Method for Nucleic Acid Sequencing with Specifiable Cell Address

Abstract

Methods and systems are provided for analyzing nucleic acids in a biological sample in a manner that preserves the spatial and / or cellular origin of the nucleic acid within the biological sample. Compositions and kits that enable the disclosed methods and systems are also provided.

Description

Technical Field

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

Background Art

[0002] Diagnostic methods that have emerged for diseases and disorders such as cancer, infectious diseases, and endotoxemia rely on next-generation sequencing (NGS) methods that provide high-resolution genetic and genomic data, enabling definitive individualized diagnoses, treatment plans, and ultimately cures for diseases that were previously untreatable. Although powerful, NGS methods are still limited by the available methods for providing nucleic acid samples to the instruments that perform the actual sequencing. For example, to identify the exact nature of mutations present in a particular tumor, multiple steps are required in the isolation of tumor tissue, isolation of nucleic acids, and sample preparation for a particular sequencing method before involving the instrument to obtain actual sequence data. Furthermore, the deconvolution and processing of sequence data in a way that enables correlation of a particular sequence with a particular cell or tissue becomes complex due to the nature of NGS techniques, which often require pooling of samples, during which spatial and cell identity information is lost.

[0003] Towards the goal of providing molecular diagnostic methods with higher spatial or tissue resolution, various methods have been proposed to address the issue of loss of cell addressability in NGS methods. For example, some techniques use unique barcodes for cell separation to identify sequences associated with individual cells after completion of the sequencing run, followed by application of unique barcodes to the nucleic acids of individual cells, and then rely on bulk sequencing. This can be achieved, for example, by exposing individual cells to lysis and hybridization mixtures such as beads or emulsions in an isolated environment. These methods may further require enrichment or processing of a partial subpopulation of target cells, such as cell sorting for circulating cells or tissue sampling for solid tumor cells, followed by dissociation and protease treatment.

[0004] Such methods can obtain cell-addressable information but face significant limitations such as the difficulty of processing solid tissues, and the throughput is limited by the ability to isolate, tag, and prepare nucleic acids for sequencing. Similarly, there are limitations related to the need to transfer the prepared library to a separate instrument, system, or location to perform the sequencing step. This results in a limitation on the sequencing throughput of approximately 50,000 cells per actual sequencing run, and when there are a large number of cells in diagnostic-related tissue samples, secretions, excretions, or exudates, or microbiome samples, severe limitations are imposed on the sensitivity and usefulness of these assays. A certain level of addressability can be achieved simply by 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 developing systematic screening methods.

[0005] Accordingly, in addition to cell-addressable sequencing methods, there is a need for compositions and methods that can increase the accuracy and throughput of cell- or spatially addressable sequencing methods that avoid the aforementioned limitations on existing techniques.

Summary of the Invention

[0006] Aspects disclosed herein provide a method for analyzing a biological sample, the method comprising: (a) detecting a multivalent binding complex formed between a target nucleic acid molecule or a target nucleic acid sequence of 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 determination in (b) is at least partially performed by analyzing the relative three-dimensional relationship between the target nucleic acid sequence and a reference point of the biological sample or a derivative thereof. In some embodiments, the method further comprises contacting the biological sample or a derivative thereof with a 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 the 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 the surface in a form sufficient to fix the relative three-dimensional relationship. In some embodiments, the method further comprises amplifying the target nucleic acid sequence 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 more when measured by: (a) contacting the surface with a fluorescently labeled nucleotide molecule comprising a nucleic acid sequence complementary to at least a portion of the capture oligonucleotide immobilized on the surface; and (b) subsequently imaging the surface using an inverted microscope and a camera under non-signal saturation conditions while the surface is immersed in a buffer.In some embodiments, the method further includes performing a nucleotide binding reaction between a nucleotide moiety conjugated to a 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 includes a fluid biological sample. In some embodiments, the origin is cancer tissue.

[0007] Aspects disclosed herein provide a method for in situ identifying at least a part of an intracellular component in 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 part 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 the tissue on the surface of a substrate. In some embodiments, the method further comprises (d) binding at least a part 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 part 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 more when measured by (a) contacting the surface with a fluorescently labeled nucleotide molecule comprising a nucleic acid sequence complementary to at least a part of the capture oligonucleotide immobilized on the surface, and (b) subsequently imaging the surface using an inverted microscope and a camera under non-signal saturation conditions while the surface is immersed in a buffer. In some embodiments, the step of detecting 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 of tumor origin.

[0008] A system for analyzing a biological sample, the biological sample including a substrate having a surface to which a polymer layer suitable for immobilizing the biological sample on the surface is bonded, the biological sample or a derivative thereof including a target nucleic acid molecule or a derivative thereof, the polymer layer being 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 a derivative thereof being configured to bind to a nucleotide moiety including a detectable label, and an image of the surface exhibiting a contrast-to-noise ratio of about 5 or more when acquired using an inverted microscope and a camera under non-signal saturation conditions while the surface is immersed in a buffer, the detectable label being a fluorescent dye. In some embodiments, the polymer layer is hydrophobic. In some embodiments, the system further includes a fixative that fixes the biological sample to the surface when the biological sample is contacted while adjacent to the surface. In some embodiments, the fixative includes formaldehyde or glutaraldehyde. In some embodiments, the target nucleic acid molecule is a concatemer. In some embodiments, the target nucleic acid molecule includes a universal sequence region including a spatial barcode sequence or a sample barcode sequence configured to retain the origin of the target nucleic acid molecule in the biological sample. In some embodiments, the image of the surface exhibits a contrast-to-noise ratio of about 10 or more when acquired. In some embodiments, the substrate is a flow cell device including 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 molecule or a derivative thereof and a detectable polymer-nucleotide conjugate in the presence of the biological sample or a derivative thereof, the signal indicating the identity of nucleotides 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 a derivative thereof and the biological sample or a 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 nucleotides 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 basecalling accuracy characterized by a Q score greater than 25 in 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, the polymer-nucleotide conjugate being configured to form a multivalent binding complex between the two or more nucleotide moieties and the target nucleic acid molecule or a derivative thereof.In some embodiments, the one or more nucleotide moieties include nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs. In some embodiments, the polymer core includes a polymer having a star, comb, cross-linked, bottlebrush, or dendrimer configuration. In some embodiments, the polymer core includes branched polyethylene glycol (PEG) molecules. In some embodiments, the system is 1.0 mm. 2 It further includes an optical imaging system including a field of view (FOV) of more than 1.0 mm.

[0010] Aspects disclosed herein provide a kit comprising: (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 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. In some embodiments, the kit includes four of the detectable polymer-nucleotide conjugates, each attached to a different nucleotide moiety.

[0011] Aspects disclosed herein include a kit comprising: (a) a substrate including a surface to which a polymer layer suitable for immobilizing a biological sample or a derivative thereof is attached; 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 including (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] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby 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 Description of the Drawings

[0013] The novel features of the invention will be described specifically together with the appended claims. To better understand the features and advantages of the invention, reference should be made to the following detailed description of exemplary embodiments in which the principles of the invention are used and the accompanying drawings.

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BEST MODE FOR CARRYING OUT THE INVENTION

[0014] This specification provides spatial and cell-addressable sequencing methods and compositions, as well as compositions, devices, and kits useful for practicing the methods and systems described herein. The methods and systems described herein can utilize polymer-nucleotide conjugates in situ in nucleotide binding reactions. The nucleotide binding reactions can be performed on hydrophilic surfaces, thereby providing numerous advantages described herein. Hybridization buffers containing polar and aprotic solvents in combination with a pH buffer are also provided herein. Additionally, optical systems useful for spatially resolving sequencing data are provided. In some embodiments, the optical systems described herein have a field of view greater than 1.0 mm 2 and.

[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 (step 701); immobilizing a biological sample containing a target nucleic acid molecule on the surface and optionally permeabilizing the biological sample (step 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 (step 703); (d) amplifying the target nucleic acid molecule to produce an amplified target nucleic acid molecule or a derivative thereof (step 704); (e) contacting the amplified target nucleic acid molecule or a 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 a derivative thereof to produce a primed target nucleic acid molecule or a derivative thereof (step 705); (f) contacting the primed target nucleic acid molecule or a derivative thereof with a polymer-nucleotide conjugate comprising two or more nucleotide moieties attached to a polymer (e.g., PEG) core labeled with a detectable label (e.g., a fluorophore) (step 706); (g) detecting a multivalent binding complex formed between the primed target nucleic acid molecule or a derivative thereof and the polymer-nucleotide conjugate (step 707); (h) washing the surface with a buffer sufficient to remove the polymer-nucleotide conjugate from the primed target nucleic acid molecule or a derivative thereof (step 708); (i) optionally incorporating a nucleotide that does not contain a detectable label but optionally contains a protecting group (e.g., azidomethyl) that interferes with the incorporation of a second nucleotide at the N+1 position on the primed target nucleic acid molecule or a derivative thereof (step 709); and (j) optionally repeating steps (f) - (j) (step 710).

[0016] Existing methods of spatially addressable array identification (also referred to herein as spatial transcriptomics technology) suffer from low sensitivity, non-specificity, and inaccurate spatial location of the target transcript. In contrast, the methods, systems, compositions, and kits described herein solve such problems, for example, by a low non-specific binding surface, a high-efficiency hybridization buffer, a method for preparing highly copious nanoballs, and seeding with multivalent molecules.

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

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

[0019] The rolling circle amplification method described herein includes a two-step method that utilizes non-catalytic divalent cations and then catalytic divalent cations to synchronize rolling circle amplification events on a surface. The rolling circle amplification reaction may follow mild conditions and a flexing amplification reaction that generates new concatemers from existing concatemers. These amplification methods together generate very compact nanoballs that contain high copy numbers of target sequences that improve sequencing signal intensity.

[0020] The throughput of the nucleic acid analysis method described herein is higher than existing methods, enabling the analysis of 50,000, 100,000, 150,000, 250,000, 500,000, 750,000, or more than 1,000,000 cells per run, and in principle enabling the detection of mutations in as few as 1 cell per million cells, thereby achieving a fairly high diagnostic sensitivity. A further advantage of the nucleic acid methods disclosed herein is that the reactions required can be carried out at a single temperature (e.g., isothermal conditions) such as, for example, 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] The multivalent molecules used during the sequencing reaction provide many advantages not offered by free nucleotides. The multivalent molecules include a core attached to multiple arms that are each tethered to a nucleotide. The multivalent molecules increase the local concentration of nucleotides near the polymerase / template binding site. The multivalent molecules further exhibit an increased duration in the formation of a stable triple complex with the polymerase and the nucleic acid template. For this reason, labeled multivalent molecules afford shorter imaging times and increased signal intensity during the sequencing reaction.

[0022] The cellular and spatial resolution of the 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 genomic applications.

[0023] Disclosed herein are optical components and system designs for high-performance fluorescence imaging methods and systems capable of providing any one or more of a larger field of view, improved optical resolution (including high-performance optical resolution), improved contrast, improved image quality, faster transition between image captures when repositioning the sample surface to capture a series of images (e.g., of different fields of view), improved duty cycle of the imaging system, and increased throughput of image acquisition and analysis.

[0024] In some examples, for example, in dual-sided (flow cell) imaging applications that include the use of thick flow cell walls (e.g., walls (or cover glasses) thicker than 700 μm) and fluid channels (e.g., fluid channel heights or thicknesses of 50 - 200 μm), the improvement in imaging performance can be achieved using a novel objective lens design that corrects for optical aberrations introduced by imaging the opposite surface of the thick cover glass and / or fluid channel from the objective lens.

[0025] In some examples, for example, in dual-sided (flow cell) imaging applications including the use of a thick flow cell wall (e.g., a wall (or cover glass) thickness greater than 700 μm) and a fluid channel (e.g., a fluid channel height or thickness of 50 - 200 μm), the improvement in imaging performance, unlike a conventional microscope tube lens that simply forms an image at an intermediate image plane, can be achieved even when using an off-the-shelf objective lens by using a novel objective lens design that corrects for optical aberrations introduced by the thick flow cell wall and / or intervening fluid layer in combination with the object.

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

[0027] In some examples, for example, the improvement in imaging performance in dual-sided (flow cell) imaging applications can be achieved by using an electro-optical phase plate in combination with an objective lens that corrects for optical aberrations introduced by a fluid layer separating the inner surfaces of the upper (proximal) and lower (distal) parts of the flow cell. In some examples, this design approach can further compensate for vibrations by a compensator that operates by movement, which can move the inside and outside of the optical path depending on, for example, which surface of the flow cell is being imaged.

[0028] A further advantage 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 with respect to the objective lens and the dichroic filter that receives the excitation beam. The excitation beam may be linearly polarized, and the orientation of the linear polarization may be such that the s-polarization is projected onto the dichroic reflecting surface of the dichroic filter. Such features can potentially improve the filtering of the excitation beam and reduce, for example, the wavefront error introduced into the emitted radiation due to surface deformation of the dichroic filter.

[0029] In this specification, we have first discussed from the perspective of fluorescence imaging (e.g., fluorescence microscopy imaging, fluorescence focus imaging, two-photon fluorescence, etc.). However, those skilled in the art will understand 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, which may include various combinations of the disclosed optical, mechanical, fluidic, thermal, electrical, and computing modules or subsystems, are disclosed. Advantages obtained by the disclosed flow cell devices, cartridges, and analysis systems include, but are not limited to, (i) a reduction in complexity and cost in the manufacturing of the devices and systems, (ii) a significant reduction in consumable costs (e.g., compared to those of currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow rate 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 a commercial disposable single lumen (e.g., a single fluid flow channel), or a multi-lumen capillary that may further include a fluid adapter, a cartridge chassis, one or more integrated fluid flow control components, or combinations 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 fluid flow controller module, a temperature regulation module, an imaging module, or any combination thereof. Design features of some of the disclosed capillary flow cell devices, cartridges, and systems include: (i) a single flow channel configuration; (ii) a simple load / unload mechanism that can be implemented between reagent flows such that the fluid interface between the system and the capillary is reliably sealed to allow for capillary repositioning and system reuse, and precise control of reaction conditions such as reagent concentration, pH, and temperature; (iii) a replaceable single fluid flow channel device or capillary flow cell cartridge with multiple flow channels that can be used interchangeably to provide a flexible system throughput; (iv) compatibility with a variety of detection methods such as fluorescence imaging, but not limited thereto.

[0032] The disclosed capillary flow cells as well as microfluidic devices and systems are described first from the perspective of their use in nucleic acid sequencing applications, but various aspects of the disclosed devices and systems can be applied to any other kind of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications, not limited to nucleic acid sequencing. It should be understood that the various aspects of the disclosed methods, devices, and systems can be evaluated individually, collectively, or in combination with each other.

[0033] The embodiments described herein provide significant advantages in the diagnosis of cancer, including circulating tumors and solid tumors, the analysis of biopsy samples, for example, the diagnosis of genetic disorders, the analysis of microbiome samples, for example, the diagnosis of disorders associated with the gut microbiota in the microbial flora, the diagnosis of disorders accompanied by secretion or exudation, or the assessment of health status or disease risk, where such risk can be evaluated 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 blood 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 (capturing) 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 DNA, circulating DNA, or organellar DNA. In some embodiments, this capture process may be followed by one or more library preparation steps, such as adding at least one adapter to a capture nucleic acid where the adapter may contain an index sequence, barcode sequence, and / or unique molecular identifier (UMI). The adapter addition step can be performed by ligation (e.g., blunt-end ligation) or by use of "sprint" oligonucleotides. These library preparation steps may result in or further include circularization of the capture nucleic acid. In some embodiments, the circularized nucleic acid molecule can be amplified, such as by rolling circle amplification (RCA), resulting in a large multi-copy nucleic acid molecule (e.g., concatemer) containing multiple tandem repeat sequences of the target sequence. In some embodiments, this large multi-copy nucleic acid can be caused to form an aggregated state by buffer conditions that favor a compact DNA state, a surface with high density capture oligonucleotides, use of a bivalent or bispecific oligonucleotide that crosslinks two or more sites within the large multi-copy nucleic acid ("clustered oligonucleotide" or "clustered oligo"), or any combination of the foregoing, or by any method known or to become known in the art for producing a compact cluster containing the large multi-copy nucleic acid.

[0035] In some embodiments, the surface used to capture nucleic acids from cells or tissues can be configured to be highly active and retain nucleic acids while simultaneously retaining unwanted proteins, lipids, carbohydrates, or other cellular debris components. Thus, the surfaces contemplated herein can bind to nucleic acids from cells or single cells in tissue lysed in contact with or in proximity to the surface. Further, this surface does not retain cellular debris and does not exhibit significant non-specific 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) is performed in contact with or in proximity to the surface, such that DNA, RNA, or other target nucleic acids released from the cell or tissue sample are captured by the surface in a substantial amount, such as a representative mass, or nearly all. The surface can be configured such that cells can 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 in contact with or in fluid communication with the surface, where reagents can later flow over the tissue in a manner that promotes in situ capture of nucleic acids from the tissue, such that nucleic acids are oriented or positioned within intact tissue, and thus 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, capture, adapter addition, circularization, amplification, and clustering of nucleic acids can be performed while the nucleic acids are attached to or in close proximity to the surface. Alternatively, one or more of the foregoing preparation steps can be performed in free solution or while attached to beads.

[0038] For example, by spatially resolved binding of cell-specific nucleic acid complements such as cell genomes or cell transcriptomes, followed by adapter addition, circularization, amplification, and clustering, the use of sequencing techniques such as sequencing methods based on binding activity as described in U.S. Patent Nos. 62 / 897,172 and 16 / 579,794 and as otherwise described herein is enabled, and the prior literature is hereby incorporated by reference in its entirety. The ability to perform cell- or tissue-addressable sequencing is further provided by advances in low-binding surfaces as disclosed in U.S. Patent Application No. 16 / 363,842, hybridization methods as disclosed in U.S. Patent Application No. 16 / 543,351, and library preparation methods as disclosed in U.S. Patent Application No. 62 / 767,943 and related International Publication WO2020 / 102766, and the content of the prior literature is hereby expressly incorporated by reference for all purposes. Thus, in some embodiments, sequence data can be obtained in a form 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 a nearly one-to-one correspondence with the cell position from which the sample originated. In some embodiments, the sequence data is in a correspondence other than a one-to-one spatial correspondence with the cell position in the original sample, but can be obtained at approximately the same position relative to other cells or sources of genetic, genomic, or transcriptomic samples within the tissue.

[0039] Solid support surface. Provided herein is a solid support comprising a surface (e.g., low non-specific binding). In some examples, the solid support comprises a non-hydrophilic surface. In some instances, the solid support comprises a hydrophilic surface. Generally, the disclosed support comprises a substrate (i.e., a support structure), one or more layers comprising a low-binding chemical modification layer attached covalently or non-covalently, 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 support the surface and / or to each other, and to crosslink all layers, may be varied such that non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction complements to the support surface is minimized or reduced relative to a monolayer of a comparison. In many cases, the formulation of the surface may be varied such that non-specific hybridization on the support surface is minimized or reduced relative to a monolayer of a comparison. The formulation of the surface may be varied such that non-specific amplification on the support surface is minimized or reduced relative to a monolayer of a comparison. The formulation of the surface may be varied such that the rate and / or yield of specific amplification on the support surface is maximized. An amplification level suitable for detection is achieved in some cases disclosed herein in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or fewer, or more than 30 amplification cycles.

[0040] Examples of materials capable of manufacturing the substrate or support structure include, but are not limited to, glass, fused silica, silicon, polymers (such as 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 consisting of a glass substrate and a plastic substrate 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 include 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., including a slide glass or a slide glass surface). Overall, the substrate or support structure may be cylindrical (e.g., including a capillary or the internal surface of a capillary), spherical (e.g., including the external surface of a non-porous bead), or irregular (e.g., including the external surface of an irregularly formed non-porous bead or particle). In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be a solid and non-porous surface. In some examples, 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 the nucleic acid hybridization and amplification reactions performed thereon may occur within the pores.

[0042] A substrate or support structure comprising one or more chemically modified layers, such as a layer of a low non-specific binding polymer, may be standalone or integrated into another structure or assembly. For example, in some instances, the substrate or support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure can include one or more surfaces in a microplate format, such as the bottom surface of a well in a microplate. As noted above, in some preferred embodiments, the substrate or support structure includes the internal surface of a capillary (such as a lumen surface). In an alternative preferred embodiment, the substrate or support structure includes the internal surface of a capillary (such as a lumen surface) etched into a planar chip.

[0043] The chemically modified layer can be applied uniformly to the surface of the substrate or support structure. Alternatively, the surface of the substrate or support structure may be non-uniformly distributed or patterned such 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 discrete chemically modified regions may include at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions, or any intermediate number falling within the ranges herein.

[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. Generally, passivation is achieved using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (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 with end groups linked to the surface using, for example, silane chemistry, with distal end groups including, but not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In some examples, two or more layers of hydrophilic polymers, such as linear polymers, branched polymers, or dendrimers, may be deposited on the surface. In some examples, the 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 ligated to the resulting surface layer at various surface densities. In some examples, for instance, both the surface functional group density and the oligonucleotide concentration may be varied to target a specific primer density range. Additionally, the primer density can be adjusted by diluting the oligonucleotide with other molecules carrying the same functional group. For example, an oligonucleotide labeled with an amine can be diluted using an amine-labeled polyethylene glycol in a reaction with a surface coated with an NHS ester to reduce the final primer density.Primers with linkers of various lengths between the hybridization region and the surface-attached functional groups can also be applied to adjust the surface density. Examples of suitable linkers include poly-T and poly-A chains at the 5' end of a primer (e.g., 0 to 20 bases), a PEG linker (e.g., 3 to 20 monomer units), and a carbon chain (e.g., C6, C12, C18, etc.). To measure the primer density, fluorescently labeled primers are tethered to the surface, and then the fluorescence reading may be compared to that of a dye solution of known concentration.

[0045] In some embodiments, the hydrophilic polymer can be a cross-linked polymer. In some embodiments, the cross-linked polymer may include a type of polymer cross-linked with other types of polymers. Examples of cross-linked polymers include polyethylene oxide (PEO), or poly(ethylene glycol) cross-linked with another polymer selected from poly(oxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (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 performing the surface passivation techniques disclosed herein, proteins, nucleic acids, and other biomolecules "do not attach" to the substrate, i.e., exhibit low non-specific binding (NSB). Examples using standard monolayer surface preparations under various glass preparation conditions are described below. Hydrophilic surfaces passivated to achieve extremely low NSB for proteins and nucleic acids require new 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, combining a new primer surface conjugation formulation (Cy3 oligonucleotide graft titration) and the resulting extremely low non-specific background (NSB functional tests performed using red and green fluorescent dyes) yields results demonstrating the feasibility of the disclosed approach. On some of the surfaces disclosed herein, the ratio of specific binding of fluorophores such as Cy3 (e.g., hybridization to ligated primers or probes) to non-specific binding (e.g., 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 described herein. On some of the surfaces disclosed herein, the ratio of specific fluorescence signal to non-specific fluorescence signal in fluorophores such as Cy3 (e.g., specific hybridization to non-specifically bound and labeled oligonucleotides, or non-specific binding (B inter ) or non-specific amplification (B intra ) of labeled oligonucleotides, or combinations thereof (B inter +B intra) in non-specific amplification for) 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 described herein.

[0047] Primer surface density is measured, and substrates containing multi-coatings of PEG and other hydrophilic polymers have been developed to add additional dimensionality to hydrophilic or amphoteric surfaces. By using hydrophilic and amphoteric surface layering techniques including, but not limited to, the polymer / copolymer materials described hereinafter, the primer packing density on the surface can be significantly increased. Conventional PEG coating techniques use single-layer primer deposition, which is generally reported for single-molecule applications but does not result in high copy numbers in nucleic acid amplification applications. As described herein, "layering" can be achieved using conventional cross-linking techniques with any compatible polymer or monomer subunit such that a surface containing two or more highly cross-linked layers can be continuously constructed. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some examples, various layers may be attached to each other 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-maleimide reactions, and ionic interactions between positively charged and negatively charged polymers. In some examples, materials with high primer density may be constructed in solution and subsequently layers may be formed on the surface in multiple steps.

[0048] The adhesion chemistry used to graft the first chemically modified layer onto the support surface will generally depend on both the material from which the support is made and the chemistry of the layer. In some examples, the first layer may be attached to the support surface by covalent bonds. In some examples, the first layer is non-covalently attached and may be adsorbed onto the surface via non-covalent interactions such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and the molecular components of the first layer. In either case, the substrate surface may be treated prior to the 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, the surface of glass or silicon can be cleaned by acid washing using a piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) and / or by oxygen plasma treatment.

[0049] The chemistry of silanes constitutes one non-limiting approach for covalently modifying silanol groups on the surface of glass or silicon to attach more reactive functional groups (such as amine or carboxyl groups), which can then be used to bind linker molecules (such as linear hydrocarbon molecules of various lengths, such as C6, C12, C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (such as 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 various PEG silanes (such as 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 choice of components used may vary to modify one or more properties of the support surface, such as functional groups and / or the surface density of 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 non-specific binding material on any of the disclosed support surfaces include polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyester, dextran, poly-lysine, and copolymers of poly-lysine, or any combination thereof, but are not limited thereto. Examples of conjugation chemistries that can be used to graft one or more layers (e.g., polymer layers) of material onto a support surface and / or crosslink layers to each other include biotin-streptavidin interactions (or variants thereof), his-tag-Ni / NTA conjugation chemistry, methoxy-ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxides, azides, hydrazides, alkynes, isocyanates, silanes, but are not limited thereto.

[0051] One or more layers of the multi-layer surface may comprise a branched polymer or 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-isopropylacrylamide) (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 poly-lysine, branched poly-glucoside, dextran.

[0052] In some examples, the branched polymer used to create any one or more layers of the multi-layer surfaces disclosed herein may comprise 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. 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 comprises PEG(8, 16, 8) (8 arms, 16 arms, 8 arms)? on PEG amine-APTES. Similar concentrations are observed for 3-layer multi-arm PEGs (8 arms, 16 arms, 8 arms) and (8 arms, 64 arms, 8 arms) on PEG-amine-APTES exposed to 8 uM primers, and 3-layer multi-arm PEGs (8 arms, 8 arms, 8 arms) using star-shaped PEG-amine to replace 16- and 64-arm PEG multilayers with comparable first, second, and third PEG layers are also contemplated.

[0054] The molecular weight of the linear, branched, or multi-branched polymers used to make any one or more of the layers of the multi-layer 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 multi-branched polymers used to make any one or more of the layers of the multi-layer 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 described in this paragraph can be combined to yield a range that is included within the present disclosure. For example, in some examples, the linear, branched, or multi-branched polymers used to make any one or more of the layers of the multi-layer surfaces disclosed herein may be in the range of about 1,500 to about 20,000 Daltons. One of ordinary skill in the art will recognize that the linear, branched, or multi-branched polymers used to make any one or more of the layers of the multi-layer surfaces disclosed herein may be any value within this range, such as about 1,260 Daltons.

[0055] In some examples, for instance, 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 deposited layer and the molecules of the previous layer may 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 may 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 the branched polymer molecules of the new layer and the molecules of the previous layer may be at most 32, at most 30, at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 covalent bond per molecule. Any of the lower and upper limits described in this paragraph can be combined to yield ranges included within the present disclosure. For example, in some examples, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may range from about 4 to about 16. One of ordinary skill in the art will recognize that the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be any value within this range, for example, about 11 in some examples, or an average of about 4.6 in other examples.

[0056] The reactive functional groups remaining after the bonding of the material layer to the support surface can be blocked, optionally, by bonding small inert molecules using a high-yield bonding chemistry. For example, when using an amine bonding chemistry to attach a new material layer to the previous layer, the remaining amine groups can be acetylated or rendered non-reactive by later bonding to a small amino acid such as glycine.

[0057] The number of layers of a low non-specific binding material, such as a hydrophilic polymer material, deposited on the surface of the disclosed low-binding support may 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 may be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any combination of the lower and upper limits described in this paragraph may form a range that is included within the scope of the present disclosure. For example, in some examples, the number of layers may be from about 2 to about 4. In some examples, all of the layers may comprise the same material. In some examples, each layer may comprise a different material. In some examples, the plurality of layers may comprise a plurality of materials. In some examples, at least one layer may comprise a branched polymer. In some examples, all of the layers may comprise a branched polymer.

[0058] One or more layers composed of a low non-specific binding material may optionally be deposited and / or conjugated onto the substrate surface using a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, or any combination thereof. In some examples, the solvents used for layer deposition and / or conjugation are alcohols (e.g., methanol, ethanol, propanol, etc.), other organic solvents (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, buffered aqueous 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 falling within or adjacent to the ranges herein, and the balance is constituted by 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 falling within or adjacent to the ranges herein, and the balance is constituted by an organic solvent. The pH of the solvent mixture used may be 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, less than 10, or greater than 10, or any value falling within or adjacent to the ranges described herein.

[0059] In some examples, one or more layers of low non-specific binding material may be deposited and / or conjugated to the substrate surface using a mixture of organic solvents, with at least one component having a dielectric constant of less than 40 and constituting 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 constitutes 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 non-specific binding supports of the present disclosure exhibit a reduction in non-specific binding of proteins, nucleic acids, and other components of hybridization and / or amplification formulations used in solid-phase nucleic acid amplification. The degree of non-specific binding exhibited by a given support surface can be evaluated 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 series of standard conditions followed by a specific rinse protocol and fluorescence imaging can be used as a qualitative tool for comparing non-specific 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 series of standard conditions followed by a specific wash protocol and fluorescence imaging can be used as a quantitative tool for comparing non-specific binding on supports containing various surface formulations. However, it is assumed that care is taken to ensure that fluorescence imaging is performed under conditions where the fluorescence signal is linearly related (or related in a predictable manner) to the number of fluorophores on the support surface (e.g., under conditions where signal saturation and / or self-quenching of the fluorophore are not issues) and an appropriate calibration standard is used. In some instances, other techniques known to those of skill in the art, such as radioisotope labeling and counting methods, may be used for the quantitative evaluation of the degree to which various support surface formulations of the present disclosure exhibit non-specific binding.

[0061] On some of the surfaces disclosed herein, the ratio of specific binding to non-specific binding of fluorophores such as Cy3 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 greater than 100, or any intermediate value within the ranges set forth herein. On some of the surfaces disclosed herein, the ratio of specific fluorescence to non-specific fluorescence of fluorophores such as Cy3 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 greater than 100, or any intermediate value within the ranges set forth herein.

[0062] As noted, in some examples, the degree of non-specific binding exhibited by the disclosed low non-specific binding support can be evaluated using a standard protocol in which the surface is contacted 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 an appropriate calibration standard. In some examples, the label may include a fluorescent label. In some examples, the label may include a radioisotope. In some examples, the label may include other detectable labels known to those of skill in the art. In some examples, the degree of non-specific binding exhibited by a given support surface formulation can thus be evaluated in terms of the number of non-specifically bound protein molecules (or other molecules) per unit area. In some examples, the low non-specific binding support of the present disclosure exhibits less than 0.001 molecules / μm2, less than 0.01 molecules / μm2, less than 0.1 molecules / μm2, less than 0.25 molecules / μm2, less than 0.5 molecules / μm2, less than 1 molecule / μm2, less than 10 molecules / μm2, less than 100 molecules / μm2, or less than 1000 molecules / μm2 of non-specific protein binding (or non-specific binding of other specific molecules, e.g., Cy3 dye). 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 / μm2 of non-specific binding. For example, some of the modified surfaces disclosed herein are contacted with a 1 uM solution of streptavidin labeled with Cy3 (GE Amersham) in phosphate buffered saline (PBS) buffer for 15 minutes, followed by rinsing three times with deionized water and exhibit less than 0.5 molecules / um2 of non-specific protein binding. Some of the modified surfaces disclosed herein exhibit non-specific binding to less than 0.25 molecules / um2 of Cy3 dye molecules.In the independent non-specific 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-propylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated on a low-binding substrate at 37 °C for 15 minutes in a 384-well plate format. Each well was rinsed 2 - 3 times with 50 μl of RNase / DNase-free deionized water and 2 - 3 times with 25 mM ACES buffer at pH 7.4. The 384-well plate was imaged on a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) instrument using a Cy3, AF555, or Cy5 filter set (according to the dye test performed) with an 800 PMT gain setting and a resolution of 50 - 100 μm. For imaging at higher resolution, images were collected with a total internal reflection fluorescence (TIRF) objective lens (20x, 0.75 NA, or 100X, 1.5 NA, Olympus), an sCMOS Andor camera (Zyla4.2), and an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) with an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors, e.g., 405, 488, 532, or 633 nm dichroic reflectors / beam splitters 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 of the modified surfaces disclosed herein exhibit non-specific binding of less than 0.25 molecules / μm2 of dye molecules.

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

[0064] Low-background surfaces consistent with the disclosure herein have a ratio of specific dye attachment (e.g., Cy3 attachment) to non-specific 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 may exhibit more than 50 specific dye molecules per non-specifically adsorbed molecule. Similarly, when exposed to excitation energy, low-background surfaces consistent with the disclosure herein and having a fluorophore, such as Cy3, attached may exhibit a ratio of specific fluorescence signal (e.g., arising from Cy3-labeled oligonucleotides attached to the surface) to non-specifically adsorbed dye fluorescence 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 an aqueous solution) of the disclosed support surface can be evaluated, for example, through measurement of the water contact angle where small water droplets are placed on the surface and the contact angle with the surface is measured using, for example, 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 low-binding support surface with hydrophilicity disclosed herein can range from about 0 to about 50 degrees. In some examples, the water contact angle of the low-binding support surface with hydrophilicity 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. Often, the contact angle is any value or less within this range, for example, 40 degrees or less. One of ordinary skill in the art will recognize that a given low-binding support surface with hydrophilicity may exhibit a water contact angle of a value within this range, for example, a water contact angle of about 27 degrees.

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

[0067] Some low-binding surfaces of the present disclosure exhibit a significant improvement in stability or durability against long-term 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 (such as oligonucleotide primers) on the surface, and monitoring the fluorescent signal before, during, or after long-term 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 evaluate the quality of the surface can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured over these periods) over an exposure period to solvents and / or high temperatures 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. In some examples, the degree of change in fluorescence used to evaluate the quality of the surface can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured over this cycle range) over 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 repetitions of exposure to solvent changes 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 in nucleic acid amplification, may exhibit an amplified signal that is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times, or more than 100 times, the signal of an adjacent unpopulated region of the surface. Similarly, some surfaces may exhibit an amplified signal that is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times, or more than 100 times, the signal of an amplified adjacent nucleic acid population region of the surface.

[0069] Fluorescent excitation energy varies between specific fluorophores and protocols and may be less than 400 nm to greater than 800 nm at the excitation wavelength, consistent with fluorophore selection or other surface use parameters disclosed herein.

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

[0071] In some examples, fluorescence images of the disclosed low-background surfaces, when used in nucleic acid hybridization or amplification applications to create clusters of hybridized or clonally amplified nucleic acid molecules (e.g., directly or indirectly labeled by 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, 200, 210, 220, 230, 240, 250, or greater than 250.

[0072] Generally, at least one of the one or more layers consisting of low-nonspecific binding material contains a functional group for covalently or non-covalently attaching an oligonucleotide molecule, such as an adapter or primer sequence, or may already contain an oligonucleotide adapter or primer sequence that is covalently or non-covalently attached when deposited on the support surface. In some examples, the oligonucleotides ligated to the polymer molecules of at least a third layer may be distributed at multiple depths throughout the layer.

[0073] In some examples, oligonucleotide adapter or primer molecules are covalently bound to the polymer in solution, for example, before binding to or depositing it on the surface. In some examples, oligonucleotide adapter or primer molecules are covalently bound to the polymer after being bound to or deposited on the polymer. In some examples, at least one hydrophilic polymer layer contains multiple covalently bound oligonucleotide adapter or primer molecules. In some examples, at least 2, at least 3, at least 4, or at least 5 layers of the hydrophilic polymer contain multiple covalently bound adapter or primer molecules.

[0074] In some examples, the oligonucleotide adapter or primer molecule may be attached to one or more layers of a hydrophilic polymer using any of a variety of suitable conjugation chemistries known to those of skill in the art. For example, the oligonucleotide adapter or primer sequence may include moieties that react 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 isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imido esters, carbodiimides, anhydrides, 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 sulfhydryl-reactive conjugation chemistries include maleimides, haloacetyls, and pyridyl disulfides.

[0075] One or more oligonucleotide molecules may be attached or ligated to the support surface. In some examples, one or more oligonucleotide adapters or primers may include a spacer sequence, an adapter sequence for the purpose of hybridizing to a template library nucleic acid sequence to which adapter ligation has been performed, 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 ligated 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 ligated to at least one layer of the surface.

[0076] The length of the ligated oligonucleotide adapter and / or primer sequence can range from about 10 nucleotides to about 100 nucleotides. In some examples, the length of the ligated oligonucleotide adapter 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 ligated oligonucleotide adapter 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 combination of the lower and upper limits described in this paragraph may form a range included within the present disclosure. For example, in some examples, the length of the ligated oligonucleotide adapter and / or primer sequence can range from about 20 to about 80 nucleotides. One of ordinary skill in the art will recognize that the length of the ligated oligonucleotide adapter and / or primer sequence may have any value within this range, such as about 24 nucleotides.

[0077] In some examples, the ligated adapter or primer sequence may include modifications designed to promote the specificity and efficiency of nucleic acid amplification when performed on a low-binding support. For example, in some examples, the primer may include a polymerase stop point such that the extension of the primer sequence between the surface conjugation point and the modification site is always in a single-stranded form and functions as a filling site for the 5’-to-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 insertion of a PEG chain into the primer backbone between two nucleotides in the 5’-terminal direction, insertion of a deoxyribonucleotide (i.e., a nucleotide having neither a purine nor a pyrimidine base), or a lesion site that can be bypassed by a helicase, but are not limited thereto.

[0078] As further discussed in the examples below, in order to "tune" the support for optimal performance when using a given amplification method, the surface density of oligonucleotide adapters or primers ligated onto the support surface, and / or the spacing of the ligated adapters or primers away from the support surface (e.g., by varying the length of the linker molecule used to ligate the adapter or primer to the surface) may be desirable to change. As noted below, adjustment of the surface density of the ligated oligonucleotide adapter or primer may affect the degree of specific and / or non-specific amplification observed on the support in a manner that varies depending on the selected amplification method. In some examples, the surface density of the ligated oligonucleotide adapter or primer can be changed by adjusting the ratio of the molecular components used to create the support surface. For example, when using an oligonucleotide primer-PEG conjugate to create the final layer of a low-binding support, the ratio of the oligonucleotide primer-PEG conjugate to the non-conjugated PEG molecules may be varied. Subsequently, the resulting surface density of the ligated primer molecules can be estimated or measured using any of a variety of techniques known to those of skill in the art. By way of example, the use of radioisotope labeling and counting methods, the covalent attachment of a cleavable molecule containing an optically detectable tag (e.g., a fluorescent tag) that can be cleaved from the support surface in a defined area, collected in a fixed amount of an appropriate solvent, and then quantified by comparison to a calibration solution of known optical tag concentration and a fluorescent signal, or the use of fluorescence imaging techniques where care is taken in 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 (ones with no significant self-quenching relative to the fluorophores on the surface), but is not limited thereto.

[0079] In some examples, the surface density that occurs on the oligonucleotide adapter or primer on the low-binding support surface of the present disclosure may range from about 100 to about 1,000,000 primer molecules per μm2. In some examples, the surface density of the oligonucleotide adapter or primer may be at least 100, 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 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, or at least 1,000,000 molecules per μm2.In some examples, the surface density of the oligonucleotide adapter or primer may be 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 450,000, 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 55,000, 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, 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 1 μm2. Any combination of the lower and upper limits described in this paragraph may form a range that is included 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 1 μm2. One of ordinary skill in the art will recognize that the surface density of the adapter or primer molecules may have any value within the foregoing range, for example, 3,800 molecules per 1 μm2 in some examples, or 455,000 molecules per 1 μm2 in other examples.In some examples, as further discussed below, the surface density of a template library nucleic acid sequence (e.g., a sample DNA molecule) that hybridizes first to an adapter or primer array on a support surface may be below that shown for the surface density of the ligated oligonucleotide primers. In some examples, as further discussed below, the surface density of a clonal amplified template library nucleic acid sequence that hybridizes to an adapter or primer array on a support surface may be in the same or different ranges as that shown for the surface density of the ligated oligonucleotide adapter or primer.

[0080] The local surface density of the adapter or primer molecules listed above does not exclude variations in the density across the surface, and thus the surface may include at least a second surface region of substantially different local density in addition to a region having an oligo density of, for example, 500,000 / μm2.

[0081] Solid support for capturing and analyzing DNA. In some embodiments, as shown in FIG. 2, the 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, each of the capture oligonucleotides comprises a single-stranded oligonucleotide. The capture oligonucleotides can be immobilized on the passivated surface by their 5' ends, or an internal portion of the capture oligonucleotide can be immobilized on the passivated surface. Each of the capture oligonucleotides can include an extendable 3' end. As shown in FIG. 2, each of the capture oligonucleotides can include a cleavable region (250) positionable near the end immobilized on the passivated surface. For example, each of the capture oligonucleotides can include a cleavable region near the 5' end. The cleavable region is cleavable by an enzyme, a chemical compound, light, or heat. In some embodiments, each of the capture oligonucleotides includes a target capture region (210) and universal sequence regions (220, 230, 240). In some embodiments, the target capture region of the capture oligonucleotide includes a sequence capable of hybridizing to at least a portion of the target nucleic acid. The target capture region can include, for example, a random nucleotide sequence or a target-specific sequence corresponding to the sequence of the known 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 a multiplex assay. In some embodiments, the universal sequence region includes a spatial barcode sequence (230) that conveys the position information of the capture oligonucleotides on a support that conveys the position information of cells within a tissue sample or a single cell. In some embodiments, the sample barcode sequence (220) can 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 that promotes circularization of the captured nucleic acid (300).In some embodiments, the universal array 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 of these. In some embodiments, the circularization anchor region (240) comprises a separate sequence that hybridizes to a portion of another type of oligonucleotide that promotes circularization of the capture nucleic acid. In some embodiments, the universal array region comprises a cleavable region that can be cleaved by an enzyme, a chemical compound, light, or heat.

[0082] Referring further to FIG. 2, in some embodiments, the surface is bound to a plurality of other types of oligonucleotides (e.g., circularization oligonucleotides (300)) that promote circularization of the captured target nucleic acid. In some embodiments, each of the circularization oligonucleotides comprises a single-stranded oligonucleotide. The circularization oligonucleotides can be immobilized on the passivated surface by their 5' ends, or an internal portion of the circularization oligonucleotide can be immobilized on 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 positioned 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 the sequencing primer sequence 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 sequence, such as the amplification primer sequence of the capture oligonucleotide. In some embodiments, the universal sequence region of the circularization oligonucleotide comprises at least one sequence that binds / hybridizes to the sample barcode sequence and / or the 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 to a portion of the circularization anchor region of the capture oligonucleotide.

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

[0084] In some embodiments, the circularized oligo may be the same as, include, or be included in the capture oligo. In some embodiments, the circularized oligonucleotide may contain distinct molecules.

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

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

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

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

[0089] In some embodiments, the low non-specific binding coating includes regions (e.g., feature portions) to which a plurality of capture and circularization oligonucleotides attached to the coating are attached to the feature portion. In some embodiments, the first feature portion is attached to a first plurality of capture oligonucleotides and a first plurality of circularization oligonucleotides, the second feature portion 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 reagents (e.g., enzymes including polymerase, polymer-nucleotide conjugate, nucleotide, and / or divalent cation) in a super-parallel manner.

[0090] In some embodiments, the cleavable region of the capture oligonucleotide is cleavable by an enzyme. In some embodiments, the cleavable region (250) shown in FIG. 2 includes 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 includes at least one 8-oxoguanine (8-oxoG) that is cleavable by a DNA-formamidopyrimidine glycosylase enzyme (Fpg). In some embodiments, the cleavable region includes a base excision site that is cleavable by endonuclease IV or endonuclease VIII. In some embodiments, the cleavable region that is cleavable by an enzyme includes a nucleotide sequence that is recognized and cleaved by a restriction endonuclease enzyme that cleaves a double-stranded or single-stranded nucleic acid strand (e.g., DNA). In some embodiments, the enzyme-cleavable region includes a glycosidic bond that is cleavable by an amylase enzyme or a peptide bond that is 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 that includes, but is not limited to, a labile chemical bond such as an ester linkage, a thiol linkage, a vicinal diol linkage, a sulfone linkage, a silyl ether linkage, a base excision, or an apurinic / apyrimidinic (AP) site. An ester linkage is cleavable by an acid, a base, or hydroxylamine. A thiol linkage can be a disulfide bond that is cleavable by glutathione or a reducing agent. A vicinal diol linkage is cleavable by sodium periodate. A sulfonate linkage is cleavable by a base. A silyl ether linkage is cleavable by an acid. A base excision or an apurinic / apyrimidinic (AP) site is cleavable by an 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 upon exposure to light, UV light, or a laser. The photocleavable moiety is cleavable upon exposure to light of any wavelength. 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 thermal exposure including a Diels-Alder linker.

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

[0094] Referring also to FIG. 4, in some embodiments herein, another type of plurality of oligonucleotides (e.g., circularized oligonucleotides (800)) in a soluble form or immobilized on a surface (e.g., a coating) is provided. The circularized oligonucleotides can promote circularization of the captured target nucleic acids. In some embodiments, each of the circularized oligonucleotides includes a single-stranded oligonucleotide. The circularized oligonucleotides can be in a soluble form or immobilized on an immobilized surface by their 5' ends, or the internal portion of the circularized oligonucleotides can be immobilized on the immobilized surface. Each of the circularized oligonucleotides can include an extendable 3' end. Each of the circularized oligonucleotides includes an adapter binding region (810). In some embodiments, the adapter binding region includes a sequencing primer binding region. In some embodiments, the adapter binding region includes an amplification primer binding region. In some embodiments, each of the circularized oligonucleotides includes 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, each of the circularized oligonucleotides includes an anchor region (830) and an anchor moiety (840).

[0095] In some embodiments, the capture oligonucleotide ((700) of FIG. 5) and the circularized oligonucleotide ((800) of FIG. 4) can be immobilized on the immobilized surface before contacting the immobilized surface with the target nucleic acid molecule (e.g., RNA) for the target molecule capture step. In an alternative embodiment, the capture oligonucleotide is immobilized on the immobilized surface before contacting the immobilized surface with the target nucleic acid molecule for the target molecule capture step, and subsequently, a plurality of circularized oligonucleotides (e.g., in a soluble form) are provided in solution and can flow onto the immobilized surface to immobilize the circularized oligonucleotides.

[0096] In some embodiments, the circularized oligo may be the same as, include, or be included within the capture oligo. In some embodiments, the circularized oligonucleotide may include distinct molecules.

[0097] In some embodiments, the cleavable region ((740) of FIG. 4) of the capture oligonucleotide is cleavable by an enzyme. In some embodiments, the cleavable region includes 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 includes at least one 8-oxoguanine (8-oxoG) that is cleavable by an RNA-formamidopyrimidine glycosylase enzyme (Fpg). In some embodiments, the cleavable region includes a base excision site that is cleavable by endonuclease IV or endonuclease VIII. In some embodiments, the cleavable region that is cleavable by an enzyme includes 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 includes a glycosidic linkage that is cleavable by an amylase enzyme, or a peptide linkage that is cleavable by a protease.

[0098] In some embodiments, the cleavable region of the capture oligonucleotide ((740) in FIG. 4) is cleavable by a chemical compound that includes, but is not limited to, labile chemical linkages including ester linkages, thiol linkages, vicinal diol linkages, sulfone linkages, silyl ether linkages, base abasic or apurinic / apyrimidinic (AP) sites. Ester linkages are cleavable by acids, bases, or hydroxylamine. Thiol linkages can be disulfide bonds cleavable by glutathione or reducing agents. Vicinal diol linkages are cleavable by sodium periodate. Sulfonate linkages are cleavable by bases. Silyl ether linkages are cleavable by acids. Base abasic or apurinic / apyrimidinic (AP) sites are cleavable by alkalis or AP endonuclease enzymes.

[0099] In some embodiments, the cleavable region of the capture oligonucleotide ((740) in FIG. 4) is cleavable by light that includes a photocleavable moiety that is cleavable upon exposure to light, UV light, or a laser. The photocleavable moiety is cleavable upon exposure to light of any wavelength. 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 FIG. 4) is cleavable by heat exposure that includes a Diels-Alder linker.

[0100] Fixation of biological samples on a surface. The present specification provides a solid support (e.g., a low non-specific binding support) further including a biological sample attached thereto. In some embodiments, the biological sample includes single cells, multiple cells, tissues, organs, organisms, or sections of these biological samples. In some embodiments, the biological sample is derived from eukaryotes (such as animals, plants, fungi, protists, etc.), archaebacteria, or eubacteria. The biological sample may be derived from prokaryotic or eukaryotic cells such as adherent or non-adherent eukaryotic cells. The biological sample may be derived from primary or immortalized cell lines from rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines.

[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 blood components (e.g., serum or plasma). In some embodiments, the biological sample is obtained from skin, heart, lung, kidney, exhaled breath, bone marrow, feces, 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, eye fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cord blood, emphatic fluid, and / or other excretions or body tissues. The biological sample may be a cell-free sample.

[0102] The biological sample may contain 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, germ cells, 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 may be derived from pathogenic cells that infect the host. In some embodiments, the cells are 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, induced-differentiated human stem cells, or a subset of cells such as 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 are consistent with the disclosure herein.

[0103] The biological sample can be extracted from an organism (e.g., by performing a biopsy) or obtained from a cell culture grown in a liquid or in a culture dish. The biological sample includes fresh, frozen, snap-frozen, or archived (e.g., formalin-fixed paraffin-embedded, FFPE) samples. The biological sample can be embedded in wax, resin, epoxy, or agarose. The biological sample can be fixed with, for example, any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton®, or glutaraldehyde. The biological sample may or may not be divided. The biological sample may or may not be stained, destained, or unstained.

[0104] In some embodiments, the biological sample may be permeabilized after being immobilized on the surface described herein to allow the nucleic acids in the sample containing the target nucleic acid molecule to move from the cells to a plurality of capture oligonucleotides immobilized on the surface. By permeabilization, agents (such as phospho-selective antibodies, nucleic acid conjugate antibodies, nucleic acid probes, primers, etc.) can penetrate into the cells and may achieve concentrations within the cells that exceed those that would normally penetrate into the cells in the absence of such permeabilization. In some embodiments, the cells may be permeabilized in the presence of at least about 60%, 70%, 80%, 90% or more methanol (or ethanol) and may be 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] The biological sample can be made permeable by contacting the biological sample with one or more permeabilizing agents including an organic solvent, a detergent, a crosslinking agent, and / or an enzyme. In some embodiments, the organic solvent includes acetone, ethanol, and methanol. In some embodiments, the detergent includes saponin, Triton® X-100, Tween20, or sodium dodecyl sulfate (SDS), or a sodium N-lauroyl sarcosinate solution. In some embodiments, the crosslinking agent includes paraformaldehyde. In some embodiments, the enzyme includes trypsin, pepsin, or a protease (such as proteinase K). In some embodiments, the target nucleic acid molecule derived from the biological sample hybridizes (is captured) to the capture oligonucleotides immobilized on the support in a form that preserves the spatial position information of the target nucleic acid molecule in the biological sample.

[0106] A biological sample can be used to generate a three-dimensional polymer matrix that includes the 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 the surfaces described herein. In some embodiments, the three-dimensional polymer matrix is porous and includes polymerized or cross-linked intracellular components that include the target nucleic acid molecule. The polymer matrix may be formed within a biological sample (e.g., a cell or tissue) by flowing one or more polymer precursors (e.g., monomers such as ethylene oxide in polyethylene glycol) into the biological sample and subjecting the one or more polymer precursors to polymerization or cross-linking. Prior to, during, or after polymer matrix formation, the location of portions (e.g., DNA, RNA, protein) within the biological sample can be fixed using, for example, a fixative (e.g., formaldehyde). The porous matrix can be manufactured in a variety of ways. For example, a polyacrylamide gel matrix can be polymerized with biotinylated DNA molecules and acridite-modified streptavidin monomers using an acrylamide:bis-acrylamide ratio appropriate for adjusting the cross-link density. Further adjustment of the size and density of the molecular sieves can be achieved by adding additional cross-linking agents such as functionalized polyethylene glycol. In addition to fixing to the surface of the biological sample, enabling the generation of a polymer matrix within the biological sample is described in PCT / US2019 / 055434, which is hereby incorporated by reference in its entirety.

[0107] A biological sample optionally contains target nucleic acid molecules 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 organellar DNA. The DNA may or may not be fragmented. In some embodiments, the target nucleic acid molecule includes RNA, including polyA RNA and / or non-poly-a RNA. The RNA includes coding and / or non-coding RNA. The 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 acid of the present disclosure has a fixed three-dimensional relationship with the biological sample after the biological sample is bound to the surface. This fixed three-dimensional relationship enables at least partial identification of the spatial origin 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 of 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. Optionally, hybridization buffer formulations are described that, in combination with the disclosed low-binding supports, result in improved hybridization rates, hybridization specificity (or stringency), and hybridization efficiency (or yield). As used herein, hybridization specificity is a measure of the ability of a ligated adapter sequence, primer sequence, or oligonucleotide sequence to hybridize correctly only to a perfectly complementary sequence, and hybridization efficiency is a measure of the percentage of the total ligated adapter sequences, primer sequences, or oligonucleotide sequences that are available for hybridization to a complementary sequence.

[0110] Improvements in hybridization specificity and / or efficiency may be achieved by optimization of the hybridization buffer formulations used with the disclosed low-binding surfaces and are described in more detail in the examples below. Examples of hybridization buffer components that may be adjusted to achieve improved performance include, but are not limited to, buffer type, organic solvent mixtures, 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, and other additives.

[0111] As a non-limiting example, suitable buffers for formulating hybridization buffers 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 a suitable buffer generally depends on the target pH of the hybridization buffer solution. Generally, the desired pH of the buffer solution is in the range of 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 combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the desired pH may be in the range of about 6.4 to about 7.2. One of ordinary skill in the art will recognize that the pH of the buffer can have any value within this range, for example, about 7.25.

[0112] Suitable surfactants for use in the hybridization buffer formulation include, but are not limited to, zwitterionic 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-oxide, 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 non-specific binding support alone or in combination with an optimized buffer formulation can result in relative hybridization rates in the range of about 2 to about 20 times faster than conventional hybridization protocols. In some examples, the relative hybridization rate can be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, at least 20 times, at least 25 times, at least 30 times, or at least 40 times faster than conventional hybridization protocols.

[0114] The disclosed use of the low non-specific binding support alone or in combination with an optimized buffer formulation can result in a total hybridization reaction time of 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or less than 5 minutes (i.e., the time required to achieve 90%, 95%, 98%, or 99% completion of the hybridization reaction) for any of these completion criteria.

[0115] The use of the disclosed low non-specific binding support alone, or in combination with an optimized buffer formulation, can result in improved hybridization specificity compared to conventional hybridization protocols. In some embodiments, the achievable hybridization specificity is better than 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 1,000 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 non-specific binding support 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 to the target oligonucleotide sequence) 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 at any of the hybridization reaction times specified above. For example, in some examples where the hybridization efficiency is less than 100%, the resulting surface density of the target nucleic acid sequence hybridized to the support surface may be less than the areal density of the oligonucleotide adapter or primer sequences on the surface.

[0117] In some examples, the use of the disclosed low non-specific binding support for nucleic acid hybridization (or amplification) applications using a conventional hybridization (or amplification) protocol, or an optimized hybridization (or amplification) protocol, can lead to reduced requirements for the input concentration of target (or sample) nucleic acid molecules in contact with the support surface. For example, in some examples, the target (or sample) nucleic acid molecules can contact the support surface at a concentration in the range of about 10 pM to about 1 μM (i.e., before annealing or amplification). In some examples, the target (or sample) nucleic acid molecules can 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 molecules can be administered at a maximum of 1 μM, a maximum of 900 nM, a maximum of 800 nM, a maximum of 700 nM, a maximum of 600 nM, a maximum of 500 nM, a maximum of 400 nM, a maximum of 300 nM, a maximum of 200 nM, a maximum of 100 nM, a maximum of 90 nM, a maximum of 80 nM, a maximum of 70 nM, a maximum of 60 nM, a maximum of 50 nM, a maximum of 40 nM, a maximum of 30 nM, a maximum of 20 nM, a maximum of 10 nM, a maximum of 1 nM, a maximum of 900 pM, a maximum of 800 pM, a maximum of 700 pM, a maximum of 600 pM, a maximum of 500 pM, a maximum of 400 pM, a maximum of 300 pM, a maximum of 200 pM, a maximum of 100 pM, a maximum of 90 pM, a maximum of 80 pM, a maximum of 70 pM, a maximum of 60 pM, a maximum of 50 pM, a maximum of 40 pM, a maximum of 30 pM, a maximum of 20 pM, or a maximum of 10 pM.Any combination of the lower and upper limits described in this paragraph can be used to form the ranges included in the present disclosure. For example, in some instances, the target (or sample) nucleic acid molecule can be administered at a concentration in the range of about 90 pM to about 200 nM. One of ordinary skill in the art will recognize that the target (or sample) nucleic acid molecule can be administered at a concentration having any value within this range, such as about 855 nM.

[0118] In another example, the volume of the biological sample that can contact the surface can be reduced compared to an equivalent biological sample analyzed using standard hybridization reagents with an equivalent surface. In some embodiments, the fluid sample containing the target (or sample) nucleic acid molecule can be in the range of about 5 μl to about 900 μl of sample volume. In some examples, the range of the sample volume is from about 5 μl to about 800 μl. In some examples, the range of the sample volume is from about 5 μl to about 700 μl. In some examples, the range of the sample volume is from about 5 μl to about 600 μl. In some examples, the range of the sample volume is from about 5 μl to about 500 μl. In some examples, the range of the sample volume is from about 5 μl to about 400 μl. In some examples, the range of the sample volume is from about 5 μl to about 300 μl. In some examples, the range of the sample volume is from about 5 μl to about 200 μl. In some examples, the range of the sample volume is from about 5 μl to about 150 μl. In some examples, the range of the sample volume is from 5 μl to about 100 μl. In some examples, the range of the sample volume is from about 5 μl to about 90 μl. In some examples, the range of the sample volume is from about 5 μl to about 85 μl. In some examples, the range of the sample volume is from about 5 μl to about 80 μl. In some examples, the range of the sample volume is from about 5 μl to about 75 μl. In some examples, the range of the sample volume is from about 5 μl to about 70 μl. In some examples, the range of the sample volume is from about 5 μl to about 65 μl. In some examples, the range of the sample volume is from about 5 μl to about 60 μl. In some examples, the range of the sample volume is from about 5 μl to about 55 μl. In some examples, the range of the sample volume is from about 5 μl to about 50 μl. In some examples, the range of the sample volume is from about 15 μl to about 150 μl. In some examples, the range of the sample volume is from about 15 μl to about 120 μl. In some examples, the range of the sample volume is from 15 μl to about 100 μl. In some examples, the range of the sample volume is from about 15 μl to about 90 μl. In some examples, the range of the sample volume is from about 15 μl to about 85 μl. In some examples, the range of the sample volume is from about 15 μl to about 80 μl. In some examples, the range of the sample volume is from about 15 μl to about 75 μl. In some examples, the range of the sample volume is 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 support alone, or in combination with an optimized hybridization buffer formulation, can result in a surface density of hybridized target (or sample) oligonucleotide molecules (i.e., prior to any subsequent solid-phase or clonal amplification reaction) in the range of about 0.0001 target oligonucleotide molecules per μm2 to about 1,000,000 target oligonucleotide molecules per μm2.In some examples, the surface density of the hybridized target oligonucleotide molecules can be 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, 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 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 per μm2.In some examples, the surface density of the hybridized target oligonucleotide molecules is 1 μm. 2 It can be a maximum of 1,000,000, a maximum of 950,000, a maximum of 900,000, a maximum of 850,000, a maximum of 800,000, a maximum of 750,000, a maximum of 700,000, a maximum of 650,000, a maximum of 600,000, a maximum of 550,000, a maximum of 500,000, a maximum of 450,000, a maximum of 400,000, a maximum of 350,000, a maximum of 300,000, a maximum of 250,000, a maximum of 200,000, a maximum of 150,000, a maximum of 100,000, a maximum of 95,000, a maximum of 90,000, a maximum of 85,000, a maximum of 80,000, a maximum of 75,000, a maximum of 70,000, a maximum of 65,000, a maximum of 60,000, a maximum of 55,000, a maximum of 50,000, a maximum of 45,000, a maximum of 40,000, a maximum of 35,000, a maximum of 30,000, a maximum of 25,000, a maximum of 20,000, a maximum of 15,000, a maximum of 10,000, a maximum of 9,500, a maximum of 9,000, a maximum of 8,500, a maximum of 8,000, a maximum of 7,500, a maximum of 7,000, a maximum of 6,500, a maximum of 6,000, a maximum of 5,500, a maximum of 5,000, a maximum of 4,500, a maximum of 4,000, a maximum of 3,500, a maximum of 3,000, a maximum of 2,500, a maximum of 2,000, a maximum of 1,500, a maximum of 1,000, a maximum of 900, a maximum of 800, a maximum of 700, a maximum of 600, a maximum of 500, a maximum of 400, a maximum of 300, a maximum of 200, a maximum of 100, a maximum of 90, a maximum of 80, a maximum of 70, a maximum of 60, a maximum of 50, a maximum of 40, a maximum of 30, a maximum of 20, a maximum of 10, a maximum of 5, a maximum of 1, a maximum of 0.5, a maximum of 0.1, a maximum of 0.05, a maximum of 0.01, a maximum of 0.005, a maximum of 0.001, a maximum of 0.0005, or a maximum of 0.0001 molecules. Any combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some cases, the surface density of the hybridized target oligonucleotide molecules is 1 μm 2 per approximately 3,000 molecules to 1 μm 2It can be in the range of about 20,000 molecules per hit. One of ordinary skill in the art will recognize that the surface density of hybridized target oligonucleotide molecules can be any value within this range, for example, 1 μm 2 and can have about 2,700 molecules per hit.

[0120] In other words, in some cases, the use of a low non-specific binding support alone or in combination with an optimized hybridization buffer formulation results in a surface density of hybridized target (or sample) oligonucleotide molecules (i.e., before any subsequent solid-phase or clonal amplification reaction is performed) in the range of about 100 hybridized target oligonucleotide molecules per mm 2 to about 1×10 2 oligonucleotide molecules per mm, or about 100 hybridized target oligonucleotide molecules per mm 7 to about 1×10 2 hybridized target oligonucleotide molecules per mm. In some examples, the areal density of hybridized target oligonucleotide molecules is 1 mm 2 to about 1×10 12 hybridized target oligonucleotide molecules. In some cases, the surface density of hybridized target oligonucleotide molecules is 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 1×10 7 、 at least 5×10 7 、 at least 1×10 8 、 at least 5×10 8 、 at least 1×10 9 、 at least 5×10 9 、 at least 1×10 10 、 at least 5×10 10 、 at least 1×10 11 、 at least 5×10 11 、 or can be the numerator of at least 1×10 12 。 In some examples, the surface density of the hybridized target oligonucleotide molecules is up to 1×10 per 1 mm 2 、 up to 5×10 12 、 up to 1×10 11 、 up to 5×10 11 、 up to 1×10 10 、 up to 5×10 10 、 up to 1×10 9 、 up to 5×109 , 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 can be the numerator. Any combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some cases, the surface density of the hybridized target oligonucleotide molecules can be from about 5,000 molecules per 1 μm 2 to about 50,000 molecules per 1 μm 2 . Those skilled in the art will recognize that the surface density of the hybridized target oligonucleotide molecules can have any value within this range, for example, about 50,700 molecules per 1 μm 2 .

[0121] In some examples, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) hybridized to an oligonucleotide adapter or primer molecule 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 is 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 falling within the ranges described herein, for example, at least 0.85 kb in length.

[0122] In some examples, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) can include a single-stranded or double-stranded multimeric nucleic acid molecule that further includes a repeat of regularly occurring monomer units. In some examples, the single-stranded or double-stranded multimeric nucleic acid molecule has a length of 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, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, or at least 20 kb, at least 30 kb, or at least 40 kb, or any intermediate value falling within the ranges described herein, for example, a length of about 2.45 kb.

[0123] In some examples, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) can include a single-stranded or double-stranded multimeric nucleic acid molecule that includes from about 2 to about 100 copies of a regularly repeating monomer unit. In some examples, the number of copies of the regularly repeating monomer 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 monomer 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 combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the number of copies of the regularly repeating monomer unit can be in the range of about 4 to about 60. One of ordinary skill in the art will recognize that the number of copies of the regularly repeating monomer 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 the hybridized target sequences with respect to the number of copies of the target sequences per unit area of the support surface can exceed the surface density of the 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 aspects of the present disclosure, nucleic acid amplification formulations are described that, in combination with the disclosed low-binding supports, provide improved amplification rates, amplification specificity, and amplification efficiency. As used herein, specific amplification refers to the amplification of template library oligonucleotide strands that are 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 oligonucleotides tethered on the surface that are successfully amplified during a given amplification cycle or amplification reaction. Nucleic acid amplification performed on the surfaces disclosed herein can provide 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 can be utilized with the disclosed low-non-specific-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, the rolling circle amplification reaction involves (1) contacting a plurality of immobilized circular nucleic acid molecules closed by a covalent bond 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 that mediates nucleotide binding but does not mediate nucleotide incorporation (e.g., strontium or barium), and optionally (iv) a plurality of amplification primers if the covalently closed circular molecule lacks a primer, to form a captured nucleotide-polymerase complex. The rolling circle amplification reaction further involves (4) performing a nucleotide polymerization reaction by contacting the captured nucleotide-polymerase complex with (i) at least one divalent cation that mediates nucleotide binding and mediates nucleotide incorporation (e.g., magnesium and / or manganese), 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 comprises a plurality of compaction oligonucleotides that can hybridize to a portion of the concatemer and collapse the concatemer into a more compact shape and size. A compaction oligonucleotide is a single-stranded nucleic acid molecule having two identical sequences separated by a short linker sequence, where the two identical sequences are reverse complementary to a portion of the concatemer. The compaction oligonucleotide can be of any length, for example, 20 to 100 nucleotides. The two identical sequence regions hybridize to the concatemer, pulling together the distal portions of the strands and causing compaction of the concatemer. In some embodiments, the compaction oligonucleotide is resistant to 3'-exonuclease digestion and / or single-strand endonuclease digestion. In some embodiments, the compaction oligonucleotide comprises any one or any combination of two or more of the following: 3'-terminal side terminal phosphorylation; at least two 3'-terminal side terminal nucleotides having phosphorothioate bonds therebetween; at least one 3'-terminal side 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 includes phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca(exo-)DNA polymerase, the Klenow fragment of Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, the reverse transcriptase of the HIV virus, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), a mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0129] In some embodiments, the amplification primer includes 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 digestion and / or single-strand endonuclease digestion. In some embodiments, the amplification primer includes any one or a combination of two or more of the following: 3'-terminal phosphorylation; at least two 3'-terminal nucleotides having phosphorothioate bonds 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 includes 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).

[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, the concatemer can contain at least 2, 10, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more copies of the repeating unit.

[0133] After the rolling circle amplification method, a multiple displacement amplification reaction using random array primers may be performed. 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, wherein each of the plurality of amplification primers is exonuclease resistant, has a 3'-extensible 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 mediates 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 includes phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca(exo-)DNA polymerase, Klenow fragment of Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, reverse transcriptase of HIV virus, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0135] In some embodiments, in a multiple displacement amplification (MDA) reaction mixture, the plurality of amplification primers includes single-stranded nucleic acid primers having a length of about 5 to 25 nucleotides. In some embodiments, the plurality of soluble amplification primers includes unprotected single-stranded nucleic acid primers. In some embodiments, the plurality of soluble amplification primers includes protected single-stranded nucleic acid primers that are resistant to 3'-exonuclease digestion and / or single-strand endonuclease digestion. In some embodiments, the plurality of soluble amplification primers includes any one or a combination of two or more of the following: 3'-terminal side terminal phosphorylation; at least two 3'-terminal side terminal nucleotides having phosphorothioate bonds therebetween; at least one 3'-terminal side 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 includes a population of primers having the same length, e.g., a length of 6 or 9 nucleotides. In some embodiments, the plurality of soluble amplification primers includes a population of primers having a mixture of different lengths, e.g., a mixture containing 6mer and 9mer primers. In some embodiments, the plurality of soluble amplification primers has a maximum of 4 6different arrays (e.g., in the case of 6-mers) or 4 9 contains a mixture of primers having random arrays that contain different arrays (e.g., in the case of 9-mers).

[0136] In some embodiments, the multiple displacement amplification (MDA) reaction mixture may further include 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 can 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 performed. The multiple displacement amplification reaction includes (1) forming a multiple displacement amplification (MDA) reaction mixture by contacting a plurality of immobilized concatemers with (i) a second plurality of polymerases having strand displacement activity, (ii) a plurality of DNA primase-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 mediates nucleotide incorporation, and (2) performing an isothermal multiple displacement amplification (MDA) reaction to generate a plurality of immobilized branched concatemers. In some embodiments, the multiple displacement amplification reaction is performed without adding amplification primers (e.g., primerless reaction).

[0139] In some embodiments, in a multiple displacement amplification (MDA) reaction mixture, a second plurality of polymerases having strand displacement activity includes phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca(exo-)DNA polymerase, Klenow fragment of Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, reverse transcriptase of HIV virus, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 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 includes 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 can 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-step amplification method involves exposing the concatemer to a nucleic acid relaxing agent (first step) and then performing a rolling circle amplification reaction during the second step. Without wishing to be bound by theory, it is hypothesized that the nucleic acid relaxing agent disrupts hydrogen bonds (e.g., denatures) in the plurality of immobilized nucleic acid concatemers, thereby relaxing the structure of the nucleic acid concatemers and increasing the number of new duplex formations between the immobilized surface capture primers and a portion of the nucleic acid concatemers, thereby increasing the opportunity to generate new concatemers from the duplexed immobilized surface capture primers. New concatemers can be generated during the rolling circle amplification reaction. By including a relaxant, nucleic acid denaturation can be induced without using a denaturing temperature or chemical.

[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 rolling circle amplification reaction mixture; (4) performing a rolling circle amplification reaction (e.g., without addition of a soluble primer) on the support to generate a plurality of double-stranded concatemers; (5) washing; and (6) repeating steps (2)-(5) at least once.

[0145] In some embodiments, the relaxation reaction mixture of step (2) can be formed using at least one nucleic acid relaxant that can interfere with hydrogen bonds in the immobilized nucleic acid concatemer. Exemplary relaxants 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,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 relaxants include sodium iodide, potassium iodide, and polyamines.

[0146] In some embodiments, the relaxation reaction mixture comprises any one or a 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 SSC in the relaxation reaction mixture can be 1x, 2x, 3x, or 4x.

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

[0149] In some embodiments, in the flexion amplification reaction mixture of step (3), the second plurality of polymerases having strand displacement activity includes 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, the reverse transcriptase of the HIV virus, or Deep Vent DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

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

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

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

[0153] In some embodiments, the bent amplification reaction mixture of step (2) may 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). In some embodiments, these accessory proteins may be omitted.

[0154] In some embodiments, in the bent amplification reaction of step (4), the temperature ramp-up conditions can be carried out from about 20°C to about 90°C. In some embodiments, in the bent amplification reaction of step (4), the temperature ramp-up conditions can be carried out 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, in the bent amplification reaction of step (4), the amplification incubation conditions can be at 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 temperatures. In some embodiments, in the bent amplification reaction of step (4), the amplification incubation conditions can be carried out for about 30 - 45 seconds, or about 45 - 60 seconds, or about 60 - 75 seconds, or about 75 - 90 seconds, or longer. In some embodiments, in the bent amplification reaction of step (4), the temperature ramp-down conditions can be carried out from about 90°C to about 20°C.

[0155] In some embodiments, in the bending amplification reaction of step (4), the temperature ramp-down condition can be carried out 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, in the washing step of step (5), the washing buffer contains 1×SSC, or 1×SSC containing cobalt hexamine. In some embodiments, steps (2)-(5) may be repeated at least once, or may be repeated up to 10 times, or may be repeated up to 15 times, or may be repeated up to 20 times, or may be repeated up to 30 or more times.

[0156] Often, improvements in amplification rate, amplification specificity, and amplification efficiency can be achieved using the disclosed low non-specific binding support 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 various ways to achieve improved performance, including, but not limited to, buffer type, buffer pH, organic solvent mixture, buffer viscosity, surfactant and zwitterionic components, ionic strength (including adjustment of monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, and selection of other additives.

[0157] Use of the disclosed low non-specific binding support alone or in combination with an optimized amplification reaction formulation can result in an increase in amplification rate compared to that obtained using conventional supports and amplification protocols. In some examples, the relative amplification rate that can be achieved is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times that of 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 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 criteria (i.e., the time required to achieve 90%, 95%, 98%, or 99% completion of the amplification reaction).

[0159] Some of the low binding support surfaces disclosed herein exhibit a ratio of specific to non-specific binding to 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 falling within the scope of this specification. Some of the surfaces disclosed herein exhibit a ratio of specific fluorescent signal to non-specific fluorescent signal to 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 falling within the scope of this specification.

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

[0161] In some examples, the use of the disclosed low non-specific binding support alone, or in combination with an optimized amplification reaction formulation, may increase specific amplification and / or decrease non-specific amplification compared to that obtained using conventional supports and amplification protocols. In some examples, the ratio of specific amplification to non-specific amplification resulting as an achievable outcome 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 examples, the use of the low non-specific binding support alone, or in combination with an optimized amplification reaction formulation, may result in increased amplification efficiency compared to that obtained using conventional supports and amplification protocols. In some examples, the achievable amplification efficiency is 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, the clonally amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) hybridized to oligonucleotide adapters or primer molecules attached to the low-binding support surface can range in length from about 0.02 kilobases (kb) to about 20 kb, or from about 0.1 kilobase (kb) to about 20 kb. In some examples, the 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, or at least 20 kb in length, or any intermediate value falling within the ranges described herein, for example, at least 0.85 kb in length.

[0164] In some instances, the clonally amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) can include single-stranded or double-stranded multimeric nucleic acid molecules that further include repeats of regularly occurring monomeric units. In some examples, the clonally amplified single-stranded or double-stranded multimeric nucleic acid molecules can be at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, or at least 20 kb in length, or any intermediate value falling within the ranges described herein, for example, about 2.45 kb in length.

[0165] In some examples, the clonally amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) can include a single-stranded or double-stranded multimeric nucleic acid molecule that includes from about 2 to about 100 copies of a regularly repeating monomer unit. In some examples, the number of copies of the regularly repeating monomer 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 monomer 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 combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the number of copies of the regularly repeating monomer unit can be in the range of about 4 to about 60. One of ordinary skill in the art will recognize that the number of copies of the regularly repeating monomer unit can have any value within this range, such as about 12. Thus, in some examples, even if the hybridization efficiency and / or amplification efficiency is less than 100%, the surface density of the clonally amplified target sequence with respect to the number of copies of the target sequence per unit area of the support surface can exceed the surface density of the oligonucleotide primer.

[0166] In some examples, the use of the disclosed low non-specific binding support alone, or in combination with an optimized amplification reaction formulation, can result in increased copy numbers of clones compared to those obtained using conventional supports and amplification protocols. In some examples, for instance, a clonally amplified target (or sample) oligonucleotide molecule contains tandem multimers of a monomeric target sequence, and the copy number of the clone can be substantially smaller compared to that obtained using conventional supports and amplification protocols. Thus, in some examples, the copy number of the clone can range from about one 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 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 per amplified colony.In some examples, the copy number of the clone can be 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 9,000, up to 8,000, up to 7,000, up to 6,000, up to 5,000, up to 4,000, up to 3,000, up to 2,000, up to 1,000, up to 500, up to 100, up to 50, up to 10, up to 5, or up to 1 molecule per amplified colony. Any combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the number of copies of the clone can be in the range of about 2,000 molecules to about 9,000 molecules. One of ordinary skill in the art will recognize that the copy number of the clone can have any value within this range, for example, in some cases about 2,220 molecules, or in other cases about 2 molecules.

[0167] In some examples, as described above, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) can include a multimeric repeat of monomeric target sequences. In some examples, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) includes multiple molecules, each of which includes a single monomeric target sequence. Thus, the use of the disclosed low non-specific binding support alone or in combination with an optimized amplification reaction formulation can result in a surface density of target sequence copies in the range of about 100 target sequence copies per mm 2 to about 1×10 2 target sequence copies per mm. In some examples, the surface density of target sequence copies is 1 mm 12 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 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 1×10 7 、 at least 5×10 7 、 at least 1×10 8 、 at least 5×10 8 、 at least 1×10 9 、 at least 5×10 9 、 at least 1x10 10 、 at least 5×10 10 、 at least 1×10 11 、 at least 5×10 11 、 or at least 1×10 12 could be. In some examples, the surface density of target sequence copies is up to 1×10 2 per 1 mm 12 at most, up to 5×10 11 at most, up to 1×10 11 at most, up to 5×10 10 at most, up to 1×10 10 at most, up to 5×10 9 at most, up to 1×10 9 at most, up to 5×108 and up to 1×10 8 and up to 5×10 7 and up to 1×10 7 and 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 target sequence copies. Any combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the surface density of the target sequence copies is from about 1,000 target sequence copies per mm 2 to about 65,000 target sequence copies per mm 2 . One of ordinary skill in the art will recognize that the surface density of the target sequence copies can have any value within this range, for example, about 49,600 target sequence copies per mm 2 .

[0168] In some examples, the use of the disclosed low non-specific binding support alone or in combination with an optimized amplification buffer formulation results in from about 100 molecules per mm 2 to about 1×10 2 per mm 12can result in a surface density of clonally amplified target (or sample) oligonucleotide molecules (or clusters) within the range of the colony. In some examples, the surface density of the clonally amplified molecules is 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 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 1×10 2 per square millimeter, and can be at least 1×10 7 、 at least 5×10 7 、 at least 1×10 8 、 at least 5×10 8 、 at least 1×10 9 、 at least 5×10 9 、 at least 1x10 10 、 at least 5×10 10 、 at least 1×10 11 、 at least 5×10 11 、 at least 1x1012 molecules. In some examples, the surface density of the clonally amplified molecules is at most 1×10 2 per square millimeter, at most 5×10 12 、 at most 1×10 11 、 at most 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 combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the surface density of the clonally amplified molecules is about 5,000 molecules per mm 2 to about 50,000 molecules per mm 2 . One of ordinary skill in the art will recognize that the surface density of the clonally amplified colonies can have any value within this range, e.g., about 48,800 molecules per mm 2 .

[0169] In some examples, the use of the disclosed low non-specific binding support alone or in combination with an optimized amplification buffer formulation is about 100 molecules per mm 2 to about 100 molecules per mm 2which can result in a surface density of clonally amplified target (or sample) oligonucleotide molecules (or clusters) in the range of colonies of about 1×10 12 . In some examples, the surface density of the clonally amplified molecules is 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 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 1×10 2 . In some examples, the surface density of the clonally amplified molecules is 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 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 1×10 7 , at least 5×10 7 , at least 1×10 8 , at least 5×10 8 , at least 1×10 9 , at least 5×10 9 , at least 1x1010, at least 5×10 10 , at least 1×10 11 , at least 5×10 11 , at least 1x1012 molecules. In some examples, the surface density of the clonally amplified molecules is at most 1×10 2 , at most 5×10 12 , at most 1×10 11 per 1 mm11 , 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 can be the numerator. Any combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the surface density of the clonally amplified molecules is about 5,000 molecules per 1 mm 2 to about 50,000 molecules per 1 mm 2 . One of ordinary skill in the art will recognize that the surface density of the clonally amplified colonies can have any value within this range, for example, about 48,800 molecules per 1 mm 2 .

[0170] In some examples, the use of the disclosed low non-specific binding support alone or in combination with an optimized amplification buffer formulation results in about 100 colonies per 1 mm 2 to about 1 mm2 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 mm2 of colonies. In some examples, the surface density of clonally amplified colonies is 1 mm2 or greater. 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 1×10 7 , at least 5 × 10 7 , at least 1 × 10 8 , at least 5 × 10 8 , at least 1 × 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 1×10 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, 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 combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some examples, the surface density of the clonally amplified colonies can be from about 5,000 colonies per 1 mm 2 to about 50,000 colonies per 1 mm 2 . One of ordinary skill in the art will recognize that the surface density of the clonally amplified colonies can be any value within this range, for example, having about 48,800 colonies per 1 mm 2 .

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

[0172] In some cases, the support surfaces and methods disclosed herein enable 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, the use of the support surfaces and methods disclosed herein enables a simplified amplification reaction. For example, in some cases, the amplification reaction is carried out using 1, 2, 3, 4, or 5 or fewer individual reagents.

[0174] In some cases, the use of the support surfaces and methods disclosed herein enables the use of a simplified temperature profile during amplification, such that the reaction is carried out at temperatures ranging from low temperatures of 15°C, 20°C, 25°C, 30°C, or 40°C to high temperatures of 40°C, 45°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, or above 80°C, for example, in the range of 20°C to 65°C.

[0175] The amplification reaction is also improved such that a sample of a lower amount of template (e.g., target or sample molecule), 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, 900 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, e.g., 500 nM, etc., is sufficient to provide a recognizable 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, e.g., nucleic acid base discrimination, nucleic acid base classification, nucleic acid base calling, nucleic acid detection applications, nucleic acid sequencing applications, nucleic acid-based (gene 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 support.

[0177] Fluorescence imaging can be carried out 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 derivatives thereof such as cyanine dye-3 (Cy3), cyanine dye-5 (Cy5), cyanine dye-7 (Cy7), etc. Examples of fluorescence imaging techniques that can be used include, but are not limited to, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, etc. Examples of fluorescence imaging devices that can be used include, but are not limited to, fluorescence microscopes equipped with image sensors or cameras, confocal fluorescence microscopes, two-photon fluorescence microscopes, or custom devices that include appropriate selections of light sources, lenses, mirrors, prisms, dichroic reflectors, apertures, and image sensors, or cameras. A non-limiting example of a fluorescence microscope equipped to acquire images of the disclosed low-binding support surface and the clonally amplified colonies (or clusters) of target nucleic acid sequences hybridized thereon is the Olympus IX83 inverted fluorescence microscope (20x, 0.75NA, 532 nm light source, set of bandpass and dichroic mirror filters optimized for 532 nm long-pass excitation, and Cy3 fluorescence emission filter, Semrock 532 nm dichroic reflector, and a camera (Andor sCMOS, Zyla 4.2) with excitation light intensity adjusted to avoid signal saturation). Often, while acquiring images, the support surface may be immersed in a buffer (e.g., 25 mM ACES, pH 7.4 buffer).

[0178] In some examples, the performance of nucleic acid hybridization and / or amplification reactions using the disclosed reaction formulations and low non-specific binding supports can be evaluated using fluorescence imaging techniques, where the contrast to noise ratio (CNR) of the image provides an important metric for evaluating amplification specificity and non-specific 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 particular feature (diffraction-limited spot, DLS) within a specified region of interest (ROI). The signal to noise ratio (SNR) is often considered a benchmark for overall signal quality, but improvement in CNR has been shown to provide a significant advantage over SNR as a benchmark for signal quality in applications that require rapid image acquisition (e.g., sequencing applications where cycle times must be minimized), as shown in the examples below. The surfaces of the present disclosure are also provided in co-pending International Application No. PCT / US2019 / 061556, which is hereby incorporated by reference in its entirety.

[0179] In most ensemble-based sequencing approaches, typically, the background term is measured as the signal associated with the "interstitial" region. In addition to the "interstitial" background (B inter ), an "intra-tissue" background (B intra ) is present within the region occupied by the amplified DNA colonies. The combination of these two background signals represents the achievable CNR, which then directly affects the optical instrument requirements, architecture cost, reagent cost, run time, cost / genome, and ultimately the accuracy and data quality for circular array-based sequencing applications. B interBackground signal arises from a variety of sources, some examples include autofluorescence from expendable flow cells, non-specific adsorption of detection molecules resulting in false fluorescent signals that may obscure signals from the ROI, and the presence of non-specific 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. Signals arising from individual DNA colonies (i.e., the (S)-B of the FOV) are averaged over time and subtracted. inter ) results in 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, and thus become 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 reduces B inter Background signals can be reduced, leading to improvements in certain nucleic acid amplifications, and non-specific amplifications that can affect background signals 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 2, 5, 10, 100, or 1000-fold improvements in CNR compared to those achieved using conventional supports and hybridization, amplification, and / or sequencing protocols. Although described herein in the context of using fluorescent imaging as a readout or detection mode, the same principles apply to the use of the disclosed low non-specific 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 support, optionally used in combination with the disclosed hybridization and / or amplification protocols, results in a solid-phase reaction that (i) has negligible non-specific binding of proteins and other reaction components (thus minimizing substrate background), (ii) exhibits negligible non-specific nucleic acid amplification products, and (iii) provides an adjustable nucleic acid amplification reaction.

[0182] A method for capturing and analyzing DNA. The present disclosure provides a method for analyzing nucleic acids in a cell- or spatially addressable manner, the method comprising: (a) providing a support comprising a low non-specific binding coating to which a plurality of capture oligonucleotides and a plurality of circularization oligonucleotides are immobilized (e.g., FIG. 2), 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 comprising a spatial barcode sequence; (iii) a circularization anchor sequence; and (iv) a cleavable region, and wherein the plurality of circularization oligonucleotides comprise: (i) a homopolymer region; (ii) a universal sequence region comprising a sequencing primer binding sequence; and (iii) a circularization anchor binding sequence, 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 fluorescence 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 has about 0.25 molecules / μm 2Shows a level of non-specific Cy3 dye absorption below which less than 5% of the target nucleic acid associates with the surface coating without hybridizing to the immobilized capture oligonucleotide. In some embodiments, when using a fluorescence imaging system under non-signal saturation conditions, the fluorescence image of the surface coating having a plurality of clonally amplified clusters of nucleic acids exhibits a contrast-to-noise ratio (CNR) of at least 20, 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 the 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 circularized oligonucleotide, and / or (iv) a cleavable region.

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

[0186] In some embodiments, the immobilized circularized oligonucleotide of step (a) can include any combination of (i) a homopolymer region, (ii) a universal sequence region that includes 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] A method for analyzing nucleic acids further includes the step of (b) contacting a cell biological sample with a low non-specific binding coating in the presence of a highly efficient hybridization buffer under conditions suitable for promoting the transfer of a target nucleic acid molecule from the cell 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 position information of the target nucleic acid molecule in the cell biological sample, and wherein the target nucleic acid includes DNA or RNA (e.g., FIG. 7).

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

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

[0190] In some embodiments, the highly efficient hybridization buffer in step (b) includes: (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of 115 and present in the highly efficient hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the highly efficient hybridization buffer formulation in the range of about 4-8; 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 the following: (i) a first polar aprotic solvent comprises 25-50% by volume of acetonitrile in the high-efficiency hybridization buffer; (ii) a second polar aprotic solvent comprises 5-10% by volume of formamide in the high-efficiency hybridization buffer; (iii) a pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) with a pH of 5-6.5; and, (iv) a crowding agent comprises 5-35% by volume of polyethylene glycol (PEG) in 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 of step (b) promotes high stringency (e.g., specificity), speed, and effectiveness of nucleic acid hybridization and increases the efficiency of subsequent amplification and sequencing steps. In some embodiments, the high-efficiency hybridization buffer significantly shortens the nucleic acid hybridization time and reduces the sample input requirements. Nucleic acid annealing is performed under isothermal conditions, and the cooling step for annealing can be eliminated.

[0193] A method for analyzing nucleic acids further comprises the step of (c) performing a primer extension reaction on an immobilized nucleic acid duplex using a target nucleic acid molecule hybridized 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 Escherichia coli DNA polymerase I, the Klenow fragment of Escherichia 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 transcription enzyme primers. In some embodiments, the reverse transcription reaction of step (a) comprises a plurality of nucleotides and an enzyme having reverse transcription activity comprising 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 MultiScribe (商標) , ThermoScript (商標) , or ArrayScript (商標) and can be a commercially available enzyme. In some embodiments, the reverse transcriptase comprises an enzyme of Superscript I, II, III, or IV. In some embodiments, the reverse transcription reaction can include an RNase inhibitor.

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

[0196] A method for analyzing nucleic acids further includes a step of cleaving the immobilized tailed target extension product to release it 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 compound, light, or heat.

[0197] A method for analyzing nucleic acids comprises the step of binding a soluble, tail-like target extension product, having an added homopolymer tail, to one of the immobilized circularized oligonucleotides under conditions appropriate for hybridizing the homopolymer region of the soluble, tail-like target extension product to the homopolymer region of the immobilized circularized oligonucleotide and for hybridizing the circularization anchor sequence of the soluble, tail-like target extension product to the circularization anchor binding sequence of the immobilized circularized oligonucleotide, thereby forming an open circular target extension product having a gap and / or a nick, such that the immobilized circularized oligonucleotide functions as a splint molecule for promoting circularization of the soluble, tail-like target extension product (e.g., FIG. 27).

[0198] A method for analyzing nucleic acids further comprises the step of (g) closing the gap, if present, by performing a gap filling primer extension reaction and closing the nick, if present, by performing a ligation reaction on the open circular target extension product, thereby forming a covalently closed circular target extension product hybridized to the immobilized circularized oligonucleotide, where the immobilized circularized oligonucleotide comprises a homopolymer region having a 3'-extendable end (e.g., FIG. 27).

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

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

[0201] In some embodiments, the rolling circle amplification reaction of step (h) includes contacting a covalently closed circularized padlock probe (e.g., a 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 appropriate to generate at least one nucleic acid concatemer, where the at least one catalytic divalent cation includes magnesium or manganese.

[0202] In some embodiments, the rolling circle amplification reaction of step (h) comprises: (1) contacting a covalently closed circular circularization padlock probe (e.g., a 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 facilitate polymerase-catalyzed nucleotide incorporation into the amplification primer, wherein the non-catalytic divalent cation comprises strontium or barium; and (2) contacting the covalently closed circular circularization padlock probe with at least one catalytic divalent cation under conditions appropriate to generate at least one nucleic acid concatemer, wherein the at least one catalytic divalent cation comprises magnesium or manganese.

[0203] In some embodiments, the rolling circle amplification reaction of step (h) is carried out at a constant temperature (e.g., isothermal) in the range 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 concatemer to form immobilized small nanoballs.

[0205] In some embodiments, the rolling circle amplification reaction of step (h) comprises a DNA polymerase having strand displacement activity selected from the group consisting of 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 virus reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0206] In some embodiments, after the rolling circle amplification reaction, a multiple displacement amplification (MDA) reaction can be performed. In some embodiments, the method further comprises performing a multiple displacement amplification (MDA) reaction before step (f), 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, after the rolling circle amplification reaction, a multiple displacement amplification (MDA) reaction can be performed. In some embodiments, the method further comprises performing a multiple displacement amplification (MDA) reaction prior to step (f), wherein the MDA reaction comprises contacting at least one nucleic acid concatemer with a DNA primase-polymerase enzyme, a DNA polymerase having strand displacement activity, a plurality of nucleotides, and a catalytic divalent cation comprising magnesium or manganese. In some embodiments, the DNA primase-polymerase comprises an enzyme having the activities of a DNA polymerase and an RNA primase. The DNA primase-polymerase enzyme can utilize deoxyribonucleotide triphosphates to synthesize a DNA primer 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 a catalytic divalent cation (e.g., magnesium and / or manganese). The DNA primase-polymerase includes enzymes that are members of primases such as DnaG (e.g., bacteria) and primases such as AEP (archaea and eukaryotes). An exemplary DNA primase-polymerase enzyme is Tth PrimPol from Thermus thermophilus HB27.

[0208] In some embodiments, after the rolling circle amplification reaction, a kink amplification reaction can be performed instead of the multiple displacement amplification (MDA) reaction. In some embodiments, the kink amplification reaction comprises: (a) forming a nucleic acid relaxation reaction mixture by contacting a nucleic acid concatemer 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 polyamine, wherein forming the nucleic acid relaxation reaction mixture is performed using a temperature ramp-up, a relaxation incubation temperature, and a temperature ramp-down; (b) washing the relaxed concatemer; (c) forming a kink amplification reaction mixture by contacting the relaxed concatemer with a strand displacement DNA polymerase, a plurality of nucleotides, and a catalytic divalent cation (in the absence of added amplification primers) to generate a double-stranded concatemer, wherein forming the kink amplification reaction mixture is performed using a temperature ramp-up, a kink incubation temperature, and a temperature ramp-down; (d) washing the double-stranded concatemer; and (e) repeating steps (a)-(d) at least once.

[0209] Methods for capturing and analyzing RNA. Methods for analyzing nucleic acids (e.g., RNA) are provided herein, the methods comprising: (a) providing a support comprising a low non-specific binding coating to which a plurality of capture oligonucleotides are immobilized (e.g., FIGS. 4 and 28), 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 comprising a spatial barcode sequence and optionally a sample barcode sequence; and (iii) a cleavable region, 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. In some embodiments, the target capture region comprises a homopolymer region having a polyT sequence.

[0210] In some embodiments, the low non-specific binding coating of step (a) exhibits a low background fluorescence 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 level of non-specific Cy3 dye absorption of less than about 0.25 molecules / μm 2 and less than 5% of the target nucleic acid associates with the surface coating without hybridizing to the immobilized capture oligonucleotide. In some embodiments, when using a fluorescence imaging system under non-signal saturation conditions, the fluorescence image of the surface coating having a plurality of 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.

[0211] A method for analyzing nucleic acids further includes the step of (b) contacting a cell biological sample with a low non-specific binding coating in the presence of a high-efficiency hybridization buffer under conditions appropriate to promote the transfer of a target nucleic acid molecule 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 position information of the target nucleic acid molecule in the cell biological sample, and wherein the target nucleic acid includes a poly-A RNA molecule. In some embodiments, a target capture region having a poly-T sequence can hybridize to poly-A RNA (e.g., FIG. 28).

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

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

[0214] In some embodiments, the high-efficiency hybridization buffer in step (b) comprises: (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4-9; (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 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding.

[0215] In some embodiments, the high-efficiency hybridization buffer in step (b) comprises: (i) the first polar aprotic solvent comprises 25-50% by volume of acetonitrile in the high-efficiency hybridization buffer; (ii) the second polar aprotic solvent comprises 5-10% by volume of formamide in the high-efficiency hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) with a pH of 5-6.5; and (iv) the crowding agent comprises 5-35% by volume of polyethylene glycol (PEG) in 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 of step (b) promotes high stringency (e.g., specificity), speed, and effectiveness of nucleic acid hybridization, and increases the efficiency of subsequent amplification and sequencing steps. In some embodiments, the high-efficiency hybridization buffer significantly shortens the nucleic acid hybridization time and reduces the sample input requirements. Nucleic acid annealing is performed under isothermal conditions, and the cooling step for annealing can be eliminated.

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

[0218] In some embodiments, the reverse transcription reaction of step (c) includes (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) includes 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 can be a commercially available enzyme including MultiScribe (商標) , ThermoScript (商標) , or ArrayScript (商標) . In some embodiments, the reverse transcriptase includes Superscript I, II, III, or IV enzyme. In some embodiments, the reverse transcription reaction can include an RNase inhibitor.

[0219] In some embodiments, a method for analyzing a nucleic acid (e.g., RNA) further includes the step of adding a nucleic acid adapter to the unimmobilized end of an immobilized target extension product, thereby generating an adapter - added immobilized double - stranded target extension product (FIG. 28). The nucleic acid adapter can be single - stranded or double - stranded. The nucleic acid adapter can be added using an RNA ligase or a DNA ligase. The 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 SplintR ligase. The double - stranded adapter can be added to the unimmobilized 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 SplintR ligase. The adapter - added immobilized double - stranded target extension product includes an immobilized capture oligonucleotide (extended via reverse transcription and with an adapter added) that hybridizes to the target nucleic acid molecule. In some embodiments, the adapter - added immobilized double - stranded target extension product is exposed to conditions that separate / remove or degrade the target nucleic acid molecule such that the adapter - added immobilized single - stranded target extension product continues to adhere to the surface.

[0220] A method for analyzing nucleic acids further includes the step of contacting an immobilized single-stranded target extension product to which an adapter has been added with a plurality of soluble circularized oligonucleotides to form a target circularized double-strand, wherein each of the soluble circularized oligonucleotides includes (i) an adapter binding region, (ii) a homopolymer region, (iii) an anchor region, and (iv) an anchor moiety, wherein the homopolymer region includes a poly-T sequence capable of hybridizing to the poly-A region of the target nucleic acid molecule, and wherein the contacting step is performed under conditions appropriate for immobilizing at least one of the soluble circularized oligonucleotides to a low non-specific binding coating proximate to the immobilized single-stranded target extension product to which the adapter has been added (e.g., FIG. 28).

[0221] In some embodiments, the adapter binding region includes a sequencing primer binding region. In some embodiments, the adapter binding region includes an amplification primer binding region. In some embodiments, the homopolymer region includes 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 includes a poly-T or poly-dT sequence. In some embodiments, the anchor region is attached to a surface, thereby enabling the generation of an immobilized circularized oligonucleotide. The adapter binding region of the immobilized circularized oligonucleotide can hybridize to the added 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] A method for analyzing nucleic acids further includes the step of cleaving a cleavable region of a target circularized double strand to release an immobilized end from a low non-specific binding coating in order to generate a released target extension product, where the added adapter region of the released target extension product continues to hybridize to the adapter binding region of the immobilized circularized oligonucleotide, and the homopolymer region of the released target extension product re-hybridizes in the homopolymer region of the immobilized circularized oligonucleotide, thereby forming an open circular target circularized double strand having a gap and / or a nick, and as a result, the immobilized circularized oligonucleotide functions as a sprint molecule for promoting circularization of the released target extension product (e.g., FIG. 8). In some embodiments, the cleavable region can be cleaved using an enzyme, a compound, light, or heat. In some embodiments, the added adapter region of the released target extension product continues to hybridize 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 re-hybridizes in the homopolymer region of the immobilized circularized oligonucleotide to form a released target extension product with an open circularized adapter having a gap or a nick. The immobilized circularized oligonucleotide can function as a sprint molecule for promoting circularization of the released target extension product and as the homopolymer region and adapter binding region of the immobilized circularized oligonucleotide that can hybridize to the ends of the released target extension product.

[0223] A method for analyzing nucleic acids further includes the step of closing a gap (if present) by performing a gap filling primer extension reaction and closing a nick (if present) by performing a ligation reaction on an open circular target circularized double strand, thereby forming a covalently closed circular target extension product hybridized to an immobilized circularized oligonucleotide, wherein the immobilized circularized oligonucleotide includes an adapter binding region having a 3'-extendable end (e.g., FIG. 28).

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

[0225] A method for analyzing nucleic acids further includes the step of performing a rolling circle amplification reaction by extending the 3'-extendable end of the adapter binding region of the immobilized circularized oligonucleotide under conditions appropriate for forming an immobilized nucleic acid concatemer molecule having a tandem repeat region including a sequencing primer binding sequence, a target sequence, and a spatial barcode sequence (e.g., FIG. 28).

[0226] In some embodiments, the rolling circle amplification reaction of step (h) comprises contacting a covalently closed circularized padlock probe (e.g., a 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 appropriate 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 a covalently closed circularized padlock probe (e.g., a 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 facilitate 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 under conditions appropriate to generate at least one nucleic acid concatemer, wherein the at least one catalytic divalent cation comprises magnesium or manganese.

[0228] In some embodiments, the rolling circle amplification reaction of step (h) is carried out at a constant temperature (e.g., isothermally) in the range 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 concatemer to form immobilized small nanoballs.

[0230] In some embodiments, the rolling circle amplification reaction of step (h) comprises a DNA polymerase having 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 Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, the reverse transcriptase of the HIV virus, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0231] In some embodiments, after the rolling circle amplification reaction, a multiple displacement amplification (MDA) reaction can be performed. In some embodiments, the method further comprises performing a multiple displacement amplification (MDA) reaction before step (f), 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, after the rolling circle amplification reaction, a multiple displacement amplification (MDA) reaction can be performed. In some embodiments, the method further comprises performing a multiple displacement amplification (MDA) reaction before step (f), wherein the MDA reaction comprises contacting at least one nucleic acid concatemer with a DNA primase-polymerase enzyme, a DNA polymerase having strand displacement activity, a plurality of nucleotides, and a catalytic divalent cation comprising magnesium or manganese. In some embodiments, the DNA primase-polymerase comprises an enzyme having the activities of a DNA polymerase and an RNA primase. The DNA primase-polymerase enzyme can utilize deoxyribonucleotide triphosphates to synthesize a DNA primer on a single-stranded DNA template in a template sequence-dependent manner, and in the presence of a catalytic divalent cation (e.g., magnesium and / or manganese), the primer strand can be extended via nucleotide polymerization (e.g., primer extension). The DNA primase-polymerase includes enzymes that are members of primases such as DNA primase-polymerase, DnaG (e.g., bacteria), and primases such as AEP (archaea and eukaryotes). An exemplary DNA primase-polymerase enzyme is Tth PrimPol from Thermus thermophilus HB27.

[0233] In certain embodiments, after the rolling circle amplification reaction, a kink amplification reaction may be performed instead of a multiple displacement amplification (MDA) reaction. In some embodiments, the kink amplification reaction comprises: (a) forming a nucleic acid relaxation reaction mixture by contacting a nucleic acid concatemer 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 polyamine, wherein forming the nucleic acid relaxation reaction mixture is performed using a temperature ramp-up, a relaxation incubation temperature, and a temperature ramp-down; (b) washing the relaxed concatemer; (c) forming a kink amplification reaction mixture by contacting the relaxed concatemer with a strand-displacing DNA polymerase, a plurality of nucleotides, and a catalytic divalent cation (in the absence of added amplification primers) to generate a double-stranded concatemer, wherein forming the kink amplification reaction mixture is performed using a temperature ramp-up, a kink incubation temperature, and a temperature ramp-down; (d) washing the double-stranded concatemer; and (e) repeating steps (a)-(d) at least once.

[0234] Methods and compositions for nucleic acid determination. Methods for analyzing nucleic acids are provided herein, and the methods include determining the sequence of a target nucleic acid (e.g., an 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 includes continuous monitoring of the incorporation of labeled nucleotides in a growing polynucleotide molecule. Sequencing can be performed by massively parallel array sequencing or single molecule sequencing.

[0235] A method for analyzing nucleic acids further includes the step of sequencing at least a portion of an immobilized nucleic acid concatemer, which includes sequencing a target sequence and a spatial barcode sequence to determine the spatial position of the target nucleic acid in a cellular biological sample.

[0236] In some embodiments, the sequencing step of step (i) includes sequencing at least a portion of the nucleic acid concatemer using an optical imaging system that includes a field of view (FOV) greater than 1.0 mm. 2

[0237] In some embodiments, the sequencing step of step (i) includes placing a cellular biological sample in a flow cell having a wall (e.g., an upper wall or a first wall and a bottom wall or a second wall) and a gap therebetween, where the gap can be filled with a body fluid, and where the flow cell is placed in a fluorescence optical imaging system. The cellular biological sample may have a thickness that requires the use of an imaging system to separately focus on the first and second surfaces of the flow cell when using a conventional imaging system. For improved imaging of the sequencing reaction of nucleic acids from the cellular biological sample, the flow cell may be placed in a high-performance fluorescence imaging system, and the high-performance fluorescence 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 fluorescence 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 step of determining the sequence in step (i) includes 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 includes two or more replicates of a nucleotide moiety connected to a core via a linker.

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

[0240] In some embodiments, the multivalent molecule includes (a) a core and (b) a plurality of nucleotide arms, the plurality of nucleotide arms including (i) a core attachment portion, (ii) a spacer including a PEG portion, (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 includes 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 includes an aliphatic chain or an oligoethylene glycol chain, wherein both linker chains have 2 to 6 subunits, and optionally, the linker includes an aromatic moiety.

[0241] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the plurality of 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, including 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, the multivalent molecule 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 sugar 2'-position, sugar 3'-position, or both 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'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethyloxycarbonyl, 3'-tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino group, 3'-phosphorothioate, 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 group, an azido group, or an azidomethyl group that can be cleaved by 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 a plurality of 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-attaching moiety comprises biotin.

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

[0250] In some embodiments, the step of sequencing in step (i) comprises: (1) contacting a plurality of immobilized concatemers with a plurality of sequencing primers, a plurality of polymerases, and a plurality of nucleotides under conditions suitable for hybridizing the sequencing primers to a sequencing primer binding sequence in a portion of the immobilized concatemer, the sequencing primers being complementary to opposite positions of nucleotides in the immobilized concatemer, and having at least one polymerase and at least one sequencing primer bound to a portion of the immobilized concatemer, wherein the incorporated nucleotide is incorporated at the 3' end of the sequencing primer; (2) detecting and identifying the incorporated nucleotides, thereby determining the sequence of the immobilized concatemer molecule; 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' position or the sugar 3' position. In some embodiments, the chain terminating moiety is an azide group, an azido group, or an azidomethyl group that is 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 may include contacting a target nucleic acid or a plurality of target nucleic acids, including multiple linked or unlinked copies of a target sequence, with a multivalent binding composition described herein. Contacting the target nucleic acid or plurality of target nucleic acids, including 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 signals from inappropriate incorporations or stepwise nucleic acid strands (i.e., elongating nucleic acid strands having one or more skipped cycles).

[0252] Methods for obtaining nucleic acid sequence information are provided herein, the methods including contacting one or more target nucleic acids with one or more polymer-nucleotide conjugates. In some embodiments, the one or more target nucleic acids include multiple linked or unlinked copies of a target sequence. In some embodiments, the method results in a reduction in the sequencing error rate, as indicated by a reduction in base misidentification, reporting of absent bases, or failure to report appropriate bases. In some embodiments, the reduction in the sequencing error rate includes a reduction of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more, compared to the error rate observed using a monovalent ligand including a free nucleotide, a labeled free nucleotide, a protein or peptide-conjugated nucleotide, or a labeled protein or peptide-conjugated nucleotide. In some embodiments, the method results in an increase in the 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 including a free nucleotide, a labeled free nucleotide, a protein or peptide-conjugated nucleotide, or a labeled protein or peptide-conjugated nucleotide. In some embodiments, the method results in an increase in the 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 including a free nucleotide, a labeled free nucleotide, a protein or peptide-conjugated nucleotide, or a labeled protein or peptide-conjugated nucleotide.

[0253] Using a polymer-nucleotide conjugate for sequencing can shorten the total time of the sequencing reaction or sequencing run. The sequencing reaction cycle, including the steps of contacting, detecting, and incorporating, is carried out with a total time in the range of 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 a maximum of 60 minutes, a maximum of 50 minutes, a maximum of 40 minutes, a maximum of 30 minutes, a maximum of 20 minutes, a maximum of 10 minutes, or a maximum of 5 minutes. Any combination of the lower and upper limits described in this paragraph can form a range included in the present disclosure. For example, in some embodiments, the sequencing reaction cycle can be carried out with a total time in the range of about 10 minutes to about 30 minutes. One of ordinary skill in the art will recognize that the sequencing cycle time can have any value within this range, for example, about 16 minutes.

[0254] Using a polymer-nucleotide conjugate for sequencing results in more accurate base calls. The disclosed compositions and methods for nucleic acid sequencing provide an average Q-score for base calling accuracy over a sequencing run in the range of 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. One of ordinary skill in the art will recognize that the average Q-score can have any value within this range, for example, about 32. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing provide a Q-score 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 provide a Q-score 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 provide a Q-score 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 provide a Q-score 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 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 a plurality of nucleotides conjugated to a particle or core (e.g., a polymer, branched polymer, dendrimer, or equivalent structure). When a 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 a more accurate determination of the bases of the target nucleic acid can be achieved.

[0256] When a polymer-nucleotide conjugate is used in place of a single conjugate or unlinked nucleotides to form a complex with a polymerase and a target nucleic acid, the local concentration of the nucleotides is increased many-fold, and the signal intensity, particularly the appropriate signal-to-mismatch, is enhanced. The polymer-nucleotide conjugates described herein can include at least one polymer-nucleotide conjugate for interaction with a target nucleic acid. The multivalent composition can also include two, three, or four different polymer-nucleotide conjugates, each having different nucleotides conjugated to the particle.

[0257] In a polymer-nucleotide conjugate having a polymer-nucleotide conjugate form or a core-nucleotide conjugate form, multiple copies of the same nucleotide can be covalently or non-covalently bound to the particle. Examples of particles include branched polymers; dendrimers; cross-linked polymer particles such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles, or other particles known in the art (e.g., nanoparticles, microparticles, etc.). In a preferred embodiment, the particle is a branched polymer.

[0258] The nucleotide can be linked to the particle or core by a linker, and the nucleotide can be attached to one end or position of the polymer. The nucleotide can be conjugated to the particle by the base of the nucleotide or the 5' end. 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 nucleotide binding moieties. In some embodiments, the nucleotide can be provided separately from a nucleotide binding moiety such as polymerase. In some embodiments, the linker does not contain a light-emitting group or a light-absorbing group.

[0259] The particle or core can also include a binding moiety. In some embodiments, the particle or core can self-associate without using separate interaction moieties. In some embodiments, the particle or core can self-associate under buffer conditions or salt conditions, such as in the case of 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., a detectable reporter moiety). 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. The label can be attached to a nucleotide (e.g., by attachment to the base of the nucleotide or the 5'-phosphate moiety), to the particle itself (e.g., to a PEG subunit), or to the core (e.g., to a streptavidin core), to the end of the polymer, to the central site, or to other positions within the polymer-nucleotide conjugate that are recognized by one of ordinary skill in the art as being sufficient to provide the composition such as a detectable particle as known in the art or as described elsewhere in this specification. 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 a PEG branch.

[0262] Suitable polymers can be characterized by repeating units having functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl, or allyl groups. The polymer may also contain one or more pre-derivatized substituents, whereby one or more specific subunits contain a site of derivatization or a branching site, 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, labels such as nucleotides, nucleosides, nucleotide analogs, fluorescent labels, radioactive labels, or spin labels, interacting moieties, additional polymer moieties, or any combination of the foregoing.

[0263] In a polymer-nucleotide conjugate (e.g., a polymer-nucleotide conjugate), the polymer can have multiple branches. The branched polymer can have various configurations including, but not limited to, star-shaped (''starburst''), aggregated star-shaped (''helter skelter''), bottle brush, or dendrimer forms. The branched polymer can radiate from a central attachment point or central portion, or can include multiple branch points, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some embodiments, each of the subunits of the polymer can optionally constitute a distinct branching point.

[0264] In a polymer-nucleotide conjugate, 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 more, 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 the polymer can be calculated from the known molecular weights of a representative number of subunits, as determined by size exclusion chromatography, or as determined by mass spectrometry, or as determined by 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 a number within the range defined by any two of these values.

[0265] In the case of a polymer-nucleotide conjugate, a branched polymer with 4, 8, 16, 32, or 64 branches can have nucleotides attached to the ends of the PEG branches, such that each end can have 0, 1, 2, 3, 4, 5, 6, or more nucleotides attached. In a non-limiting example, a branched polymer with PEG arms between 3 and 128 attached to the polymer branches terminates with one or more nucleotides, such that each end can have 0, 1, 2, 3, 4, 5, 6, or more nucleotides or nucleotide analogs attached. 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 be provided with a moiety that includes a nucleotide (e.g., a residue of adenine, thymine, uracil, cytosine, guanine, 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 may or may not be able to bind to a polymerase-template-primer complex, or may be incorporated into the nucleic acid strand that is elongating during a polymerase reaction. In some embodiments, the nucleotide moiety includes a chain-terminating moiety that blocks the incorporation of subsequent nucleotides during a polymerase-mediated reaction. In some embodiments, the nucleotide moiety can be unblocked (reversibly blocked), such that subsequent nucleotides cannot be incorporated into the nucleic acid strand that is elongating during a polymerase reaction until such a block is removed, and after such a block is removed, subsequent nucleotides can be incorporated into the nucleic acid strand that is elongating during a polymerase reaction.

[0267] The polymer-nucleotide conjugate can further have a binding moiety in each branch or subset of branches. Some examples of binding moieties include, but are not limited to, biotin, avidin, streptavidin, polyhistidine domain, complementary pair of nucleic acid domains, nucleic acid domains that form G-quadruplexes, 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, protein G. The binding moiety can be any interaction molecule or fragment thereof known in the art that binds between or promotes the interaction between proteins, between a protein and a ligand, between a protein and a nucleic acid, between nucleic acids, or between small molecule interaction domains or moieties.

[0268] In some embodiments, the polymer-nucleotide conjugate may include one or more elements of an interacting moiety. Exemplary interacting 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; lectin and a cognate polysaccharide; an ion chelating moiety, a complementary nucleic acid, a nucleic acid for triple-strand or triple-helix interaction; a nucleic acid capable of forming a G-quadruplex, and the like. One of ordinary skill in the art can readily recognize that many pairs of moieties exist and are commonly used because of their property of interacting strongly and specifically with each other. Thus, any such complementary pair or set is considered suitable for this purpose when 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 interacting moiety is attached to one molecule or a multivalent ligand, and another element of the interacting moiety is attached to a separate molecule or multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of the interacting moiety are attached to a single molecule or multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of the interacting moiety are attached to separate arms of a single molecule or multivalent ligand, or to positions on a single molecule or multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of the interacting moiety are attached to the same arm of a single molecule or multivalent ligand, or to positions on a single molecule or multivalent ligand. In some embodiments, a composition containing one element of the interacting moiety and a composition containing another element of the interacting moiety may be mixed simultaneously or sequentially. In some embodiments, intermolecular or interparticle interactions disclosed herein enable the association or aggregation of multiple molecules or particles, resulting in, for example, an increase in a detectable signal.In some embodiments, a fluorescent signal, a colorimetric signal, or a radioactive signal is enhanced. In other embodiments, other interacting moieties disclosed herein or known in the art are contemplated. In some embodiments, the compositions provided herein are provided such that one or more molecules comprising a first interacting moiety, such as, for example, one or more imidazole moieties or pyridine moieties, and one or more additional molecules comprising a second interacting moiety, such as, for example, a histidine residue, are mixed simultaneously or sequentially. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, or more imidazole moieties or pyridine moieties. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, or more stidine residues. In such embodiments, the intermolecular or interparticle interactions provided can be facilitated by the presence of divalent cations such as nickel, manganese, magnesium, calcium, strontium, etc. In some embodiments, for example, a (His)3 group can interact with a (His)3 group on another molecule or particle via coordination of nickel ions or manganese ions.

[0269] The polymer-nucleotide conjugate can contain one or more buffers, salts, ions, or additives. In some embodiments, representative additives include, but are not limited to, betaine, spermidine, surfactants 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 Pluronic® series polymers, arginine, histidine, imidazole, or any combination thereof, or any substance known in the art as a DNA "relaxer" (a compound that modifies the conformational dynamics of a DNA molecule such that the persistence length of the DNA is altered, the number of junctions or crossovers within the polymer is altered, or the accessibility of sites within the strand to the DNA-binding moiety is increased).

[0270] The polymer-nucleotide conjugate can contain zwitterionic compounds as additives. Further representative additives are found in Lorenz, T.C. J Vis Exp (63), e3998, doi:10.3791 / 3998 (2012), which is hereby incorporated by reference for its disclosure of additives for promoting nucleic acid binding or dynamics, or for promoting processes involving manipulation, use, or storage of nucleic acids.

[0271] In some embodiments, the multivalent composition comprises at least one cation, and the cations include, but are 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, formation of secondary or tertiary structures, base pairing, surface association, peptide association, protein binding, etc.

[0272] When the polymer-nucleotide conjugate is used to replace a conjugate or unligated nucleotide to form a complex with polymerase and a target nucleic acid, the local concentration of the nucleotide is increased many-fold, and the signal intensity, particularly the appropriate signal-to-mismatch, is enhanced. The present disclosure contemplates contacting the polymer-nucleotide conjugate with polymerase and a primed target nucleic acid to determine the formation of a ternary complex.

[0273] Due to the increased local concentration of nucleotides on the polymer-nucleotide conjugate, the binding between the polymerase, the primed target strand, and the nucleotide is more favorable when the nucleotide is complementary to the next base of the target nucleic acid. The formed binding complex has a longer duration, which helps 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 is such that the formed binding complex remains stable during the washing step, and when other reaction mixtures and non-corresponding nucleotide analogs are washed away, the signal is maintained at a high intensity. After the imaging step, 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 steps 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 robust and controllable means for establishing and maintaining a ternary enzyme complex (e.g., during sequencing), as well as highly improved means by which the presence of said complex can be identified and / or measured, and means by which the persistence of said complex can be controlled. This provides an important solution to problems such as determination of the identity of N+1 bases in nucleic acid sequencing applications.

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

[0276] In various embodiments, the polymerases suitable for the binding interactions (e.g., during sequencing) described herein can include any polymerase known or potentially known in the art. Exemplary polymerases include, but are not limited to, the following: 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 DNA polymerase III alpha and epsilon; reverse transcriptases such as 9°N polymerase, HIV type M or O reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, or telomerase. Further non-limiting examples of DNA polymerases include those derived from various Archaea genera such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, which include polymerases known in the art such as Vent™, Deep Vent™, Pfu, KOD, Pfx, Therminator™, Tgo polymerase, etc.In some embodiments, the polymerase is Klenow polymerase.

[0277] The ternary complex has a longer duration when the nucleotide on the polymer-nucleotide conjugate is complementary to the target nucleic acid than when it is non-complementary. The ternary complex also has a longer duration when the nucleotide of the polymer-nucleotide conjugate is complementary to the target nucleic acid than when it is an un-conjugated or non-ligated complementary nucleotide. 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, or in a range defined by two or more of these values.

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

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

[0280] Dissociation of the ternary complex can be controlled by changing the buffer conditions. After the imaging step, a buffer with increased salt content is used to cause dissociation of the ternary complex, such that the labeled polymer-nucleotide conjugate can be washed away, providing a means to attenuate or terminate the signal, such as during the transition between one sequencing cycle and the next. This dissociation may be achieved, in some embodiments, by washing the complex with a buffer lacking the required metal or cofactor. In some embodiments, the wash buffer may include one or more compositions for maintaining pH adjustment. In some embodiments, the wash buffer may include one or more monovalent cations, such as sodium. In some embodiments, the wash buffer lacks or is substantially lacking in divalent cations, e.g., has no or substantially no strontium, calcium, magnesium, or manganese. In some embodiments, the wash buffer further includes a chelating agent, such as, for example, EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole. In some embodiments, the wash buffer may maintain the pH of the environment at the same level as that of the binding complex. In some embodiments, the wash buffer may increase or decrease the pH of the environment relative to the level seen for the binding complex. In some embodiments, the pH may be within a range defined by 2-4, 2-7, 5-8, 7-9, 7-10, or less than 2, or greater than 10, or within a range defined by any two of the values provided herein.

[0281] The addition of certain ions can affect the binding of polymerase to a primed target nucleic acid, the formation of a ternary complex, the dissociation of the ternary complex, or the incorporation of one or more nucleotides into the elongating nucleic acid, such as during a polymerase reaction. In some embodiments, the relevant anions may include chloride ions, acetate ions, gluconate ions, sulfate ions, or phosphate ions. In some embodiments, the ions can be included in the compositions of the present disclosure by the addition of one or more acids, bases, or salts, such as NiCl2, CoCl2, MgCl2, MnCl2, SrCl2, CaCl2, CaSO4, SrCO3, BaCl2. Representative salts, ions, solutions, and conditions can be found in Remington: The Science and Practice of Pharmacy, 20th. Edition, Gennaro, A.R., Ed. (2000), and in particular, Chapter 17, and the relevant disclosures regarding salts, ions, salt solutions, and ionic solutions are hereby incorporated by reference in their entirety.

[0282] The present disclosure contemplates contacting a polymerase-nucleotide conjugate with one or more polymerases. The contacting can optionally be performed 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 hybridizes to a nucleic acid primer. In some embodiments, the target nucleic acid is a double-stranded nucleic acid. In some embodiments, the contacting includes contacting the polymer-nucleotide conjugate with one polymerase. In some embodiments, the contacting includes contacting one or more nucleotides included in the composition with a plurality of polymerases. The polymerase can be bound to a single nucleic acid molecule.

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

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

[0285] When a catalytically inactive polymerase is used to assist the nucleic acid in interacting with the multivalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex so that it can be detected by fluorescence or by other methods disclosed herein or otherwise known in the art. The released polymer-nucleotide conjugate may 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 performed 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 performed in a solution lacking either calcium or magnesium, or both calcium and magnesium, and in a separate step, without regard to the order of the steps, one of calcium or magnesium, or both calcium and magnesium, is added to the solution. In some embodiments, contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is performed in a solution lacking strontium or barium, and in a separate step, without regard to the order of the steps, includes adding strontium to the solution.

[0287] Disclosed herein are polymer-nucleotide conjugates and their use in the analysis of nucleic acids, including sequencing or other bioassay applications. An increase in the binding of nucleotides to an enzyme (e.g., polymerase) or enzyme complex can be achieved by increasing the effective concentration of the nucleotides. The increase can be accomplished by increasing the concentration of the nucleotides in free solution or by increasing the amount of nucleotides in the vicinity of the relevant binding site. The increase can also be achieved by physically confining a plurality of nucleotides into a limited volume, thereby resulting in a local increase in concentration and thereby enabling the structure to bind to the binding site with a higher apparent binding activity than that observed with individual nucleotides that are not conjugated, ligated, or otherwise restricted. One exemplary means of achieving such confinement is to provide a polymer-nucleotide conjugate in which a plurality of 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 include a plurality of nucleotide moieties attached to a particle. In some embodiments, the plurality of nucleotide moieties are composed of nucleotide moieties of the same type (e.g., having the same or similar base pairing characteristics). When the plurality of nucleotide moieties are complementary to adjacent nucleotides in a target nucleic acid to be identified, the polymer-nucleotide conjugate forms a binding complex (multivalent binding complex) between at least two nucleotide moieties and the next nucleotides in at least two copies of the target nucleic acid sequence. In some embodiments, the multivalent binding complex includes two or more polymerases associated with a primed template of a target nucleic acid molecule. The multivalent binding complexes described herein exhibit increased stability and longer duration compared to binding complexes formed using single, un-conjugated, un-ligated nucleotides. When bound to a polymerase, the multivalent binding complex can withstand a washing step, such that the signal intensity remains high throughout the imaging and washing steps of the workflow; see, for example, FIG. 7. The polymer core of the polymer-nucleotide conjugate can be labeled with two or more detectable labels, which at least partially contribute to an enhanced signal that can be detected.

[0289] In some embodiments, for example, as shown in FIGS. 5A and 5B, at least one polymer-nucleotide conjugate includes two or more copies of a nucleotide moiety connected to the core by a linker. In some embodiments, for example, as shown in FIGS. 5A-D and FIGS. 6A-B, the polymer-nucleotide conjugate includes (a) a core and (b) a plurality of nucleotide arms, where each nucleotide arm includes (i) a core attachment portion, (ii) a spacer including a PEG portion, (iii) a linker, and (iv) a nucleotide unit.

[0290] In some embodiments, the spacer is attached to a linker, where the linker is attached to a 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 a 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 (FIGS. 6A and 6B). In some embodiments, the polymer-nucleotide conjugate comprises a core attached to a plurality of nucleotide arms, and where the plurality of 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 where each individual 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 both the sugar 2'- and 3'-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 termination 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 termination moiety is selected from the group consisting of 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azide, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethyloxycarbonyl, 3'-tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino group, 3'-phosphorothioate, and 3-O-benzyl, or derivatives thereof. In some embodiments, the chain termination moiety comprises an azide group, an azido group, or an azidomethyl group.

[0293] In some embodiments, the chain termination moiety is cleavable / removable from the nucleotide arm, for example, by a chemical compound, light, or heating. In some embodiments, the chain termination moiety contains an alkyl, alkenyl, alkynyl, or allyl group that is cleavable by tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), by piperidine, or by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain termination moiety contains an aryl or benzyl group that is cleavable by Pd / C. In some embodiments, the chain termination moiety contains an amine, amide, keto, isocyanate, phosphate, thio, or disulfide group that is cleavable by a phosphine or by a thiol group containing beta-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the chain termination moiety contains a carbonate group that is cleavable by potassium carbonate (K2CO3) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH). In some embodiments, the chain termination moiety contains a urea or silyl group that is cleavable by tetrabutylammonium fluoride, by pyridine-HF, by ammonium fluoride, or by triethylamine trihydrofluoride. In some embodiments, the chain termination moiety is an azide group, an azido group, or an azidomethyl group that is cleavable by a phosphine compound. In some embodiments, the phosphine compound contains an induced tri-alkylphosphine moiety or an induced tri-arylphosphine moiety. In some embodiments, the phosphine compound contains tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0294] In some embodiments, the polymer-nucleotide conjugate comprises a core attached to a plurality of nucleotide arms, where the core or nucleotide bases comprise a label. In some embodiments, the label is a detectable reporter moiety. The polymer-nucleotide conjugate may 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, radiolabels, or other labels such that the composition can be made detectable by methods known in the art of macromolecules or molecular interactions. The detectable reporter moiety may 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 terminus of the polymer, to the central portion, or to other positions within the polymer-nucleotide conjugate, which will be recognized by one of ordinary skill in the art as sufficient to make the composition, such as the particle, detectable by methods known in the art or as otherwise described herein. In some embodiments, one or more labels are provided to correspond to or to differentiate a particular polymer-nucleotide conjugate. The detectable reporter moiety can be a fluorophore. In some embodiments, the core can be a moiety such as avidin and the core attachment 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 the foregoing patent application are hereby expressly incorporated herein by reference for all purposes.

[0296] A polymer-nucleotide conjugate can be used to localize a detectable signal to an active region of a biochemical interaction, such as a site of a protein-nucleic acid interaction, a nucleic acid hybridization reaction, or an enzymatic reaction such as a polymerase reaction. For example, the polymer-nucleotide conjugates described herein can be used to identify the site of a base that binds to a template or the site of base incorporation in nucleic acid strand extension during a polymerase reaction and to provide base discrimination for sequencing and array-based applications. Increased binding between a target nucleic acid and a nucleotide in a multivalent binding composition provides an enhanced signal when the nucleotide is complementary to the target nucleic acid, which greatly improves basecalling accuracy and shortens imaging times.

[0297] In addition, the use of polymer-nucleotide conjugates enables a sequencing signal to be generated from a given sequence within a cluster region containing multiple copies of the target sequence. Sequencing methods that include multiple copies of a target sequence (e.g., a concatemer) have the advantage that the signal can be amplified by the presence of multiple simultaneous sequencing reactions that each provide their own signal within a defined region. The presence of multiple signals within a defined region also reduces the impact of any single skipped cycle by virtue of the fact that a large number of accurate basecall signals can overwhelm the signals of a small number of skipped or inaccurate basecalls, thereby providing a method for reducing phasing errors and / or improving read length in a sequencing reaction.

[0298] The polymer-nucleotide conjugates disclosed herein and their use result in one or more of the following: (i) a stronger signal for better basecall 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; (iv) increased sequencing speed and shortened sequencing time; (v) reduced phasing errors; and (vi) improved read length in sequencing reactions.

[0299] In a series of iterative sequencing reactions, it will sometimes happen that one or more sites fail to incorporate a nucleotide during a given cycle, which in turn will lead to one or more sites not being synchronized with the majority of the elongating nucleic acid strand. A person of ordinary skill in the art will understand this. Under circumstances where the sequencing signal is derived from a reaction occurring on a single copy of the target nucleic acid, these failures to incorporate will result in individual errors in the output sequence. Using polymer-nucleotide conjugates for sequencing can reduce this type of error in the sequencing reaction. For example, the use of a multivalent substrate that is capable of binding to the polymerase-template primer complex or of being incorporated into the elongating strand can reduce the frequency of "skipped" cycles where a base is not incorporated by increasing the probability of recombination in the premature dissociation of the ternary polymerase complex. Thus, in some embodiments, the present disclosure contemplates that the use of the multivalent substrates disclosed herein involves nucleotides having a free or reversibly modified 5' phosphate, diphosphate, or triphosphate moiety, and where the nucleotides are connected to the particle or polymer via a labile or cleavable linkage as disclosed herein. In some embodiments, the present disclosure contemplates that as a result of using the multivalent substrates disclosed herein, the intrinsic error rate due to skipped incorporations is reduced.

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

[0301] When each sequencing cycle proceeds to completion, each reaction within the defined region yields the same signal. However, as described elsewhere herein, in a series of repeating sequencing reactions, sometimes one or more sites will fail to incorporate a nucleotide during a given cycle, thereby resulting in one or more sites becoming out of sync with the majority of the growing nucleic acid strand. This problem, referred to as "phasing," results in degradation of the sequencing signal because the signal is contaminated with spurious signals from sites that have skipped one or more cycles. This in turn causes a likelihood of error in base calling. The progressive accumulation of skipped cycles over multiple cycles also decreases the effective read length due to the progressive degradation of the sequencing signal in each cycle. It is another object of the present disclosure to provide a method for reducing phasing errors and / or improving the read length in a sequencing reaction.

[0302] The sequencing method of the present invention can include contacting a target nucleic acid or a plurality of target nucleic acids, including linked or unlinked multiple copies of a target sequence, with a multivalent binding composition described herein. Contacting the target nucleic acid or plurality of target nucleic acids, including linked or unlinked multiple 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, and thus suppresses signals from inappropriate incorporations or stepwise nucleic acid strands (i.e., growing nucleic acid strands having one or more skipped cycles).

[0303] A method for obtaining nucleic acid sequence information may include contacting the target nucleic acid or multiple target nucleic acids, which include multiple linked copies or unlinked copies of a target sequence, with one or more polymer-nucleotide conjugates. This method results in a decrease in the sequencing err...

Claims

1. A method, which is a method for in situ sequencing of a target nucleic acid sequence, comprising: (a) contacting the cell or tissue with a detectable nucleotide conjugate under ex vivo conditions suitable for forming a binding complex within the cell or tissue, wherein the cell or tissue comprises a plurality of nucleic acid molecules comprising the primed target nucleic acid sequence, the detectable nucleotide conjugate comprises at least two nucleotide moieties, and the binding complex is formed between (i) the at least two nucleotide moieties of the detectable nucleotide conjugate and (ii) at least one nucleotide of each of at least two of the target nucleic acid sequences of the plurality of nucleic acid molecules; (b) detecting the binding complex; (c) identifying the target nucleic acid sequence by performing (a) to (b) on at least two other nucleotides of the at least two of the target nucleic acid sequences of the plurality of nucleic acid molecules. A method comprising the above steps.

2. The method according to claim 1, wherein the cell or tissue is immobilized on the inner surface of a flow cell.

3. The method according to claim 2, wherein the inner surface of the flow cell comprises one or more hydrophilic polymer layers.

4. The method according to claim 3, wherein the one or more hydrophilic polymer layers comprise a polymer containing polyethylene glycol (PEG).

5. The method according to claim 4, wherein the one or more hydrophilic polymer layers comprise a branched polymer.

6. The method according to claim 3, wherein the one or more hydrophilic polymer layers comprise a branched polymer.

7. The method according to claim 2, wherein the inner surface has a water contact angle of 45 degrees or less.

8. The method according to claim 1, further comprising permeabilizing the tissue or lysing the cells prior to the contacting step of (a).

9. The image of the inner surface has a contrast-to-noise ratio (CNR) of (1) contacting the inner surface with a fluorescently labeled nucleotide molecule comprising a nucleic acid sequence complementary to at least a part of a capture oligonucleotide immobilized on the inner surface, and (2) subsequently imaging the inner surface using an inverted microscope and a camera under non-signal saturation conditions while the inner surface is immersed in a buffer solution. The method according to claim 2, which exhibits a CNR of about 10 or more when measured by

10. The method according to claim 1, wherein the step of identifying the target nucleic acid sequence in (c) is performed with the accuracy of base calling characterized by a Q score exceeding 25 in at least 80% of the identified nucleotides.

11. The method according to claim 1, further comprising the step of determining the spatial position of the target nucleic acid sequence in the cell or the tissue.

12. The method according to claim 1, further comprising the step of determining the cell type of the cell, at least partially based on the step of identifying the target nucleic acid sequence in (c).

13. The method according to claim 1, further comprising the step of determining the tissue type of the tissue, at least partially based on the step of identifying the target nucleic acid sequence in (c).

14. The detectable nucleotide conjugate is (i) a common core, and (ii) at least two nucleotide moieties bound to the common core including a polymer, a micelle, a liposome, a microparticle, a nanoparticle, or a quantum dot The method according to claim 1, comprising.

15. The method according to claim 14, wherein the common core is spherical.

16. The method according to claim 14, wherein the detectable nucleotide conjugate further comprises a detectable moiety.

17. The method according to claim 16, wherein the detectable moiety is bound to the common core.

18. In (a), the detectable nucleotide conjugate is included in a mixture of a plurality of detectable nucleotide conjugates, and each of the plurality of detectable nucleotide conjugates contains different types of nucleotide moieties. The method according to claim 1.

19. The method according to claim 18, wherein the different types of the nucleotide moieties include at least three different types of the nucleotide moieties.

20. The method according to claim 1, wherein the at least two nucleotide moieties do not include a protecting group bound thereto.

21. The method according to claim 1, wherein the plurality of nucleic acid molecules contain a protecting group sufficient to prevent incorporation of the at least two nucleotide moieties into at least two of the target nucleic acid sequences.

22. The method according to claim 1, wherein the tissue is a cancer tissue.

23. The method according to claim 1, wherein the cell is a cancer cell.

24. The method according to claim 1, wherein the step of detecting the binding complex comprises imaging the cell or the tissue using one or more image sensors.

25. The method according to claim 24, wherein the one or more image sensors comprise a photodetector array.

26. The method according to claim 1, further comprising a step of washing the cell or the tissue after (b) to remove the detectable nucleotide conjugate from the cell or the tissue.

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