, the universal sequence region of the circularization oligonucleotide comprises at least one sequence that binds / hybridizes to a universal primer sequence, such as a sequencing primer sequence of a capture oligonucleotide. In some embodiments, the universal sequence region of the circularization oligonucleotide comprises at least one sequence that binds / hybridizes to a universal sequence, such as an amplification primer sequence of a capture oligonucleotide. In some embodiments, the universal sequence region of the circularization oligonucleotide comprises at least one sequence that binds / hybridizes to a sample barcode sequence and / or a spatial barcode sequence of the capture oligonucleotide. In some embodiments, the circularization oligonucleotide comprises a separate sequence (e.g., a circularization anchor binding sequence) that binds / hybridizes with a portion of the circularization anchor region of the capture oligonucleotide.

[0083] In some embodiments, the capture oligonucleotide ((200) in FIG. 2) and the circularizing oligonucleotide ((300) in FIG. 3) can be immobilized on the passivated surface before contacting the passivated surface with the target nucleic acid molecule for the target molecule capture step. In an alternative embodiment, the capture oligonucleotide can be immobilized on the passivated surface before contacting the passivated surface with the target nucleic acid molecule for the target molecule capture step, and then a plurality of circularizing oligonucleotides (e.g., in soluble form) can be provided in solution and flowed to the passivated surface to immobilize the circularizing oligonucleotides.

[0084] In some embodiments, the circularizing oligo may be the same as, comprise, or be contained within the capture oligo, hi some embodiments, the circularizing oligo may comprise a separate molecule.

[0085] The present disclosure provides a low-binding support having a coating that provides a low non-specific binding surface for proteins, carbohydrates, lipids, cell debris, or dye molecules from solution. In some embodiments, a tissue sample or cells, or a single cell, can be disposed on the surface of the support (left side of Figure 3). In some embodiments, the low non-specific binding surface includes multiple regions (e.g., features) located at different predetermined positions on the support (right side of Figure 3). The different features on the support can be located in non-overlapping or overlapping positions on the support. The features can be configured to assume any shape, such as a circle, oval, square, rectangle, or polygon. The features can be arranged in a grid pattern with rows and columns, or arranged in rows or columns. In some embodiments, a given feature includes multiple capture oligonucleotides and multiple circularization oligonucleotides immobilized on the coating. The multiple features include at least a first and a second feature.

[0086] In some embodiments, the first feature comprises a plurality of first capture oligonucleotides having a first target capture region, a first spatial barcode sequence, a first sample barcode sequence, and a first cleavable region, and a plurality of first circularization oligonucleotides having a first circularization anchor binding sequence, a first amplification primer binding sequence, and a first sequencing primer binding sequence. In some embodiments, the first capture oligonucleotide also comprises a first amplification primer binding sequence and / or a first amplification primer binding sequence. In some embodiments, the first circularization oligonucleotide also comprises a sequence capable of binding / hybridizing to the first spatial barcode sequence and / or a sequence capable of binding to the first sample barcode sequence.

[0087] In some embodiments, the second feature comprises a plurality of second capture oligonucleotides having a second target capture region, a second spatial barcode sequence, a second sample barcode sequence, and a second cleavable region, and a plurality of second circularization oligonucleotides having a second circularization anchor binding sequence, a second amplification primer binding sequence, and a second sequencing primer binding sequence. In some embodiments, the second capture oligonucleotide also comprises a second amplification primer binding sequence and / or a second amplification primer binding sequence. In some embodiments, the second circularization oligonucleotide also comprises a sequence capable of binding / hybridizing to the second spatial barcode sequence and / or a sequence capable of binding to the second sample barcode sequence.

[0088] In some embodiments, the sequence of the first target capture region in the first feature is the same as or different from the sequence of the second target capture region in the second feature. In some embodiments, the first spatial barcode sequence in the first feature is different from the second spatial barcode sequence in the second feature. In some embodiments, the first sample barcode sequence in the first feature is the same as or different from the second sample barcode sequence in the second feature. The first amplification primer binding sequence in the first feature can be the same as the second amplification primer binding sequence in the second feature. The first sequencing primer binding sequence in the first feature can be the same as the second sequencing primer binding sequence in the second feature. The first cleavable region in the first feature is cleavable by the same or different conditions (e.g., the same enzyme, chemical compound, light, or heat) as the second cleavable region in the second feature.

[0089] In some embodiments, the low nonspecific binding coating comprises a plurality of regions (e.g., features) in which features are attached to a plurality of capture and circularization oligonucleotides attached to the coating. In some embodiments, a first feature is attached to a first plurality of capture oligonucleotides and a first plurality of circularization oligonucleotides, a second feature is attached to a second plurality of capture oligonucleotides and a second plurality of circularization oligonucleotides, and the first and second capture oligonucleotides and the first and second circularization oligonucleotides are in fluid communication with each other so that the capture and circularization oligonucleotides can react with a reagent (e.g., a polymerase, a polymer-nucleotide conjugate, a nucleotide, and / or an enzyme comprising a divalent cation) in a massively parallel manner.

[0090] In some embodiments, the cleavable region of the capture oligonucleotide is enzymatically cleavable. In some embodiments, the cleavable region (250) shown in FIG. 2 comprises at least one uracil base or a poly-uracil sequence, which is cleavable using a uracil DNA glycosylase (UDG) enzyme or a DNA glycosylase-lyase endonuclease VIII (e.g., the commercially available enzyme USER™). In some embodiments, the cleavable site comprises at least one 8-oxoguanine (8-oxoG) cleavable by a DNA-formamidopyrimidine glycosylase enzyme (Fpg). In some embodiments, the cleavable region comprises an abasic site cleavable by endonuclease IV or endonuclease VIII. In some embodiments, the enzymatically cleavable cleavable region comprises a nucleotide sequence recognized and cleaved by a restriction endonuclease enzyme that cleaves double-stranded or single-stranded nucleic acid strands (e.g., DNA). In some embodiments, the enzyme-cleavable region comprises a glycosidic bond cleavable by an amylase enzyme or a peptide bond cleavable by a protease.

[0091] As shown in FIG. 2, in some embodiments, the cleavable region (250) of the capture oligonucleotide is cleavable by a chemical compound, including a labile chemical bond, including, but not limited to, an ester linkage, a thiol linkage, a vicinal diol linkage, a sulfone linkage, a silyl ether linkage, an abasic or apurinic / apyrimidinic (AP) site. The ester linkage is cleavable by acid, base, or hydroxylamine. The thiol linkage can be a disulfide bond cleavable by glutathione or a reducing agent. The vicinal diol linkage is cleavable by sodium periodate. The sulfonate linkage is cleavable by base. The silyl ether linkage is cleavable by acid. The abasic or apurinic / apyrimidinic (AP) site is cleavable by alkali or an AP endonuclease enzyme.

[0092] In some embodiments, the cleavable region (250) of the capture oligonucleotide is cleavable by light, including a photocleavable moiety that is cleavable by exposure to light, UV light, or a laser. The photocleavable moiety is cleavable by exposure to any wavelength of light. The photocleavable moiety includes 3-amino-3-(2-nitrophenyl)propionic acid (ANP), dicoumarin, 6-bromo-7-alkoxycoumarin-4-ylmethoxycarbonyl, phenacyl ester derivatives, or 8-quinolinylbenzenesulfonate. The photocleavable moiety includes a bimane-based linker, a bis-arylhydrazone-based linker, or an ortho-nitrobenzyl (ONB) linker. In some embodiments, the cleavable region (250) of the capture oligonucleotide is cleavable by heat exposure, including a Diels-Alder linker.

[0093] Support for capturing and analyzing RNA. Figure 4 provides a support (700) comprising a plurality of immobilized oligonucleotides. This support can be used to capture and analyze target nucleic acids, such as RNA molecules. In some embodiments, the support comprises a passivated surface (e.g., coating or layer) (Figure 1) as disclosed elsewhere herein, whereby the surface provides low or no binding to proteins, carbohydrates, lipids, cell debris, or solution-borne dye molecules. In some embodiments, the surface has attached thereto a plurality of oligonucleotides (e.g., capture oligonucleotides, (700) in Figure 4) for capturing target nucleic acids. In some embodiments, each of the capture oligonucleotides comprises a single-stranded oligonucleotide. The capture oligonucleotides can be immobilized to the passivated surface by their 5' ends, or an internal portion of the capture oligonucleotide can be immobilized to the passivated surface. Each of the capture oligonucleotides can comprise an extendable 3' end. As shown in Figure 4, each of the capture oligonucleotides can comprise a cleavable region (740) that can be positioned near the end immobilized to the passivated surface. For example, each capture oligonucleotide can include a cleavable region near its 5' end. The cleavable region can be cleaved by an enzyme, a chemical compound, light, or heat. In some embodiments, each capture oligonucleotide includes a target capture region (710) and a universal sequence region (720, 730). In some embodiments, the target capture region of the capture oligonucleotide includes a sequence that can hybridize to at least a portion of a target nucleic acid. The target capture region can include, for example, a homopolymer sequence (e.g., poly-T or poly-dT) corresponding to a known sequence of the target nucleic acid, a random nucleotide sequence, or a target-specific sequence. In some embodiments, the universal sequence region includes a sample barcode sequence (720) that can be used to distinguish target nucleic acids from different sample sources in multiplex assays.In some embodiments, the universal sequence region comprises a spatial barcode sequence (730) that conveys location information of a cell within a tissue sample, or a single cell, on a support, conveying location information of the capture oligonucleotide. In some embodiments, the sample barcode sequence (720) may be upstream or downstream of the spatial barcode sequence (730). In some embodiments, the universal sequence region of the capture oligonucleotide comprises at least one sequence that binds / hybridizes to a universal primer sequence, such as a sequencing primer sequence and / or an amplification primer sequence. In some embodiments, the capture oligonucleotide comprises a cleavable region (740) that is cleavable by an enzyme, a chemical compound, light, or heat.

[0094] Also referring to FIG. 4 , some embodiments herein provide another type of multiple oligonucleotides (e.g., circularizing oligonucleotides (800)) in soluble form or immobilized on a surface (e.g., a coating). The circularizing oligonucleotides can promote circularization of captured target nucleic acids. In some embodiments, the circularizing oligonucleotides each comprise a single-stranded oligonucleotide. The circularizing oligonucleotides can be in soluble form or immobilized on a passivated surface by their 5′ ends, or an internal portion of the circularizing oligonucleotide can be immobilized on a passivated surface. Each of the circularizing oligonucleotides can comprise an extendable 3′ end. Each of the circularizing oligonucleotides comprises an adapter binding region (810). In some embodiments, the adapter binding region comprises a sequencing primer binding region. In some embodiments, the adapter binding region comprises an amplification primer binding region. In some embodiments, each of the circularizing oligonucleotides comprises a homopolymer region ((830) in FIG. 4 ). The homopolymer region may be selected from the group consisting of poly-T, poly-dT, poly-A, poly-dA, poly-C, poly-dC, poly-G, and poly-dG. In some embodiments, the circularizing oligonucleotides each comprise an anchor region (830) and an anchor portion (840).

[0095] In some embodiments, the capture oligonucleotide (700 in FIG. 5) and the circularization oligonucleotide (800 in FIG. 4) can be immobilized on the passivated surface before contacting the passivated surface with a target nucleic acid molecule (e.g., RNA) for the target molecule capture step. In an alternative embodiment, the capture oligonucleotide can be immobilized on the passivated surface before contacting the passivated surface with a target nucleic acid molecule for the target molecule capture step, followed by providing a plurality of circularization oligonucleotides (e.g., in soluble form) in solution and flowing them to the passivated surface to immobilize the circularization oligonucleotides.

[0096] In some embodiments, the circularizing oligo may be the same as, comprise, or be contained within the capture oligo, hi some embodiments, the circularizing oligonucleotide may comprise a separate molecule.

[0097] In some embodiments, the cleavable region of the capture oligonucleotide (740 in Figure 4) is enzymatically cleavable. In some embodiments, the cleavable region comprises at least one uracil base or a poly-uracil sequence, which is cleavable using a uracil RNA glycosylase (UDG) enzyme or an RNA glycosylase-lyase endonuclease VIII (e.g., the commercially available enzyme USER™). In some embodiments, the cleavable site comprises at least one 8-oxoguanine (8-oxoG) cleavable by an RNA-formamidopyrimidine glycosylase enzyme (Fpg). In some embodiments, the cleavable region comprises an abasic site cleavable by endonuclease IV or endonuclease VIII. In some embodiments, the enzymatically cleavable cleavable region comprises a nucleotide sequence that is recognized and cleaved by a restriction endonuclease enzyme that cleaves double-stranded or single-stranded nucleic acid strands (e.g., RNA). In some embodiments, the enzyme-cleavable region comprises a glycosidic linkage cleavable by an amylase enzyme or a peptidic linkage cleavable by a protease.

[0098] In some embodiments, the cleavable region of the capture oligonucleotide (740 in Figure 4) is cleavable by a chemical compound containing a labile chemical bond, including, but not limited to, an ester linkage, a thiol linkage, a vicinal diol linkage, a sulfone linkage, a silyl ether linkage, an abasic or apurinic / apyrimidinic (AP) site. An ester linkage can be cleaved by acid, base, or hydroxylamine. A thiol linkage can be a disulfide bond cleavable by glutathione or a reducing agent. A vicinal diol linkage can be cleaved by sodium periodate. A sulfonate linkage can be cleaved by base. A silyl ether linkage can be cleaved by acid. An abasic or apurinic / apyrimidinic (AP) site can be cleaved by alkali or an AP endonuclease enzyme.

[0099] In some embodiments, the cleavable region of the capture oligonucleotide ((740) in Figure 4) is cleavable by light, including a photocleavable moiety that is cleavable by exposure to light, UV light, or a laser. The photocleavable moiety is cleavable by exposure to any wavelength of light. The photocleavable moiety includes 3-amino-3-(2-nitrophenyl)propionic acid (ANP), dicoumarin, 6-bromo-7-alkoxycoumarin-4-ylmethoxycarbonyl, phenacyl ester derivatives, or 8-quinolinylbenzenesulfonate. The photocleavable moiety includes a bimane-based linker, a bis-arylhydrazone-based linker, or an ortho-nitrobenzyl (ONB) linker. In some embodiments, the cleavable region of the capture oligonucleotide ((740) in Figure 4) is cleavable by heat exposure, including a Diels-Alder linker.

[0100] Immobilization of a biological sample on a surface. Provided herein are solid supports (e.g., low non-specific binding supports) further comprising a biological sample attached thereto. In some embodiments, the biological sample comprises a single cell, multiple cells, a tissue, an organ, an organism, or a section of these biological samples. In some embodiments, the biological sample is derived from a eukaryote (such as an animal, plant, fungus, or protist), an archaebacterium, or a eubacterium. The biological sample may be derived from a prokaryotic or eukaryotic cell, such as an adherent or non-adherent eukaryotic cell. The biological sample may be derived from a primary or immortalized cell line derived from a rodent, porcine, feline, canine, bovine, equine, primate, or human cell line.

[0101] The biological sample may be a solid sample, such as a tissue biopsy. The biological sample may be a fluid sample, such as blood or a blood component (e.g., serum or plasma). In some embodiments, the biological sample is obtained from skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, interstitial fluid from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestinal tract, brain, cavity fluids, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice, digestive fluid, tears, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, fingernail, skin cells, plasma, nasal swab or nasopharyngeal washing, cerebrospinal fluid, umbilical cord blood, emphatic fluid, and / or other excretions or body tissues. The biological sample may be an acellular sample.

[0102] The biological sample may include cells. The cells described herein may be white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells derived from the heart, lung, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine. The cells may be normal or healthy cells. Alternatively, or in combination, the cells may be abnormal cells, such as cancer cells, or derived from pathogenic cells infecting the host. In some embodiments, the cells belong to a subset of cells such as immune cells (e.g., T cells, cytotoxic (killer) T cells, helper T cells, αβ T cells, γδ T cells, T cell precursors, B cells, B cell precursors, immune stem cells, myeloid progenitor cells, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and / or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells induced to differentiate, or rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, circulating bone marrow cells, progenitor cells, foam cells, mesenchymal cells, or trophoblasts). Other cells are contemplated and consistent with the disclosure herein.

[0103] Biological samples can be extracted from an organism (e.g., by performing a biopsy) or obtained from cell cultures grown in liquid or culture dishes. Biological samples include fresh, frozen, flash-frozen, or archived (e.g., formalin-fixed paraffin-embedded, FFPE) samples. Biological samples can be embedded in wax, resin, epoxy, or agar. Biological samples can be fixed, for example, with any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton®, or glutaraldehyde. Biological samples can be sectioned or unsectioned. Biological samples can be stained, destained, or unstained.

[0104] In some embodiments, a biological sample may be permeabilized after being immobilized on a surface described herein to allow nucleic acids within the sample, including target nucleic acid molecules, to migrate from the cells to multiple capture oligonucleotides immobilized on the surface. Permeabilization may allow agents (such as phospho-selective antibodies, nucleic acid-conjugated antibodies, nucleic acid probes, primers, etc.) to enter the cells and achieve concentrations within the cells that exceed those that would normally penetrate the cells without such permeabilization. In some embodiments, cells may be permeabilized in the presence of at least about 60%, 70%, 80%, 90% or more methanol (or ethanol) and incubated on ice for a period of time. The incubation period may be at least about 10, 15, 20, 25, 30, 35, 40, 50, or 60 minutes or more.

[0105] A biological sample can be permeabilized by contacting the biological sample with one or more permeabilizing agents, including organic solvents, detergents, crosslinking agents, and / or enzymes. In some embodiments, the organic solvent includes acetone, ethanol, and methanol. In some embodiments, the detergent includes saponin, Triton® X-100, Tween 20, or sodium dodecyl sulfate (SDS), or N-lauroyl sarcosine sodium salt solution. In some embodiments, the crosslinking agent includes paraformaldehyde. In some embodiments, the enzyme includes trypsin, pepsin, or a protease (e.g., proteinase K). In some embodiments, target nucleic acid molecules from a biological sample are hybridized (captured) to capture oligonucleotides immobilized on a support in a manner that preserves the spatial location of the target nucleic acid molecules in the biological sample.

[0106] A biological sample can be used to generate a three-dimensional polymer matrix containing cells and intracellular components (e.g., nucleic acid molecules) of the biological sample. The three-dimensional polymer matrix can be covalently or non-covalently bound to a surface described herein. In some embodiments, the three-dimensional polymer matrix is ​​porous and contains polymerized or crosslinked intracellular components, including target nucleic acid molecules. The polymer matrix can be formed within a biological sample (e.g., cells or tissue) by flowing one or more polymer precursors (e.g., monomers such as ethylene oxide for polyethene glycol) into the biological sample and polymerizing or crosslinking the one or more polymer precursors. Before, during, or after polymer matrix formation, the location of moieties (e.g., DNA, RNA, proteins) within the biological sample can be fixed using, for example, a fixative (e.g., formaldehyde). Porous matrices can be produced by various methods. For example, a polyacrylamide gel matrix can be polymerized with biotinylated DNA molecules and acrydite-modified streptavidin monomers using an appropriate acrylamide:bis-acrylamide ratio to adjust the crosslink density. Further control over the size and density of the molecular sieves can be achieved by adding additional cross-linking agents, such as functionalized polyethylene glycol, allowing for immobilization to the surface of biological samples as well as the creation of polymer matrices within the biological sample, as described in PCT / US2019 / 055434, which is incorporated herein by reference in its entirety.

[0107] The biological sample optionally contains a target nucleic acid molecule to be analyzed using the systems, methods, and compositions described herein. In some embodiments, the target nucleic acid includes naturally occurring nucleic acid, recombinant nucleic acid, and / or synthetic nucleic acid. The target nucleic acid includes linear and / or circular forms. In some embodiments, the target nucleic acid may be DNA. In some embodiments, the target nucleic acid may be genomic DNA. In some embodiments, the target nucleic acid may be viral DNA. In some embodiments, the target nucleic acid may be cell-free DNA (cfDNA). In some embodiments, the DNA is methylated DNA, methylated or unmethylated DNA, and / or organelle DNA. The DNA may be fragmented and / or unfragmented. In some embodiments, the target nucleic acid molecule includes RNA, including poly-A RNA and / or non-poly-a RNA. The RNA includes coding and / or non-coding RNA. RNA includes tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), antisense RNA, non-coding RNA, and / or protein-coding RNA.

[0108] The target nucleic acids of the present disclosure have a fixed three-dimensional relationship with the biological sample after the biological sample is bound to a surface, which at least partially enables identification of spatial and cellular origin within the biological sample after nucleic acid identification using the systems and methods described herein.

[0109] Target nucleic acid capture and preparation. Provided herein is a method for hybridizing a target nucleic acid to a capture oligonucleotide bound to a surface (e.g., a low non-specific binding surface) in the presence of a biological sample. In some cases, hybridization buffer formulations are described that, in combination with the low-binding support of the present disclosure, result in improved hybridization speed, hybridization specificity (or stringency), and hybridization efficiency (or yield). As used herein, hybridization specificity is a measure of the ability of a tethered adapter sequence, primer sequence, or oligonucleotide sequence to hybridize correctly only to a completely complementary sequence, and hybridization efficiency is a measure of the percentage of the total of commonly available tethered adapter sequences, primer sequences, or oligonucleotide sequences that hybridize to complementary sequences.

[0110] Improved hybridization specificity and / or efficiency may be achieved by optimizing the hybridization buffer formulation used with the disclosed low-binding surfaces, as described in more detail in the examples below. Examples of hybridization buffer components that can be adjusted to achieve improved performance include, but are not limited to, buffer type, organic solvent mixture, buffer pH, buffer viscosity, surfactants, and zwitterionic components, ionic strength (including adjustment of both monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, other additives, etc.

[0111] As non-limiting examples, suitable buffers for use in formulating the hybridization buffer may include, but are not limited to, phosphate-buffered saline (PBS), succinate, citrate, histidine, acetate, Tris, TAPS, MOPS, PIPES, HEPES, MES, and the like. The selection of an appropriate buffer generally depends on the target pH of the hybridization buffer solution. Generally, the desired pH of the buffer solution ranges from about pH 4 to about pH 8.4. In some embodiments, the buffer pH can be at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.2, at least 6.4, at least 6.6, at least 6.8, at least 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least 8.0, at least 8.2, or at least 8.4. In some embodiments, the buffer pH can be at most 8.4, at most 8.2, at most 8.0, at most 7.8, at most 7.6, at most 7.4, at most 7.2, at most 7.0, at most 6.8, at most 6.6, at most 6.4, at most 6.2, at most 6.0, at most 5.5, at most 5.0, at most 4.5, or at most 4.0. Any of the lower and upper limits listed in this paragraph can be combined to form ranges within the present disclosure; for example, in some instances, the desired pH can range from about 6.4 to about 7.2. One of skill in the art will recognize that the buffer pH can have any value within this range, such as about 7.25.

[0112] Suitable surfactants for use in hybridization buffer formulations include, but are not limited to, zitterionic surfactants (e.g., 1-dodecanoyl-sn-glycero-3-phosphocholine, 3-(4-tert-butyl-1-pyridinio)-1-propanesulfonate, 3-(N,N-dimethylmyristylammonio)propanesulfonate, 3-(N,N-dimethylmyristylammonio)propanesulfonate, ASB-C80, C7BzO, CHAPS, CHAPS hydrate, CHAPSO, DDMAB, dimethylethylammonium propanesulfonate, N,N-dimethyldodecylamine N-oxides, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, or N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), and anionic, cationic, and nonionic surfactants. Examples of nonionic surfactants include poly(oxyethylene) ethers, related polymers (e.g., Brij®, TWEEN®, TRITON®, TRITON® X-100, and IGEPAL® CA-630), bile salts, and glycoside surfactants.

[0113] Use of the disclosed low nonspecific binding supports, alone or in combination with optimized buffer formulations, can result in relative hybridization rates ranging from about 2-fold to about 20-fold faster than conventional hybridization protocols. In some examples, the relative hybridization rate can be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold faster than conventional hybridization protocols.

[0114] Use of the disclosed low nonspecific binding supports alone or in combination with optimized buffer formulations can result in total hybridization reaction times (i.e., the time required to achieve 90%, 95%, 98%, or 99% completion of the hybridization reaction) of less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes for any of these completion metrics.

[0115] The use of the disclosed low non-specific binding supports alone or in combination with optimized buffer formulations can result in improved hybridization specificity compared to conventional hybridization protocols. In some embodiments, hybridization specificity that can be achieved is one base mismatch in 10 hybridization events, one base mismatch in 20 hybridization events, one base mismatch in 30 hybridization events, one base mismatch in 40 hybridization events, one base mismatch in 50 hybridization events, one base mismatch in 75 hybridization events, one base mismatch in 100 hybridization events, one base mismatch in 200 hybridization events, one base mismatch in 300 hybridization events, one base mismatch in 400 hybridization events, one base mismatch in 500 hybridization events, one base mismatch in 600 hybridization events, one base mismatch in 700 hybridization events, one base mismatch in 800 hybridization events, one base mismatch in 900 hybridization events, one base mismatch in 1 ... In one embodiment, the hybridization rate is better than one base mismatch in 1000 hybridization events, one base mismatch in 800 hybridization events, one base mismatch in 900 hybridization events, one base mismatch in 1,000 hybridization events, one base mismatch in 2,000 hybridization events, one base mismatch in 3,000 hybridization events, one base mismatch in 4,000 hybridization events, one base mismatch in 5,000 hybridization events, one base mismatch in 6,000 hybridization events, one base mismatch in 7,000 hybridization events, one base mismatch in 8,000 hybridization events, one base mismatch in 9,000 hybridization events, or one base mismatch in 10,000 hybridization events.

[0116] In some examples, the use of the disclosed low nonspecific binding supports alone or in combination with an optimized buffer formulation can result in improved hybridization efficiency (e.g., the fraction of available oligonucleotide primers on the support surface that successfully hybridize with target oligonucleotide sequences) compared to conventional hybridization protocols. In some examples, the hybridization efficiency that can be achieved is better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% for any of the input target oligonucleotide concentrations specified below and for any of the hybridization reaction times specified above. For example, in some examples where hybridization efficiency is less than 100%, the resulting surface density of target nucleic acid sequences hybridized to the support surface may be less than the areal density of oligonucleotide adapter or primer sequences on the surface.

[0117] In some instances, use of the disclosed low nonspecific binding supports for nucleic acid hybridization (or amplification) applications using conventional or optimized hybridization (or amplification) protocols can lead to reduced input concentration requirements for target (or sample) nucleic acid molecules contacted with the support surface. For example, in some instances, target (or sample) nucleic acid molecules can be contacted with the support surface at a concentration ranging from about 10 pM to about 1 μM (i.e., before annealing or amplification). In some examples, the target (or sample) nucleic acid molecule may be administered at at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, or at least 1 μM. In some examples, the target (or sample) nucleic acid molecule has a concentration of at most 1 μM, at most 900 nM, at most 800 nM, at most 700 nM, at most 600 nM, at most 500 nM, at most 400 nM, at most 300 nM, at most 200 nM, at most 100 nM, at most 90 nM, at most 80 nM, at most 70 nM, at most 60 nM, at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, It may be administered at up to 10 nM, up to 1 nM, up to 900 pM, up to 800 pM, up to 700 pM, up to 600 pM, up to 500 pM, up to 400 pM, up to 300 pM, up to 200 pM, up to 100 pM, up to 90 pM, up to 80 pM, up to 70 pM, up to 60 pM, up to 50 pM, up to 40 pM, up to 30 pM, up to 20 pM, or up to 10 pM.Any of the lower and upper limits described in this paragraph can be combined to form ranges within the present disclosure, e.g., in some instances, the target (or sample) nucleic acid molecule can be administered at a concentration ranging from about 90 pM to about 200 nM. One of skill in the art will recognize that the target (or sample) nucleic acid molecule can be administered at any concentration within this range, e.g., about 855 nM.

[0118] In another example, the volume of a biological sample that may contact the surface may be reduced compared to an equivalent biological sample analyzed using an equivalent surface with standard hybridization reagents. In some embodiments, a fluid sample containing target (or sample) nucleic acid molecules may have a sample volume range of about 5 μl to about 900 μl. In some examples, the sample volume range is about 5 μl to about 800 μl. In some examples, the sample volume range is about 5 μl to about 700 μl. In some examples, the sample volume range is about 5 μl to about 600 μl. In some examples, the sample volume range is about 5 μl to about 500 μl. In some examples, the sample volume range is about 5 μl to about 400 μl. In some examples, the sample volume range is about 5 μl to about 300 μl. In some examples, the sample volume range is about 5 μl to about 200 μl. In some examples, the sample volume range is about 5 μl to about 150 μl. In some examples, the sample volume ranges from 5 μl to about 100 μl. In some examples, the sample volume ranges from about 5 μl to about 90 μl. In some examples, the sample volume ranges from about 5 μl to about 85 μl. In some examples, the sample volume ranges from about 5 μl to about 80 μl. In some examples, the sample volume ranges from about 5 μl to about 75 μl. In some examples, the sample volume ranges from about 5 μl to about 70 μl. In some examples, the sample volume ranges from about 5 μl to about 65 μl. In some examples, the sample volume ranges from about 5 μl to about 60 μl. In some examples, the sample volume ranges from about 5 μl to about 55 μl. In some examples, the sample volume ranges from about 5 μl to about 50 μl. In some examples, the sample volume ranges from about 15 μl to about 150 μl. In some examples, the sample volume ranges from about 15 μl to about 120 μl. In some examples, the sample volume ranges from 15 μl to about 100 μl. In some examples, the sample volume ranges from about 15 μl to about 90 μl. In some examples, the sample volume ranges from about 15 μl to about 85 μl. In some examples, the sample volume ranges from about 15 μl to about 80 μl. In some examples, the sample volume ranges from about 15 μl to about 75 μl. In some examples, the sample volume ranges from about 15 μl to about 70 μl.In some examples, the sample volume ranges from about 15 μl to about 65 μl. In some examples, the sample volume ranges from about 15 μl to about 60 μl. In some examples, the sample volume ranges from about 15 μl to about 55 μl. In some examples, the sample volume ranges from about 15 μl to about 50 μl.

[0119] In some examples, use of the disclosed low non-specific binding supports alone or in combination with optimized hybridization buffer formulations can result in surface densities of hybridized target (or sample) oligonucleotide molecules (i.e., before performing any subsequent solid-phase or clonal amplification reactions) ranging from about 0.0001 target oligonucleotide molecules per μm to about 1,000,000 target oligonucleotide molecules per μm.In some examples, the surface density of hybridized target oligonucleotide molecules is at least 0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, 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, at least 100, at least 200, at least 300 per μm , at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least The number of molecules may be at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules.In some instances, the surface density of hybridized target oligonucleotide molecules is 1 μm. 2 Up to 1,000,000, Up to 950,000, Up to 900,000, Up to 850,000, Up to 800,000, Up to 750,000, Up to 700,000, Up to 650,000, Up to 600,000, Up to 550,000, Up to 500,000, Up to 450,000, Up to 400,000, Up to 350,000, Up to 300,000, Up to 250,000, Up to 200,000 0, up to 150,000, up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000 00, up to 10,000, up to 9,500, up to 9,000, up to 8,500, up to 8,000, up to 7,500, up to 7,000, up to 6,500, up to 6,000, up to 5,500, up to 5,000, up to 4,500, up to 4,000, up to 3,500, up to 3,000, up to 2,500, up to 2,000, up to 1,500, up to 1,000, up to 900, up to 800, up to The surface density of hybridized target oligonucleotide molecules can be at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 5, at most 1, at most 0.5, at most 0.1, at most 0.05, at most 0.01, at most 0.005, at most 0.001, at most 0.0005, or at most 0.0001 molecules. Any of the lower and upper limits set forth in this paragraph can be combined to form ranges within the disclosure; for example, in some cases, the surface density of hybridized target oligonucleotide molecules can be greater than 1 μm 2 Approximately 3,000 molecules per 1 μm 2Those skilled in the art will appreciate that the surface density of hybridized target oligonucleotide molecules can be anywhere within this range, e.g., 1 μm 2 It will be appreciated that the number of molecules per molecule may be approximately 2,700.

[0120] In other words, in some cases, the use of low nonspecific binding supports alone or in combination with optimized hybridization buffer formulations can reduce the 2 Approximately 100 hybridized target oligonucleotide molecules per ~1 mm 2 Approximately 1 x 10 7 of oligonucleotide molecules, or 1 mm 2 Approximately 100 hybridized target oligonucleotide molecules per ~1 mm 2 Approximately 1 x 10 12 This can result in a surface density of hybridized target (or sample) oligonucleotide molecules (i.e., before any subsequent solid phase or clonal amplification reactions are performed) in the range of 1 mm of hybridized target oligonucleotide molecules. In some examples, the surface density of hybridized target oligonucleotide molecules is in the range of 1 mm 2at least 100, at least 500, at least 1,000, at least 4,000, at least 5,000, at least 6,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95, 000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 x 10 7 , at least 5 × 10 7 , at least 1 x 10 8 , at least 5 × 10 8 , at least 1 x 10 9 , at least 5 × 10 9 , at least 1 x 10 10 , at least 5 × 10 10 , at least 1 x 10 11 , at least 5 × 10 11 , or at least 1 × 10 12 In some examples, the surface density of hybridized target oligonucleotide molecules can be 1 mm 2 Maximum of 1 x 10 12 , up to 5 × 10 11 , up to 1 × 10 11 , up to 5 × 10 10 , up to 1 × 10 10 , up to 5 × 10 9 , up to 1 × 109 , up to 5 × 10 8 , up to 1 × 10 8 , up to 5 × 10 7 , up to 1 × 10 7 , up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000 , up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 molecules. Any of the lower and upper limits set forth in this paragraph can be combined to form ranges within the disclosure; for example, in some cases, the surface density of hybridized target oligonucleotide molecules is greater than or equal to 1 μm 2 Approximately 5,000 molecules per 1 μm 2 Those skilled in the art will appreciate that the surface density of hybridized target oligonucleotide molecules can be anywhere within this range, e.g., 1 μm 2 It will be appreciated that the number of molecules per molecule may be approximately 50,700.

[0121] In some examples, the target (or sample) oligonucleotide molecules (or nucleic acid molecules) hybridized to oligonucleotide adapter or primer molecules attached to the surface of a low-binding support can range in length from about 0.02 kilobases (kb) to about 20 kb, or from about 0.1 kilobases (kb) to about 20 kb. In some examples, the target oligonucleotide molecule can be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb in length, at least 0.2 kb in length, at least 0.3 kb in length, at least 0.4 kb in length, at least 0.5 kb in length, at least 0.6 kb in length, at least 0.7 kb in length, at least 0.8 kb in length, at least 0.9 kb in length, at least 1 kb in length, at least 2 kb in length, at least 3 kb in length, at least 4 kb in length, at least 5 kb in length, at least 6 kb in length, at least 7 kb in length, at least 8 kb in length, at least 9 kb in length, at least 10 kb in length, at least 15 kb in length, at least 20 kb in length, at least 30 kb in length, or at least 40 kb in length, or any intermediate value spanning the ranges described herein, e.g., at least 0.85 kb in length.

[0122] In some examples, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a single-stranded or double-stranded multimeric nucleic acid molecule further comprising repeating regularly occurring monomeric units. In some examples, single-stranded or double-stranded multimeric nucleic acid molecules can be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb in length, at least 0.2 kb in length, at least 0.3 kb in length, at least 0.4 kb in length, at least 0.5 kb in length, at least 1 kb in length, at least 2 kb in length, at least 3 kb in length, at least 4 kb in length, at least 5 kb in length, at least 6 kb in length, at least 7 kb in length, at least 8 kb in length, at least 9 kb in length, at least 10 kb in length, at least 15 kb in length, or at least 20 kb in length, at least 30 kb in length, or at least 40 kb in length, or any intermediate value spanning the ranges described herein, e.g., about 2.45 kb in length.

[0123] In some examples, a target (or sample) oligonucleotide molecule (or nucleic acid molecule) can comprise a single-stranded or double-stranded multimeric nucleic acid molecule comprising about 2 to about 100 copies of a regularly repeating monomeric unit. In some examples, the number of copies of the regularly repeating monomeric unit can be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, and at least 100. In some examples, the number of copies of the regularly repeating monomeric unit can be at most 100, at most 95, at most 90, at most 85, at most 80, at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, or at most 2. Any of the lower and upper limits listed in this paragraph can be combined to form ranges within the disclosure; for example, in some examples, the number of copies of the regularly repeating monomeric unit can range from about 4 to about 60. One of skill in the art will recognize that the number of copies of the regularly repeating monomeric unit can have any value within this range, for example, about 17. Thus, in some cases, even if the hybridization efficiency is less than 100%, the surface density of hybridized target sequences, in terms of the number of copies of target sequences per unit area of ​​support surface, may exceed the surface density of oligonucleotide primers.

[0124] As used herein, the phrase "nucleic acid surface amplification" (NASA) is used interchangeably with the phrase "solid-phase nucleic acid amplification" (or simply "solid-phase amplification"). In some embodiments of the present disclosure, nucleic acid amplification formulations are described that, in combination with the disclosed low-binding supports, result in improved amplification rates, amplification specificity, and amplification efficiency. As used herein, specific amplification refers to the amplification of template library oligonucleotide strands tethered to a solid support, either covalently or non-covalently. As used herein, non-specific amplification refers to the amplification of primer dimers or other non-template nucleic acids. As used herein, amplification efficiency is a measure of the percentage of tethered oligonucleotides on the support surface that are successfully amplified during a given amplification cycle or amplification reaction. Nucleic acid amplification performed on the surfaces disclosed herein can result in amplification efficiencies of at least 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%, such as 98% or 99%.

[0125] Any of a variety of thermal cycling or isothermal nucleic acid amplification schemes may be used with the disclosed low-binding supports. Examples of nucleic acid amplification methods that may be utilized with the disclosed low-nonspecific binding supports include, but are not limited to, polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, or single-stranded binding (SSB) protein-dependent amplification.

[0126] In some embodiments, a rolling circle amplification reaction includes: (1) forming captured nucleotide-polymerase complexes by contacting a plurality of immobilized, covalently closed, circular nucleic acid molecules with: (i) a first plurality of polymerases having strand displacement activity; (ii) a plurality of nucleotides (e.g., one type of nucleotide or a mixture of dATP, dGTP, dCTP, and dTTP); (iii) a non-catalytic divalent cation (e.g., strontium or barium) that mediates nucleotide binding but not nucleotide incorporation, and, optionally, (iv) a plurality of amplification primers when the covalently closed circular molecules lack primers. The rolling circle amplification reaction further includes (4) performing a nucleotide polymerization reaction by contacting the captured nucleotide-polymerase complex with (i) at least one divalent cation (e.g., magnesium and / or manganese) that mediates nucleotide binding and mediates nucleotide incorporation, and (ii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP) under conditions suitable for performing an isothermal rolling circle amplification reaction to generate a plurality of immobilized concatemers.

[0127] In some embodiments, the rolling circle amplification reaction further includes a plurality of compaction oligonucleotides that hybridize to portions of the concatemers and collapse the concatemers into a more compact shape and size. Compaction oligonucleotides are single-stranded nucleic acid molecules with two identical sequences separated by a short linker sequence, where the two identical sequences are reverse-complementary to portions of the concatemers. Compaction oligonucleotides can be of any length, for example, 20 to 100 nucleotides. The two identical sequence regions hybridize to the concatemers, drawing the distal portions of the concatemers together and causing compaction of the concatemers. In some embodiments, the compaction oligonucleotides are resistant to 3' exonuclease degradation and / or single-strand endonuclease degradation. In some embodiments, the compaction oligonucleotide comprises any one or any combination of two or more of the following: 3'-terminal phosphorylation; at least two 3'-terminal nucleotides having a phosphorothioate linkage therebetween; at least one 3'-terminal nucleotide having a 2'-O methyl moiety; and / or at least one 3'-terminal nucleotide having a 2' fluoro base.

[0128] In some embodiments, in the captured nucleotide-polymerase mixture of step (c), the first plurality of polymerases having strand displacement activity include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., Expedeon's MagniPhi), mutant EquiPhi29 DNA polymerase (e.g., Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., 4basebio).

[0129] In some embodiments, the amplification primer comprises a single-stranded nucleic acid primer having a length of about 5 to 25 nucleotides. In some embodiments, the amplification primer is resistant to 3' exonuclease degradation and / or single-stranded endonuclease degradation. In some embodiments, the amplification primer comprises any one or a combination of two or more of the following: 3'-terminal phosphorylation; at least two 3'-terminal nucleotides having a phosphorothioate linkage therebetween; at least one 3'-terminal nucleotide having a 2'-O-methyl moiety; and / or at least one 3'-terminal nucleotide having a 2'-fluoro base.

[0130] In some embodiments, the rolling circle amplification reaction further comprises at least one accessory protein or enzyme, including a helicase, a single-stranded binding (SSB) protein, or a recombinase (e.g., T4 uvsX) and / or a recombinase accessory factor (e.g., T4 uvsY or T4 gp32).

[0131] In some embodiments, the isothermal rolling circle amplification reaction can be performed at a temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

[0132] In some embodiments, a concatemer may comprise at least 2, 10, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more copies of a repeating unit.

[0133] The rolling circle amplification method may be followed by a multiple displacement amplification reaction using random sequence primers. The multiple displacement amplification reaction includes: (1) contacting a plurality of immobilized concatemers with (i) a second plurality of polymerases having strand displacement activity, and (ii) a plurality of soluble amplification primers, each of which is exonuclease-resistant, has a 3'-extendable end, and contains a random sequence capable of hybridizing to a portion of a single-stranded circular nucleic acid template, (iii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP), and (iv) at least one divalent cation (e.g., magnesium and / or manganese) that mediates nucleotide binding and nucleotide incorporation, to form a multiple displacement amplification (MDA) reaction mixture; and (2) performing an isothermal multiple displacement amplification (MDA) reaction to generate a plurality of immobilized branched concatemers.

[0134] In some embodiments, in a multiple displacement amplification (MDA) reaction mixture, the second plurality of polymerases having strand displacement activity include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., Expedeon's MagniPhi), mutant EquiPhi29 DNA polymerase (e.g., Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., 4basebio).

[0135] In some embodiments, in a multiple displacement amplification (MDA) reaction mixture, the plurality of amplification primers comprises single-stranded nucleic acid primers having a length of about 5 to 25 nucleotides. In some embodiments, the plurality of soluble amplification primers comprises unprotected single-stranded nucleic acid primers. In some embodiments, the plurality of soluble amplification primers comprises protected single-stranded nucleic acid primers that are resistant to 3' exonuclease degradation and / or single-stranded endonuclease degradation. In some embodiments, the plurality of soluble amplification primers comprises any one or a combination of two or more of the following: 3'-terminal phosphorylation; at least two 3'-terminal nucleotides having a phosphorothioate linkage therebetween; at least one 3'-terminal nucleotide having a 2'-O-methyl moiety; and / or at least one 3'-terminal nucleotide having a 2'-fluoro base. In some embodiments, the plurality of soluble amplification primers comprises a population of primers having the same length, e.g., 6 or 9 nucleotides in length. In some embodiments, the plurality of soluble amplification primers comprises a population of primers having a mixture of different lengths, e.g., a mixture including 6-mer and 9-mer primers. In some embodiments, the plurality of soluble amplification primers comprises up to 4 6different sequences (e.g., in the case of a 6-mer) or 4 9 The primers contain a mixture of primers with random sequences, each containing a different sequence (e.g., in the case of a 9-mer).

[0136] In some embodiments, the multiple displacement amplification (MDA) reaction mixture may further comprise at least one accessory protein or enzyme, including a helicase, a single-stranded binding (SSB) protein, or a recombinase (e.g., T4 uvsX) and / or a recombinase accessory factor (e.g., T4 uvsY or T4 gp32).

[0137] In some embodiments, the isothermal multiple displacement amplification (MDA) reaction may be performed at a temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C.

[0138] After the rolling circle amplification method, a multiple displacement amplification reaction using a primase-polymerase enzyme can be carried out.The multiple displacement amplification reaction includes: (1) contacting a plurality of immobilized concatemers with (i) a second plurality of polymerases having strand displacement activity, (ii) a plurality of DNA primerase-polymerase enzymes, (iii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP), and (iv) at least one divalent cation (e.g., magnesium and / or manganese) that mediates nucleotide binding and nucleotide incorporation, thereby forming a multiple displacement amplification (MDA) reaction mixture; and (2) carrying out an isothermal multiple displacement amplification (MDA) reaction to generate a plurality of immobilized branched concatemers.In some embodiments, the multiple displacement amplification reaction is carried out without adding amplification primers (e.g., primerless reaction).

[0139] In some embodiments, in a multiple displacement amplification (MDA) reaction mixture, the second plurality of polymerases having strand displacement activity include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., Expedeon's MagniPhi), mutant EquiPhi29 DNA polymerase (e.g., Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., 4basebio).

[0140] In some embodiments, the plurality of DNA primase-polymerase enzymes includes an enzyme from Thermus thermophilus HB27 (e.g., Tth PrimPol enzyme).

[0141] In some embodiments, the multiple displacement amplification (MDA) reaction mixture further comprises at least one accessory protein or enzyme, including a helicase, a single-stranded binding (SSB) protein, or a recombinase (e.g., T4 uvsX) and / or a recombinase accessory factor (e.g., T4 uvsY or T4 gp32).

[0142] In some embodiments, the isothermal multiple displacement amplification (MDA) reaction may be performed at a temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C.

[0143] Another embodiment of the two-stage amplification method involves exposing the concatemers to a nucleic acid relaxing agent (first stage) and then performing a bending amplification reaction during the second stage. Without wishing to be bound by theory, it is hypothesized that the nucleic acid relaxing agent disrupts hydrogen bonds (e.g., denaturation) in multiple immobilized nucleic acid concatemers, thereby relaxing the structure of the nucleic acid concatemers and increasing the number of new duplex formations between immobilized surface-captured primers and portions of the nucleic acid concatemers, thereby increasing the chance of generating new concatemers from the duplexed immobilized surface-captured primers. New concatemers can be generated during the bending amplification reaction. The inclusion of a relaxing agent can cause nucleic acid denaturation without the use of denaturing temperatures or denaturing chemicals.

[0144] In some embodiments, the amplification method includes (1) performing on-support rolling circle amplification to generate a plurality of single-stranded concatemers; (2) forming a relaxation reaction mixture; (3) forming a flexure amplification reaction mixture; (4) performing a flexure amplification reaction on a support (e.g., without the addition of soluble primers) to generate a plurality of double-stranded concatemers; (5) washing; and (6) repeating steps (2) through (5) at least once.

[0145] In some embodiments, the relaxation reaction mixture of step (2) can be formed using at least one nucleic acid relaxation agent capable of disrupting hydrogen bonds in the immobilized nucleic acid concatemers. Exemplary relaxation agents include nucleic acid denaturants, chaotropic compounds, amide compounds, aprotic compounds, primary alcohols, and ethylene glycol derivatives. Chaotropic compounds include urea, guanidine hydrochloride, or guanidine thiocyanate. Amide compounds include formamide, acetamide, or N-dimethylformamide (DMF). Aprotic compounds include acetonitrile, DMSO (dimethyl sulfoxide), 1,4-dioxane, or tetrahydrofuran. Primary alcohols include 1-propanol, ethanol, or methanol. Ethylene glycol derivatives include 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, or 2-methoxyethanol. Other relaxation agents include sodium iodide, potassium iodide, and polyamines.

[0146] In some embodiments, the relaxation reaction mixture comprises any one or combination of two or more selected from the group consisting of urea, guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide, N,N-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide), 1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, 2-methoxyethanol, sodium iodide, potassium iodide, and / or polyamines.

[0147] In some embodiments, the relaxation reaction mixture comprises formamide and SSC. In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, and SSC. In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, and MES (2-(4-morpholino)-ethanesulfonic acid). In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, guanidinium hydrochloride, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, urea, and HEPES. In some embodiments, the amount of SSC in the relaxation reaction mixture can be 1x, 2x, 3x, or 4x.

[0148] In some embodiments, in forming the relaxation reaction mixture in step (2), the temperature ramp-up conditions can be performed from about 20° C. to about 70° C., the relaxation incubation conditions can be performed at a temperature of about 40-70° C., and the temperature ramp-down conditions can be performed from about 70° C. to about 20° C. One skilled in the art will recognize that the temperature ramp-up conditions, relaxation incubation temperature conditions, and temperature ramp-down conditions can be varied.

[0149] In some embodiments, in the bent amplification reaction mixture of step (3), the second plurality of polymerases having strand displacement activity include a large fragment of Bst DNA polymerase (e.g., exonuclease minus), phi29 DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca (exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., Expedeon's MagniPhi), a mutant EquiPhi29 DNA polymerase (e.g., Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., 4basebio).

[0150] In some embodiments, in the flexure amplification reaction mixture of step (2), the concentration (e.g., total concentration) of the third plurality of nucleotides can promote a nucleotide polymerization reaction, for example, the concentration (e.g., total concentration) of the third plurality of nucleotides is about 0.1 to 10 mM.

[0151] In some embodiments, the third plurality of nucleotides in the flexure amplification reaction mixture of step (2) comprises a mixture of two or more nucleotides selected from the group consisting of dATP, dGTP, dCTP, and dTTP.

[0152] In some embodiments, in the bending amplification reaction mixture of step (2), the at least one divalent cation that mediates nucleotide binding and nucleotide polymerization comprises a catalytic divalent cation. In some embodiments, the catalytic divalent cation comprises magnesium and / or manganese. The concentration of the catalytic divalent cation in the amplification reaction mixture can be about 1 to 20 mM.

[0153] In some embodiments, the flexure amplification reaction mixture of step (2) may include at least one accessory protein or enzyme, including a helicase, a single-strand binding (SSB) protein, or a recombinase (e.g., T4 uvsX) and / or a recombinase accessory factor (e.g., T4 uvsY or T4 gp32). In some embodiments, these accessory proteins may be omitted.

[0154] In some embodiments, the temperature ramp-up conditions in the flexure amplification reaction of step (4) can be performed from about 20°C to about 90°C. In some embodiments, the temperature ramp-up conditions in the flexure amplification reaction of step (4) can be performed for about 5-15 seconds, or about 15-30 seconds, or about 30-45 seconds, or about 45-60 seconds, or longer. In some embodiments, the amplification incubation conditions in the flexure amplification reaction of step (4) can be about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C, or higher. In some embodiments, the amplification incubation conditions for the flexural amplification reaction of step (4) can be about 30-45 seconds, about 45-60 seconds, about 60-75 seconds, or about 75-90 seconds, or longer. In some embodiments, the temperature ramp-down conditions for the flexural amplification reaction of step (4) can be from about 90°C to about 20°C.

[0155] In some embodiments, the temperature ramp-down conditions in the bending amplification reaction of step (4) can be carried out for about 5 to 15 seconds, about 15 to 30 seconds, about 30 to 45 seconds, or about 45 to 60 seconds, or longer. In some embodiments, in the washing step of step (5), the wash buffer comprises 1x SSC or 1x SSC containing cobalt hexamine. In some embodiments, steps (2) to (5) can be repeated at least once, up to 10 times, up to 15 times, up to 20 times, or up to 30 or more times.

[0156] Often, improvements in amplification rate, amplification specificity, and amplification efficiency can be achieved using the disclosed low nonspecific binding supports alone or in combination with formulations of amplification reaction components. In addition to including nucleotides, one or more polymerases, helicases, single-stranded binding proteins, etc. (or any combination thereof), the amplification reaction mixture can be adjusted in a variety of ways to achieve improved performance, including, but not limited to, selection of buffer type, buffer pH, organic solvent mixture, buffer viscosity, detergent and zwitterionic components, ionic strength (including adjustment of monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, other additives, etc.

[0157] The use of the disclosed low non-specific binding supports alone or in combination with optimized amplification reaction formulations can result in increased amplification rates compared to those obtained using conventional supports and amplification protocols. In some examples, the relative amplification rates that can be achieved can be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, or at least 20-fold higher than those achieved using conventional supports and amplification protocols for any of the above amplification methods.

[0158] In some examples, use of the disclosed low non-specific binding supports alone or in combination with optimized buffer formulations can result in amplification reaction times (i.e., the time required to achieve 90%, 95%, 98%, or 99% completion of the amplification reaction) of 180 minutes, 120 minutes, 90 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, or 10 seconds for any of these completion metrics.

[0159] Some low binding support surfaces disclosed herein exhibit a ratio of specific to non-specific binding for a fluorophore, such as Cy3, of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value spanning the ranges herein. Some surfaces disclosed herein exhibit a ratio of specific to non-specific fluorescent signal for a fluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value spanning the ranges herein.

[0160] In some examples, the use of the disclosed low non-specific binding supports alone or in combination with an optimized amplification buffer formulation can enable fast amplification reaction times (i.e., the time required to achieve 90%, 95%, 98%, or 99% completion of the amplification reaction) of 60, 50, 40, 30, 20, or 10 minutes or less. Similarly, the use of the disclosed low non-specific binding supports alone or in combination with an optimized buffer formulation can enable the amplification reaction to be completed in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or fewer cycles, or 30 or fewer, in some cases.

[0161] In some examples, the use of the disclosed low non-specific binding supports alone or in combination with optimized amplification reaction formulations may increase specific amplification and / or decrease non-specific amplification compared to that obtained using conventional supports and amplification protocols. In some examples, the resulting specific amplification to non-specific amplification ratio that can be achieved is at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, or 1,000:1.

[0162] In some instances, the use of low nonspecific binding supports alone or in combination with optimized amplification reaction formulations can result in increased amplification efficiencies compared to those obtained using conventional supports and amplification protocols. In some instances, amplification efficiencies that can be achieved are better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% at any of the amplification reaction times specified above.

[0163] In some examples, clonally amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) hybridized to oligonucleotide adapter or primer molecules attached to the surface of a low-binding support can range in length from about 0.02 kilobases (kb) to about 20 kb, or from about 0.1 kilobases (kb) to about 20 kb. In some examples, clonally amplified target oligonucleotide molecules can be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb in length, at least 0.2 kb in length, at least 0.3 kb in length, at least 0.4 kb in length, at least 0.5 kb in length, at least 1 kb in length, at least 2 kb in length, at least 3 kb in length, at least 4 kb in length, at least 5 kb in length, at least 6 kb in length, at least 7 kb in length, at least 8 kb in length, at least 9 kb in length, at least 10 kb in length, at least 15 kb in length, or at least 20 kb in length, or any intermediate value spanning the ranges described herein, e.g., at least 0.85 kb in length.

[0164] In some instances, clonally amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) may comprise single-stranded or double-stranded multimeric nucleic acid molecules further comprising regularly occurring repeating monomer units. In some instances, clonally amplified single-stranded or double-stranded multimeric nucleic acid molecules may be at least 0.1 kb long, at least 0.2 kb long, at least 0.3 kb long, at least 0.4 kb long, at least 0.5 kb long, at least 1 kb long, at least 2 kb long, at least 3 kb long, at least 4 kb long, at least 5 kb long, at least 6 kb long, at least 7 kb long, at least 8 kb long, at least 9 kb long, at least 10 kb long, at least 15 kb long, or at least 20 kb long, or any intermediate value spanning the ranges described herein, for example, about 2.45 kb long.

[0165] In some examples, clonally amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) can comprise single-stranded or double-stranded multimeric nucleic acid molecules comprising from about 2 to about 100 copies of regularly repeating monomeric units. In some examples, the number of copies of regularly repeating monomeric units can be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, and at least 100. In some examples, the number of copies of the regularly repeating monomeric unit can be at most 100, at most 95, at most 90, at most 85, at most 80, at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, or at most 2. Any of the lower and upper limits listed in this paragraph can be combined to form ranges within the disclosure; for example, in some examples, the number of copies of the regularly repeating monomeric unit can range from about 4 to about 60. One of skill in the art will recognize that the number of copies of the regularly repeating monomeric unit can have any value within this range, for example, about 12. Thus, in some instances, the surface density of clonally amplified target sequences, in terms of the number of copies of target sequences per unit area of ​​support surface, may exceed the surface density of oligonucleotide primers, even if the hybridization and / or amplification efficiency is less than 100%.

[0166] In some examples, the use of the disclosed low nonspecific binding supports, alone or in combination with optimized amplification reaction formulations, can result in increased clonal copy numbers compared to those obtained using conventional supports and amplification protocols. In some examples, for example, clonally amplified target (or sample) oligonucleotide molecules contain concatenated multimeric repeats of monomeric target sequences, and the clonal copy numbers can be substantially smaller than those obtained using conventional supports and amplification protocols. Thus, in some examples, the clonal copy numbers can range from about 1 molecule to about 100,000 molecules (e.g., target sequence molecules) per amplified colony. In some examples, the copy number of the clone is at least 1, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 15,000, at least The number of molecules may be at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, or at least 100,000 molecules.In some instances, the copy number of clones is up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to The copy number of a clone can be as high as 30,000, as high as 25,000, as high as 20,000, as high as 15,000, as high as 10,000, as high as 9,000, as high as 8,000, as high as 7,000, as high as 6,000, as high as 5,000, as high as 4,000, as high as 3,000, as high as 2,000, as high as 1,000, as high as 500, as high as 100, as high as 50, as high as 10, as high as 5, or as high as 1 molecule. Any of the lower and upper limits described in this paragraph can be combined to form ranges within the disclosure; for example, in some instances, the copy number of a clone can range from about 2,000 molecules to about 9,000 molecules. One of skill in the art will recognize that the copy number of a clone can have any value within this range, for example, about 2,220 molecules in some cases, or about 2 molecules in other cases.

[0167] In some instances, as described above, the amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) may contain multiple concatenated multimeric repeats of a monomeric target sequence. In some instances, the amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) contain multiple molecules, each of which contains a single monomeric target sequence. Thus, the use of the disclosed low nonspecific binding supports, alone or in combination with an optimized amplification reaction formulation, can reduce the number of nonspecific binding reactions by 1 mm. 2 Approximately 100 target sequence copies per ~1 mm 2 Approximately 1 x 10 12 In some examples, the surface density of target sequence copies can be in the range of 1 mm 2at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 110,000, at least 120,000, at least 130,000, at least 140,000, at least 150,000, at least 160,000, at least 170,000, at least 180,000, at least 190,000, at least 210,000, at least 220,000, at least 230,000, at least 240,000, at least 250,000, at least 260,000, at least 270,000, at least 280,000, at least 290,000, at least 300,000, at least 310,000, at least 320,000, at least 330,000, at least 340,000, at least 350,000, at least 360,000, at least 370,000, at least 00, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 x 10 7 , at least 5 × 10 7 , at least 1 x 10 8 , at least 5 × 10 8 , at least 1 x 10 9 , at least 5 × 10 9 , at least 1x10 10 , at least 5 × 10 10 , at least 1 x 10 11 , at least 5 × 10 11 , or at least 1 × 10 12 In some instances, the surface density of target sequence copies can be greater than 1 mm 2 Maximum of 1 x 10 12 , up to 5 × 10 11 , up to 1 × 10 11 , up to 5 × 10 10 , up to 1 × 10 10 , up to 5 × 10 9 , up to 1 × 10 9 , up to 5 × 108 , up to 1 × 10 8 , up to 5 × 10 7 , up to 1 × 10 7 , up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, up to The surface density of target sequence copies can be at most 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 copies of the target sequence. Any of the lower and upper limits set forth in this paragraph can be combined to form ranges within the disclosure; for example, in some examples, the surface density of copies of the target sequence can be greater than 1 mm 2 Approximately 1,000 target sequence copies per ~1 mm 2 Those skilled in the art will appreciate that the surface density of target sequence copies may range from about 65,000 copies per mm to about 65,000 copies per mm. 2 It is recognized that the number of copies of the target sequence per vector can be approximately 49,600.

[0168] In some instances, the use of the disclosed low non-specific binding supports alone or in combination with optimized amplification buffer formulations can reduce the 2 Approximately 100 molecules per ~1mm 2 Approximately 1 x 10 12This can result in a surface density of clonally amplified target (or sample) oligonucleotide molecules (or clusters) in the range of colonies of 1 mm. In some examples, the surface density of clonally amplified molecules is in the range of 1 mm. 2 per at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 1 00,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 x 10 7 , at least 5 × 10 7 , at least 1 x 10 8 , at least 5 × 10 8 , at least 1 x 10 9 , at least 5 × 10 9 , at least 1x10 10 , at least 5 × 10 10 , at least 1 x 10 11 , at least 5 × 10 11 , can be at least 1 x 10 molecules. In some instances, the surface density of clonally amplified molecules can be at least 1 x 10 molecules per mm. 2 Maximum of 1 x 10 12 , up to 5 × 10 11 , up to 1 × 10 11, up to 5 × 10 10 , up to 1 × 10 10 , up to 5 × 10 9 , up to 1 × 10 9 , up to 5 × 10 8 , up to 1 × 10 8 , up to 5 × 10 7 , up to 1 × 10 7 , up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000 , up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 molecules. Any of the lower and upper limits listed in this paragraph can be combined to form ranges within the disclosure; for example, in some examples, the surface density of clonally amplified molecules is greater than 1 mm 2 Approximately 5,000 molecules per ~1mm 2 Those skilled in the art will appreciate that the surface density of clonally amplified colonies may range from about 50,000 molecules per mm 2 It will be appreciated that the number of molecules per molecule may be approximately 48,800.

[0169] In some instances, the use of the disclosed low non-specific binding supports alone or in combination with optimized amplification buffer formulations can reduce the 2 Approximately 100 molecules per ~1mm 2Approximately 1 x 10 12 This can result in a surface density of clonally amplified target (or sample) oligonucleotide molecules (or clusters) in the range of colonies of 1 mm. In some examples, the surface density of clonally amplified molecules is in the range of 1 mm. 2 per at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 1 00,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 x 10 7 , at least 5 × 10 7 , at least 1 x 10 8 , at least 5 × 10 8 , at least 1 x 10 9 , at least 5 × 10 9 , at least 1x10, at least 5x10 10 , at least 1 x 10 11 , at least 5 × 10 11 , can be at least 1 x 10 molecules. In some instances, the surface density of clonally amplified molecules can be at least 1 x 10 molecules per mm. 2 Maximum of 1 x 10 12 , up to 5 × 10 11 , up to 1 × 1011 , up to 5 × 10 10 , up to 1 × 10 10 , up to 5 × 10 9 , up to 1 × 10 9 , up to 5 × 10 8 , up to 1 × 10 8 , up to 5 × 10 7 , up to 1 × 10 7 , up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000 , up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 molecules. Any of the lower and upper limits listed in this paragraph can be combined to form ranges within the disclosure; for example, in some examples, the surface density of clonally amplified molecules is greater than 1 mm 2 Approximately 5,000 molecules per ~1mm 2 Those skilled in the art will appreciate that the surface density of clonally amplified colonies may range from about 50,000 molecules per mm 2 It will be appreciated that the number of molecules per molecule may be approximately 48,800.

[0170] In some instances, the use of the disclosed low non-specific binding supports alone or in combination with optimized amplification buffer formulations can reduce the 2 Approximately 100 colonies per ~1mm2 Approximately 1 x 10 12 This can result in a surface density of clonally amplified target (or sample) oligonucleotide colonies (or clusters) in the range of 1 mm. In some examples, the surface density of clonally amplified colonies is in the range of 1 mm. 2 per at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 1 00,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 x 10 7 , at least 5 × 10 7 , at least 1 x 10 8 , at least 5 × 10 8 , at least 1 x 10 9 , at least 5 × 10 9 , at least 1x10, at least 5x10 10 , at least 1x10, at least 5x10 11 In some instances, the surface density of clonally amplified colonies may be at least 1 x 10 colonies. 2 Maximum of 1 x 10 12 , up to 5 × 10 11, up to 1 × 10 11 , up to 5 × 10 10 , up to 1 × 10 10 , up to 5 × 10 9 , up to 1 × 10 9 , up to 5 × 10 8 , up to 1 × 10 8 , up to 5 × 10 7 , up to 1 × 10 7 , up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, The surface density of clonally amplified colonies may be up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 colonies. Any of the lower and upper limits listed in this paragraph can be combined to form ranges within the disclosure; for example, in some examples, the surface density of clonally amplified colonies is greater than or equal to 100 colonies per mm. 2 Approximately 5,000 colonies per ~1mm 2 Those skilled in the art will appreciate that the surface density of clonally amplified colonies may range from about 50,000 colonies per mm 2 It is recognized that the number of colonies per culture may be approximately 48,800.

[0171] In some cases, the use of a low non-specific binding support alone or in combination with an optimized amplification reaction formulation can result in signals from amplified, labeled nucleic acid populations (e.g., fluorescent signals) having a coefficient of variation of 50% or less, e.g., 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less than 5%.

[0172] In some cases, the support surfaces and methods disclosed herein allow for amplification at elevated extension temperatures such as 15°C, 20°C, 25°C, 30°C, 40°C, or higher, or, for example, about 21°C or 23°C.

[0173] In some cases, use of the support surfaces and methods disclosed herein allows for simplified amplification reactions, for example, in some cases, amplification reactions are performed using no more than 1, 2, 3, 4, or 5 separate reagents.

[0174] In some cases, the use of the support surfaces and methods disclosed herein allows for the use of simplified temperature profiles during amplification, such that reactions are carried out at temperatures ranging from as low as 15°C, 20°C, 25°C, 30°C, or 40°C, to as high as 40°C, 45°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, or greater than 80°C, e.g., in the range of 20°C to 65°C.

[0175] Amplification reactions can also be performed with lower amounts of template (e.g., target or sample molecules), e.g., 1 pM, 2 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 90 pM, The improved method is such that 0 pM, 1,000 pM, 2,000 pM, 3,000 pM, 4,000 pM, 5,000 pM, 6,000 pM, 7,000 pM, 8,000 pM, 9,000 pM, 10,000 pM, or greater than 10,000 pM, such as 500 nM, of sample is sufficient to produce a discernible signal on the surface. In an exemplary embodiment, an input of about 100 pM is sufficient to generate a signal for reliable signal determination.

[0176] The disclosed solid-phase nucleic acid amplification reaction formulations and low non-specific binding supports can be used in any of a variety of nucleic acid analysis applications, such as nucleic acid base discrimination, nucleic acid base typing, nucleic acid base calling, nucleic acid detection applications, nucleic acid sequencing applications, and nucleic acid-based (genetic and genomic) diagnostic applications. In many of these applications, fluorescence imaging techniques can be used to monitor hybridization, amplification, and / or sequencing reactions performed on the low-binding supports.

[0177] Fluorescence imaging can be performed using any of a variety of fluorophores, fluorescence imaging techniques, and fluorescence imaging devices known to those skilled in the art. Examples of suitable fluorescent dyes that can be used (e.g., by conjugation to nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and their derivatives, including cyanine derivatives such as cyanine dye-3 (Cy3), cyanine dye-5 (Cy5), and cyanine dye-7 (Cy7). Examples of fluorescence imaging techniques that can be used include, but are not limited to, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, and the like. Examples of fluorescence imaging devices that can be used include, but are not limited to, a fluorescence microscope equipped with an image sensor or camera, a confocal fluorescence microscope, a two-photon fluorescence microscope, or a custom instrument that includes an appropriate selection of light sources, lenses, mirrors, prisms, dichroic reflectors, apertures, and an image sensor or camera. A non-limiting example of a fluorescence microscope equipped to acquire images of the disclosed low-binding support surfaces and clonally amplified colonies (or clusters) of target nucleic acid sequences hybridized thereto is an Olympus IX83 inverted fluorescence microscope (equipped with a 20x, 0.75 NA, 532 nm light source, a set of bandpass and dichroic mirror filters optimized for 532 nm long-pass excitation and Cy3 fluorescence emission filters, a Semrock 532 nm dichroic reflector, and a camera (Andor sCMOS, Zyla 4.2) with excitation light intensity adjusted to avoid signal saturation). Often, the support surface may be immersed in a buffer solution (e.g., 25 mM ACES, pH 7.4 buffer) while acquiring images.

[0178] In some examples, the performance of nucleic acid hybridization and / or amplification reactions using the disclosed reaction formulations and low nonspecific binding supports can be evaluated using fluorescence imaging techniques, where the image contrast-to-noise ratio (CNR) provides an important metric for evaluating amplification specificity and nonspecific binding on the support. CNR is generally defined as follows: CNR = (signal - background) / noise. The background term is typically considered to be the signal measured for the interstitial region surrounding a specific feature (diffraction-limited spot, DLS) within a specified region of interest (ROI). While signal-to-noise ratio (SNR) is often considered a benchmark of overall signal quality, improvements in CNR can be shown to provide significant advantages over SNR as a benchmark of signal quality in applications requiring rapid image acquisition (e.g., sequencing applications where cycle time must be minimized), as shown in the following examples. Surfaces of the present disclosure are also provided in co-pending International Application No. PCT / US2019 / 061556, which is incorporated herein by reference in its entirety.

[0179] In most ensemble-based sequencing approaches, the background term is typically measured as the signal associated with the "stromal" region. "Stroma" background (B inter ) plus the "intra-organizational" background (B intra ) are present within the region occupied by the amplified DNA colonies. The combination of these two background signals indicates the achievable CNR, which subsequently directly impacts the optical equipment requirements, architecture cost, reagent cost, runtime, cost per genome, and ultimately, accuracy and data quality for circular array-based sequencing applications. interBackground signals arise from a variety of sources; some examples include autofluorescence from the expendable flow cell, nonspecific adsorption of detection molecules resulting in false fluorescent signals that can obscure signals from the ROI, and the presence of nonspecific DNA amplification products (e.g., those resulting from primer dimers). In a typical next-generation sequencing (NGS) application, this background signal in the current field of view (FOV) is averaged over time and subtracted. The signal arising from individual DNA colonies (i.e., the (S)-B in the FOV) is then subtracted. inter ) provide recognizable features that can be classified. In some instances, background (B intra ) may contribute to confounding fluorescent signals that are not specific to the target of interest but are present in the same ROI, thus making them much more difficult to average and subtract.

[0180] As demonstrated in the examples below, performing nucleic acid amplification on low binding substrates of the present disclosure results in a reduction in non-specific binding, resulting in a B inter Background signal can be reduced, leading to improvements in specific nucleic acid amplification, and nonspecific amplification, which can affect background signal arising from both interstitial and intratissue regions, can be reduced. In some examples, the disclosed low-binding support surfaces, optionally used in combination with the disclosed hybridization and / or amplification reaction formulations, can lead to a 2-, 5-, 10-, 100-, or 1000-fold improvement in CNR compared to that achieved using conventional supports and hybridization, amplification, and / or sequencing protocols. While described herein in the context of using fluorescent imaging as a readout or detection mode, the same principles apply equally to the use of the disclosed low-nonspecific binding supports and nucleic acid hybridization and amplification formulations for other detection modes, including optical and non-optical detection modes.

[0181] The disclosed low-binding supports, optionally used in combination with the disclosed hybridization and / or amplification protocols, result in solid-phase reactions that (i) exhibit negligible non-specific binding of proteins and other reaction components (thus minimizing substrate background), (ii) exhibit negligible non-specific nucleic acid amplification products, and (iii) provide tunable nucleic acid amplification reactions.

[0182] The present disclosure provides a method for analyzing nucleic acids in a cellular or spatially addressable manner, the method comprising: (a) providing a support (e.g., FIG. 2 ) comprising a low non-specific binding coating on which a plurality of capture oligonucleotides and a plurality of circularization oligonucleotides are immobilized, wherein the plurality of capture oligonucleotides comprise: (i) a target capture region that hybridizes to at least a portion of a target nucleic acid molecule, (ii) a universal sequence region that includes a spatial barcode sequence, (iii) a circularization anchor sequence, and (iv) a cleavable region, wherein the plurality of circularization oligonucleotides comprise: (i) a homopolymer region, (ii) a universal sequence region that includes a sequencing primer binding sequence, and (iii) a circularization anchor binding sequence, and wherein the low non-specific binding coating comprises at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less.

[0183] In some embodiments, the low non-specific binding coating of step (a) exhibits a low background fluorescent signal or a high contrast-to-noise ratio (CNR) compared to surfaces known in the art. In some embodiments, the low non-specific binding coating exhibits a low background fluorescent signal or a high contrast-to-noise ratio (CNR) of about 0.25 molecules / μm 2In some embodiments, when using a fluorescent imaging system under non-signal saturating conditions, fluorescent images of surface coatings bearing multiple clonally amplified clusters of nucleic acids exhibit a contrast-to-noise ratio (CNR) of at least 20, and a high contrast-to-noise ratio (CNR) of at least 50.

[0184] In some embodiments, the immobilized capture oligonucleotide of step (a) can include any combination of the following: (i) a target capture region that hybridizes to at least a portion of a target nucleic acid molecule, (ii) a universal sequence region that includes a spatial barcode sequence, (iii) a circularization anchor sequence that binds to a portion of the circularization oligonucleotide, and / or (iv) a cleavable region.

[0185] In some embodiments, the target capture region of the immobilized capture oligonucleotide in step (a) comprises a target-specific sequence or a random sequence.

[0186] In some embodiments, the immobilized circularization oligonucleotide of step (a) may comprise any combination of (i) a homopolymer region, (ii) a universal sequence region comprising a sequencing primer binding sequence, and / or (iii) a circularization anchor binding sequence that binds to the circularization anchor sequence of the capture oligonucleotide.

[0187] The method for analyzing nucleic acids further includes (b) contacting the cellular biological sample with a low non-specific binding coating in the presence of a high-efficiency hybridization buffer under conditions suitable to promote the transfer of target nucleic acid molecules from the cellular biological sample to one of the immobilized capture oligonucleotides, thereby forming an immobilized target nucleic acid duplex, wherein the target nucleic acid molecule is immobilized to the low non-specific binding coating in a manner that preserves the spatial location information of the target nucleic acid molecule in the cellular biological sample, wherein the target nucleic acid comprises DNA or RNA (e.g., Figure 7).

[0188] In some embodiments, the cellular biological sample of step (b) comprises a cellular biological sample that is fresh, frozen, fresh frozen, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE).

[0189] In some embodiments, the cellular biological sample of step (b) is subjected to a permeabilization reaction to promote the transfer of cellular nucleic acid molecules (e.g., DNA and / or RNA) containing the target nucleic acid molecule from the cellular biological sample to one of the immobilized capture oligonucleotides.

[0190] In some embodiments, the high efficiency hybridization buffer of step (b) comprises: (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 and present in the high efficiency hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the high efficiency hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding.

[0191] In some embodiments, the high-efficiency hybridization buffer of step (b) comprises: (i) the first polar aprotic solvent comprises 25-50% acetonitrile by volume of the high-efficiency hybridization buffer; (ii) the second polar aprotic solvent comprises 5-10% formamide by volume of the high-efficiency hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises 5-35% polyethylene glycol (PEG) by volume of the high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0192] In some embodiments, the high-efficiency hybridization buffer in step (b) promotes high stringency (e.g., specificity), speed, and effectiveness of nucleic acid hybridization, increasing the efficiency of subsequent amplification and sequencing steps. In some embodiments, the high-efficiency hybridization buffer significantly shortens nucleic acid hybridization time and reduces sample input requirements. Nucleic acid annealing can be performed under isothermal conditions, eliminating the need for a cooling step for annealing.

[0193] The method for analyzing nucleic acids further comprises (c) performing a primer extension reaction on the immobilized nucleic acid duplex using the hybridized target nucleic acid molecule as a template, thereby forming an immobilized target extension product. In some embodiments, the primer extension reaction comprises contacting the immobilized nucleic acid duplex with a plurality of nucleotides and a polymerase. In some embodiments, the polymerase comprises E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase.

[0194] In some embodiments, the primer extension reaction of step (c) can be a reverse transcription reaction comprising (i) a reverse transcriptase, (ii) a plurality of nucleotides, and (iii) a plurality of reverse transcriptase primers. In some embodiments, the reverse transcription reaction of step (a) comprises a plurality of nucleotides and an enzyme having reverse transcription activity, including reverse transcriptase from AMV (avian myeloblastosis virus), M-MLV (Moloney murine leukemia virus), or HIV (human immunodeficiency virus). In some embodiments, the reverse transcriptase is a MultiScribe (商標) , ThermoScript (商標) , or ArrayScript (商標) In some embodiments, the reverse transcriptase comprises a Superscript I, II, III, or IV enzyme. In some embodiments, the reverse transcription reaction may include an RNase inhibitor.

[0195] The method for analyzing nucleic acids further includes (d) performing a non-template tailing reaction on the immobilized target extension products under conditions suitable for adding a homopolymeric tail to the immobilized target extension products, thereby forming immobilized tailed target extension products (e.g., Figure 27). In some embodiments, the non-template tailing reaction includes contacting the immobilized target extension products with a plurality of nucleotides and a polymerase, wherein the polymerase is Taq polymerase, Tfi DNA polymerase, 3' exonuclease minus large (Klenow) fragment, or 3' exonuclease minus-T4 polymerase.

[0196] The method for analyzing nucleic acids further comprises (e) cleaving the immobilized tailed target extension product to release the immobilized tailed target extension product from the low-binding coating, thereby forming a soluble tailed target extension product. In some embodiments, the cleavable region can be cleaved using an enzyme, a chemical compound, light, or heat.

[0197] The method for analyzing nucleic acids further includes (f) binding the soluble tailed target extension product to one of the immobilized circularization oligonucleotides under conditions suitable for hybridizing the added homopolymeric tail of the soluble tailed target extension product to the homopolymeric region of the immobilized circularization oligonucleotide and suitable for hybridizing the circularization anchor sequence of the soluble tailed target extension product to the circularization anchor binding sequence of the immobilized circularization oligonucleotide, thereby forming an open circular target extension product having a gap and / or a nick, such that the immobilized circularization oligonucleotide functions as a splint molecule to promote circularization of the soluble tailed target extension product (e.g., FIG. 27).

[0198] The method for analyzing nucleic acids further includes (g) performing a gap-filling primer extension reaction to close gaps (if present) and a ligation reaction on the open circular target extension product to close nicks (if present), thereby forming covalently closed circular target extension products that are hybridized to an immobilized circularizing oligonucleotide, wherein the immobilized circularizing oligonucleotide comprises a homopolymer region with a 3' extendable end (e.g., Figure 27).

[0199] In some embodiments, forming a covalently closed circular target extension product in step (g) comprises a polymerase-mediated gap-filling reaction, an enzymatic ligation reaction, or a polymerase-mediated gap-filling reaction and an enzymatic ligation reaction. In some embodiments, the polymerase-mediated gap-filling reaction comprises contacting the open circular target molecule with a DNA polymerase and a plurality of nucleotides, wherein the DNA polymerase comprises E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase. In some embodiments, the enzymatic ligation reaction comprises the use of a ligase enzyme, including T3, T4, T7, or Taq DNA ligase enzyme. In some embodiments, forming a covalently closed circular target molecule comprises contacting the open circular target molecule with CircLigase or CircLigase II enzyme.

[0200] The method for analyzing nucleic acids further includes (h) performing a rolling circle amplification reaction using the 3' extendable end of the homopolymer region of the immobilized circularized oligonucleotide under conditions suitable to form an immobilized nucleic acid concatemer molecule having a tandem repeat region comprising a sequencing primer binding sequence, a target sequence, and a spatial barcode sequence (e.g., Figure 27).

[0201] In some embodiments, the rolling circle amplification reaction of step (h) comprises contacting the covalently closed circularized padlock probe (e.g., the circularized nucleic acid template molecule) with an amplification primer, a DNA polymerase, a plurality of nucleotides, and at least one catalytic divalent cation under conditions suitable to generate at least one nucleic acid concatemer, wherein the at least one catalytic divalent cation comprises magnesium or manganese.

[0202] In some embodiments, the rolling circle amplification reaction of step (h) comprises: (1) contacting the covalently closed circularized padlock probe (e.g., the circularized nucleic acid template molecule) with an amplification primer, a DNA polymerase, a plurality of nucleotides, and at least one non-catalytic divalent cation that does not promote polymerase-catalyzed nucleotide incorporation into the amplification primer, wherein the non-catalytic divalent cation comprises strontium or barium; and (2) contacting the covalently closed circularized padlock probe with at least one catalytic divalent cation, wherein the at least one catalytic divalent cation comprises magnesium or manganese, under conditions suitable to produce at least one nucleic acid concatemer.

[0203] In some embodiments, the rolling circle amplification reaction of step (h) is carried out at a constant temperature (eg, isothermal) ranging from room temperature to about 50°C, or from room temperature to about 65°C.

[0204] In some embodiments, the rolling circle amplification reaction of step (h) can be carried out in the presence of a plurality of compaction oligonucleotides that compress the size and / or shape of the immobilized concatemers to form small immobilized nanoballs.

[0205] In some embodiments, the rolling circle amplification reaction of step (h) comprises a DNA polymerase with strand displacement activity selected from the group consisting of phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., Expedeon's MagniPhi), or mutant EquiPhi29 DNA polymerase (e.g., Thermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g., 4basebio).

[0206] In some embodiments, the rolling circle amplification reaction may be followed by a multiple displacement amplification (MDA) reaction. In some embodiments, the method further comprises, before step (f), carrying out a multiple displacement amplification (MDA) reaction, wherein the MDA reaction comprises contacting at least one nucleic acid concatemer with at least one amplification primer comprising a random sequence, a DNA polymerase having strand displacement activity, a plurality of nucleotides, and a catalytic divalent cation comprising magnesium or manganese.

[0207] In some embodiments, a multiple displacement amplification (MDA) reaction can be performed after the rolling circle amplification reaction. In some embodiments, the method further includes a step of performing a multiple displacement amplification (MDA) reaction before step (f), wherein the MDA reaction includes contacting at least one nucleic acid concatemer with a DNA primerase-polymerase enzyme, a DNA polymerase with strand displacement activity, a plurality of nucleotides, and catalytic divalent cations, including magnesium or manganese. In some embodiments, the DNA primerase-polymerase includes an enzyme with DNA polymerase and RNA primase activity. The DNA primerase-polymerase enzyme can utilize deoxyribonucleotide triphosphates to synthesize DNA primers on a single-stranded DNA template in a template sequence-dependent manner, and can extend the primer strand via nucleotide polymerization (e.g., primer extension) in the presence of catalytic divalent cations (e.g., magnesium and / or manganese). DNA primerase-polymerase includes enzymes that are members of primases, such as DnaG (e.g., bacterial) and primases, such as AEP (archaeal and eukaryotic). An exemplary DNA primase-polymerase enzyme is Tth PrimPol from Thermus thermophilus HB27.

[0208] In certain embodiments, a rolling circle amplification reaction can be followed by a flexure amplification reaction instead of a multiple displacement amplification (MDA) reaction. In some embodiments, the flexural amplification reaction comprises: (a) forming a nucleic acid relaxation reaction mixture by contacting nucleic acid concatemers with one compound or a combination of two or more compounds selected from the group consisting of formamide, acetonitrile, ethanol, guanidine hydrochloride, urea, potassium iodide, and / or a polyamine, wherein forming the nucleic acid relaxation reaction mixture is carried out using a temperature ramp-up, a relaxation incubation temperature, and a temperature ramp-down; (b) washing the relaxed concatemers; (c) forming a flexural amplification reaction mixture by contacting the relaxed concatemers (in the absence of added amplification primers) with a strand-displacing DNA polymerase, a plurality of nucleotides, and catalytic divalent cations to generate double-stranded concatemers, wherein forming the flexural amplification reaction mixture is carried out using a temperature ramp-up, a flexural incubation temperature, and a temperature ramp-down; (d) washing the double-stranded concatemers; and (e) repeating steps (a)-(d) at least once.

[0209] Methods for capturing and analyzing RNA. Provided herein are methods for analyzing nucleic acids (e.g., RNA), the methods comprising: (a) providing a support (e.g., FIGS. 4 and 28) comprising a low non-specific binding coating to which a plurality of capture oligonucleotides are immobilized, wherein the plurality of capture oligonucleotides comprise: (i) a target capture region that hybridizes to at least a portion of a target nucleic acid molecule; (ii) a universal sequence region that comprises a spatial barcode sequence and, optionally, a sample barcode sequence; and (iii) a cleavable region, wherein the low non-specific binding coating comprises at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less. In some embodiments, the target capture region comprises a homopolymer region having a poly-T sequence.

[0210] In some embodiments, the low non-specific binding coating of step (a) exhibits a low background fluorescent signal or a high contrast-to-noise ratio (CNR) compared to surfaces known in the art. In some embodiments, the low non-specific binding coating exhibits a low background fluorescent signal or a high contrast-to-noise ratio (CNR) of about 0.25 molecules / μm 2 In some embodiments, a fluorescent image of a surface coating bearing multiple clonally amplified clusters of nucleic acids exhibits a contrast-to-noise ratio (CNR) of at least 20, or a contrast-to-noise ratio (CNR) of at least 50 or higher, when using a fluorescent imaging system under non-signal-saturating conditions.

[0211] The method for analyzing nucleic acids further includes (b) contacting the cellular biological sample with a low non-specific binding coating in the presence of a high-efficiency hybridization buffer under conditions suitable for promoting the transfer of the target nucleic acid molecule from the cellular biological sample to one of the immobilized capture oligonucleotides, thereby forming an immobilized target nucleic acid duplex, wherein the target nucleic acid molecule is immobilized on the low non-specific binding coating in a manner that preserves the spatial location information of the target nucleic acid molecule in the cellular biological sample, and wherein the target nucleic acid comprises a poly-A RNA molecule. In some embodiments, the target capture region having a poly-T sequence can hybridize to poly-A RNA (e.g., Figure 28).

[0212] In some embodiments, the cellular biological sample of step (b) comprises a cellular biological sample that is fresh, frozen, fresh frozen, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE).

[0213] In some embodiments, the cellular biological sample of step (b) is subjected to a permeabilization reaction to promote the transfer of cellular nucleic acid molecules (e.g., DNA and / or RNA) containing the target nucleic acid molecule from the cellular biological sample to one of the immobilized capture oligonucleotides.

[0214] In some embodiments, the high efficiency hybridization buffer of step (b) comprises: (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the high efficiency hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the high efficiency hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding.

[0215] In some embodiments, the high-efficiency hybridization buffer of step (b) comprises: (i) the first polar aprotic solvent comprises 25-50% acetonitrile by volume of the high-efficiency hybridization buffer; (ii) the second polar aprotic solvent comprises 5-10% formamide by volume of the high-efficiency hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises 5-35% polyethylene glycol (PEG) by volume of the high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0216] In some embodiments, the high-efficiency hybridization buffer in step (b) promotes high stringency (e.g., specificity), speed, and effectiveness of nucleic acid hybridization, increasing the efficiency of subsequent amplification and sequencing steps. In some embodiments, the high-efficiency hybridization buffer significantly shortens nucleic acid hybridization time and reduces sample input requirements. Nucleic acid annealing can be performed under isothermal conditions, eliminating the need for a cooling step for annealing.

[0217] The method for analyzing nucleic acids further includes (c) performing a reverse transcription reaction on the immobilized duplex using the hybridized target nucleic acid molecule as a template, thereby forming an immobilized target extension product (e.g., cDNA) (e.g., Figure 28).

[0218] In some embodiments, the reverse transcription reaction of step (c) comprises (i) a reverse transcriptase, (ii) a plurality of nucleotides, and (iii) a plurality of reverse transcriptase primers. In some embodiments, the reverse transcription reaction of step (a) comprises a plurality of nucleotides and an enzyme having reverse transcription activity, including a reverse transcriptase from AMV (avian myeloblastosis virus), M-MLV (Moloney murine leukemia virus), or HIV (human immunodeficiency virus). In some embodiments, the reverse transcriptase is a MultiScribe (商標) , ThermoScript (商標) , or ArrayScript (商標) In some embodiments, the reverse transcriptase comprises Superscript I, II, III, or IV enzyme. In some embodiments, the reverse transcription reaction may include an RNase inhibitor.

[0219] In some embodiments, the method for analyzing nucleic acids (e.g., RNA) further comprises (d) adding a nucleic acid adapter to the non-immobilized end of the immobilized target extension product, thereby generating an adapted, immobilized, double-stranded target extension product (Figure 28). The nucleic acid adapter can be single-stranded or double-stranded. The nucleic acid adapter can be added using RNA ligase or DNA ligase. A single-stranded adapter can be added to the 3' end of one strand of the immobilized target extension product using T4 RNA ligase, KOD ligase, Circligase, or Splint® ligase. A double-stranded adapter can be added to the non-immobilized end of the immobilized target extension product using T4 DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9 degrees N) DNA ligase, Ampligase, or Splint® ligase. The adapted, immobilized, double-stranded target extension products comprise an immobilized capture oligonucleotide (extended via reverse transcription and adapted) that is hybridized to a target nucleic acid molecule. In some embodiments, the adapted, immobilized, double-stranded target extension products are exposed to conditions that separate / remove or degrade the target nucleic acid molecule such that the adapted, immobilized, single-stranded target extension products remain attached to the surface.

[0220] The method for analyzing nucleic acids further includes (e) contacting the immobilized, adapted, single-stranded target extension products with a plurality of soluble circularizing oligonucleotides to form target circularized duplexes, wherein the soluble circularizing oligonucleotides each comprise (i) an adapter binding region, (ii) a homopolymer region, (iii) an anchor region, and (iv) an anchor moiety, wherein the homopolymer region comprises a poly-T sequence capable of hybridizing to a poly-A region of the target nucleic acid molecule, and wherein the contacting is performed under conditions suitable to immobilize at least one of the soluble circularizing oligonucleotides to the low nonspecific binding coating in proximity to the immobilized, adapted, single-stranded target extension products (e.g., Figure 28).

[0221] In some embodiments, the adapter binding region comprises a sequencing primer binding region. In some embodiments, the adapter binding region comprises an amplification primer binding region. In some embodiments, the homopolymer region comprises a polynucleotide sequence selected from the group consisting of poly-T, poly-dT, poly-A, poly-dA, poly-C, poly-dC, poly-G, and poly-dG. In some embodiments, the homopolymer region comprises a poly-T or poly-dT sequence. In some embodiments, the anchor region can be attached to a surface, thereby generating an immobilized circularized oligonucleotide. The adapter binding region of the immobilized circularized oligonucleotide can hybridize to the attached adapter sequence of the immobilized single-stranded target extension product to which the adapter has been added. The homopolymer region of the immobilized circularized oligonucleotide can hybridize to the homopolymer region (e.g., poly-A) of the immobilized single-stranded target extension product to which the adapter has been added.

[0222] The method for analyzing nucleic acids further includes (f) cleaving the cleavable region of the target circularized duplex to release the immobilized end from the low nonspecific binding coating to generate a released target extension product, wherein the added adapter region of the released target extension product remains hybridized to the adapter binding region of the immobilized circularized oligonucleotide, and the homopolymer region of the released target extension product can rehybridize with the homopolymer region of the immobilized circularized oligonucleotide, thereby forming an open circular target circularized duplex having a gap and / or a nick, such that the immobilized circularized oligonucleotide functions as a splint molecule to promote circularization of the released target extension product (e.g., Figure 8). In some embodiments, the cleavable region can be cleaved using an enzyme, a chemical compound, light, or heat. In some embodiments, the added adapter region of the released target extension product remains hybridized to the immobilized single-stranded target extension product to which the adapter has been added. In some embodiments, the homopolymer region of the released target extension product can rehybridize with the homopolymer region of the immobilized circularization oligonucleotide to form a target extension product to which a gapped or nick-containing open circularization adaptor has been added. The immobilized circularization oligonucleotide can function as a splint molecule to promote circularization of the released target extension product, and as the homopolymer region and adaptor binding region of the immobilized circularization oligonucleotide can hybridize to the termini of the released target extension product.

[0223] The method for analyzing nucleic acids further includes (g) performing a gap-filling primer extension reaction to close gaps (if present) and a ligation reaction on the open circular target circularized duplex to close nicks (if present), thereby forming covalently closed circular target extension products that are hybridized to an immobilized circularized oligonucleotide, wherein the immobilized circularized oligonucleotide comprises an adapter binding region with a 3' extendable end (e.g., Figure 28).

[0224] In some embodiments, forming a covalently closed circular target extension product in step (g) comprises a polymerase-mediated gap-filling reaction, an enzymatic ligation reaction, or a polymerase-mediated gap-filling reaction and an enzymatic ligation reaction. In some embodiments, the polymerase-mediated gap-filling reaction comprises contacting the open circular target molecule with a DNA polymerase and a plurality of nucleotides, wherein the DNA polymerase comprises E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase. In some embodiments, the enzymatic ligation reaction comprises the use of a ligase enzyme, including T3, T4, T7, or Taq DNA ligase enzyme. In some embodiments, forming a covalently closed circular target molecule comprises contacting the open circular target molecule with CircLigase or CircLigase II enzyme.

[0225] The method for analyzing nucleic acids further includes (h) performing a rolling circle amplification reaction by extending the 3' extendable end of the adapter binding region of the immobilized circularized oligonucleotide under conditions suitable to form an immobilized nucleic acid concatemer molecule having a tandem repeat region comprising a sequencing primer binding sequence, a target sequence, and a spatial barcode sequence (e.g., Figure 28).

[0226] In some embodiments, the rolling circle amplification reaction of step (h) comprises contacting the covalently closed circularized padlock probe (e.g., the circularized nucleic acid template molecule) with an amplification primer, a DNA polymerase, a plurality of nucleotides, and at least one catalytic divalent cation under conditions suitable to generate at least one nucleic acid concatemer, wherein the at least one catalytic divalent cation comprises magnesium or manganese.

[0227] In some embodiments, the rolling circle amplification reaction of step (h) comprises: (1) contacting the covalently closed circularized padlock probe (e.g., the circularized nucleic acid template molecule) with an amplification primer, a DNA polymerase, a plurality of nucleotides, and at least one non-catalytic divalent cation that does not promote polymerase-catalyzed nucleotide incorporation into the amplification primer, wherein the non-catalytic divalent cation comprises strontium or barium; and (2) contacting the covalently closed circularized padlock probe with at least one catalytic divalent cation, wherein the at least one catalytic divalent cation comprises magnesium or manganese, under conditions suitable to produce at least one nucleic acid concatemer.

[0228] In some embodiments, the rolling circle amplification reaction of step (h) is carried out at a constant temperature (eg, isothermal) ranging from room temperature to about 50°C, or from room temperature to about 65°C.

[0229] In some embodiments, the rolling circle amplification reaction of step (h) can be carried out in the presence of a plurality of compaction oligonucleotides that compress the size and / or shape of the immobilized concatemers to form small immobilized nanoballs.

[0230] In some embodiments, the rolling circle amplification reaction of step (h) comprises a DNA polymerase with strand displacement activity selected from the group consisting of phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., Expedeon's MagniPhi), or mutant EquiPhi29 DNA polymerase (e.g., Thermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g., 4basebio).

[0231] In some embodiments, the rolling circle amplification reaction may be followed by a multiple displacement amplification (MDA) reaction. In some embodiments, the method further comprises, before step (f), carrying out a multiple displacement amplification (MDA) reaction, wherein the MDA reaction comprises contacting at least one nucleic acid concatemer with at least one amplification primer comprising a random sequence, a DNA polymerase having strand displacement activity, a plurality of nucleotides, and a catalytic divalent cation comprising magnesium or manganese.

[0232] In some embodiments, a multiple displacement amplification (MDA) reaction can be performed after the rolling circle amplification reaction. In some embodiments, the method further includes a step of performing a multiple displacement amplification (MDA) reaction before step (f), wherein the MDA reaction includes contacting at least one nucleic acid concatemer with a DNA primerase-polymerase enzyme, a DNA polymerase with strand displacement activity, a plurality of nucleotides, and catalytic divalent cations, including magnesium or manganese. In some embodiments, the DNA primerase-polymerase includes an enzyme with DNA polymerase and RNA primase activity. The DNA primerase-polymerase enzyme can utilize deoxyribonucleotide triphosphates to synthesize DNA primers on a single-stranded DNA template in a template sequence-dependent manner, and can extend the primer strand via nucleotide polymerization (e.g., primer extension) in the presence of catalytic divalent cations (e.g., magnesium and / or manganese). DNA primerase-polymerase includes enzymes that are members of primases, such as DnaG (e.g., bacterial) and primases, such as AEP (archaeal and eukaryotic). An exemplary DNA primase-polymerase enzyme is Tth PrimPol from Thermus thermophilus HB27.

[0233] In certain embodiments, a rolling circle amplification reaction can be followed by a flexure amplification reaction instead of a multiple displacement amplification (MDA) reaction. In some embodiments, the flexural amplification reaction comprises: (a) forming a nucleic acid relaxation reaction mixture by contacting nucleic acid concatemers with one compound or a combination of two or more compounds selected from the group consisting of formamide, acetonitrile, ethanol, guanidine hydrochloride, urea, potassium iodide, and / or a polyamine, wherein forming the nucleic acid relaxation reaction mixture is carried out using a temperature ramp-up, a relaxation incubation temperature, and a temperature ramp-down; (b) washing the relaxed concatemers; (c) forming a flexural amplification reaction mixture by contacting the relaxed concatemers (in the absence of added amplification primers) with a strand-displacing DNA polymerase, a plurality of nucleotides, and catalytic divalent cations to generate double-stranded concatemers, wherein forming the flexural amplification reaction mixture is carried out using a temperature ramp-up, a flexural incubation temperature, and a temperature ramp-down; (d) washing the double-stranded concatemers; and (e) repeating steps (a)-(d) at least once.

[0234] Methods and compositions for nucleic acid determination. Provided herein are methods for analyzing nucleic acids, comprising determining the sequence of a target nucleic acid (e.g., immobilized concatemer) referred to herein. Sequencing can be targeted sequencing. Sequencing can be whole genome sequencing. Whole genome sequencing can include massively parallel sequencing ("next generation sequencing" or "second generation sequencing"). In some embodiments, sequencing is performed by ligation. In some embodiments, sequencing involves continuous monitoring of the incorporation of labeled nucleotides in growing polynucleotide molecules. Sequencing can be performed by massively parallel array sequencing or single molecule sequencing.

[0235] The method for analyzing nucleic acids further includes (i) sequencing at least a portion of the immobilized nucleic acid concatemers, including sequencing the target sequence and the spatial barcode sequence, to determine the spatial location of the target nucleic acid in the cellular biological sample.

[0236] In some embodiments, the sequencing step of step (i) comprises sequencing a 1.0 mm 2 The method includes sequencing at least a portion of the nucleic acid concatemers using an optical imaging system comprising a field of view (FOV) greater than 100 nm.

[0237] In some embodiments, the sequencing step (i) includes placing the cellular biological sample in a flow cell having walls (e.g., a top or first wall and a bottom or second wall) and a gap therebetween, where the gap can be filled with a biological fluid, and where the flow cell is placed in a fluorescent optical imaging system. The cellular biological sample has a thickness that, when using conventional imaging systems, would require the imaging system to focus separately on the first and second surfaces of the flow cell. For improved imaging of nucleic acid sequencing reactions from the cellular biological sample, the flow cell may be placed in a high-performance fluorescent imaging system, where the high-performance fluorescent imaging system includes two or more tube lenses designed to provide optimal imaging performance of the first and second surfaces of the flow cell at two or more fluorescent wavelengths. In some embodiments, the high-performance imaging system further includes a focusing device configured to refocus the optical system while acquiring images of the first and second surfaces of the flow cell. In some embodiments, the high-performance imaging system is configured to image two or more fields of view on at least one of the first flow cell surface or the second flow cell surface.

[0238] In some embodiments, the sequencing step of step (i) comprises contacting a plurality of nucleic acid concatemers with a plurality of sequencing primers, a plurality of polymerases, and a plurality of multivalent molecules, wherein each of the multivalent molecules comprises two or more copies of a nucleotide moiety connected to a core via a linker.

[0239] In some embodiments, the multivalent molecule comprises multiple nucleotides attached to a particle (or core), such as a polymer, branched polymer, dendrimer, micelle, liposome, microparticle, nanoparticle, quantum dot, or other suitable particle known in the art.

[0240] In some embodiments, the multivalent molecule comprises (a) a core and (b) a plurality of nucleotide arms, the plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms. In some embodiments, the spacer is attached to the linker. In some embodiments, 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, wherein 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, wherein both linker chains have 2 to 6 subunits, and optionally, the linker comprises an aromatic moiety.

[0241] In some embodiments, the multivalent molecule comprises a core to which are attached multiple nucleotide arms, wherein the multiple nucleotide arms have the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0242] In some embodiments, the multivalent molecule further comprises a plurality of multivalent molecules comprising a mixture of multivalent molecules having two or more different types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0243] In some embodiments, a multivalent molecule comprises a core to which are attached multiple nucleotide arms, and wherein each nucleotide arm comprises a nucleotide unit having a chain-terminating moiety (e.g., a blocking moiety) at the sugar 2' position, the sugar 3' position, or the sugar 2' and 3' positions.

[0244] In some embodiments, the chain-terminating moiety comprises an azide group, an azido group, or an azidomethyl group. In some embodiments, the chain-terminating moiety is selected from the group consisting of 3'-deoxynucleotides, 2',3'-dideoxynucleotides, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'butyl, 3'-tertbutyl, 3'-fluorenylmethyloxycarbonyl, 3'tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino groups, 3'-phosphorothioates, and 3-O-benzyl, or derivatives thereof.

[0245] In some embodiments, the chain-terminating moiety is cleavable / removable from the nucleotide unit.

[0246] In some embodiments, the chain-terminating moiety is an azide, azido, or azidomethyl group cleavable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0247] In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, wherein the core is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[0248] In some embodiments, the core of the multivalent molecule comprises an avidin-like moiety and the core-attached moiety comprises biotin.

[0249] In some embodiments, the sequencing step of step (i) comprises: (1) contacting a plurality of nucleic acid concatemers with (i) a plurality of polymerases, (ii) at least one multivalent molecule comprising two or more copies of a nucleotide moiety connected to a core via a linker, and (iii) a plurality of sequencing primers that hybridize to a portion of the concatemers, under conditions suitable for binding at least one polymerase and at least one sequencing primer to a portion of one of the nucleic acid concatemer molecules and suitable for binding at least one of the nucleotide moieties of the multivalent molecule to the 3' end of the sequencing primer opposite the complementary nucleotide in the concatemer molecule, wherein the bound nucleotide moiety is not incorporated into the sequencing primer; and (2) detecting and identifying the bound nucleotide moiety of the multivalent molecule, thereby identifying the nucleotide moiety of the concatemer molecule. determining the sequence; (3) optionally repeating steps (1) and (2) at least once; (4) contacting the concatemer molecules with (i) a plurality of polymerases and (ii) a plurality of nucleotides under conditions suitable for binding at least one polymerase to at least a portion of the concatemer molecules and suitable for binding at least one of the plurality of nucleotides to the 3' end of a hybridized sequencing primer opposite the complementary nucleotide in the concatemer molecules; (5) optionally detecting the incorporated nucleotide, wherein the bound nucleotide is incorporated into the hybridized sequencing primer; (6) optionally identifying the incorporated nucleotide, thereby determining or confirming the sequence of the concatemer; and (7) repeating steps (1) through (6) at least once.

[0250] In some embodiments, the sequencing step of step (i) comprises: (1) contacting a plurality of immobilized concatemers with a plurality of sequencing primers hybridized at the sequencing primer binding sequence, a plurality of polymerases, and a plurality of nucleotides under conditions suitable for binding at least one polymerase and at least one sequencing primer to a portion of the immobilized concatemers and suitable for binding at least one nucleotide to the 3' end of the sequencing primer opposite the complementary nucleotide in the immobilized concatemers, wherein the binding nucleotide is incorporated into the 3' end of the sequencing primer; (2) detecting and identifying the incorporated nucleotide, thereby determining the sequence of the immobilized concatemer molecules; and (3) optionally repeating steps (1) and (2) at least once. In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a chain-terminating moiety at the sugar 2' or sugar 3' position. In some embodiments, the chain-terminating moiety is an azide, azido, or azidomethyl group cleavable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0251] The sequencing method can include contacting a target nucleic acid or a plurality of target nucleic acids, comprising multiple linked or unlinked copies of a target sequence, with the multivalent binding composition described herein.Contacting the target nucleic acid or a plurality of target nucleic acids, comprising multiple linked or unlinked copies of a target sequence, with one or more polymer-nucleotide conjugates results in a substantially increased local concentration of the appropriate nucleotide being examined in a given sequencing cycle, thus suppressing the signal from improper incorporation or stepwise nucleic acid strands (i.e., extending nucleic acid strands with one or more skipped cycles).

[0252] Methods for obtaining nucleic acid sequence information are provided herein, comprising contacting a target nucleic acid or multiple target nucleic acids with one or more polymer-nucleotide conjugates. In some embodiments, the target nucleic acid or multiple target nucleic acids comprise multiple linked or unlinked copies of the target sequence. In some embodiments, the methods result in a reduced sequencing error rate, as indicated by a reduction in base misidentifications, reporting of absent bases, or failure to report the correct base. In some embodiments, the reduction in sequencing error rate may comprise a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more reduction compared to the error rate observed using monovalent ligands comprising free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides. In some embodiments, the methods result in an increase in average read length of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more compared to the average read length observed using a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide-bound nucleotides, or labeled protein or peptide-bound nucleotides. In some embodiments, the methods result in an increase in average read length of 10, 20, 25, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 500 nucleotides, or more compared to the average read length observed using a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide-bound nucleotides, or labeled protein or peptide-bound nucleotides.

[0253] The use of polymer-nucleotide conjugates for sequencing can shorten the total time of a sequencing reaction or sequencing run. A sequencing reaction cycle, including the contacting, detecting, and incorporating steps, is carried out for a total time ranging from about 5 minutes to about 60 minutes. In some embodiments, a sequencing reaction cycle is carried out for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, a sequencing reaction cycle is carried out for up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes, up to 10 minutes, or up to 5 minutes. Any of the lower and upper limits described in this paragraph can be combined to form ranges within the present disclosure; for example, in some embodiments, a sequencing reaction cycle can be carried out for a total time ranging from about 10 minutes to about 30 minutes. Those skilled in the art will recognize that the sequencing cycle time can have any value within this range, such as about 16 minutes.

[0254] The use of polymer-nucleotide conjugates for sequencing results in more accurate base readouts. The disclosed compositions and methods for nucleic acid sequencing result in an average Q-score of base-calling accuracy across sequencing runs ranging from about 20 to about 50. In some embodiments, the average Q-score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q-score can have any value within this range, such as about 32. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides.In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides.

[0255] The present disclosure relates to polymer-nucleotide conjugates, each of which has multiple nucleotides conjugated to a particle or core (e.g., a polymer, a branched polymer, a dendrimer, or an equivalent structure). When the polymer-nucleotide conjugate is contacted with a polymerase and a primed target nucleic acid, a ternary complex can be formed that can be detected, and more accurate determination of the bases of the target nucleic acid can be achieved.

[0256] When polymer-nucleotide conjugates are used instead of single conjugates or untethered nucleotides to form complexes with polymerase and target nucleic acid, the local concentration of nucleotides is increased many times, and signal intensity, particularly appropriate signal pair mismatch, is enhanced.The polymer-nucleotide conjugates described herein can comprise at least one polymer-nucleotide conjugate for target nucleic acid interaction.Multivalent compositions can also comprise two, three, or four different polymer-nucleotide conjugates, each of which has a different nucleotide conjugated to a particle.

[0257] In the polymer-nucleotide conjugate having polymer-nucleotide conjugate form or core-nucleotide conjugate form, multiple copies of the same nucleotide can be covalently or non-covalently bound to particle.Examples of particle can include branched polymer; dendrimer; cross-linked polymer particle such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particle; glass particle; ceramic particle; metal particle; quantum dot; liposome; emulsion particle or other particle (for example, nanoparticle, microparticle, etc.) known in the art.In a preferred embodiment, particle is branched polymer.

[0258] The nucleotide may be linked to the particle or core by a linker, and the nucleotide may be attached to one end or position of the polymer. The nucleotide may be conjugated to the particle by the base or 5' end of the nucleotide. In some polymer-nucleotide conjugates, one nucleotide is attached to one end or position of the polymer. In some polymer-nucleotide conjugates, multiple nucleotides are attached to one end or position of the polymer. The conjugated nucleotide is sterically accessible to one or more proteins, one or more enzymes, and a nucleotide-binding moiety. In some embodiments, the nucleotide may be provided separately from the nucleotide-binding moiety, such as a polymerase. In some embodiments, the linker does not include a light-releasing group or a light-absorbing group.

[0259] The particles or cores may also include binding moieties. In some embodiments, the particles or cores may self-assemble without the use of separate interacting moieties. In some embodiments, the particles or cores may self-assemble due to buffer or salt conditions, as in, for example, calcium-mediated interactions of hydroxyapatite particles, lipid- or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metal (such as iron or gold) nanoparticles.

[0260] The polymer-nucleotide conjugate may have one or more labels (e.g., detectable reporter moieties). Examples of labels include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or other labels that can render the composition detectable by methods known in the art of macromolecules or molecular interactions. Labels can be attached to the nucleotide (e.g., by attachment to the base or 5' phosphate site of the nucleotide), the particle itself (e.g., to a PEG subunit) or to the core (e.g., to a streptavidin core), at the end of the polymer, at a central location, or at any other location within the polymer-nucleotide conjugate recognized by a skilled artisan as sufficient to render the composition, such as a particle, detectable by methods known in the art or described elsewhere herein. In some embodiments, one or more labels are provided to match or distinguish a particular polymer-nucleotide conjugate.

[0261] One example of a polymer-nucleotide conjugate (e.g., a polymer-nucleotide conjugate) is a polymer-nucleotide conjugate. Examples of branched polymers include polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyglycine, polyvinyl acetate, dextran, or other such polymers. In one embodiment, the polymer is PEG. In another embodiment, the polymer can be PEG-branched.

[0262] Suitable polymers may feature repeating units with functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl, or allyl groups. Polymers may also contain one or more pre-derivatized substituents, such that one or more particular subunits contain a site for derivatization or branching, regardless of whether other subunits contain the same position, substituent, or moiety. The pre-derivatized substituents may further comprise, or may further comprise, for example, a nucleotide, a nucleoside, a nucleotide analog, a label such as a fluorescent label, a radioactive label, or a spin label, an interactive moiety, an additional polymer moiety, or the like, or any combination of the foregoing.

[0263] In polymer-nucleotide conjugates (e.g., polymer-nucleotide conjugates), the polymer can have multiple branches. Branched polymers can have a variety of configurations, including, but not limited to, star-like ("star-shaped") morphology, aggregated star-like ("helter skelter") morphology, bottle brush, or dendrimer. Branched polymers can emanate from a central attachment point or central portion, or can include multiple branch points, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some embodiments, each subunit of the polymer can constitute an optional, separate branch point.

[0264] In polymer-nucleotide conjugates, the length and size of the branches may vary depending on the type of polymer. In some branched polymers, the branches may be between 1 and 1,000 nm, between 1 and 100 nm, between 1 and 200 nm, between 1 and 300 nm, between 1 and 400 nm, between 1 and 500 nm, between 1 and 600 nm, between 1 and 700 nm, between 1 and 800 nm, or between 1 and 900 nm, or longer, or may have a length within or between any of the values ​​disclosed herein. In some branched polymers, the branches may have a size corresponding to an apparent molecular weight of 1K, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 30K, 50K, 80K, 100K, or any value within the range defined by any two of the foregoing. The apparent molecular weight of a polymer can be calculated from the known molecular weights of a representative number of subunits, as determined by size exclusion chromatography, mass spectrometry, or other methods known in the art. The polymer can have multiple branches. The number of branches in the polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128, or more, or within a range defined by any two of these values.

[0265] In the case of polymer-nucleotide conjugates, branched polymers with 4, 8, 16, 32, or 64 branches can have nucleotides attached to the termini of the PEG branches, such that each terminus can have 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In one non-limiting example, a branched polymer with between 3 and 128 PEG arms attached to a polymer branch can terminate with one or more nucleotides, such that each terminus can have 0, 1, 2, 3, 4, 5, 6, or more nucleotides or nucleotide analogs. In some embodiments, the branched polymer or dendrimer has an even number of arms. In some embodiments, the branched polymer or dendrimer has an odd number of arms.

[0266] In a polymer-nucleotide conjugate, each branch or subset of branches of the polymer can have attached thereto a moiety containing a nucleotide (e.g., an adenine, thymine, uracil, cytosine, or guanine residue, or a derivative or mimetic thereof), and the moiety can bind to a polymerase, reverse transcriptase, or other nucleotide-binding domain. Optionally, the nucleotide moiety can bind to the polymerase-template-primer complex but cannot be incorporated, or can be incorporated into a growing nucleic acid chain during a polymerase reaction. In some embodiments, the nucleotide moiety includes a chain-terminating moiety that blocks incorporation of a subsequent nucleotide during a polymerase-mediated reaction. In some embodiments, the nucleotide moiety can be unblocked (reversibly blocked), such that a subsequent nucleotide cannot be incorporated into a growing nucleic acid chain during a polymerase reaction until such block is removed, after which a subsequent nucleotide can be incorporated into a growing nucleic acid chain during a polymerase reaction.

[0267] The polymer-nucleotide conjugate can further have a binding moiety in each branch or a subset of branches. Some examples of binding moieties include, but are not limited to, biotin, avidin, streptavidin, polyhistidine domains, complementary paired nucleic acid domains, G-tetrad-forming nucleic acid domains, calmodulin, maltose-binding protein, cellulase, maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and its derivatives, benzylcysteine ​​and its derivatives, antibodies, epitopes, protein A, and protein G. The binding moiety can be any interacting molecule or fragment thereof known in the art that binds to or promotes interactions between proteins, proteins and ligands, proteins and nucleic acids, nucleic acids, or small molecule interacting domains or moieties.

[0268] In some embodiments, the polymer-nucleotide conjugate may contain one or more elements of an interactive moiety. Exemplary interactive moieties include, for example, biotin and avidin; SNAP-benzylguanosine; an antibody or FAB and an epitope; IgG FC and Protein A, Protein G, Protein A / G, or Protein L; maltose-binding protein and maltose; a lectin and a cognate polysaccharide; an ion-chelating moiety; complementary nucleic acids; nucleic acids in triplex or triple-helix interactions; nucleic acids capable of forming G-quadruplexes; and the like. Those skilled in the art will readily recognize that many pairs of moieties exist and are commonly used due to their property of interacting strongly and specifically with each other. Therefore, any such complementary pair or set is considered suitable for this purpose in constructing or envisioning the compositions of the present disclosure. In some embodiments, the compositions disclosed herein may include compositions in which one element of the interactive moiety is also attached to one molecule or multivalent ligand, and another element of the interactive moiety is attached to a separate molecule or multivalent ligand. In some embodiments, the compositions disclosed herein can include compositions in which both or all elements of the interactive moiety are attached to a single molecule or a multivalent ligand. In some embodiments, the compositions disclosed herein can include compositions in which both or all elements of the interactive moiety are attached to separate arms of a single molecule or a multivalent ligand, or to positions on a single molecule or a multivalent ligand. In some embodiments, the compositions disclosed herein can include compositions in which both or all elements of the interactive moiety are attached to the same arm of a single molecule or a multivalent ligand, or to positions on a single molecule or a multivalent ligand. In some embodiments, a composition containing one element of the interactive moiety and a composition containing another element of the interactive moiety can be mixed simultaneously or sequentially. In some embodiments, the molecular or particle-to-particle interactions disclosed herein enable association or aggregation of multiple molecules or particles, resulting in, for example, an increased detectable signal.In some embodiments, the fluorescent, colorimetric, or radioactive signal is enhanced. Other embodiments contemplate other interactive moieties disclosed herein or known in the art. In some embodiments, the compositions provided herein are provided by simultaneously or sequentially mixing one or more molecules comprising a first interactive moiety, e.g., one or more imidazole or pyridine moieties, with one or more additional molecules comprising a second interactive moiety, e.g., histidine residues. In some embodiments, the compositions comprise one, two, three, four, five, six, or more imidazole or pyridine moieties. In some embodiments, the compositions comprise one, two, three, four, five, six, or more histidine residues. In such embodiments, interactions between provided molecules or particles may be facilitated by the presence of divalent cations such as nickel, manganese, magnesium, calcium, or strontium. In some embodiments, for example, a (His)3 group may interact with a (His)3 group on another molecule or particle through the coordination of a nickel or manganese ion.

[0269] The polymer-nucleotide conjugate may include one or more buffers, salts, ions, or additives. In some embodiments, exemplary additives may include, but are not limited to, betaine, spermidine, detergents such as Triton® X-100, Tween 20, SDS, or NP-40, ethylene glycol, polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol, methylcellulose, heparin, heparan sulfate, glycerol, sucrose, 1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymers such as the Pluronic® series polymers, arginine, histidine, imidazole, or any combination thereof, or any substance known in the art as a DNA "relaxer" (a compound that acts to alter the conformational dynamics of a DNA molecule, such as by altering the DNA persistence length, altering the number of intrapolymer junctions or crossovers, or increasing the accessibility of sites within the strand to DNA-binding moieties).

[0270] The polymer-nucleotide conjugates can include zwitterionic compounds as additives. Further exemplary additives can be found in Lorenz, TCJ Vis. Exp. (63), e3998, doi:10.3791 / 3998 (2012), which is incorporated herein by reference for its disclosure of additives for enhancing nucleic acid binding or kinetics, or for enhancing processes involving the manipulation, use, or storage of nucleic acids.

[0271] In some embodiments, the multivalent binding composition comprises at least one cation, which may include, but is not limited to, sodium, magnesium, strontium, barium, potassium, manganese, calcium, lithium, nickel, cobalt, or other cations known in the art to promote nucleic acid interactions such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, and the like.

[0272] When a polymer-nucleotide conjugate is used to replace a conjugated or untethered nucleotide to form a complex with a polymerase and a target nucleic acid, the local concentration of the nucleotide is increased many-fold, enhancing signal strength, particularly a suitable signal versus a mismatch. The present disclosure contemplates contacting a polymer-nucleotide conjugate with a polymerase and a primed target nucleic acid to determine the formation of a ternary binding complex.

[0273] Due to the increased local concentration of nucleotides on the polymer-nucleotide conjugate, binding between the polymerase and the primed target strand and the nucleotide becomes more favorable when the nucleotide is complementary to the next base in the target nucleic acid. The formed binding complex has a longer duration, thereby helping to shorten the imaging process. The high signal intensity resulting from the use of the polymer-nucleotide conjugate is maintained throughout the entire binding and imaging process. The strong binding between the polymerase, the primed target strand, and the nucleotide or nucleotide analog allows the formed binding complex to remain stable during the washing process, and the signal remains high even after the other reaction mixture and non-corresponding nucleotide analogs are washed away. After the imaging process, the binding complex may be destabilized, and then the primed target nucleic acid may be extended by one base. After extension, the binding and imaging process may be repeated again using the polymer-nucleotide conjugate to determine the identity of the next base.

[0274] The compositions and methods of the present disclosure provide a robust and controllable means of establishing and maintaining ternary enzyme complexes (e.g., during sequencing), as well as greatly improved means by which the presence of the complexes can be identified and / or measured, and by which the persistence of the complexes can be controlled, providing an important solution to problems such as determining the identity of the N+1 base in nucleic acid sequencing applications.

[0275] Without wishing to be bound by any particular theory, it has been observed that the multivalent binding compositions disclosed herein bind to polymerase-nucleotide complexes to form ternary binding complexes at a rate that is significantly slower than the association rate known to be achieved by nucleotides in free solution, but that is time-dependent. Thus, their on-rate (Kon) is substantially and surprisingly slower than the on-rate of a single nucleotide or a nucleotide not attached to a multivalent ligand complex. Importantly, however, the rate (Koff) of the multivalent ligand complex is substantially slower than that observed for nucleotides in free solution. Thus, the multivalent ligand complexes disclosed herein provide surprising and beneficial improvements in the persistence of ternary polymerase-polynucleotide-nucleotide complexes (especially for complexes formed with free nucleotides) over currently available methods and reagents, enabling significant improvements in imaging quality for, for example, nucleic acid sequencing applications. Importantly, this property of the multivalent substrates disclosed herein confers controllable formation of visible ternary complexes so that subsequent visualization, modification, or processing steps can proceed essentially without regard to complex dissociation—i.e., the complexes can be formed, imaged, modified, or otherwise used as desired, and will remain stable until the user performs an active dissociation step, such as exposing the complex to a dissociation buffer.

[0276] In various embodiments, polymerases suitable for binding interactions (e.g., during sequencing) as described herein can include any polymerase known or that can be known in the art. Exemplary polymerases include, but are not limited to, Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; bacteriophage polymerases such as T4, RB69, and phi29 bacteriophage DNA polymerases, Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III, and Escherichia coli (E. coli) DNA polymerase III alpha and epsilon; 9°N polymerase, HIV M or O reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney Murine Leukemia virus (MMUV) reverse transcriptase. Reverse transcriptases, such as Mycobacterium tuberculosis (MMLV) reverse transcriptase, or telomerase. Further non-limiting examples of DNA polymerases can include those derived from various archaeal genera such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, including polymerases known in the art, such as Vent™, Deep Vent™, Pfu, KOD, Pfx, Therminator™, and Tgo polymerases.In some embodiments, the polymerase is Klenow polymerase.

[0277] Ternary complexes have a longer duration when the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid compared to non-complementary nucleotides. Ternary complexes also have a longer duration when the nucleotides of the polymer-nucleotide conjugate are complementary to the target nucleic acid compared to unconjugated or untethered complementary nucleotides. For example, in some embodiments, the ternary complex may have a duration of less than 1 second, more than 1 second, more than 2 seconds, more than 3 seconds, more than 5 seconds, more than 10 seconds, more than 15 seconds, more than 20 seconds, more than 30 seconds, more than 60 seconds, more than 120 seconds, more than 360 seconds, more than 3600 seconds, or more than 1 second, or a range of times defined by two or more of these values.

[0278] The duration can be measured by observing the onset and / or duration of the binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in the binding complex, thereby allowing a signal from the label to be detected during the duration of the binding complex.

[0279] It has been observed that different duration ranges can be achieved depending on the type of salt and ion. For example, it has been shown that complexes formed in the presence of magnesium form more quickly than complexes formed with other ions. It has also been observed that, for example, in the presence of strontium, complexes form readily and dissociate completely or substantially completely when the ions are removed or washed with a buffer lacking one or more components of the composition, such as a polymer, one or more nucleotides, and / or one or more interacting moieties, or with a buffer containing a chelating agent that may cause or promote removal of divalent cations from the multivalent reagent-containing complex. Thus, in some embodiments, the disclosed compositions include magnesium. In some embodiments, the disclosed compositions include calcium. In some embodiments, the disclosed compositions include strontium or barium. In some embodiments, the disclosed compositions include cobalt. In some embodiments, the disclosed compositions include MgCl2. In some embodiments, the disclosed compositions include CaCl2. In some embodiments, the disclosed compositions include SrCl2. In some embodiments, the disclosed compositions include CoCl2. In some embodiments, the compositions contain no or substantially no magnesium. In some embodiments, the compositions contain no or substantially no calcium. In some embodiments, methods of the disclosure provide for contacting one or more nucleic acids with one or more of the compositions disclosed herein, wherein the compositions lack either calcium or magnesium, or both calcium and magnesium.

[0280] Dissociation of the ternary complex can be controlled by changing buffer conditions. After the imaging step, a buffer with increased salt content is used to induce dissociation of the ternary complex, allowing the labeled polymer-nucleotide conjugate to be washed away and providing a means to attenuate or terminate the signal, such as during the transition between one sequencing cycle and the next. In some embodiments, this dissociation can be achieved by washing the complex with a buffer lacking necessary metals or cofactors. In some embodiments, the wash buffer can include one or more compositions for maintaining pH regulation. In some embodiments, the wash buffer can include one or more monovalent cations, such as sodium. In some embodiments, the wash buffer is devoid or substantially devoid of divalent cations, e.g., completely or substantially free of strontium, calcium, magnesium, or manganese. In some embodiments, the wash buffer further includes a chelating agent, such as EDTA, EGTA, nitrilotriacetic acid, polyhistidine, or imidazole. In some embodiments, the wash buffer may maintain the pH of the environment at the same level as that of the bound complex. In some embodiments, the wash buffer may raise or lower the pH of the environment relative to the level found for the bound complex. In some embodiments, the pH may be within a range of 2-4, 2-7, 5-8, 7-9, 7-10, or less than 2 or more than 10, or within a range defined by any two of the values ​​provided herein.

[0281] The addition of certain ions can affect polymerase binding to a primed target nucleic acid, ternary complex formation, ternary complex dissociation, or incorporation of one or more nucleotides into an elongating nucleic acid, such as during a polymerase reaction. In some embodiments, relevant anions can include chloride, acetate, gluconate, sulfate, or phosphate. In some embodiments, ions can be included in the disclosed compositions by the addition of one or more acids, bases, or salts, such as NiCl, CoCl, MgCl, MnCl, SrCl, CaCl, CaSO, SrCO, BaCl, etc. Representative salts, ions, solutions, and conditions can be found in Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, AR, Ed. (2000), which is incorporated herein by reference in its entirety, particularly Chapter 17 and related disclosures on salts, ions, salt solutions, and ionic solutions.

[0282] The present disclosure contemplates contacting a polymerase-nucleotide conjugate with one or more polymerases. The contacting can optionally occur in the presence of one or more target nucleic acids. In some embodiments, the target nucleic acid is a single-stranded nucleic acid. In some embodiments, the target nucleic acid is hybridized to a nucleic acid primer. In some embodiments, the target nucleic acid is a double-stranded nucleic acid. In some embodiments, the contacting comprises contacting the polymer-nucleotide conjugate with one polymerase. In some embodiments, the contacting comprises contacting one or more nucleotides comprising the composition with multiple polymerases. The polymerases can be bound to a single nucleic acid molecule.

[0283] The bond between the target nucleic acid and the polymer-nucleotide conjugate is provided in the presence of a catalytically inactive polymerase. In one embodiment, the polymerase may be catalytically inactive by mutation. In one embodiment, the polymerase may be catalytically inactive by chemical modification. In some embodiments, the polymerase may be catalytically inactive due to the absence of a required substrate, ion, or cofactor. In some embodiments, the polymerase enzyme may be catalytically inactive due to the lack of magnesium ions.

[0284] The binding between the target nucleic acid and the polymer-nucleotide conjugate occurs in the presence of a polymerase, wherein the binding solution, reaction solution, or buffer lacks a catalytic ion, such as magnesium or manganese. Alternatively, the binding between the target nucleic acid and the polymer-nucleotide conjugate occurs in the presence of a polymerase, wherein the binding solution, reaction solution, or buffer contains a non-catalytic ion, such as strontium, barium, or calcium.

[0285] When a catalytically inactive polymerase is used to assist nucleic acids in interacting with a multivalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex so that the complex can be detected by fluorescence or other methods disclosed herein or otherwise known in the art. The released polymer-nucleotide conjugates can optionally be washed away prior to detection of the ternary binding complex.

[0286] Contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is carried out in a solution containing either calcium or magnesium, or both calcium and magnesium. Alternatively, contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is carried out in a solution lacking either calcium or magnesium, or both calcium and magnesium, and adding either calcium or magnesium to the solution in a separate step, regardless of the order of the steps. In some embodiments, contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is carried out in a solution lacking strontium or barium, and adding strontium to the solution in a separate step, regardless of the order of the steps.

[0287] Disclosed herein are polymer-nucleotide conjugates and their use in the analysis of nucleic acids, including sequencing and other bioassay applications. Increased binding of nucleotides to enzymes (e.g., polymerases) or enzyme complexes can be achieved by increasing the effective concentration of nucleotides. This can be achieved by increasing the concentration of nucleotides in free solution or by increasing the amount of nucleotides in the vicinity of the relevant binding site. This can also be achieved by physically confining many nucleotides to a limited volume, resulting in a local increase in concentration and allowing the structure to bind to the binding site with a higher apparent binding activity than observed with individual nucleotides that are not conjugated, tethered, or otherwise restricted. One exemplary means of achieving such confinement is by providing a polymer-nucleotide conjugate in which multiple nucleotides are bound to a polymer, branched polymer, dendrimer, micelle, liposome, microparticle, nanoparticle, quantum dot, or other suitable particle known in the art.

[0288] The polymer-nucleotide conjugates disclosed herein can contain multiple nucleotide moieties attached to a particle. In some embodiments, the multiple nucleotide moieties are composed of the same type (e.g., having identical or similar base-pairing properties). When the multiple nucleotide moieties are complementary to adjacent nucleotides in the target nucleic acid to be identified, the polymer-nucleotide conjugate forms a binding complex (a multivalent binding complex) between at least two nucleotide moieties and the next nucleotide in at least two copies of the target nucleic acid sequence. In some embodiments, the multivalent binding complex comprises two or more polymerases associated with a primed template of the target nucleic acid molecule. The multivalent binding complexes described herein exhibit increased stability and longer duration than binding complexes formed using a single, untethered nucleotide. When bound to a polymerase, the multivalent binding complex can withstand washing steps, such that signal intensity remains high throughout the imaging and washing steps of the workflow; see, e.g., Figure 7. The polymer core of the polymer-nucleotide conjugate can be labeled with two or more detectable labels, which contributes at least in part to the enhanced signal that can be detected.

[0289] In some embodiments, at least one polymer-nucleotide conjugate comprises two or more copies of a nucleotide moiety connected to a core by a linker, e.g., as shown in Figures 5A and 5B. In some embodiments, a polymer-nucleotide conjugate comprises (a) a core and (b) a plurality of nucleotide arms, wherein each nucleotide arm comprises (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, e.g., as shown in Figures 5A-D and 6A-B.

[0290] In some embodiments, the spacer is attached to a linker, where 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. In some embodiments, 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, where both linker chains have 2 to 6 subunits, and optionally, the linker comprises an aromatic moiety (Figures 6A and 6B). In some embodiments, the polymer-nucleotide conjugate comprises a core attached to multiple nucleotide arms, where the multiple nucleotide arms have the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the low-binding support further comprises a mixture of polymer-nucleotide conjugates having two or more different types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0291] In some embodiments, the polymer-nucleotide conjugate comprises a core attached to a plurality of nucleotide arms, and wherein each nucleotide arm comprises a nucleotide unit having a chain-terminating moiety (e.g., a blocking moiety) at the 2' sugar position, the 3' sugar position, or the 2' and 3' sugar positions. In some embodiments, the chain-terminating moiety is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0292] In some embodiments, the chain-terminating moiety comprises a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the chain-terminating moiety is selected from the group consisting of 3'-deoxynucleotides, 2',3'-dideoxynucleotides, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'butyl, 3'-tertbutyl, 3'-Fluorenylmethyloxycarbonyl, 3'tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino groups, 3'-phosphorothioates, and 3-O-benzyl, or derivatives thereof. In some embodiments, the chain-terminating moiety comprises an azide group, an azido group, or an azidomethyl group.

[0293] In some embodiments, the chain-terminating moiety is cleavable / removable from the nucleotide arm by, for example, chemical compounds, light, or heat. In some embodiments, the chain-terminating moiety comprises an alkyl, alkenyl, alkynyl, or aryl group cleavable by tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain-terminating moiety comprises an aryl or benzyl group cleavable by Pd / C. In some embodiments, the chain-terminating moiety comprises an amine, amide, keto, isocyanate, phosphate, thio, or disulfide group cleavable by a phosphine or a thiol group, including beta-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the chain-terminating moiety comprises a carbonate group cleavable by potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating moiety comprises a urea or silyl group cleavable by tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the chain-terminating moiety is an azide, azido, or azidomethyl group cleavable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0294] In some embodiments, the polymer-nucleotide conjugate comprises a core attached to multiple nucleotide arms, wherein the core or nucleotide base comprises a label. In some embodiments, the label is a detectable reporter moiety. The polymer-nucleotide conjugate can have one or more labels. Examples of detectable reporter moieties include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or other labels that can render the composition detectable by methods known in the art of macromolecules or molecular interactions. A detectable reporter moiety can be attached to the nucleotide (e.g., by attachment to the 5' phosphate moiety of the nucleotide), to the particle itself (e.g., to a PEG subunit), to the terminal end of the polymer, to the central portion, or to another location within the polymer-nucleotide conjugate, which will be recognized by those skilled in the art as being sufficient to render the composition, such as a particle, detectable by methods known in the art or as described elsewhere herein. In some embodiments, one or more labels are provided to correspond to or differentiate between specific polymer-nucleotide conjugates. The detectable reporter moiety can be a fluorophore. In some embodiments, the core can be a moiety such as avidin and the core-attached moiety can be a biotin moiety.

[0295] Exemplary polymer-nucleotide conjugates and methods of use are described in U.S. Application No. 16 / 579,794, filed September 23, 2019, the contents of which are expressly incorporated herein by reference for all purposes.

[0296] Polymer-nucleotide conjugates (polymer-nucleotide conjugates) can be used to localize detectable signals at active regions of biochemical interactions, such as protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions such as polymerase reactions. For example, the polymer-nucleotide conjugates described herein can be used to identify the site of base binding to a template or the site of base incorporation in nucleic acid chain elongation during a polymerase reaction, and to provide base discrimination for sequencing and array-based applications. The increased binding between the target nucleic acid and the nucleotide in the multivalent binding composition provides an enhanced signal when the nucleotide is complementary to the target nucleic acid, which greatly improves the accuracy of base calling and shortens imaging times.

[0297] In addition, the use of polymer-nucleotide conjugates allows sequencing signals to be generated from a given sequence in cluster regions that contain multiple copies of target sequences.The sequencing method that includes multiple copies of target sequences (for example, concatemers) has the advantage that the signal can be amplified by the existence of multiple simultaneous sequencing reactions in a defined region, each of which provides its own signal.The existence of multiple signals in a defined region can also reduce the impact of any single skipped cycle, due to the fact that the signal of a large number of correct base calls can surpass the signal of a small number of skipped or inaccurate base calls, thereby providing a method for reducing phasing errors and / or improving read length in sequencing reactions.

[0298] The polymer-nucleotide conjugates disclosed herein and their use provide one or more of the following: (i) stronger signals for better base calling accuracy compared to conventional nucleic acid amplification and sequencing methods; (ii) enabling greater discrimination of sequence-specific signals from background signals; (iii) reduced requirements regarding the amount of starting material needed; (iv) increased sequencing speed and reduced sequencing time; (v) reduced phasing errors; and (vi) improved read length in sequencing reactions.

[0299] Those skilled in the art will understand that in a series of repeated sequencing reactions, sometimes one or more sites will fail to incorporate nucleotides during a given cycle, resulting in one or more sites being out of sync with the majority of the elongating nucleic acid chain.Under the circumstances where sequencing signals are derived from the reaction occurring on a single copy of target nucleic acid, these incorporation failures will result in individual errors in the output sequence.The use of polymer-nucleotide conjugates for sequencing can reduce this kind of error in sequencing reactions.For example, the use of multivalent substrates that can bind to polymerase-template-primer complexes or can be incorporated into elongating chains can reduce the frequency of "skipped" cycles in which no base is incorporated, by increasing the probability of recombination in the premature dissociation of ternary polymerase complexes. Thus, in some embodiments, the present disclosure contemplates the use of multivalent substrates disclosed herein comprising nucleotides having free or reversibly modified 5' phosphate, diphosphate, or triphosphate moieties, and wherein the nucleotides are connected to particles or polymers via labile or cleavable linkages as disclosed herein. In some embodiments, the present disclosure contemplates that the use of multivalent substrates disclosed herein results in a reduced inherent error rate due to skipped incorporation.

[0300] The present disclosure also contemplates a sequencing reaction in which a sequencing signal derived from or related to a given sequence is derived from or occurs within a definable region containing multiple copies of the target sequence. Sequencing methods that incorporate multiple copies of a target sequence have the advantage that the signal can be amplified by the presence of multiple simultaneous sequencing reactions within a defined region, each providing its own signal. The presence of multiple signals within a defined region also reduces the impact of any single skipped cycle, due to the fact that the signals of a large number of correct base calls can outweigh the signals of a small number of skipped or incorrect base calls. The present disclosure further contemplates incorporating free, unlabeled nucleotides during extension reactions, or during separate portions of extension cycles, to provide incorporation at sites that may have been skipped in the previous cycle. For example, unlabeled blocked nucleotides may be added during or after an incorporation cycle to be incorporated into skipped sites. The unlabeled blocked nucleotides can be of the same type(s) as the multivalently bound substrate or nucleotides attached to the substrate that are or were present in a particular cycle, or can be a mixture containing one, two, three, four or more types of unlabeled blocked nucleotides.

[0301] When each sequencing cycle is completed, each reaction within a defined region produces the same signal. However, as described elsewhere herein, in a series of repeated sequencing reactions, sometimes one or more sites will fail to incorporate nucleotides in a given cycle, resulting in one or more sites being out of sync with the majority of the elongating nucleic acid chain. This problem, called "phasing," leads to degradation of sequencing signals because the signals are contaminated with spurious signals from sites that skip one or more cycles. This, in turn, leads to the possibility of errors in base discrimination. The progressive accumulation of skipped cycles over multiple cycles also reduces the effective read length due to the progressive deterioration of sequencing signals in each cycle. Another objective of the present disclosure is to provide a method for reducing phasing errors and / or improve the read length in sequencing reactions.

[0302] The sequencing method of the present invention can include contacting a target nucleic acid or multiple target nucleic acids containing multiple linked or unlinked copies of a target sequence with a multivalent binding composition described herein. Contacting the target nucleic acid or multiple target nucleic acids containing multiple linked or unlinked copies of a target sequence with one or more polymer-nucleotide conjugates results in a substantially increased local concentration of the appropriate nucleotide being interrogated in a given sequencing cycle, thus suppressing signals from improperly incorporated or phased nucleic acid strands (i.e., extending nucleic acid strands with one or more skipped cycles).

[0303] A method for obtaining nucleic acid sequence information can include contacting a target nucleic acid or multiple target nucleic acids, wherein the target nucleic acid or multiple target nucleic acids comprise multiple linked or unlinked copies of a target sequence, with one or more polymer-nucleotide conjugates. This method results in a reduced sequencing error rate, as indicated by a reduction in base misidentifications, reporting of non-existent bases, or failure to report correct bases. In some embodiments, the reduction in sequencing error rate can include a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more reduction in the error rate observed using a monovalent ligand, including free nucleotides, labeled free nucleotides, nucleotides bound to proteins or peptides, and nucleotides bound to labeled proteins or peptides.

[0304] A method for obtaining nucleic acid sequence information can include contacting a target nucleic acid or a plurality of target nucleic acids, wherein the template nucleic acid or plurality of target nucleic acids comprises multiple linked or unlinked copies of a target sequence, with one or more polymer-nucleotide conjugates, which results in an increase in average read length of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more, including free nucleotides, labeled free nucleotides, nucleotides bound to proteins or peptides, and nucleotides bound to labeled proteins or peptides, as compared to the error rate observed using monovalent ligands.

[0305] A method for obtaining nucleic acid sequence information includes contacting a target nucleic acid or multiple target nucleic acids, wherein the target nucleic acid or multiple target nucleic acids comprise multiple linked or unlinked copies of a target sequence, with one or more polymer-nucleotide conjugates, which results in an increase in average read length of 10, 20, 25, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 500, or more nucleotides compared to the average read length observed using monovalent ligands comprising free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides.

[0306] The use of polymer-nucleotide conjugates for sequencing effectively shortens sequencing time. A sequencing reaction cycle, including the contacting, detecting, and incorporating steps, is carried out for a total time ranging from about 5 minutes to about 60 minutes. In some embodiments, the sequencing reaction cycle is carried out for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, the sequencing reaction cycle is carried out for up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes, up to 10 minutes, or up to 5 minutes. Any of the lower and upper limits described in this paragraph can be combined to form ranges within the present disclosure; for example, in some embodiments, the sequencing reaction cycle can be carried out for a total time ranging from about 10 minutes to about 30 minutes. Those skilled in the art will recognize that the sequencing cycle time can have any value within this range, such as about 16 minutes.

[0307] The use of polymer-nucleotide conjugates for sequencing results in more accurate base readouts. The disclosed compositions and methods for nucleic acid sequencing result in an average Q-score of base call accuracy across a sequencing run ranging from about 20 to about 50. In some embodiments, the average Q-score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q-score can have any value within this range, such as about 32. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing result in a Q-score of greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified termin...

Claims

1. 1. A method of analyzing a biological sample, comprising: (a) detecting a multivalent binding complex formed between a target nucleic acid sequence of a target nucleic acid molecule or a derivative thereof and a detectable polymer-nucleotide conjugate in the presence of a biological sample or a derivative thereof; (b) determining the origin of the target nucleic acid sequence in the biological sample or a derivative thereof; A method comprising:

2. 2. The method of claim 1, wherein the determining step (b) is performed at least in part by analyzing the relative three-dimensional relationship between the target nucleic acid sequence and a reference point of the biological sample or derivative thereof.

3. 10. The method of claim 1, further comprising contacting the biological sample or a derivative thereof with the detectable polymer-nucleotide conjugate in the presence of the biological sample.

4. The method of claim 3 , further comprising binding at least a portion of the target nucleic acid sequence to a capture oligonucleotide molecule bound to a surface of a substrate.

5. 5. The method of claim 4, wherein the surface has a water contact angle of 45 degrees or less.

6. The step of binding comprises hybridizing in the presence of a hybridization buffer, the hybridization buffer comprising: (a) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (b) a second polar aprotic solvent having a dielectric constant of 115 or less; The method of claim 4, comprising:

7. 5. The method of claim 4, further comprising immobilizing the biological sample or derivative thereof on the surface in a manner sufficient to fix the relative three-dimensional relationship.

8. 5. The method of claim 4, further comprising amplifying the target nucleic acid sequence on the surface of the substrate, optionally using rolling circle amplification.

9. an image of the surface in the presence of the biological sample or derivative thereof; (a) contacting the surface with a fluorescently labeled nucleotide molecule comprising a nucleic acid sequence complementary to at least a portion of a capture oligonucleotide immobilized on the surface; and (b) following (a), imaging the surface while it is immersed in buffer using an inverted microscope and camera under non-signal saturating conditions; 5. The method of claim 4, wherein the image exhibits a contrast-to-noise ratio of about 5 or greater, as measured by

10. The method of claim 1, further comprising the step of performing a nucleotide coupling reaction between the nucleotide moiety attached to the polymer-nucleotide conjugate and the target nucleic acid molecule or a derivative thereof.

11. 10. The method of claim 1, wherein the target nucleic acid molecule or derivative thereof is a deoxyribonucleic acid (DNA) molecule.

12. The method of claim 1 , wherein the biological sample or derivative thereof comprises a fluid biological sample.

13. The method of claim 1 , wherein the source is cancer tissue.

14. 1. A method for identifying at least a portion of an intracellular component in a cell or tissue in situ, comprising: (a) detecting a signal from a multivalent binding complex between the intracellular component or a derivative thereof and a detectable polymer-nucleotide conjugate; (b) processing at least the signal detected in (a) to identify at least some of the intracellular components or derivatives thereof; A method comprising:

15. The method of claim 14, wherein the intracellular component or derivative thereof is a nucleic acid.

16. 16. The method of claim 15, wherein the nucleic acid is DNA.

17. The method of claim 14, further comprising the step of (c) immobilizing the cells or tissue on the surface of a substrate.

18. 20. The method of claim 17, further comprising the step of: (d) binding at least a portion of the intracellular components to capture molecules bound to the surface.

19. 18. The method of claim 17, further comprising the step of permeabilizing the tissue or lysing the cells prior to detection in (a).

20. 18. The method of claim 17, wherein the surface has a water contact angle of 45 degrees or less.

21. The binding step in (d) comprises hybridizing the capture molecule to at least a portion of the intracellular component in the presence of a hybridization buffer, the hybridization buffer comprising: (a) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (b) a second polar aprotic solvent having a dielectric constant of 115 or less; 20. The method of claim 18, comprising:

22. The image of the surface (a) contacting the surface with a fluorescently labeled nucleotide molecule comprising a nucleic acid sequence complementary to at least a portion of a capture oligonucleotide immobilized on the surface; and (b) following (a), imaging the surface while it is immersed in buffer using an inverted microscope and camera under non-signal saturating conditions; 15. The method of claim 14, wherein the image exhibits a contrast-to-noise ratio of about 5 or greater, as measured by

23. 15. The method of claim 14, wherein the step of detecting the signal from the multivalent binding complex in (a) comprises performing a nucleotide binding reaction between a nucleotide moiety attached to the polymer-nucleotide conjugate and the intracellular component or a derivative thereof.

24. 15. The method of claim 14, wherein the tissue is from a tumor.

25. 1. A system for analyzing a biological sample, the biological sample comprising a substrate comprising a surface having attached thereto a polymer layer suitable for immobilizing the biological sample on the surface; the biological sample or a derivative thereof comprises a target nucleic acid molecule or a derivative thereof; the polymer layer is configured to bind to (i) the biological sample or a derivative thereof, or (ii) the target nucleic acid molecule or a derivative thereof; the target nucleic acid molecule or derivative thereof is configured to bind to a nucleotide moiety comprising a detectable label; and A system wherein an image of the surface exhibits a contrast-to-noise ratio of about 5 or greater when acquired using an inverted microscope and camera under non-signal-saturating conditions while the surface is immersed in a buffer solution, and the detectable label is a fluorescent dye.

26. 26. The system of claim 25, wherein the polymer layer is hydrophobic.

27. 26. The system of claim 25, further comprising a fixative that fixes the biological sample to the surface when contacted while adjacent to the surface.

28. 26. The system of claim 25, wherein the fixative comprises formaldehyde or glutaraldehyde.

29. 26. The system of claim 25, wherein the target nucleic acid molecule is a concatemer.

30. 26. The system of Claim 25, wherein the target nucleic acid molecule comprises a universal sequence region comprising a spatial barcode sequence or a sample barcode sequence configured to preserve the origin of the target nucleic acid molecule in the biological sample.

31. 26. The system of claim 25, wherein the image of the surface, when acquired, exhibits a contrast-to-noise ratio of about 10 or greater.

32. 26. The system of claim 25, wherein the substrate is a flow cell device comprising a first flow channel and optionally a second flow channel.

33. 33. The system of claim 32, wherein the substrate is a reflective, transparent, or translucent planar substrate.

34. 26. The system of claim 25, wherein the flow cell device is a capillary flow cell device.

35. 1. A system for analyzing nucleic acid sequence information in a biological sample or a derivative thereof, comprising: (a) detecting a signal from a multivalent binding complex formed between a target nucleic acid sequence of a target nucleic acid molecule or a derivative thereof and a detectable polymer-nucleotide conjugate in the presence of the biological sample or a derivative thereof, wherein the signal indicates the identity of a nucleotide in the target nucleic acid sequence; (b) determining the origin of the target nucleic acid sequence in the biological sample; and A system including one or more computer processors programmed to perform the steps of:

36. 36. The system of claim 35, wherein the one or more computer processors are programmed to determine the origin of the target nucleic acid sequence in (b) by analyzing the relative three-dimensional relationship between the target nucleic acid molecule or derivative thereof and the biological sample or derivative thereof.

37. 36. The system of claim 35, further comprising a database configured to store three-dimensional data related to the origin of the target nucleic acid sequence.

38. 38. The system of claim 37, wherein the database is further configured to store sequencing data comprising the identities of the nucleotides in the target nucleic acid sequences.

39. 39. The system of claim 38, wherein (b) is performed by correlating the sequencing data and the three-dimensional data.

40. 36. The system of claim 35, wherein the one or more computer processors are programmed to identify the target nucleic acid sequence in less than 60 minutes by repeating (a)-(b).

41. 36. The system of Claim 35, wherein the one or more computer processors are programmed to perform (a)-(b) with a base calling accuracy characterized by a Q-score of greater than 25 for at least 80% of identified nucleotides.

42. the detectable polymer-nucleotide conjugate is (a) a polymer core; (b) two or more nucleotide moieties attached to the polymer core, wherein the polymer-nucleotide conjugate is configured to form a multivalent binding complex between the two or more nucleotide moieties and the target nucleic acid molecule or derivative thereof; 36. The system of claim 35, comprising:

43. 43. The system of claim 42, wherein the one or more nucleotide moieties comprise a nucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog.

44. 43. The system of claim 42, wherein the polymer core comprises a polymer having a star, comb, cross, bottlebrush, or dendrimer configuration.

45. 43. The system of claim 42, wherein the polymer core comprises branched polyethylene glycol (PEG) molecules.

46. 1.0 mm ２ 36. The system of claim 35, further comprising an optical imaging system comprising a field of view (FOV).

47. (a)(i) a polymer core, and (ii) two or more nucleotide moieties attached to said polymer core a detectable polymer-nucleotide conjugate comprising: (b) instructions for identifying at least a portion of the intracellular component in a cell or tissue in situ by contacting the detectable polymer-nucleotide conjugate with the intracellular component under conditions sufficient to form a multivalent binding complex between the two or more nucleotide moieties and the intracellular component; Kit including:

48. 48. The kit of claim 47, comprising four of said detectable polymer-nucleotide conjugates, each having a different nucleotide moiety attached thereto.

49. (a) a substrate comprising a surface having attached thereto a polymer layer suitable for immobilizing a biological sample or a derivative thereof; (b) instructions for determining a target nucleic acid sequence and its origin in the biological sample or derivative on the surface; Kit including:

50. (a)(i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; and (ii) a second polar aprotic solvent having a dielectric constant of 115 or less; a hybridization buffer comprising: (b) instructions for hybridizing at least a portion of the target nucleic acid sequence to at least a portion of a capture oligonucleotide bound to the surface; 50. The kit of claim 49, further comprising:

Images