Imaging system hardware

Abstract

A sample holder includes a first member having a first holding mechanism configured to hold a first substrate including a sample, a second member having a second holding mechanism configured to hold a second substrate including a reagent medium, and an alignment mechanism connected to at least one of the first and second members and configured to align the first and second members such that, when the first and second members are aligned, the sample contacts at least a portion of the reagent medium.

Description

[0001] Cross-reference to related applications

[0002] This application claims U.S. Provisional Patent Application No. 62 / 777,521, filed December 10, 2018; U.S. Provisional Patent Application No. 62 / 779,342, filed December 13, 2018; U.S. Provisional Patent Application No. 62 / 779,348, filed December 13, 2018; U.S. Provisional Patent Application No. 62 / 788,867, filed January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,871, filed January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,885, filed January 6, 2019; and U.S. Provisional Patent Application No. 62 / 788,897, filed January 6, 2019. U.S. Provisional Patent Application No. 62 / 788,905, filed January 6, 2019; U.S. Provisional Patent Application No. 62 / 788,906, filed February 27, 2019; U.S. Provisional Patent Application No. 62 / 811,495, filed February 28, 2019; U.S. Provisional Patent Application No. 62 / 812,219, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,439, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,444, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,448, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,448, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,448, filed March 15, 2019; 49. U.S. Provisional Patent Application No. 62 / 819,453, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,456, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,458, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,467, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,470, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,477, filed March 15, 2019; ... U.S. Provisional Patent Application No. 62 / 819,478, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,486, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 819,495, filed March 15, 2019; U.S. Provisional Patent Application No. 62 / 822,554, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,565, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,566, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822, filed March 22, 2019.575, U.S. Provisional Patent Application No. 62 / 822,592, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,605, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,606, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,610, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,618, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,622, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,627, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,632, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,649, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,680, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 822,722, filed March 22, 2019; U.S. Provisional Patent Application No. 62 / 839,212, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,219, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,223, filed April 26, 2019; and U.S. Provisional Patent Application No. 62 / 839, filed April 26, 2019. U.S. Provisional Patent Application No. 62 / 839,294, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,320, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,346, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,526, filed April 26, 2019; U.S. Provisional Patent Application No. 62 / 839,575, filed April 2, 2019; U.S. Provisional Patent Application No. 62 / 842,463, filed May 2, 2019; U.S. Provisional Patent Application No. 62 / 858,331, filed June 7, 2019; and U.S. Provisional Patent Application No. 62 / 858,331, filed June 13, 2019. U.S. Provisional Patent Application No. 62 / 860,993, filed October 22, 2019; U.S. Provisional Patent Application No. 62 / 924,241, filed October 24, 2019; U.S. Provisional Patent Application No. 62 / 925,578, filed October 24, 2019; U.S. Provisional Patent Application No. 62 / 925,550, filed October 24, 2019; U.S. Provisional Patent Application No. 62 / 931,779, filed November 6, 2019; U.S. Provisional Patent Application No. 62 / 931,587, filed November 6, 2019; U.S. Provisional Patent Application No. 62 / 933,318, filed November 8, 2019; and U.S. Provisional Patent Application No. 62 / 933, filed November 8, 2019.Priority claims to U.S. Provisional Patent Application No. 62 / 933,878, filed November 11, 2019; U.S. Provisional Patent Application No. 62 / 934,356, filed November 12, 2019; U.S. Provisional Patent Application No. 62 / 934,766, filed November 13, 2019; U.S. Provisional Patent Application No. 62 / 934,883, filed November 13, 2019; U.S. Provisional Patent Application No. 62 / 935,043, filed November 13, 2019; U.S. Provisional Patent Application No. 62 / 937,668, filed November 19, 2019; U.S. Provisional Patent Application No. 62 / 939,488, filed November 22, 2019; and U.S. Provisional Patent Application No. 62 / 941,581, filed November 27, 2019.

[0003] The contents of each of these applications are included in this article by quoting their full text. Background Technology

[0004] Cells within a target tissue exhibit differences in morphology and / or function due to varying levels of analytes (e.g., gene and / or protein expression) within different cells. A cell's specific location within the tissue (e.g., its position relative to neighboring cells or its position relative to the tissue microenvironment) can influence, for example, cell morphology, differentiation, fate, viability, proliferation, behavior, and signal transduction and crosstalk with other cells in the tissue.

[0005] Spatial heterogeneity has been previously studied, but the techniques used in those studies only provided data on a small amount of analyte in intact tissue or partial tissue contact, or a large amount of analyte data for single cells, but could not provide information on the location of single cells in the parent biological sample (e.g., tissue sample).

[0006] Furthermore, imaging systems used for spatial analyte data exhibit inherent variability in resolution and sensitivity. This is largely attributable to variations in the manufacturers of imaging system components, in addition to differences in the arrangement of imaging equipment, the differences between various types of imaging devices, and the various image acquisition software. Image quality is also affected by variations in the image acquisition performed by the user. This problem becomes more pronounced when attempting to image samples with unknown fluorescence intensities or when the samples are imaged by users with varying levels of experience.

[0007] In addition, various processing protocols are used in laboratory settings to prepare samples for analysis. These protocols can be performed in test tubes, on glass slides, or more generally, on samples supported by a substrate. Some protocols are performed at stable, controlled temperatures to ensure the fidelity of the samples and protocol reagents. Other protocols involve temperature cycling and other steps in which the temperature of the sample is adjusted in a controlled manner. To heat the sample and its supporting substrate during the protocol, thermal cyclers, hot plates, or other heating devices can be used. For example, thermal cyclers can be used as part of polymerase chain reaction protocols for nucleic acid amplification and transcriptional and reverse transcription sequence analysis. For example, controlled heating of samples in thermal cyclers and other heating devices can also facilitate temperature-sensitive reactions for restriction enzyme digestion and rapid diagnostics.

[0008] In addition, biological samples can be placed on solid supports for analysis to identify or characterize analytes within the sample, such as DNA, RNA, or other genetic material. Printed instructions can help improve sample placement on solid supports. Summary of the Invention

[0009] Control slides, methods, and systems used to evaluate the quality and resolution of imaging devices and systems can be implemented using a variety of substrates. As used herein, the term "substrate" refers to a support having a surface (e.g., slides, hydrogels, membranes, layers, porous membranes, flow cells, solid materials, etc.).

[0010] As used herein, “substrate” unless preceded by the modifiers “chemical” or “sequence analysis” refers to a component having at least one surface that typically serves to provide physical support for the biological samples, analytes, and / or any other chemical and / or physical components, reagents, and structures described herein. Substrate can be formed from a variety of solid materials, gel-based materials, colloidal materials, semi-solid materials (e.g., at least partially cross-linked materials), fully or partially cured materials, and materials undergoing phase transitions or transitions to provide physical support. Examples of substrates that can be used in the methods and systems described herein include, but are not limited to, glass slides (e.g., slides formed from various types of glass, slides formed from various polymers), hydrogels, layers and / or thin films, membranes (e.g., porous membranes), flow cells, cuvettes, wafers, and plates. In some embodiments, the substrate may optionally include functional elements such as grooves, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings, as will be discussed in further detail below.

[0011] This section describes examples of such substrates and methods of using such substrates. However, it should be understood that, in general, the various steps and techniques discussed herein can be performed using a variety of different apparatus and system components, not all of which are explicitly described.

[0012] Furthermore, various embodiments of this disclosure relate to control slides and associated substrates, methods of using control slides, and control slide systems preferably intended for evaluating image quality and / or resolution. More specifically, embodiments include control slides and their substrates, methods, and systems for testing and evaluating the quality and / or resolution of imaging systems used in spatial gene expression technologies. In some embodiments, a significant advantage of the control slides provided herein is their ability to assess the viability of an imaging system prior to imaging and / or analyzing a sample. For example, an imaging system may be sufficient or insufficient to detect a sample at a predetermined or required resolution. A control slide or substrate consistent with this disclosure can be imaged prior to sample processing to determine whether the imaging system provides sufficient resolution. As another example, conventional imaging acquisition methods (e.g., in spatial gene expression technologies) may offer no other means of verifying resolution besides imaging and / or analyzing the sample, which can be quite expensive and inefficient. In other words, conventional methods for obtaining spatial gene expression images, for example, do not allow users to verify the resolution and / or quality of images prior to imaging and analyzing the sample. However, control slides and / or substrates consistent with embodiments of this disclosure may be more efficient in this case because they allow users to qualitatively and / or quantitatively assess the compatibility of their imaging systems and optimize their image acquisition without wasting experimental resources. Such control slides and / or substrates will be suitable for testing and / or calibrating a variety of imaging devices and systems, including those not involving spatial gene expression imaging and analysis.

[0013] In some embodiments, an additional advantage of the control slides and substrates provided herein is that they provide users with imaging capabilities to evaluate the image quality of samples with more than one histochemical staining and / or more than one fluorescent staining. For example, a user may have a sample containing two or more fluorophores. Therefore, by simultaneously imaging a substrate containing two or more fluorescent markers, a user can evaluate the image quality of the imaging system and make any necessary adjustments to the parameters of all fluorescence channels. That is, imaging capability can be the ability of the imaging device to adequately image the sample. In some embodiments, an additional advantage of the substrate is that it provides users with rapid reference to the substrate region through multiple reference markers and glyphs. For example, a user utilizing a microscope slide used in spatial gene expression methods (e.g., a microscope slide with a spatially barcoded array) may be able to efficiently align the substrate region with the region of interest in the microscope slide (e.g., the region containing the spatially barcoded array or the sample) during image processing. Thus, the user can correctly align the sample region and / or the array region with the substrate region. Furthermore, multiple reference markers allow for rapid identification of orientation and slide placement. Therefore, the control slides and substrates within the scope of this disclosure can reduce experimental time and improve the calibration of imaging systems. Furthermore, by arranging and positioning fiducials that match the gene expression slides, users can use the control slides as a reference for automating image acquisition.

[0014] The apparatus described herein can provide consistent and uniform heating to the surface of a substrate. Uniform heating is also crucial for ensuring that the preparative reactions carried out on the substrate-supported sample occur according to the established protocol and achieve the desired results.

[0015] Furthermore, if the substrate is heated without an upper lid, heating to temperatures above room temperature can cause condensation to form on the upper surface of the closed substrate pores. This condensation can alter the composition of the reaction mixture within the substrate pores, inhibit the preparation reaction, and / or produce unpredictable results. The apparatus described in this disclosure can be used to reduce or prevent condensation formation in the substrate pores.

[0016] Certain types of thermal cyclers and heating devices are specifically designed for certain types of substrates, such as porous substrates. Loading other types of substrates (such as standard microscope slides) into such devices can result in uneven heating of the substrate. The devices described in this disclosure can be used to support substrates within heating devices not designed for such substrates, ensuring adequate and uniform heat transfer to the substrate. Specifically, these devices can be used to adjust thermal cyclers designed to accept porous substrates so that other types of substrates can be effectively heated within the thermal cycler as part of a sample preparation protocol.

[0017] In some embodiments, the apparatus of this disclosure allows the surface of the substrate to directly contact the surface of the heating device (e.g., a thermal cycler), thereby allowing uniform heating throughout the substrate. That is, in some embodiments, it is not necessary to position other substrates or housing elements between the heat source and the substrate to be heated. Furthermore, because the apparatus of the present invention allows the surface of the substrate to directly contact the surface of the heating device (e.g., a thermal cycler), the user can more easily control the temperature of the substrate, and the temperature of the substrate can be heated to the desired temperature in a shorter time compared to using apparatuses that do not allow surface contact between the substrate and the heating device (e.g., a thermal cycler). Therefore, samples (e.g., biological samples) on the substrate can be heated uniformly and in a controlled manner.

[0018] In some embodiments, another advantage of the described devices is that they can be designed as one-piece designs that facilitate setup and reduce the time spent by the user assembling the device and substrate. In some embodiments, the user can easily insert the substrate into the device without securing multiple components. For example, the user can use a single optional tool, such as a blade, to assist in inserting or removing the substrate from the device. In some embodiments, the user does not use any tools to assist in inserting or removing the substrate from the device.

[0019] In some implementations, the device may be a disposable device that can be discarded after use, thereby preventing any contamination or sterility issues of the substrate-supported sample (e.g., a biological sample). For example, the device may be sterilized and pre-packaged for the user to reduce the risk of sample contamination.

[0020] This disclosure further describes means for holding or supporting substrates. Specifically, the described means includes first and second members that respectively receive first and second substrates. In some embodiments, the means of this disclosure can be used to clamp first and second substrates together for spatial transcriptomics applications. In some embodiments, the first substrate may support a sample (e.g., a biological substrate) on its surface. In some embodiments, the second substrate may include a plurality of barcoded probes and / or permeabilizers.

[0021] The described apparatus for holding or supporting the substrate further includes an alignment mechanism connected to at least one of the components and aligning the first and second components. Therefore, the apparatus of this disclosure can advantageously align the first and second substrates with any sample, barcode probe, or permeabilizer that may be present on the surfaces of the first and second substrates. That is, the apparatus of this disclosure can facilitate the analysis of samples (e.g., biological samples) by bringing the first and second substrates into aligned contact with each other. The alignment of the first and second substrates is critical in spatial transcriptomics applications because samples (e.g., biological samples) may require alignment with barcode regions of the substrates.

[0022] Current methods for aligning biological samples with barcoded regions in spatial transcriptomics analysis involve the user carefully placing the biological sample onto a substrate comprising multiple barcoded probes. Therefore, in some embodiments, the described apparatus has the advantage of providing the user with alignment tools to align the sample with the barcoded region. The apparatus of this disclosure can reduce user error during the assay analysis process, thereby also reducing sample analysis costs. In some embodiments, another advantage of the apparatus of this disclosure is the reduction of the number of aberrations or imaging defects that may arise due to user errors in aligning the biological sample with the barcoded region of the substrate. In some embodiments, the apparatus of this disclosure allows for sample pre-screening targeting regions of interest. In some embodiments, the apparatus of this disclosure allows for the examination of archived samples.

[0023] In one aspect, this disclosure relates to a substrate comprising an array disposed on a substrate surface. The array includes a plurality of non-metallic fluorescent markers. Among the plurality of non-metallic fluorescent markers, the non-metallic fluorescent markers include fluorescent probes. The substrate includes a plurality of metallic reference markers arranged in a frame pattern on the surface, forming a perimeter surrounding the array.

[0024] In some embodiments, the metallic reference marker includes gold. In some embodiments, the metallic reference marker includes nanoparticles. In some embodiments, the frame pattern is rectangular or square. In some embodiments, the metallic reference markers among the plurality of metallic reference markers have a size of at least 0.1 mm. In some embodiments, the maximum size of the non-metallic fluorescent marker is less than 0.1 mm. In some embodiments, the array includes a first non-metallic fluorescent marker comprising a first fluorescent probe having a first average concentration, and a second non-metallic fluorescent marker comprising a second fluorescent probe having a second average concentration; wherein the first average concentration is different from the second average concentration. In some embodiments, the first average concentration is from about 0.01 micromolar (μM) to about 100 μM.

[0025] In some embodiments, the plurality of non-metallic fluorescent markers includes a first subset of non-metallic fluorescent markers having a first fluorescence intensity peak at a first excitation wavelength and a second subset of non-metallic fluorescent markers having a second fluorescence intensity peak at a second excitation wavelength. In some embodiments, the first excitation wavelength is different from the second excitation wavelength. In some embodiments, the plurality of metallic reference markers do not fluoresce at the first excitation wavelength. In some embodiments, the plurality of metallic reference markers do not fluoresce at the second excitation wavelength. In some embodiments, the first excitation wavelength is from about 560 nanometers (nm) to about 610 nm. In some embodiments, the second excitation wavelength is from about 600 nanometers (nm) to about 700 nm. In some embodiments, the first subset of the plurality of non-metallic fluorescent markers fluoresces at the first excitation wavelength, while the second subset of the plurality of non-metallic fluorescent markers does not fluoresce in a detectable manner at the first excitation wavelength.

[0026] In some embodiments, the fluorescent probe is conjugated to an oligonucleotide. In some embodiments, the fluorescent probe includes tetramethylrhodamine (TRITC), red fluorescent protein (DsRed), a dye having an absorption wavelength that peaks at about 590 nm, anthocyanin-3 (Cy3), a dye having an absorption wavelength that peaks at about 650 nm, anthocyanin-5 (Cy5), or combinations thereof.

[0027] In another aspect, this disclosure relates to a substrate comprising an array including a plurality of fluorescent markers disposed on a surface of the substrate, the plurality of fluorescent markers including a first subset of fluorescent markers having a first fluorescent probe, and a second subset of fluorescent markers having a second fluorescent probe. The first fluorescent probe is different from the second fluorescent probe.

[0028] In some embodiments, the first or second fluorescent probe comprises a fluorescent dye, a fluorescent protein, or a combination thereof. In some embodiments, the first or second fluorescent probe comprises a fluorescent dye selected from the group consisting of acridine dyes, fluoroketone dyes, anthocyanin dyes, fluorescein, oxazine dyes, phenanthridine dyes, rhodamine dyes, and combinations thereof. In some embodiments, the first or second fluorescent probe comprises a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), tagged blue fluorescent protein (TagBFP), cerulean, cyan fluorescent protein (CFP), venus, citrine, yellow fluorescent protein (YFP), monomer-enhanced green fluorescent protein (EGFP), mCherry, mKate2, photoactivated green fluorescent protein (PA-GFP), photoactivated mCherry (PA-mCherry), fluorescent protein fusion proteins, and combinations thereof. In some embodiments, the plurality of fluorescent markers are non-metallic. In some embodiments, the fluorescent markers among the plurality of fluorescent markers have a maximum size of less than 0.1 mm.

[0029] In another aspect, this disclosure relates to a substrate comprising an array including a plurality of fluorescent markers disposed on a surface of the substrate, and a plurality of reference markers arranged in a frame pattern on the surface to form a perimeter surrounding the array. The fluorescent markers among the plurality of fluorescent markers include fluorescent dyes.

[0030] In some embodiments, the fluorescent dye is coupled to an oligonucleotide. In some embodiments, the fluorescent dye is selected from the group consisting of acridine dyes, fluoroketone dyes, anthocyanin dyes, fluorescein, oxazine dyes, phenanthridine dyes, rhodamine dyes, and combinations thereof. In some embodiments, the fluorescent dye is selected from the group consisting of tetramethylrhodamine (TRITC), red fluorescent protein (DsRed), dyes having an absorption wavelength that peaks at about 590 nm, anthocyanin-3 (Cy3), dyes having an absorption wavelength that peaks at about 650 nm, anthocyanin-5 (Cy5), and combinations thereof.

[0031] In another aspect, this disclosure relates to a substrate comprising an array of fluorescent markers disposed on a surface of the substrate, and a plurality of reference markers arranged in a frame pattern on the surface to form a perimeter surrounding the array. The fluorescent markers among the plurality of fluorescent markers include fluorescent probes. The minimum spacing between the fluorescent markers and the reference markers among the plurality of reference markers is at least 50 micrometers.

[0032] In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is at least 100 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is at least 500 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is at least 1,000 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is at least 1,500 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is at least 2,000 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is from about 50 to about 3,000 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is from about 500 to about 2,000 micrometers. In some embodiments, the minimum spacing between the fluorescent marker and the reference marker is from about 1,000 to about 1,500 micrometers.

[0033] In another aspect, this disclosure relates to a substrate comprising a plurality of fluorescent markers disposed on a surface of the substrate, and a plurality of reference markers arranged on the surface. The plurality of reference markers includes a first subset of reference markers arranged in a first pattern and a second subset of reference markers arranged in a second pattern. The second pattern differs from the first pattern.

[0034] In some embodiments, the plurality of reference markers further includes a third subset of reference markers arranged in a third pattern adjacent to the first and second patterns on the surface. In some embodiments, the third subset of reference markers is a graphic symbol. In some embodiments, the third pattern is a geometric shape. In some embodiments, the third pattern is a circle, hourglass, hexagon, square, rectangle, triangle, pentagon, heptagon, octagon, nonagon, decagon, ellipse, or regular polygon. In some embodiments, the first pattern forms a first side and the second pattern forms a second side. In some embodiments, the third pattern forms a third side, wherein the third pattern is different from the first and second patterns. In some embodiments, the plurality of reference markers further includes a fourth subset of reference markers arranged in a fourth pattern forming a fourth side, wherein the fourth pattern is different from the first, second, and third patterns. In some embodiments, the first, second, third, and fourth sides are arranged to form a frame on the surface of the substrate. In some embodiments, the first subset of reference markers is spaced apart from each other with a period different from that of the second subset of reference markers. In some embodiments, a first subset of reference markers is spaced apart from each other on the surface of the substrate with a higher periodicity than a second subset of reference markers. In some embodiments, the first subset of reference markers is spaced apart from each other on the surface of the substrate with a lower periodicity than a second subset of reference markers. In some embodiments, the first subset of reference markers is arranged on the surface of the substrate in three staggered rows forming a two-dimensional lattice structure.

[0035] In another respect, this disclosure relates to a glass slide comprising any of the aforementioned substrates.

[0036] In another aspect, this disclosure relates to a method for evaluating the imaging capability of an imaging system. The method includes identifying a frame comprising a plurality of metallic reference markers disposed on a substrate surface, irradiating the substrate with radiation of a first wavelength, measuring light emitted by an array disposed on a surface within a perimeter defined by the frame, wherein the array comprises a plurality of non-metallic fluorescent markers, wherein the non-metallic fluorescent markers among the plurality of non-metallic fluorescent markers include fluorescent probes, and determining the presence or absence of the non-metallic fluorescent markers on a visual representation of a target object, including relative dimensional information in at least two orthogonal spatial dimensions, wherein the visual representation is generated by the imaging system based on the light emitted by the array.

[0037] In another aspect, this disclosure relates to a system comprising at least one array including a plurality of non-metallic fluorescent markers disposed on a substrate surface, wherein the non-metallic fluorescent markers among the plurality of non-metallic fluorescent markers include fluorescent probes, a plurality of metallic reference markers arranged on the surface in at least one frame pattern forming a perimeter around the array, and a computing device including a processor operatively coupled to a microscope, and a non-transitory computer-readable storage medium having a computer program including instructions executable by the processor, causing the processor to generate a visual representation of a target object including relative dimensional information in at least two orthogonal spatial dimensions, wherein the target object includes at least one frame pattern and at least one array.

[0038] In another aspect, this disclosure relates to a support device for a substrate, the substrate including a sample area, the support device including a plate, the plate including a platform, a plurality of members connected to a first surface of the platform, and a support member connected to a second surface of the platform, and a substrate holder including a substrate mount and an attachment mechanism for engaging the substrate holder to the support member. The substrate holder is configured such that when the substrate is secured by the substrate mount and the substrate holder is engaged to the support member, at least 60% of the sample area is covered by the support member.

[0039] In some embodiments, the dimensions of the components among the plurality of members are designed to be received by a region of the heating device. In some embodiments, the region of the heating device includes a heat transfer element configured to transfer heat to pores in the porous substrate. In some embodiments, the heat transfer element includes a recess in the heating member. In some embodiments, the plurality of members are formed in a two-dimensional array on a first surface, and the plurality of members are spaced apart such that they are aligned with pores in the porous substrate. In some embodiments, the substrate mount includes a recess formed in a substrate holder. In some embodiments, the substrate mount includes at least one fastener configured to secure the substrate within the recess. In some embodiments, the attachment mechanism includes an orifice configured to receive a support member. In some embodiments, the attachment mechanism includes one or more extensions configured to engage with corresponding recesses in the support member. In some embodiments, the sample area includes a plurality of pores in the substrate. In some embodiments, the substrate holder includes a recess sized to receive a gasket. In some embodiments, the substrate holder includes a gasket positioned to form an hermetically sealed seal between the substrate holder and the substrate when the substrate is secured by the substrate mount. In some embodiments, the sample area includes a plurality of holes in the substrate, and the gasket includes a plurality of apertures, wherein the apertures are positioned such that they align with the holes when the substrate is secured by a substrate mount. In some embodiments, the gasket is configured to prevent fluid transfer between the apertures when the substrate is secured by a substrate mount.

[0040] In some embodiments, the substrate holder includes a plurality of orifices, wherein one of the orifices is aligned with an orifice in the gasket. In some embodiments, the sample area is completely covered by a support member. In some embodiments, the substrate holder is configured such that at least a portion of the substrate contacts the support member when the substrate is secured by a substrate mount and the substrate holder is coupled to the support member. In some embodiments, the portion of the substrate contacting the support member includes at least a portion of the sample area or a portion of the substrate on the side of the substrate opposite to the sample area. In some embodiments, the support member contacts the entire substrate. In some embodiments, the attachment mechanism is configured such that the substrate holder is coupled to the support member in a single orientation.

[0041] In some embodiments, the substrate holder includes a first member, a second member, and an engagement mechanism configured to secure the first member to the second member. A substrate mount is located within either the first or second member. In some embodiments, the engagement mechanism is adjustable. In some embodiments, the engagement mechanism includes one or more wing screws. In some embodiments, the first member includes a substrate mount, wherein the substrate mount includes a recess formed in the first member. In some embodiments, the first member includes at least one fastener configured to secure the substrate within the recess. In some embodiments, the first member includes an attachment mechanism, wherein the attachment mechanism includes an aperture configured to receive a support member. In some embodiments, the second member includes a recess sized to receive a gasket. In some embodiments, the second member includes a gasket positioned to form an airtight seal between the substrate holder and the substrate when the substrate is secured by the substrate mount and the first member is secured to the second member.

[0042] In some embodiments, the sample area includes a plurality of holes in the substrate, and the gasket includes a plurality of orifices, wherein the orifices of the plurality of orifices are positioned such that the orifices align with the holes when the substrate is secured by a substrate mount. In some embodiments, when the substrate is secured by a substrate mount and a first member is secured to a second member, the gasket is configured to prevent fluid transfer between the gasket orifices. In some embodiments, the second member includes a plurality of orifices, wherein the orifices of the plurality of orifices are aligned with the orifices of the plurality of orifices of the gasket.

[0043] In another aspect, this disclosure relates to a method for incubating a sample disposed on a sample area of ​​a substrate. The method includes mounting the substrate on a support device, positioning the substrate and the support device in a heating device, and activating the heating device to transfer heat to the sample. The support device includes a plate comprising a platform, a plurality of members connected to a first surface of the platform, and a support member connected to a second surface of the platform, and a substrate holder including a substrate mounting member and an attachment mechanism for engaging the substrate holder to the support member. The substrate holder is configured such that when the substrate is secured by the substrate mounting member and the substrate holder is engaged to the support member, at least 60% of the sample area is covered by the support member.

[0044] In another aspect, this disclosure relates to a support device for a substrate including a sample area. The support device includes a plate comprising a platform, a plurality of members connected to a first surface of the platform, and a support member connected to a second surface of the platform. A substrate mounting member, including the first and second surfaces, is attached to the support member via the first surface. A substrate holder, including an attachment mechanism, is also included to attach the substrate mounting member to the substrate holder. The second surface of the substrate mounting member is a substrate for receiving a sample.

[0045] In some embodiments, the substrate mount is a glass slide. In some embodiments, when the substrate holder is attached to the support member, at least 60% of the sample area is covered by the support member. In some embodiments, when the substrate holder is attached to the support member, at least 75% of the sample area is covered by the support member. In some embodiments, when the substrate holder is attached to the support member, at least 90% of the sample area is covered by the support member. In some embodiments, when the substrate holder is attached to the support member, the sample area is completely covered by the support member. In some embodiments, the dimensions of the members among the plurality of members are designed to be received by a region of a heating device. In some embodiments, the region of the heating device includes a heat transfer element configured to transfer heat to pores in the porous substrate. In some embodiments, the heat transfer element includes a recess in the heating member. In some embodiments, the plurality of members are formed in a two-dimensional array on a first surface, and the plurality of members are spaced apart such that they are aligned with pores in the porous substrate. In some embodiments, the attachment mechanism includes fasteners configured to engage the substrate mount. In some embodiments, the attachment mechanism includes orifices configured to receive the support member. In some embodiments, the attachment mechanism includes one or more tabs configured to engage with a substrate mount. In some embodiments, the sample area includes a plurality of holes in the substrate. In some embodiments, the substrate holder includes a gasket positioned to form an hermetically tight seal between the substrate holder and the substrate when the substrate is secured by the substrate mount. In some embodiments, the sample area includes a plurality of holes in the substrate, and wherein the gasket includes a plurality of orifices, wherein an orifice of the plurality of orifices is positioned such that an orifice of the gasket aligns with a hole when the substrate is secured by the substrate mount. In some embodiments, the gasket is configured to prevent fluid transfer between the orifices of the gasket when the substrate is secured by the substrate mount. In some embodiments, the substrate holder includes a plurality of orifices, wherein an orifice of the plurality of orifices of the substrate holder aligns with an orifice of the gasket. In some embodiments, the sample area is completely covered by a support member. In some embodiments, the substrate holder is configured such that at least a portion of the substrate contacts the support member when the substrate is held by the substrate mount and the substrate holder is coupled to the support member. In some embodiments, the portion of the substrate contacting the support member includes at least a portion of the sample area or a portion of the substrate on the side of the substrate opposite to the sample area. In some embodiments, the support member contacts the entire substrate. In some embodiments, the attachment mechanism is configured such that the substrate holder is coupled to the support member in a single direction.

[0046] In another aspect, this disclosure relates to a support device for a substrate including a sample area. The support device includes a substrate mount having a first surface and a second surface, the first surface being coupled to a support member. A substrate holder includes an attachment mechanism for coupling the substrate mount to the substrate holder, a gasket, a plurality of ribs extending orthogonally to the bottom surface of the substrate holder, and an engagement mechanism configured to secure the substrate mount to the gasket. The second surface of the substrate mount is the substrate for receiving the sample, and the gasket is located between the substrate mount and the bottom surface of the substrate holder.

[0047] In some embodiments, the engagement mechanism is adjustable. In some embodiments, the engagement mechanism includes one or more tabs. In some embodiments, the engagement mechanism includes one or more latches. In some embodiments, the substrate holder includes at least one fastener configured to secure the substrate mount within a recess. In some embodiments, the substrate holder includes a recess sized to receive a pad. In some embodiments, the pad is positioned such that an airtight seal is formed between the substrate holder and the substrate holder when the substrate holder is secured by the substrate holder. In some embodiments, the sample area includes a plurality of holes on the substrate mount, and the pad includes a first set of orifices, wherein the orifices in the first set of orifices are positioned such that the orifices are aligned with the holes when the substrate mount is secured by the substrate holder. In some embodiments, the pad is configured to prevent fluid transfer between the first set of orifices when the substrate mount is secured by the substrate holder. In some embodiments, the substrate holder includes a second set of orifices, wherein the orifices in the second set of orifices are aligned with the orifices in the first set of orifices.

[0048] In another aspect, this disclosure relates to a method for incubating a sample disposed on a sample area of ​​a substrate. The method includes mounting the substrate on a support device, positioning the substrate and support device in a heating device, and activating and heating the device to transfer heat to the sample. The support device includes a plate comprising a platform, a plurality of members connected to a first surface of the platform, and a support member connected to a second surface of the platform. A substrate mounting member includes both the first and second surfaces, the first surface being coupled to the support member. A substrate holder includes an attachment mechanism for coupling the substrate mounting member to the substrate holder. The second surface of the substrate mounting member includes a substrate for receiving the sample.

[0049] In some embodiments, the substrate mount is a glass slide. In some embodiments, when the substrate holder is attached to the support member, at least 60% of the sample area is covered by the support member.

[0050] In another aspect, this disclosure relates to a support device for a substrate including a sample area. The support device includes a substrate mount having a first surface and a second surface, the second surface of which is a substrate configured to receive a sample; a substrate holder including a first portion configured to receive a pad, the first portion including a plurality of ribs extending from a surface of the substrate holder; and a second portion configured to receive the substrate mount. The first and second portions are hinged together such that when the substrate holder is in a closed state, the first portion is configured to fold over the second portion to secure the substrate mount between the first and second portions.

[0051] In some embodiments, the substrate mount is a glass slide. In some embodiments, the substrate holder includes a gasket disposed between a first portion and a second portion of the substrate holder. In some embodiments, the first portion of the substrate holder includes a releasable engagement mechanism configured to secure the first portion to the second portion when the substrate holder is in a closed state. In some embodiments, a first surface of the substrate mount engages with at least one of a plurality of ribs extending from a surface of the substrate holder. In some embodiments, the second portion defines a cavity formed in the substrate holder, the cavity being configured to receive the substrate mount. In some embodiments, the second portion defines a cavity configured to receive the substrate mount. In some embodiments, the second portion defines an opening within the cavity, wherein when the substrate holder is in a closed state, the opening exposes at least a portion of one side of the substrate mount.

[0052] In another aspect, this disclosure relates to a sample holder comprising a first component and a second component, the first component including a first retaining mechanism configured to retain a first substrate comprising a sample, the second component including a second retaining mechanism configured to retain a second substrate comprising a reagent medium, and an alignment mechanism connected to one or both of the first and second components and configured to align the first and second components such that when the first and second components are aligned, the sample contacts at least a portion of the reagent medium.

[0053] In some embodiments, the alignment mechanism includes a rotary actuator connected to the first and second components. In some embodiments, the alignment mechanism includes one or more connectors located on one or both of the first and second components, and one or more receivers located on one or both of the first and second components, wherein the one or more receivers are positioned to engage with the one or more connectors. In some embodiments, the rotary actuator includes a hinge. In some embodiments, the rotary actuator includes a folding member. In some embodiments, the rotary actuator includes at least one arm. In some embodiments, the first holding mechanism includes a recess sized to receive a first substrate. In some embodiments, the sample holder further includes a pad located within the recess, the pad being configured to maintain an interference fit between the recess and the first substrate. In some embodiments, the first holding mechanism includes one or more members configured to apply force to the first substrate to maintain contact between the first substrate and the first member.

[0054] In some embodiments, the second holding mechanism includes a recess sized to receive a second substrate. In some embodiments, the second holding mechanism includes one or more members configured to apply force to the second substrate to maintain contact between the second substrate and the second member. In some embodiments, the reagent medium includes at least one of the following: a solution containing a permeabilizer, a solid permeabilizer, and a hydrogel compound containing a permeabilizer. In some embodiments, the solution containing a permeabilizer includes greater than about 2 w / v% sodium dodecyl sulfate (SDS). In some embodiments, the solution containing a permeabilizer includes about 8 w / v% to about 12 w / v% SDS. In some embodiments, the solution containing a permeabilizer includes proteinase K. In some embodiments, the solution containing a permeabilizer includes greater than 2 w / v% N-lauroyl sarcosine or its sodium salt. In some embodiments, the first member includes an opening positioned such that the opening is aligned with a sample region of the first substrate when the first substrate is held. In some embodiments, the second component includes at least one opening positioned such that when the first substrate is held and the first and second components are aligned by an alignment mechanism, the opening of the at least one opening is at least partially aligned with a sample region of the first substrate.

[0055] In some embodiments, the sample holder further includes one or more boundary surfaces formed by at least one opening and a reagent orifice formed by the rear surface of a second substrate, wherein reagent solution added to the reagent orifice is contained within the boundary surface and permeates through the back surface of the second substrate. In some embodiments, the back surface of the second substrate faces the front surface of the second substrate facing the sample on the first substrate. In some embodiments, the sample holder further includes a first adjustment mechanism connected to a first member and configured to translate the first substrate in at least one direction parallel to the surface of the first substrate supporting the sample. In some embodiments, an alignment mechanism is configured to maintain a gap between the first and second substrates when the first and second substrates are aligned. In some embodiments, the alignment mechanism is configured to maintain this gap such that at least a portion of the sample on the first substrate contacts at least a portion of the reagent medium on the second substrate. In some embodiments, the gap between the first and second substrates is 50 μm–1 mm when measured in a direction orthogonal to the surface of the first substrate supporting the sample.

[0056] In some embodiments, the spacing between the first and second substrates is 50 μm to 500 μm. In some embodiments, the alignment mechanism is configured to keep the first and second substrates substantially parallel when aligned, such that the angle between the first and second substrates is two degrees or less. In some embodiments, this angle is 0.5 degrees or less. In some embodiments, the sample holder further includes one or more spacer members connected to one or both of the first and second members, the first and second members being positioned such that the one or more spacer members are located between the first and second members when the first and second substrates are aligned. In some embodiments, the sample holder further includes a second adjustment mechanism configured to adjust the spacing distance in a direction orthogonal to the surface of the first substrate supporting the sample. In some embodiments, the second adjustment mechanism is a component of the alignment mechanism. In some embodiments, the second adjustment mechanism is connected to one or both of the first and second members.

[0057] In another aspect, this disclosure relates to a support device for a substrate including a sample. The support device includes any of the sample holders described above; and a plate including a platform, a plurality of members connected to a first surface of the platform, and support members connected to a second surface of the platform. The plate is configured to be connected to the sample holder.

[0058] In some embodiments, the sample holder and plate are configured such that when the sample holder and plate are connected, at least 75% of the area of ​​the first substrate in contact with the sample is covered by a support member. In some embodiments, the dimensions of the members among the plurality of members are designed to be received by a region of a heating device. In some embodiments, the region of the heating device includes a heat transfer element configured to transfer heat to pores in the porous substrate. In some embodiments, the heat transfer element includes a recess in the heating member. In some embodiments, the support device further includes an attachment mechanism configured to attach the support member to the sample holder. In some embodiments, the attachment mechanism includes an opening and a recess formed in the first member of the sample holder and configured to receive one or both of the openings and recesses of the support member.

[0059] In some embodiments, the attachment mechanism includes one or more extension members connected to a first member of the sample holder and configured to engage a corresponding recess in a support member. In some embodiments, the attachment mechanism includes one or more extension members connected to a support member and configured to engage a corresponding recess in a first member of the sample holder. In some embodiments, a plurality of members are formed in a two-dimensional array on a first surface of the platform, and the members are spaced apart such that they are aligned with holes in a porous substrate. In some embodiments, the area of ​​the first substrate in contact with the sample is completely covered by the support member. In some embodiments, the sample holder and plate are configured such that at least a portion of the first substrate contacts the support member when the sample holder and plate are connected. In some embodiments, the portion of the first substrate in contact with the support member includes a portion of the substrate located on the side of the first substrate opposite to the area of ​​the first substrate in contact with the sample. In some embodiments, the portion of the first substrate in contact with the support member includes at least 90% of the surface of the substrate opposite to the surface of the first substrate in contact with the sample. In some embodiments, the attachment mechanism is configured such that the sample holder is coupled to the support member in a single direction.

[0060] In another aspect, this disclosure relates to a method for providing visual guidance for the location of a printed array, comprising placing a biological sample on a solid support using information markers with printed guidance, and analyzing the sample, wherein the printed guidance provides visual guidance for the location of the printed array.

[0061] On the other hand, this disclosure relates to a method for placing biological samples on an array, including placing the biological sample on a solid support using information tags with printed instructions, analyzing the biological sample, wherein the printed instructions provide visual guidance on the location of the printed array; and removing the information tags from the solid support.

[0062] In some embodiments, the information marker is transparent. In some embodiments, the step of removing the information marker occurs before the step of analyzing the biological sample. In some embodiments, the solid support is a glass slide. In some embodiments, the biological sample is a tissue section. In some embodiments, the information marker is removable. In some embodiments, the information marker is mechanically adhered. In some embodiments, the information marker is printed with ink. In some embodiments, the ink is white ink, black ink, colored ink, fluorescent ink, or a combination thereof. In some embodiments, the information marker is matte. In some embodiments, the information marker is glossy. In some embodiments, the information marker includes holes or cutouts within the information marker. In some embodiments, the information marker is thermally and electrically conductive. In some embodiments, the printed guide is a reference marker. In some embodiments, the reference marker includes a frame surrounding the array and a point identifying the center of the array. In some embodiments, the information marker contains metadata. In some embodiments, the information marker occupies a portion of the solid support. In some embodiments, the information marker occupies all of the solid support.

[0063] In one aspect, a method for generating a spatial RNA integrity number at a location on an array includes: (a) contacting a tissue sample stained with a histological stain with an array, wherein the array includes a capture probe attached to a location on the array, and the capture probe includes a capture domain specifically bound to a bioanalyte from the tissue sample; (b) generating a cDNA molecule from the bioanalyte specifically bound to the capture domain; (c) labeling the cDNA by hybridizing a labeled oligonucleotide probe to the cDNA; (e) generating an image of the labeled cDNA and an image of the histological stain, and using the image of the labeled cDNA and the image of the histological stain to generate a spatial RNA integrity number at the location on the array.

[0064] In some embodiments, the tissue sample includes tissue sections, regions within tissue, or single cells within tissue. In some embodiments, the histological staining agents are hematoxylin and eosin. In some embodiments, the capture domain comprises a poly(T) sequence. In some embodiments, the bioanalyte is 18S rRNA. In some embodiments, the labeled oligonucleotide probes are fluorescently labeled. In some embodiments, step (c) includes hybridizing at least four different labeled oligonucleotide probes with cDNA. In some embodiments, at least four labeled oligonucleotide probes hybridize sequentially at different sites within the cDNA. In some embodiments, the labeled oligonucleotide probes are fluorescently labeled. In some embodiments, the step of generating an image of the labeled cDNA includes measuring the fluorescence signal of each of the at least four labeled oligonucleotide probes. In some embodiments, the fluorescence signal from each of the at least four labeled oligonucleotide probes is used to generate the spatial RNA integrity number at the location described on the array.

[0065] On the other hand, a method for determining process deviations in a spatial analysis workflow includes: (a) providing a substrate containing one or more test analytes, wherein the one or more test analytes are arranged in known amounts at known locations on the substrate; (b) contacting the substrate with an array containing one or more capture probes, wherein the capture probes include spatial barcodes and capture fields, under conditions that allow one or more capture probes to interact with one or more test analytes; (c) detecting one or more test analytes interacting with one or more capture probes; and (d) determining, based on the detection in step (c), whether the spatial analysis workflow accurately detects the presence, quantity, location, or combination thereof of one or more test analytes, thereby determining a process deviation in the spatial analysis workflow.

[0066] In some embodiments, the substrate comprises a semi-porous material. In some embodiments, the semi-porous material comprises at least one of nitrocellulose membranes, hydrogels, nylon filters, or combinations thereof. In some embodiments, one or more analytes comprise at least one of nucleic acids, proteins, lipids, or combinations thereof. In some embodiments, one or more analytes are RNA. In some embodiments, one or more analytes are arranged on the substrate in a defined pattern. In some embodiments, the defined pattern comprises one or more dots.

[0067] All publications, patents, and patent applications available on the Internet mentioned in this specification are incorporated herein by reference as if each individual publication, patent, patent application, and information item were incorporated herein by reference separately and individually. In the event of any conflict between the publications, patents, patent applications, and information items incorporated by reference and the disclosure contained in this specification, this specification shall prevail and / or take precedence over any conflicting material.

[0068] When a value is described as a range, it should be understood that the description includes the disclosure of all possible subranges within that range, as well as the disclosure of a specific numerical value within that range, regardless of whether the specific numerical value or specific subrange is explicitly stated.

[0069] When the term “each” refers to a group of items, it is intended to identify a single item in that group, but not necessarily every item in that group, unless otherwise explicitly stated, or unless the context of the usage clearly indicates otherwise.

[0070] This document describes various embodiments of the features of the present invention. However, it should be understood that these embodiments are provided by way of example only, and many variations, modifications, and substitutions can be made by those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure. Attached Figure Description

[0071] The following figures illustrate certain embodiments of the features and advantages of the invention. These embodiments are not intended to limit the scope of the appended claims in any way. Like reference numerals in the figures denote like elements.

[0072] Figure 1 An exemplary spatial analysis workflow is shown.

[0073] Figure 2 An exemplary spatial analysis workflow is shown.

[0074] Figure 3 An exemplary spatial analysis workflow is shown.

[0075] Figure 4 An exemplary spatial analysis workflow is shown.

[0076] Figure 5 An exemplary spatial analysis workflow is shown.

[0077] Figure 6 This is a schematic diagram illustrating an example of a barcode-based capture probe as described in this article.

[0078] Figure 7This is a schematic diagram illustrating a lysable capture probe, in which the lysed capture probe can enter non-permeable cells and bind to the target analyte within the sample.

[0079] Figure 8 This is a schematic diagram illustrating an exemplary multi-space barcode feature.

[0080] Figure 9 This is a schematic diagram of an exemplary analyte trapping agent.

[0081] Figure 10 This is a schematic diagram depicting an exemplary interaction between the feature-immobilized capture probe 1024 and the analyte capture agent 1026.

[0082] Figure 11A , 11B Figures 1 and 11C are schematic diagrams illustrating how streptavidin cell tags can be used to generate spatially barcoded cells or cell contents in an array-based system.

[0083] Figure 12 This is a schematic diagram showing the arrangement of barcode features within the array.

[0084] Figure 13 This is a schematic diagram showing a side view of an anti-diffusion medium (e.g., a cap).

[0085] Figure 14A and 14B This is a diagram showing the unfolding of the electrophoretic transfer system. Figure 14A and side view Figure 14B The schematic diagram shows that the electrophoretic transfer system is configured to guide transcript analytes to a spatially barcode-capture probe array.

[0086] Figure 15 This is a schematic diagram illustrating an exemplary workflow scheme utilizing an electrophoretic transfer system.

[0087] Figure 16 An example of a microfluidic channel structure 1600 for dividing a dissociated sample (e.g., biological particles or individual cells from the sample) is shown.

[0088] Figure 17A An example of a microfluidic channel structure 1700 is shown, which is used to deliver a bead carrying a spatial barcode to a droplet.

[0089] Figure 17B A cross-sectional view of another example of a microfluidic channel structure 1750 with geometric features for controlled partitioning is shown.

[0090] Figure 17C An example of a workflow diagram is shown.

[0091] Figure 18 It is a schematic diagram depicting cell tagging using the covalent coupling of the analyte binding portion to the cell surface or the non-covalent interaction with cell membrane elements.

[0092] Figure 19 This is a schematic diagram depicting cell spiking using cell-penetrating peptides or delivery systems.

[0093] Figure 20A This is a schematic diagram illustrating an exemplary, non-limiting, and non-exhaustive process for “pixelating” a sample, in which a small portion of the sample is moved into an individual partition or well by cutting, imprinting, microdissecting, or transferring the sample using a hollow needle or microneedle.

[0094] Figure 20B This is a schematic diagram depicting multi-needle pixilation, in which a set of needles passes through the sample on a scaffold and into nanopores containing gel beads and reagents below. Once the needles enter the nanopores, cells are ejected.

[0095] Figure 21 A schematic diagram of an exemplary, non-limiting, non-exhaustive workflow for dissociating spatially barcoded samples for analysis by droplet or flow cell analysis methods is shown.

[0096] Figure 22A This is a schematic diagram illustrating an exemplary sample processing apparatus that can be used to implement the various steps and methods described herein.

[0097] Figure 22B This is a schematic diagram illustrating an example imaging device that can be used to obtain images of biological samples, analytes, and feature arrays.

[0098] Figure 22C yes Figure 22A and Figure 22B A schematic diagram of an example of the control unit of the device.

[0099] Figure 23A This is a schematic diagram showing an example of a sample slide (e.g., a control slide).

[0100] Figure 23B It is a display Figure 23A A schematic diagram of an example of a sample slide for additional measurements not shown in the image.

[0101] Figure 24A This is a schematic diagram illustrating an example of a sample slide and a magnified view of a selected area.

[0102] Figure 24B This is an explanation Figure 24AA schematic diagram illustrating an example of the concentration range of fluorescent markers described herein.

[0103] Figure 25A This is a bright-field micrograph of the sample slide.

[0104] Figure 25B These are fluorescence micrographs of sample slides acquired using an excitation wavelength of 594 nanometers (nm).

[0105] Figure 25C These are fluorescence micrographs of sample slides acquired using a 647nm excitation wavelength.

[0106] Figure 25D Through merger Figure 25B and 25C The image shown is a fluorescence micrograph of the sample slide obtained.

[0107] Figure 25E yes Figure 25D A magnified view of the fluorescence micrograph shown.

[0108] Figure 26A This is a schematic diagram illustrating an example of a sample slide and a magnified view of a selected area.

[0109] Figure 26B This is an explanation Figure 26A A schematic diagram illustrating an example of the concentration range of fluorescent markers described herein.

[0110] Figure 26C yes Figure 26A Fluorescence micrograph of the array shown.

[0111] Figure 27A This is a schematic diagram illustrating an example of a sample slide and an enlarged view of the size calibration array and colorimetric dynamic range array.

[0112] Figure 27B These are fluorescence micrographs of a colorimetric dynamic range array obtained using a 488nm excitation wavelength.

[0113] Figure 27C These are fluorescence micrographs of a colorimetric dynamic range array obtained using a 594 nm excitation wavelength.

[0114] Figure 27D These are fluorescence micrographs of a colorimetric dynamic range array obtained using a 647nm excitation wavelength.

[0115] Figure 28A This is a schematic diagram illustrating a sample slide that includes the sample and a colorimetric dynamic range array.

[0116] Figure 28B These are fluorescence micrographs of sample slides acquired using a 488nm excitation wavelength.

[0117] Figure 28C These are fluorescence micrographs of sample slides acquired using a 594nm excitation wavelength.

[0118] Figure 28D These are fluorescence micrographs of sample slides acquired using a 647nm excitation wavelength.

[0119] Figures 29A-29D An example of a glass slide including reference markers is shown.

[0120] Figure 30 Examples of different patterns within the reference marker frame are shown.

[0121] Figure 31 This is a perspective view of an apparatus including a plate and a substrate holder for heating a substrate, according to some embodiments provided herein.

[0122] Figure 32 This is based on some of the implementation methods provided in this article. Figure 31 Top perspective view of the board.

[0123] Figure 33 This is based on some of the implementation methods provided in this article. Figure 31 Bottom perspective view of the board.

[0124] Figure 34 This is based on some of the implementation methods provided in this article. Figure 31 Exploded view of the substrate holder.

[0125] Figure 35 This is based on some of the implementation methods provided in this article. Figure 34 Top perspective view of the bottom component of the substrate holder.

[0126] Figure 36 This is based on some of the implementation methods provided in this article. Figure 35 Bottom perspective view of the bottom component.

[0127] Figure 37 Based on some implementation methods provided in this article, and Figure 31 board connection Figure 35 Perspective view of the bottom component.

[0128] Figure 38 This is based on some implementation methods provided herein, and is connected to the second board implementation method. Figure 35 Perspective view of the bottom component.

[0129] Figure 39 Based on some implementation methods provided in this article, and Figure 35 Front perspective view of the fasteners used with the bottom components.

[0130] Figure 40 This is based on some implementation methods provided in this article. Figure 39 Rear perspective view of the fastener.

[0131] Figure 41 Based on some implementation methods provided in this article, and Figure 34 A perspective view of the liner used with the substrate retainer.

[0132] Figure 42 This is based on some implementation methods provided in this article. Figure 34 Top perspective view of the top component of the substrate retainer.

[0133] Figure 43 This is based on some implementation methods provided in this article. Figure 42 Bottom perspective view of the top component.

[0134] Figure 44A , 44B Figures 44C and 44C show substrate holders for heating substrates according to some embodiments provided herein. Figure 44A This is a perspective view of the substrate holder. Figure 44B yes Figure 44A Exploded view of the substrate holder. Figure 44C yes Figure 44A A partial perspective view of the substrate holder.

[0135] Figure 45A This is based on some implementation methods provided in this article. Figure 44A Top perspective view of the substrate holder. Figure 45B This is based on some implementation methods provided in this article. Figure 44A Bottom perspective view of the substrate holder. Figure 45C This is based on some implementation methods provided in this article. Figure 44A Side perspective view of the substrate holder.

[0136] Figure 46 This is a perspective view of a liner according to some embodiments provided herein.

[0137] Figure 47 This is a top perspective view of a substrate loader tool according to some embodiments provided herein.

[0138] Figures 48A-48B It is inserted into some implementation methods provided in this article. Figure 47 Side and top perspective views of the substrate holder in the substrate loader tool.

[0139] Figure 49A -C is a perspective view of another example of a substrate loader tool according to some implementations provided herein.

[0140] Figure 50A -C is a perspective view of a substrate retainer tool according to some embodiments provided herein.

[0141] Figure 51 This is a schematic diagram showing two substrates that support the sample and the feature array, respectively.

[0142] Figure 52A This is a schematic top view of an example of a sample holder.

[0143] Figure 52B-52F This is a schematic side view of an example of a sample holder.

[0144] Figure 53 This is a schematic side view of an example of a sample holder.

[0145] Figure 54 This is a schematic diagram showing the substrates aligned at right angles.

[0146] Figure 55 This is a schematic diagram showing the reference markings on the substrate holder.

[0147] Figure 56 This is a schematic diagram illustrating the workflow associated with the analysis of multiple samples on a single substrate.

[0148] Figure 57A This is a schematic diagram showing the workflow of using anisotropic permeation layers.

[0149] Figure 57B This is a schematic partial view of a sample in contact with a feature array in the presence of an anisotropic permeation layer.

[0150] Figure 57C yes Figure 57B A schematic enlarged view of the part.

[0151] Figure 58 This is a schematic diagram showing the workflow of using a feature array on top of a hydrogel layer containing a permeabilizing agent.

[0152] Figure 59 This is a schematic diagram illustrating the workflow of using a permeable fluid layer to analyze samples.

[0153] Figure 60A-60C From Figure 59 Images of samples obtained through the workflow.

[0154] Figure 60D It is a display Figure 60B and Figure 60C A graph comparing the image intensities between the two.

[0155] Figures 61A-61C From Figure 59The workflow yielded another image of the sample.

[0156] Figure 61D It is a display Figure 61B and Figure 61C A chart comparing the signal-to-noise ratios of the two.

[0157] Figure 62 This is a schematic diagram showing the workflow of analyzing samples using a feature array, which consists of beads immersed in a permeation solution.

[0158] Figure 63 This is a schematic diagram illustrating the workflow of analyzing samples using a hydrogel layer injected with a permeation solution.

[0159] Figure 64A It is based on Figure 63 The workflow analysis consists of a series of images of different tissue samples.

[0160] Figure 64B It is a display Figure 64A A graph comparing signal strength between mid-bottom row images.

[0161] Figure 65 The sample design displays printed guide markers to help users place tissues during cryosectioning. Dots indicate the center of the array, while numbers and letters represent individual wells where tissue samples can be placed.

[0162] Figure 66 This shows three variations before applying transparent static adhesive information markers to the slides. These variations differ in size but are made of the same material. Variation 1 shows clear information markers without any markings, Variation 2 shows clear information markers with white lettering, and Variation 3 shows clear information markers with black lettering.

[0163] Figure 67 This image shows slides with each of the three information markers applied when scanned at a PMT gain of 600 and 5 pixels / micrometer in the red and green channels. The image shows the difference in background in each channel with the information markers. The image also shows the visibility of the letters on each information marker when applied to the slide in each channel.

[0164] Figure 68 An example analytical workflow using a dried permeation reagent is shown.

[0165] Figure 69 An example analytical workflow is shown using the first and second components with temperature control using an example sample holder.

[0166] Figure 70AA schematic diagram showing an example electrophoretic migration of an analyte using the gap between the sample and the surface of a first substrate is shown.

[0167] Figure 70B A schematic diagram showing an example electrophoretic migration of analytes using the gap between the sample and the coating is displayed.

[0168] Figure 70C This diagram illustrates an example of electrophoretic migration of the analyte, where the sample is in contact with a coating on a second substrate.

[0169] Figure 70D This diagram shows an example of electrophoretic migration of the analyte, with the sample in contact with a first substrate.

[0170] Figure 71 An example setup for sample electrophoretic permeation is shown.

[0171] Figure 72A A perspective view of the top surface of an example substrate holder is shown.

[0172] Figure 72B A perspective view of the bottom surface of an example substrate holder is shown.

[0173] Figure 73A A top view of an example substrate holder in the open position is shown.

[0174] Figure 73B A side view of an example substrate holder in the open position is shown.

[0175] Figure 73C A side view of an example locking mechanism for an example substrate retainer is shown.

[0176] Figure 74A An exploded perspective view of an example substrate holder and glass slide is shown.

[0177] Figure 74B A perspective view of an example substrate holder and glass slide in the open position is shown.

[0178] Figure 75A Tissue sections of invasive ductal carcinoma are shown, annotated by a pathologist.

[0179] Figure 75B An organizational diagram showing points colored by unsupervised clustering is displayed.

[0180] Figure 75C It is a tSNE graph of points colored by unsupervised clustering.

[0181] Figure 75D A gene expression heatmap showing the genes with the greatest variation among the nine clusters is displayed.

[0182] Figure 75E The expression levels of genes corresponding to human epidermal growth factor receptor 2 (Her2), estrogen receptor (ER), and progesterone receptor (PR) were shown in tissue sections.

[0183] Figure 75F The gene expression levels of the most differentially expressed genes from each of the nine clusters on each graph are shown.

[0184] Figure 75G The gene expression levels of the most differentially expressed genes from each of the nine clusters on a single graph are shown.

[0185] Figure 75H This is a graph showing the expression levels of the most differentially expressed genes from each of the nine clusters in invasive ductal cell carcinoma (IDC) and normal breast tissue.

[0186] Figure 75I The expression of KRT14 in IDC and matched normal tissues is shown.

[0187] Figure 75J This is a graph showing the extracellular matrix gene expression levels in IDC and normal tissues.

[0188] The various reference numerals in the accompanying figures indicate similar elements. Detailed Implementation

[0189] I. Introduction

[0190] This invention describes apparatus, systems, methods, and compositions for spatial analysis of biological samples. This section describes certain general terms, analytes, sample types, and preparation steps, which will be referenced later in the invention.

[0191] (a) Spatial Analysis

[0192] Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from single-celled or multicellular organisms (e.g., mammals). Tissues and cells obtained from mammals (e.g., humans) often have different levels of analytes (e.g., gene and / or protein expression), which can lead to differences in cell morphology and / or function. The location of cells within a tissue can influence cell fate, behavior, morphology, and signal transduction and crosstalk with other cells in the tissue. Information about differences in intracellular analyte levels (gene and / or protein expression) in mammalian tissues can also help physicians select or administer treatments effective against single-celled or multicellular organisms (e.g., mammals) based on detected differences in intracellular analyte levels in tissues. Differences in intracellular analyte levels in mammalian tissues can also provide information on how tissues (e.g., healthy and diseased tissues) function and / or develop. Differences in intracellular analyte levels in mammalian tissues can also provide information on different disease pathogenesis mechanisms within tissues and on the mechanisms of action of treatments within tissues. Differences in intracellular analyte levels in mammalian tissues can also provide information on drug resistance mechanisms and the development of drug resistance mechanisms in mammalian tissues. Differences in the presence or absence of analytes in different cells within tissues of multicellular organisms (e.g., mammals) can provide information about the mechanisms and development of drug resistance in multicellular organism tissues.

[0193] The spatial analysis methods presented herein are used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells of mammalian tissues or within single cells of mammals. For example, spatial analysis methods can be used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells of tissue section samples, from which data can be reconstructed to generate a three-dimensional map of analyte levels (e.g., gene and / or protein expression) of tissue samples obtained from mammals, for example at a certain degree of spatial resolution (e.g., single-cell resolution).

[0194] Spatial heterogeneity in developmental systems is typically investigated through RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subsets, and subsequent genome profiling (e.g., RNA-seq). However, these methods rely on a relatively small set of predefined markers, thus introducing selection bias that limits discovery. These previous methods also depend on prior knowledge. Traditionally, spatial RNA analysis has relied on staining a limited amount of RNA material. In contrast, single-cell RNA sequencing allows for in-depth profiling of cellular gene expression, including non-coding RNA, but established methods isolate cells from their native spatial context.

[0195] Current spatial analysis methods provide a wealth of analyte level and / or expression data for multiple analytes in a sample with high spatial resolution, while preserving the natural spatial environment. Spatial analysis methods include, for example, the use of capture probes comprising a spatial barcode (e.g., a nucleic acid sequence providing information about the location of the capture probe within a cellular or tissue sample (e.g., mammalian cell or mammalian tissue sample)) and a capture domain capable of binding to an analyte (e.g., a protein and / or nucleic acid) produced by or present in the cell. As described herein, the spatial barcode can be a nucleic acid having a unique sequence, a unique fluorophore or a unique combination of fluorophores, a unique amino acid sequence, a unique heavy metal or a unique combination of heavy metals, or any other unique detectable reagent. The capture domain can be any reagent capable of binding to an analyte produced by and / or present in the cell (e.g., a nucleic acid capable of hybridizing with: cellular nucleic acids (e.g., mRNA, genomic DNA, mitochondrial DNA, or miRNA), a substrate containing the analyte, a binding chaperone of the analyte, or an antibody specifically bound to the analyte). The capture probe may also include a nucleic acid sequence complementary to a universal forward and / or universal reverse primer sequence. The capture probe may also include cleavage sites (e.g., cleavage recognition sites of restriction endonucleases), light-labile bonds, thermosensitive bonds, or chemically sensitive bonds.

[0196] A variety of different methods can be used to detect the binding of analytes to capture probes, such as nucleic acid sequencing, fluorophore detection, nucleic acid amplification, nucleic acid ligation detection, and / or nucleic acid lysis product detection. In some examples, detection is used to associate a specific spatial barcode with a specific analyte produced by and / or present in cells (e.g., mammalian cells).

[0197] The capture probe can be attached to a surface, such as a solid array, beads, or coverslip. In some examples, the capture probe is not attached to a surface. In some examples, the capture probe may be encapsulated within, embedded in, or layered on a surface of a permeable composition (e.g., any of the substrates described herein). For example, the capture probe may be encapsulated or disposed within a permeable bead (e.g., a gel bead). In some examples, the capture probe may be encapsulated within, embedded in, or layered on a surface of a substrate (e.g., any of the exemplary substrates described herein, such as a hydrogel or porous membrane).

[0198] In some embodiments, cells or tissue samples comprising cells are contacted with a capture probe attached to a substrate (e.g., the surface of the substrate), and the cells or tissue sample are permeated to release analytes from the cells and bind to the capture probe attached to the substrate. In some examples, a variety of methods (e.g., electrophoresis, chemical gradients, pressure gradients, fluid flow, or magnetic fields) can be used to actively guide the analytes released from the cells to the capture probe attached to the substrate.

[0199] In other examples, a variety of methods can be used to guide the capture probe to interact with the cell or tissue sample, including lipid anchors in the capture probe, reagents that can specifically bind to or form covalent bonds with membrane proteins in the capture probe, fluid flow, pressure gradient, chemical gradient, or magnetic field.

[0200] The non-restrictive aspects of spatial analysis methods are discussed in WO 2011 / 127099, WO 2014 / 210233, WO 2014 / 210225, WO 2016 / 162309, WO 2018 / 091676, WO 2012 / 140224, WO 2014 / 060483, U.S. Patent No. 10,002,316, U.S. Patent No. 9,727,810, U.S. Patent Application Publication No. 2017 / 0016053, Rodriques et al., Science 363(6434): 1463-1467, 2019; WO 2018 / 045186, Lee et al., Nat.Protoc.10(3): 442-458, 2015; WO 2016 / 007839, WO 2018 / 045181, WO 2014 / 163886, Trejo et al., PLoS ONE 14(2): e0212031, 2019, US Patent Application Publication No. 2018 / 0245142, Chen et al., Science 348(6233): aaa6090, 2015, Gao et al., BMC Biol.15:50, 2017, WO 2017 / 144338, WO 2018 / 107054, WO 2017 / 222453, WO 2019 / 068880, WO 2011 / 094669, US Patent No. 7,709,198, US Patent No. 8,604,182, US Patent No. 8,951,726, US Patent No. 9,783,841, US Patent No. 10,041,949, WO 2016 / 057552, WO 2017 / 147483, WO 2018 / 022809, WO 2016 / 166128, WO 2017 / 027367, WO 2017 / 027368, WO2018 / 136856, WO 2019 / 075091, US Patent No. 10,059,990, WO 2018 / 057999, WO 2015 / 161173, and Gupta et al., Nature The methods described in Biotechnol.36:1197-1202, 2018, and may be used herein in any combination. Further non-limiting aspects of the spatial analysis methods are described herein.

[0201] (b) General terms

[0202] Specific terminology is used in this invention to explain various aspects of the described apparatuses, systems, methods, and compositions. This section includes explanations of certain terms that appear in later sections of this invention. If the descriptions in this section conflict with their usage in other sections of this invention, the definitions in this section shall prevail.

[0203] (i) Barcode

[0204] A barcode is a label or identifier that conveys or is capable of conveying information (e.g., information about analytes, beads, and / or capture probes in a sample). A barcode can be part of the analyte or independent of it. A barcode can be attached to the analyte. A particular barcode may be unique relative to other barcodes.

[0205] Barcodes can take many different forms. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. Barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. For example, barcodes can be added to fragments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before or during sequencing. Barcodes can allow for the identification and / or quantification of individual sequencing reads (e.g., barcodes can be or can include a unique molecular identifier or "UMI").

[0206] Barcodes can spatially resolve molecular components present in a biological sample, for example, at single-cell resolution (e.g., a barcode may be or may include a "spatial barcode"). In some embodiments, a barcode includes both a UMI and a spatial barcode. In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode may include two or more polynucleotide sequences separated by one or more non-barcode sequences (e.g., sub-barcodes).

[0207] (ii) Nucleic acids and nucleotides

[0208] The terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and include naturally occurring substances or their functional analogs. Particularly useful nucleic acid functional analogs are capable of hybridizing with nucleic acids in a sequence-specific manner (e.g., capable of hybridizing with two nucleic acids such that a link can occur between the two hybridized nucleic acids) or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures may have other backbone linkages, including any of the various linkages known in the art. Naturally occurring nucleic acids typically have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0209] Nucleic acids may comprise any of a nucleotide having any of the various analogs of these sugar moieties known in the art. Nucleic acids may include natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acid may have one or more bases selected from adenine (A), thymine (T), cytosine (C), or guanine (G), and ribonucleic acid may have one or more bases selected from uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-natural bases that may be included in nucleic acids or nucleotides are known in the art.

[0210] (iii) Probes and targets

[0211] "Probe" or "target", when used in relation to nucleic acids or nucleic acid sequences, means a semantic identifier of a nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the nucleic acid or sequence beyond what is explicitly indicated.

[0212] (iv) Oligonucleotides and Polynucleotides

[0213] The terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polymers of nucleotides with a length of about 2 to about 500 nucleotides. Oligonucleotides can be prepared synthetically, enzymatically (e.g., by polymerization), or using a "split-pool" method. Oligonucleotides may comprise ribonucleotide monomers (i.e., may be oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some examples, oligonucleotides may comprise combinations of deoxyribonucleotide monomers and ribonucleotide monomers (e.g., random or ordered combinations of deoxyribonucleotide monomers and ribonucleotide monomers). For example, the length of an oligonucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400 to 500 nucleotides. An oligonucleotide may include (e.g., covalently or non-covalently) one or more functional portions attached to a multimeric structure. For example, an oligonucleotide may include one or more detectable labels (e.g., radioisotopes or fluorophores).

[0214] (v) object

[0215] "Object" is an animal, such as a mammal (like a human or ape), or a bird (like a bird), or other organism, such as a plant. Examples of objects include, but are not limited to, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, and primates (i.e., humans or non-human primates); plants such as Arabidopsis thaliana, maize, sorghum, oats, wheat, rice, rapeseed, or soybeans; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila melanogaster, mosquitoes, fruit flies, or bees; arachnids such as spiders; fish such as zebrafish; reptiles; amphibians such as frogs or Xenopus laevis; Dictyostelium discoideum; and fungi such as Pneumocystis carinii. The species *Carinii*, *Takifugu rubripes*, yeast, *Saccharomyces cerevisiae* or *Schizosaccharomyces pombe*; or *Plasmodium falciparum*.

[0216] (vi) Genome

[0217] "Genome" generally refers to genomic information from an object, such as at least part or all of the genetic information encoded by the object's genes. A genome can include coding regions (e.g., regions that encode proteins) and non-coding regions. A genome can include sequences of some or all of the object's chromosomes. For example, the human genome typically has 46 chromosomes in total. Some or all of these sequences can constitute the genome.

[0218] (vii) Adapter, Connector, and Label

[0219] "Adaptor," "adapter," and "tag" are terms that may be used interchangeably in this disclosure and refer to a substance that can be coupled to a polynucleotide sequence using any of a number of different techniques, including (but not limited to) ligation, hybridization, and tagging (in a process referred to as "tagging"). An adaptor can also be a nucleic acid sequence with added functionality, such as a spacer sequence, primer sequence / site, barcode sequence, or unique molecular identifier sequence.

[0220] (viii) Heterozygium, hybridization, annealing, and chain recombination.

[0221] The terms “hybridization,” “hybridization,” “annealing,” and “chain recombination” are used interchangeably in this disclosure and refer to the pairing of essentially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved through any process in which nucleic acid sequences are linked to essentially complementary or fully complementary sequences by base pairing to form a hybridization complex. For the purpose of hybridization, two nucleic acid sequences are “essentially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the individual bases in each other are complementary.

[0222] (ix) primers

[0223] A primer is a single-stranded nucleic acid sequence with a 3′ end that can be used as a chemical substrate for nucleic acid polymerases in nucleic acid extension reactions. RNA primers are formed from RNA nucleotides and are used for RNA synthesis, while DNA primers are composed of DNA nucleotides and are used for DNA synthesis. Primers may also include RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers may also include other natural or synthetic nucleotides with other functionalities as described herein. In some examples, DNA primers can be used to initiate RNA synthesis and vice versa (e.g., RNA primers can be used to initiate DNA synthesis). Primer lengths can vary. For example, primers can be from about 6 bases to about 120 bases. For example, primers may include up to about 25 bases.

[0224] (x) Primer extension

[0225] "Primer extension" refers to any method in which two nucleic acid sequences (e.g., constant regions from each of two different capture probes) are joined (e.g., hybridized) by overlapping their respective complementary terminal nucleic acid sequences (e.g., 3′ ends). Following this ligation, another nucleic acid sequence can be used as an extension template to perform one or both-end extension (e.g., enzyme extension). Enzyme extension can be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0226] (xi) Neighborhood connection

[0227] "Proximity joining" is a method of joining two (or more) nucleic acid sequences that are adjacent to each other by enzymatic means (e.g., ligases). In some embodiments, proximity joining may include a "gap filling" step, which involves incorporating one or more nucleic acids across a distance spanning two nucleic acid molecules of interest using a polymerase based on the nucleic acid sequence of a template nucleic acid molecule (see, for example, U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference).

[0228] Various methods can be used for proximity ligation of nucleic acid molecules, including (but not limited to) "sticky-end" and "blunt-end" ligation. Furthermore, single-stranded ligation can be used for proximity ligation of single-stranded nucleic acid molecules. Sticky-end proximity ligation involves hybridizing the complementary single-stranded sequences between the two nucleic acid molecules to be ligated before the ligation event occurs. Blunt-end proximity ligation typically does not include hybridization from the complementary regions of each nucleic acid molecule because both nucleic acid molecules lack single-stranded overhangs at the ligation site.

[0229] (xii) Nucleic acid extension

[0230] Nucleic acid extension typically involves incorporating one or more nucleic acids (e.g., A, g, C, T, U, nucleotide analogs or derivatives thereof) into a molecule (e.g., but not limited to nucleic acid sequences) in a template-dependent manner, such that consecutive nucleic acids are incorporated by an enzyme (e.g., polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. For example, by using a complementary nucleic acid sequence as a template for nucleic acid synthesis, a new nucleic acid molecule can be synthesized using primers that hybridize with the complementary nucleic acid sequence. Similarly, the 3′ polyadenylated tail of an mRNA transcript that hybridizes with a poly(dT) sequence (e.g., a capture domain) can be used as a template for the single-stranded synthesis of the corresponding cDNA molecule.

[0231] (xiii) PCR amplification

[0232] “PCR amplification” refers to the generation of copies of genetic material, including DNA and RNA sequences, using polymerase chain reaction (PCR). Suitable reagents and conditions for performing PCR are described, for example, in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of which are incorporated herein by reference. In typical PCR amplification, the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers for primer extension, and reagents for the reaction. Oligonucleotide primers are of sufficient length to hybridize with complementary genetic material under annealing conditions. The length of primers typically depends on the length of the amplified domain, but is usually at least 4, 5, 6, 8, 9, 10 base pairs (bp), at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 bp, and can be as long as 40 bp or more, with primer length generally between 18 and 50 bp. Genetic material can be contacted with a single primer or a pair of primers (forward and reverse primers), depending on whether primer extension, linear, or exponential amplification of the genetic material is required.

[0233] In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In some embodiments, the DNA polymerase is derived from bacteria; for example, the DNA polymerase is a bacterial DNA polymerase. For instance, the DNA polymerase can be derived from bacteria of the genera *Escherichia coli*, *Bacillus*, *Thermophilus*, or *Thermococcus*.

[0234] Suitable examples of usable DNA polymerases include, but are not limited to: Escherichia coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, and VENT. TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot-start Taq DNA polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase DNA polymerase, Quick DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-fidelity DNA polymerases, including platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase.

[0235] The term "DNA polymerase" includes not only naturally occurring enzymes but also all modified derivatives thereof, as well as derivatives of naturally occurring DNA polymerases. For example, in some embodiments, DNA polymerases may be modified to remove 5′–3′ exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that can be used include, but are not limited to, mutants that retain at least partial function, such as DNA polymerase activity with wild-type sequences. Under different reaction conditions, such as temperature, template concentration, primer concentration, etc., mutations can affect the enzyme's activity profile, such as increasing or decreasing the polymerization rate. Mutations or sequence modifications may also affect the enzyme's exonuclease activity and / or thermal stability.

[0236] In some embodiments, PCR amplification may include, for example, but not limited to, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, isothermal amplification, and / or loop-mediated amplification.

[0237] In some embodiments, PCR amplification uses a single primer complementary to the 3′ tag of the target DNA fragment. In some embodiments, PCR amplification uses first and second primers, wherein at least the 3′ terminal portion of the first primer is complementary to at least a portion of the 3′ tag of the target nucleic acid fragment, and wherein the at least 3′ terminal portion of the second primer displays at least a portion of the sequence of the 5′ tag of the target nucleic acid fragment. In some embodiments, the 5′ terminal portion of the first primer is not complementary to the 3′ tag of the target nucleic acid fragment, and the 5′ terminal portion of the second primer does not display at least a portion of the sequence of the 5′ tag of the target nucleic acid fragment. In some embodiments, the first primer includes a first universal sequence and / or the second primer includes a second universal sequence.

[0238] In some implementations (e.g., when PCR amplification amplifies captured DNA), DNA ligases can be used to ligate PCR amplification products to other sequences. DNA ligase activity can be provided by one or more different DNA ligases. In some implementations, the DNA ligase is derived from bacteria; for example, the DNA ligase is a bacterial DNA ligase. In some implementations, the DNA ligase is derived from viruses (e.g., bacteriophages). For example, the DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Thermococcus spp. (strain 9oN) DNA ligase (9oN™ DNA ligase, available from New England Biolabs in Ipswich, Massachusetts), and Ampligase. TM (Available from Epicentre Biotechnologies, Madison, Wisconsin). Derivatives, such as sequence-modified derivatives and / or their mutants, may also be used.

[0239] In some implementations, genetic material is amplified via reverse transcription polymerase chain reaction (RT-PCR). The desired reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which include, but are not limited to: M-MLV, MuLV, AMV, HIV, and ArrayScript. TM MultiScribe TM ThermoScript TM ,and Reverse transcriptases include enzymes I, II, III, and IV. The term "reverse transcriptase" encompasses not only naturally occurring enzymes but also all their modified derivatives, as well as derivatives of naturally occurring reverse transcriptases.

[0240] Furthermore, reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases, including mutants that retain at least some functional (e.g., reverse transcriptase) activity while retaining the wild-type sequence. The reverse transcriptase can be provided as part of a composition comprising other components, such as stabilizing components that enhance or improve the activity of the reverse transcriptase, such as RNase inhibitors or inhibitors of DNA-dependent DNA synthesis, for example, actinomycin D. Sequence-modified derivatives or mutants of many reverse transcriptases, such as M-MLV, and compositions comprising unmodified and modified enzymes, such as ArrayScript, are also available. TM MultiScribe TM ThermoScript TM ,and Enzymes I, II, III, and IV are commercially available.

[0241] Some reverse transcriptases (such as avian myeloblastosis virus (AMV) reverse transcriptase and Moroni murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use enzymes (reverse transcriptases) capable of using both RNA and ssDNA as templates for the extension reaction, such as AMV or MMLV reverse transcriptases.

[0242] In some implementations, RNA and / or DNA quantification is performed by real-time PCR (also known as quantitative PCR or qPCR) using techniques well-known in the art, such as, but not limited to, TAQMAN. TM "or Or on the capillary (" (Capillary). In some embodiments, the quantification of genetic material is determined by light absorbance and real-time PCR. In some embodiments, the quantification of genetic material is determined by digital PCR. In some embodiments, the analyzed gene may correspond to expression (mRNA) and quantity (DNA) compared with a reference nucleic acid extract (DNA and RNA) to compare the expression level of the target nucleic acid.

[0243] (xiv) antibody

[0244] An antibody is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a Y-shaped molecular structure. Naturally occurring antibodies, known as immunoglobulins, belong to the immunoglobulin class, including IgG, IgM, IgA, IgD, and IgE. Antibodies can also be synthesized artificially. For example, recombinant antibodies, as monoclonal antibodies, can be synthesized using synthetic genes by recovering the antibody gene from a source cell, amplifying it into a suitable vector, and introducing the vector into a host to induce the host to express the recombinant antibody. Generally, recombinant antibodies can be cloned from any antibody-producing animal species using suitable oligonucleotide primers and / or hybridization probes. Recombinant technology can be used to generate antibodies and antibody fragments, including non-endogenous substances.

[0245] Synthetic antibodies can be obtained from non-immunoglobulin sources. For example, antibodies can be produced from nucleic acids (e.g., aptamers) and non-immunoglobulin scaffolds (e.g., peptide aptamers) with hypervariable loops inserted to form antigen-binding sites. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue permeability.

[0246] Antibodies may also include affinity proteins, which are affinity agents typically having a molecular weight of about 12-14 kDa. Affinity proteins typically bind to targets (e.g., target proteins) with high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive proteins. In some embodiments, the affinity protein is derived from a cysteine ​​protease inhibitor and includes a peptide ring and a variable N-terminal sequence that provides a binding site.

[0247] Antibodies can also include single-domain antibodies (V) H H domain and VNAR domain), scFv and Fab fragments.

[0248] (xv) affinity group

[0249] A “generic affinity” is a molecule or part of a molecule that has a high affinity or preference for association or binding with another particular or specific molecule or part. Association or binding with another particular or specific molecule or part can be achieved through non-covalent interactions, such as hydrogen bonds, ionic forces, and van der Waals interactions. For example, an affinity group can be biotin, which has a high affinity or preference for association or binding with proteavidin or streptavidin. For example, an affinity group can also refer to avidin or streptavidin, which has an affinity for biotin. Other examples of affinity groups and the particular or specific molecules or parts they bind to or associate with include, but are not limited to, antibodies or antibody fragments and their respective antigens, such as digoxigenin and anti-digoxigenin antibodies, lectins and carbohydrates (e.g., sugars, monosaccharides, disaccharides, or polysaccharides), and receptors and receptor ligands.

[0250] Any pair of affinity groups and the specific or particular molecule or part thereof that binds to or associates with them can reverse their effects, for example, between a first molecule and a second molecule, in the first example the first molecule is characterized as the affinity group of the second molecule, and in the second example the second molecule is characterized as the affinity group of the first molecule.

[0251] (xvi) Markers, detectable markers and optical markers.

[0252] The terms “detectable label,” “optical label,” and “label” are used interchangeably herein to refer to the directly or indirectly detectable portion associated (e.g., coupled) with a molecule to be detected (e.g., a capture probe or analyte). A detectable label may be directly detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, may be indirectly detectable, for example, by catalyzing a chemical change in a chemical substrate compound or composition that makes it directly detectable. Detectable labels may be suitable for small-scale detection and / or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.

[0253] Detectable markers can be detected qualitatively (e.g., optically or spectroscopically) or quantitatively. Qualitative detection typically involves detection methods that confirm the presence or occurrence of the detectable marker, while quantitative detection typically involves detection methods that have quantifiable (e.g., digitally reportable) values, such as intensity, duration, polarization, and / or other properties. In some embodiments, the detectable marker is incorporated into a feature or a capture probe associated with the feature. For example, a detectably tagged feature may include a fluorescent, colorimetric, or chemiluminescent marker attached to a bead (see, for example, Rajeswali et al., J. Microbiol Methods 139: 22-28, 2017, and Forcucci et al., J. Biomed Opt. 10: 105010, 2015, the entire contents of which are incorporated herein by reference).

[0254] In some implementations, multiple detectable markers can be attached to the feature to be detected, the capture probe, or the composition. For example, the detectable markers can be incorporated during nucleic acid polymerization or amplification (e.g., For example Any suitable detectable marker can be used. In some embodiments, the detectable marker is a fluorophore. For example, the fluorophore may be from the following group: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA). Alexa Alexa Alexa Alexa Allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, aniline blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, O-Feulgen (Auramine), BCECF (High pH), BFP (Blue Fluorescent Protein), BFP / GFP FRET, BOBO TM -1 / BO-PRO TM -1, BOBO TM -3 / BO-PRO TM -3、 BTC, Calcein, Calcein Blue, Calcium Crimson TM Calcium Green-1 TM Calcium Orange TM , White, 5-Carboxyfluorescein (5-FAM), 5-Carboxynaphthalenefluorescein, 6-Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow TM CCF2 (GeneBlaze) TM CFP (cyan fluorescent protein), CFP / YFP FRET, chromopycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC-methyl Cychrome (PE-Cy5), dansylamide, dansyl cadaverine, dansyl chloride, DAPI, dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4ANEPPS, Di-8ANEPPS, DM-NERF (4.5-6.5pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, Alcohol, Eosin, Erythromycin, Ethidium bromide, Ethidium homodimer-1 (EthD-1), Europium(III) chloride, 5-FAM (5-carboxyfluorescein), Quick Blue, fluorescein dT phosphoramide, FITC, Fluoro-3, Fluo-4 Fluoro-Gold TM (High pH), Fluoro-Gold TM (low pH), F1uoro-Jade, 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red TM (High calcium), Fura Red TM / Fluo-3, GeneBLAzer TM (CCF2), GFP redshift (rsGFP), GFP wild-type, GFP / BFP-FRET, GFP / DsRed-FRET, Hoechst 33342&33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5-indoleacetic acid, Indo-1 (high calcium), Indo-1 (low calcium), indole dicarboxycyanine, indole tricarboxycyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751(+DNA), LDS 751(+RNA), LOLO TM -1 / LO-PRO TM -1, Lucifer Yellow, LysoSensor TM Blue (pH5), LysoSensor TM Green (pH 5), LysoSensor TM Yellow / Blue (pH 4.2) green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM Marina 4-Methylumbelliferone, Miteradine green, orange, Red, NBD (amine), Nile Red, Oregon Oregon Oregon Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (polydinophyll-chlorophyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1, POPO TM -3 / PO-PRO TM -3, Propidium iodide (PI), PyMPO, Pyrene, Pyrrolidone Y, Quantum Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein (DsRed), Halogen, RH 414, Rhod-2, Rhodamine B, Rhodamine Green TM Rhodamine Red TM Rhodamine phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS (High pH) (High pH) (Low pH), Sodium Green TM , blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), Texas Texas (NHS ester), thiazolinone anthocyanin, thiazolinone orange, Tricolor (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein) 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705. (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye (NHS ester) and Dy 750 (NHS ester).

[0255] As described above, in some embodiments, the detectable marker is or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze a chemiluminescent reaction given a suitable chemical substrate (e.g., an oxidizing agent plus a chemiluminescent compound). Many families of compounds are known to provide chemiluminescence under a variety of conditions. Non-limiting examples of families of chemiluminescent compounds include 2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and dimethylamino[ca]benzene analogs. These compounds are luminescent in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of families of chemiluminescent compounds include, for example, 2,4,5-triphenylimidazolium, p-dimethylamino and -methoxy substituents, oxalates (e.g., oxaloyl active esters), p-nitrophenyl, N-alkyl acridine esters, luciferin, luster, or acridine esters.

[0256] (xvii) Template-converting oligonucleotides

[0257] A template-transfer oligonucleotide is an oligonucleotide that hybridizes with an untemplated nucleotide added by a reverse transcriptase (e.g., an enzyme with terminal transferase activity) during reverse transcription. In some embodiments, the template-transfer oligonucleotide hybridizes with an untemplated poly(C) nucleotide added by a reverse transcriptase. In some embodiments, the template-transfer oligonucleotide adds a common 5′ sequence to full-length cDNA used for cDNA amplification.

[0258] In some embodiments, template-converting oligonucleotides add a common sequence to the 5′ end of the RNA being reverse transcribed. For example, the template-converting oligonucleotide can hybridize with an untemplated poly(C)nucleotide added to the end of a cDNA molecule and provide a template for reverse transcriptase to continue replicating to the 5′ end of the template-converting oligonucleotide, thereby generating full-length cDNA capable of further amplification. In some embodiments, once the full-length cDNA molecule is generated, the template-converting oligonucleotide can be used as a primer in the cDNA amplification reaction.

[0259] In some embodiments, template-converting oligonucleotides are added before, during, or after reverse transcription or other terminal transferase-based reactions. In some embodiments, the capture probe includes template-converting oligonucleotides. In some embodiments, sample analysis methods using template-converting oligonucleotides may involve generating nucleic acid products from analytes in tissue samples, followed by further processing of the nucleic acid products using template-converting oligonucleotides.

[0260] The template-converting oligonucleotide may include a hybridization region and a template region. The hybridization region may include any sequence capable of hybridizing with a target. In some embodiments, the hybridization region may, for example, include consecutive G bases complementary to a C base overhang at the 3′ end of the cDNA molecule. Consecutive G bases may include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. The template sequence may include any sequence of the cDNA to be incorporated. In other embodiments, the hybridization region may include at least one base in addition to at least one G base. In other embodiments, hybridization may include non-G bases. In some embodiments, the template region includes at least one (e.g., at least 2, 3, 4, 5, or more) tag sequence and / or functional sequence. In some embodiments, the template region and the hybridization region are separated by a spacer.

[0261] In some embodiments, the template region includes a barcode sequence. The barcode sequence may function as a spatial barcode and / or as a unique molecular identifier. The template-transforming oligonucleotide may include deoxyribonucleic acid; ribonucleic acid; modified nucleic acids, including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reversed dT, 5-methyl dC, 2′-deoxyinosine, superT (5-hydroxybutyl-2′-deoxyuridine), superG (8-aza-7-dezoguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, e.g., UNA-A, UNA-U, UNA-C, UNA-g), Iso-dG, Iso-dC, 2′ fluorobases (e.g., fluorine C, fluorine U, fluorine A, and fluorine G), or any combination thereof.

[0262] In some embodiments, the template-converting oligonucleotide may be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides long or longer. In some embodiments, the template-converting oligonucleotide may be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides long or longer.

[0263] (xviii) Clamping oligonucleotides

[0264] A "sandwich oligonucleotide" is an oligonucleotide that acts as a "sandwich" when hybridizing with other polynucleotides, keeping the polynucleotides adjacent to each other so they can be linked together. In some embodiments, the sandwich oligonucleotide is DNA or RNA. A sandwich oligonucleotide may include a nucleotide sequence that is partially complementary to the nucleotide sequence of two or more different oligonucleotides. In some embodiments, the sandwich oligonucleotide assists in linking a "donor" oligonucleotide and a "recipient" oligonucleotide. Generally, RNA ligase, DNA ligase, or other types of ligase are used to link two nucleotide sequences together.

[0265] In some embodiments, the length of the splice oligonucleotide is between 10 and 50 oligonucleotides, for example, between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides. In some embodiments, the length of the splice oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25 nucleotides.

[0266] (c) Analytes

[0267] The apparatus, systems, methods, and compositions described in this disclosure can be used to detect and analyze a wide variety of analytes. For the purposes of this invention, "analyte" can include any biological substance, structure, part, or component to be analyzed. The term "target" can similarly refer to the analyte of interest.

[0268] Analytes can be broadly classified into two categories: nucleic acid analytes and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidated variants of proteins, hydroxylated variants of proteins, methylated variants of proteins, ubiquitinated variants of proteins, sulfated variants of proteins, viral capsid proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte may be an organelle (e.g., the cell nucleus or mitochondria).

[0269] Cell surface features corresponding to the analyte may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) of cell surface proteins, gap junctions, and adhesion junctions.

[0270] Analytes can originate from specific cell types and / or specific subcellular regions. For example, analytes can originate from the cytoplasm, nucleus, mitochondria, microsomes, and more generally, any other compartment, organelle, or part of the cell. Permeabilizers specifically targeting certain cell compartments and organelles can be used to selectively release analytes from the cell for analysis.

[0271] Examples of nucleic acid analytes include DNA analytes, such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids.

[0272] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (miRNA), and viral RNA. RNA can be a transcript (e.g., present in tissue sections). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA longer than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). RNA can be double-stranded or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16S rRNA or 23S rRNA).

[0273] Other examples of analytes include mRNA and cell surface features (e.g., using markers described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cellular methylation status, mRNA and available chromatin (e.g., ATAC-seq, DNase-seq, and / or micrococcal enzyme-seq), mRNA and metabolites (e.g., using markers described herein), barcoded markers (e.g., oligonucleotide-labeled antibodies described herein), and V(D)J sequences of immune cell receptors (e.g., T cell receptors), mRNA and perturbators (e.g., CRISPR crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein). In some embodiments, the perturbator may be a small molecule, antibody, drug, aptamer, miRNA, physical environment (e.g., temperature change), or any other known perturbator.

[0274] The analyte may include a nucleic acid molecule having at least a portion of a nucleic acid sequence encoding a V(D)J sequence of an immune cell receptor (e.g., TCR or BCR). In some embodiments, the nucleic acid molecule is cDNA first generated from the reverse transcription of the corresponding mRNA using a poly(T)-containing primer. The generated cDNA can then be barcoded using a capture probe having a barcoded sequence (and optionally a UMI sequence) that hybridizes to at least a portion of the generated cDNA. In some embodiments, a template-converting oligonucleotide hybridizes to a poly(C) tail added to the 3′ end of the cDNA via reverse transcriptase. The original mRNA template and template-converting oligonucleotide can be denatured from the cDNA, and then the barcoded capture probe can hybridize with the cDNA to generate a complement to the cDNA. Other methods and compositions suitable for barcoding cDNA generated from mRNA transcripts, including those encoding V(D)J regions of immune cell receptors, are described in PCT patent application PCT / US2017 / 057269 filed October 18, 2017, and U.S. Patent Application Serial No. 15 / 825,740 filed November 29, 2017, both of which are incorporated herein by reference in their entirety. V(D)J analysis can also be performed using one or more markers that bind to specific surface features of immune cells and are associated with barcode sequences. One or more markers may include MHC or MHC multimers.

[0275] As described above, the analyte may include nucleic acids capable of functioning as components of a gene editing reaction, such as gene editing based on regularly clustered, spaced short palindromic repeats (CRISPR). Therefore, the capture probe may include a nucleic acid sequence complementary to the analyte (e.g., a sequence that can hybridize to CRISPR RNA (crRNA), single-guide RNA (sgRNA), or an adaptor sequence engineered into crRNA or sgRNA).

[0276] In some implementations, analytes can be extracted from live cells. Processing conditions can be adjusted to ensure that the biological sample remains active during analysis and that the analyte is extracted (or released) from the live cells of the sample. Live cell-derived analytes can be obtained only once from the sample, or they can be obtained periodically from a sample that continues to remain viable.

[0277] Generally, the systems, apparatus, methods, and compositions described herein can be used to analyze any number of analytes. For example, the number of analytes analyzed may be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1000, at least about 10000, at least about 10000, or at least about 100000 or more different analytes present in the sample region or within the individual characteristics of the substrate. Methods for performing multiple analyses to analyze two or more different analytes will be discussed in later sections of this invention.

[0278] (d) biological samples

[0279] (i) Types of biological samples

[0280] "Biological samples" are obtained from an object for analysis using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and typically include cells and / or other biological material from the object. In addition to the objects described above, biological samples can be obtained from non-mammalian organisms (e.g., plants, insects, spiders, nematodes (e.g., *Caenorhabditis elegans*), fungi, amphibians, or fish (e.g., zebrafish)). Biological samples can also be obtained from prokaryotes, such as bacteria like *Escherichia coli*, *Staphylococci*, or *Mycoplasma pneumoniae*; archaea; viruses such as hepatitis C virus or human immunodeficiency virus; or viroids. Biological samples can also be obtained from eukaryotes, such as patient-derived organoids (PDOs) or patient-derived xenografts (PDXs). Biological samples can include organoids, miniaturized and simplified versions of organs generated in vitro that can display realistic microscopic anatomy in three dimensions. Organoids can generate one or more self-organizing cells, embryonic stem cells, and / or induced pluripotent stem cells, and due to their self-renewal and differentiation capabilities, they can self-organize in three-dimensional culture. In some embodiments, organoids are brain organoids, intestinal organoids, gastric organoids, tongue organoids, thyroid organoids, thymus organoids, testicular organoids, liver organoids, pancreatic organoids, epithelial organoids, lung organoids, kidney organoids, gastrula, heart organoids, or retinal organoids. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals with or suspected of having a disease (e.g., cancer), individuals with pre-treatment for a disease, and / or individuals requiring or suspected of requiring treatment.

[0281] Biological samples may be derived from homogeneous cultures or populations of the objects or organisms described herein, or alternatively from a collection of several different organisms in, for example, a community or ecosystem.

[0282] Biological samples may include one or more diseased cells. Diseased cells may exhibit altered metabolic properties, gene expression, protein expression, and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells may originate from solid tumors, hematologic malignancies, cell lines, or be obtained as circulating tumor cells.

[0283] Biological samples can also include fetal cells. For example, procedures such as amniocentesis can be performed to obtain fetal cell samples from the maternal circulation. Fetal cell sequencing can be used to identify any of many genetic disorders, including, for example, aneuploidy, such as Down syndrome, Edwards syndrome, and Patau syndrome. Furthermore, cell surface features of fetal cells can be used to identify any of a variety of diseases or conditions.

[0284] Biological samples can also include immune cells. Sequence analysis of the immune function of these cells, including genomics, proteomics, and cell surface characteristics, can provide a wealth of information to help understand the state and function of the immune system. For example, determining the status of minimal residual disease (MRD) (e.g., negative or positive) in patients with multiple myeloma (MM) after autologous stem cell transplantation is considered a predictor of MRD in MM patients (see, for example, U.S. Patent Application Publication No. 2018 / 0156784, which is incorporated herein by reference in its entirety).

[0285] Examples of immune cells in biological samples include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and helper T cells), natural killer cells, cytokine-induced killer (CIK) cells, myeloid cells such as granulocytes (basophils, eosinophils, neutrophils / multi-segmented neutrophils), monocytes / macrophages, mast cells, platelets / megakaryocytes, and dendritic cells.

[0286] Biological samples can include any number of macromolecules, such as cellular macromolecules and organelles (e.g., mitochondria and the nucleus). Biological samples can be nucleic acid samples and / or protein samples. Biological samples can be carbohydrate samples or lipid samples. Biological samples can be obtained as tissue samples, such as tissue sections, biopsies, core biopsies, needle aspiration, or fine-needle aspiration. Samples can be liquid samples, such as blood samples, urine samples, or saliva samples. Samples can be skin samples, colon samples, buccal swabs, histological samples, histopathological samples, plasma or serum samples, tumor samples, live cells, cultured cells, clinical samples, such as whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.

[0287] Cell-free biological samples may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from bodily samples such as blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0288] As described above, a biological sample may include a single analyte of interest or multiple analytes of interest. Methods for performing multiplex analyses to analyze two or more different analytes in a single biological sample are discussed later in this invention.

[0289] (ii) Preparation of biological samples

[0290] Various steps can be performed to prepare biological samples for analysis. Unless otherwise stated, the preparation steps described below can generally be combined in any way to appropriately prepare a specific sample for analysis.

[0291] (1) Tissue section

[0292] Biological samples can be obtained from a subject (e.g., via surgical biopsy, whole-subject section), grown in vitro as cell populations on a growth substrate or culture dish, or prepared as tissue sections or tissue slides. The grown samples can be thin enough to be analyzed without further processing steps. Alternatively, grown samples and samples obtained via biopsy or sectioning can be prepared into thin tissue sections using mechanical cutting equipment (e.g., a vibrating blade microtome). As another alternative, in some embodiments, thin tissue sections can be prepared by applying a biological sample tough imprint to a suitable substrate material.

[0293] The thickness of a tissue section can be a fraction of the largest cross-sectional size of the cells (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections thicker than the largest cross-sectional cell size can also be used. For example, cryogenic sections, such as 10–20 micrometers thick, can be used.

[0294] More generally, the thickness of tissue sections typically depends on the method used to prepare the sections and the physical properties of the tissue; therefore, sections with a variety of thicknesses can be prepared and used. For example, tissue section thicknesses can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 micrometers. Thicker sections, such as at least 70, 80, 90, or 100 micrometers or greater, can also be used if desired or convenient. Typically, tissue section thicknesses are between 1-100 micrometers, 1-50 micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1-15 micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6 micrometers; however, as mentioned above, sections with thicknesses greater than or less than these ranges can also be analyzed.

[0295] Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by sequentially slicing the biopsy sample using a slicing blade. In this way, spatial information between consecutive sections can be preserved, and sections can be analyzed sequentially to obtain three-dimensional information about the biological sample.

[0296] (2) Freezing

[0297] In some embodiments, biological samples (e.g., tissue sections as described above) can be prepared by deep freezing at temperatures suitable for maintaining or preserving the integrity (e.g., physical properties) of the tissue structure. For example, this temperature may be below -20°C, or below -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, or -200°C. Frozen tissue samples can be sliced ​​(e.g., thinly sliced) onto a substrate surface using any number of suitable methods. For example, tissue samples can be prepared using a cryostat (e.g., a cryostat) set at a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. For example, this temperature may be below -15°C, below -20°C, or below -25°C. Samples can be flash-frozen in isopentane and liquid nitrogen. Frozen samples can be stored in sealed containers before embedding.

[0298] (3) Formaldehyde fixation and paraffin embedding

[0299] In some embodiments, formaldehyde fixation and paraffin embedding (FFPE) can be used to prepare biological samples, which is an established method. In some embodiments, formaldehyde fixation and paraffin embedding can be used to prepare cell suspensions and other non-tissue samples. After fixing the sample and embedding it in paraffin or resin blocks, the sample can be sectioned as described above. Prior to analysis, the paraffin embedding material can be removed from the tissue sections by incubating them in a suitable solvent (e.g., xylene) followed by rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes) (e.g., deparaffining).

[0300] (4) Fix

[0301] As an alternative to formaldehyde fixation, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, samples can be fixed by immersion in ethanol, methanol, acetone, formaldehyde (e.g., 2% formaldehyde), paraformaldehyde-triton, glutaraldehyde, or combinations thereof.

[0302] In some implementations, acetone fixation is used with freshly frozen samples, which may include, but are not limited to, cortical tissue, mouse olfactory bulbs, human brain tumors, post-mortem human brains, and breast cancer samples. In some implementations, a compatible fixation method is selected and / or optimized based on the desired workflow. For example, formaldehyde fixation may be chosen to be compatible with workflows using IHC / IF protocols for protein visualization. As another example, methanol fixation may be chosen for workflows that prioritize RNA / DNA library quality. In some applications, acetone fixation may be chosen to permeabilize the tissue. When performing acetone fixation, a pre-permeabilization step (described below) may be omitted. Alternatively, acetone fixation may be combined with a permeabilization step.

[0303] (5) Embedding

[0304] As an alternative to paraffin embedding, biological samples can be embedded in any of a variety of other embedding materials to provide a substrate for the sample prior to sectioning and other processing steps. Generally, the embedding material is removed before analyzing tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylates), epoxy resins, and agar.

[0305] (6) Staining

[0306] To facilitate visualization, biological samples can be stained using a variety of staining agents and techniques. In some embodiments, any number of biological staining agents can be used to stain the samples, including but not limited to acridine orange, Bismarck brown, carmine, Coomassie blue, cresol purple, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Höller's stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or saffron.

[0307] Samples can be stained using known staining techniques, including Can-Grunwald staining, Giemsa staining, hematoxylin and eosin (H&E), Jenner's staining, Leishman staining, Masson's trichrome staining, Papanicolaou staining, Romanowsky staining, silver staining, Sudan staining, Wright's staining, and / or PAS staining. PAS staining is typically performed after fixation in formaldehyde or acetone.

[0308] In some embodiments, biological samples may be stained using detectable markers (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes) as described elsewhere herein. In some embodiments, biological samples are stained using only one type of staining agent or one technique. In some embodiments, staining includes biological staining techniques such as H&E staining. In some embodiments, staining includes identifying analytes using fluorescently conjugated antibodies. In some embodiments, biological samples are stained using two or more different types of staining agents or two or more different staining techniques. For example, a biological sample may be prepared by staining and imaging using one technique (e.g., H&E staining and bright-field imaging) and then staining and imaging the same biological sample using another technique (e.g., IHC / IF staining and fluorescence microscopy).

[0309] In some embodiments, biological samples can be destained. Methods for decontaminating or destaining biological samples are known in the art and generally depend on the nature of the staining agent applied to the sample. For example, H&E staining can be destained by washing the sample in HCl. In some embodiments, destaining may include one, two, three, or more washes in HCl. In some embodiments, destaining may include adding HCl to a downstream solution (e.g., a permeation solution). As another example, in some embodiments, one or more immunofluorescent staining agents are applied to the sample via antibody conjugation. These staining agents can be removed using techniques such as disulfide bond cleavage by washing with reducing agents and detergents, hygroscopic salt treatment, antigen recovery solution treatment, and acidic glycine buffer treatment. For example, methods for multiple staining and destaining are described in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6: 8390, Pirici et al., J. Histochem. Cytochem. 2009; 57: 567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57: 899-905, the entire contents of which are incorporated herein by reference.

[0310] (7) Hydrogel embedding

[0311] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue section) is embedded in a hydrogel. In some embodiments, hydrogel subunits are injected into a biological sample, and the polymerization of the hydrogel is initiated by external or internal stimuli. As described herein, a “hydrogel” can comprise a cross-linked 3D network of hydrophilic polymer chains. A “hydrogel subunit” can be a polymerizable (e.g., cross-linked) hydrophilic monomer, molecular precursor, or polymer that can form a three-dimensional (3D) hydrogel network.

[0312] Hydrogels can swell in the presence of water. In some embodiments, the hydrogel comprises natural materials. In some embodiments, the hydrogel comprises synthetic materials. In some embodiments, the hydrogel comprises hybrid materials, for example, the hydrogel material comprises components having both synthetic and natural polymers. Any material used in hydrogels or hydrogels containing peptide-based materials as described herein can be used. Encapsulating a sample in this manner typically involves contacting a biological sample with the hydrogel such that the biological sample is surrounded by the hydrogel. For example, the sample can be encapsulated by contacting the sample with a suitable polymeric material and activating the polymeric material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.

[0313] In some embodiments, biological samples are immobilized in a hydrogel by crosslinking the polymeric material forming the hydrogel. Crosslinking can be performed chemically and / or photochemically, or by any other hydrogel-forming method known in the art. For example, biological samples can be immobilized in a hydrogel by crosslinking with polyacrylamide. Furthermore, analytes from biological samples can be immobilized in a hydrogel by crosslinking (e.g., with polyacrylamide).

[0314] The composition of hydrogels and their application to biological samples generally depend on the nature and preparation of the biological sample (e.g., sliced, unsliced, fresh or frozen tissue, type of fixation). A hydrogel can be any suitable hydrogel in which the biological sample becomes anchored or embedded within the hydrogel after it has been formed on the sample. Non-limiting examples of hydrogels are described herein or are known in the art. As an example, where the biological sample is a tissue slice, the hydrogel may comprise a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) promoter solution. As another example, when the biological sample consists of cells (e.g., cultured cells or cells isolated from a tissue sample), the cells may be incubated with both the monomer solution and the APS / TEMED solution. For cells, the hydrogel forms in compartments, including but not limited to devices for culturing, maintaining, or transporting cells. For example, the hydrogel may be formed using a monomer solution plus APS / TEMED, added to the compartments to a depth between about 0.1 μm and about 5 mm.

[0315] In some embodiments, the hydrogel includes a connector that allows for the anchoring of biological samples to the hydrogel. In some embodiments, the hydrogel includes a connector that allows for the anchoring of bioanalytes to the hydrogel. In this case, the connector can be added to the hydrogel before, during, or after hydrogel formation. Non-limiting examples of connectors for anchoring nucleic acids to hydrogels may include 6-((acryloyl)amino)hexanoic acid (acryloyl-XSE) (available from Thermo Fisher Scientific, Waltham, MA), Label-IT amine (available from MirusBio, Madison, Wisconsin), and Label X.

[0316] In some embodiments, functionalizing chemistry may be used. In some embodiments, functionalizing chemistry includes hydrogel tissue chemistry (HTC). Any hydrogel tissue framework suitable for HTC (e.g., synthetic or natural) can be used to anchor biomolecules and modulate functionalization. Non-limiting examples of methods using HTC framework variants include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, hydrogel formation within the biological sample is permanent. For example, biomolecules can permanently adhere to the hydrogel, enabling multi-round interrogation. In some embodiments, hydrogel formation within the biological sample is reversible.

[0317] In some embodiments, other reagents are added to the hydrogel subunits before, during, and / or after polymerization. These other reagents may include, but are not limited to, oligonucleotides (e.g., capture probes), endonucleases for DNA fragmentation, DNA fragmentation buffers, DNA polymerases, and dNTPs for amplifying nucleic acids and attaching barcodes to the amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, transposases, ligases, proteinase K, and DNases. Other reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers, and converting oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, during, and / or after polymerization.

[0318] In some embodiments, HTC reagent is added to the hydrogel before, during, and / or after polymerization. In some embodiments, cell spiking agents are added to the hydrogel before, during, and / or after polymerization. In some embodiments, cell penetrating agents are added to the hydrogel before, during, and / or after polymerization.

[0319] In some embodiments, biological samples are embedded in a hydrogel to facilitate sample transfer to another location (e.g., to an array). For example, archived biological samples (e.g., FFPE tissue sections) can be transferred from storage to a spatial array for spatial analysis. In some embodiments, biological samples on a substrate can be covered with any prepolymer solution described herein. In some embodiments, the prepolymer solution can be polymerized to form a hydrogel above and / or around the biological sample. Hydrogel formation can occur in a manner sufficient to anchor (e.g., embed) the biological sample into the hydrogel. After hydrogel formation, the biological sample is anchored (e.g., embedded) into the hydrogel, wherein separating the hydrogel from the substrate (e.g., a glass slide) causes the biological sample to separate from the substrate along with the hydrogel. The biological sample contained in the hydrogel can then be contacted with the spatial array, and spatial analysis of the biological sample can be performed.

[0320] Any kind of characteristic can determine the transfer conditions required for a given biological sample. Non-limiting examples of characteristics that may affect transfer conditions include the sample (e.g., thickness, fixation, and cross-linking) and / or the analyte of interest (different conditions for preserving and / or transferring different analytes (e.g., DNA, RNA, and proteins)).

[0321] In some implementations, the hydrogel is removed after the biological sample has been in contact with the spatial array. For example, the methods described herein may include event-dependent (e.g., light- or chemically) depolymerizing hydrogels, wherein the hydrogel depolymerizes upon application of an event (e.g., an external stimulus). In one example, the biological sample may be anchored to a DTT-sensitive hydrogel, wherein the addition of DTT causes the hydrogel to depolymerize and release the anchored biological sample.

[0322] Hydrogels embedded in biological samples can be removed by any suitable method. For example, electrophoretic tissue removal methods can be used to remove biomacromolecules from hydrogel-embedded samples. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixation medium, methylcellulose, or other semi-solid medium) before or after hydrogel removal.

[0323] In some embodiments, the hydrogel chemistry can be tuned to specifically bind (e.g., retain) specific types of analytes (e.g., RNA, DNA, proteins, etc.). In some embodiments, the hydrogel includes connectors that allow biological samples to be anchored to the hydrogel. In some embodiments, the hydrogel includes connectors that allow biological analytes to be anchored to the hydrogel. In this case, the connectors can be added to the hydrogel before, during, or after hydrogel formation. Non-limiting examples of connectors for anchoring nucleic acids to the hydrogel may include 6-((acryloyl)amino)hexanoic acid (acryloyl-X SE), Label-IT amine, and Label X. Non-limiting examples of characteristics that may affect transfer conditions include the sample (e.g., thickness, fixation, and crosslinking) and / or the analyte of interest (different conditions for preserving and / or transferring different analytes (e.g., DNA, RNA, and proteins)).

[0324] Other methods and aspects of hydrogel embedding of biological samples are described, for example, in Chen et al., Science 347(6221): 543-548, 2015, the entire contents of which are incorporated herein by reference.

[0325] (8) Transfer of biological samples

[0326] In some embodiments, a hydrogel is used to transfer a biological sample immobilized on a substrate (e.g., a biological sample prepared using methanol fixation or formalin fixation and paraffin embedding (FFPE)) to a spatial array. In some embodiments, the hydrogel is formed over the biological sample on a substrate (e.g., a glass slide). For example, hydrogel formation may occur in a manner sufficient to anchor (e.g., embed) the biological sample into the hydrogel. After hydrogel formation, the biological sample is anchored to (e.g., embedded into) the hydrogel, wherein separating the hydrogel from the substrate causes the biological sample to separate from the substrate along with the hydrogel. The biological sample can then be contacted with the spatial array, thereby allowing spatial analysis of the biological sample. In some embodiments, the hydrogel is removed after the biological sample has been contacted with the spatial array. For example, the methods described herein may include event-dependent (e.g., photo- or chemically) depolymerizing hydrogels, wherein the hydrogel depolymerizes upon application of an event (e.g., an external stimulus). In one example, the biological sample may be anchored on a DTT-sensitive hydrogel, wherein the addition of DTT causes the hydrogel to depolymerize and release the anchored biological sample. The hydrogel can be any suitable hydrogel in which the biological sample becomes anchored or embedded in the hydrogel after it has formed on the sample. Non-limiting examples of hydrogels are described herein or are known in the art. In some embodiments, the hydrogel includes a connector that allows the biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel includes a connector that allows the bioanalyte to be anchored to the hydrogel. In this case, the connector can be added to the hydrogel before, during, or after its formation. Non-limiting examples of connectors for anchoring nucleic acids to a hydrogel may include 6-((acryloyl)amino)hexanoic acid (acryloyl-XSE) (available from Thermo Fisher Scientific, Waltham, MA), Label-IT amine (available from MirusBio, Madison, Wisconsin), and Label X. Any kind of property can determine the transfer conditions required for a given biological sample. Non-limiting examples of characteristics that may affect transfer conditions include the sample (e.g., thickness, fixation, and crosslinking) and / or the analyte of interest (different conditions for preserving and / or transferring different analytes (e.g., DNA, RNA, and proteins)). In some embodiments, hydrogel formation can occur in a manner sufficient to anchor (e.g., embed) the analyte in the biological sample into the hydrogel. In some embodiments, the hydrogel can indent (e.g., shrink) along with the anchored analyte present in the biological sample (e.g., embedded in the hydrogel). In some embodiments, the hydrogel can expand (e.g., expand isometrically) along with the anchored analyte present in the biological sample (e.g., embedded in the hydrogel). In some embodiments, the hydrogel can be indented (e.g., shrink) and subsequently expand along with the anchored analyte present in the biological sample (e.g., embedded in the hydrogel).

[0327] (9) Isometric Expansion

[0328] In some implementations, biological samples embedded in hydrogels can be expanded isometrically. Available isometric expansion methods include hydration, a preparative step in expanded microscopy, as described in the following references: Chen et al., Science 347(6221): 543-548, 2015; Asano et al. Current Protocols. 2018, 80: 1, doi: 10.1002 / cpcb.56; and Gao et al. BMC Biology. 2017, 15: 50, doi: 10.1186 / s12915-017-0393-3; and Wassie, AT et al., Expandation microscopy: principles and uses in biological research, Nature Methods, 16(1): 33-41 (2018), each incorporated herein by reference in its entirety.

[0329] Generally, the steps used for isometric expansion of biological samples can depend on the characteristics of the sample (e.g., the thickness of the tissue section, fixation, cross-linking) and / or the analyte of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to the gel).

[0330] Isometric expansion can be achieved by anchoring one or more components of a biological sample to a gel, followed by gel formation, protein hydrolysis, and swelling. Isometric expansion of the biological sample can occur before or after the biological sample is fixed to the substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate before contacting the expanded biological sample with a spatial barcode array (e.g., a spatially barcode-coded capture probe on the substrate).

[0331] In some embodiments, proteins in a biological sample are anchored to a swellable gel (e.g., a polyelectrolyte gel). Antibodies can be directed to proteins before, after, or during anchoring to the swellable gel. DNA and / or RNA in a biological sample can also be anchored to the swellable gel via suitable adapters. Examples of such adapters include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X-SE) (available from Thermo Fisher Scientific, Waltham, MA), Label ITamine (available from Mirus Bio, Madison, Wisconsin), and Label X (described, for example, in Chen et al., Nat. Methods 13:679-684, 2016, the entire contents of which are incorporated herein by reference).

[0332] Isometric scaling of a sample can improve the spatial resolution of subsequent analyses. For example, isometric scaling of biological samples can lead to increased resolution in spatial analyses (e.g., single-cell analyses). The increased resolution in a spatial profile can be determined by comparing isometrically scaled samples with non-isometrically scaled samples.

[0333] In some embodiments, the biological sample is isometrically extended to at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x of its non-extended amount. In some embodiments, the sample is isometrically extended to at least 2 times and less than 20 times its non-extended amount.

[0334] In some embodiments, the biological sample embedded in the hydrogel is equidistantly extended to at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x of its non-extended amount. In some embodiments, the biological sample embedded in the hydrogel is equidistantly extended to at least 2x and less than 20x of its non-extended amount.

[0335] (10) Substrate attachment

[0336] In some embodiments, biological samples may be attached to a hydrogel substrate. Examples of substrates suitable for this purpose are described in detail below. The attachment of biological samples can be irreversible or reversible, depending on the nature of the sample and subsequent steps in the analytical method.

[0337] In some embodiments, the sample can be reversibly attached to the substrate by applying a suitable polymer coating to the substrate and bringing the sample into contact with the polymer coating. The sample can then be separated from the substrate using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are an example of polymers suitable for this purpose.

[0338] More generally, in some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate sample adhesion to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0339] (11) Non-aggregated cells

[0340] In some implementations, the biological sample corresponds to cells (e.g., from cell cultures or tissue samples). In a cell sample containing multiple cells, the multiple individual cells may naturally not aggregate. For example, the cells may be derived from cell suspensions and / or isolated or deaggregated cells from tissues or tissue sections.

[0341] Alternatively, the cells in the sample may aggregate and can be deaggregated into multiple individual cells using techniques such as enzymes or mechanical methods. Examples of enzymes used for enzymatic deaggregation include, but are not limited to, dispersases, collagenases, trypsin, or combinations thereof. Mechanical deaggregation can be performed, for example, using a tissue homogenizer.

[0342] In some embodiments involving non-aggregated or deaggregated cells, the cells are distributed on a substrate such that at least one cell occupies a different spatial feature on the substrate. The cells may be immobilized on the substrate (e.g., to prevent lateral cell diffusion). In some embodiments, a cell immobilizer may be used to immobilize non-aggregated or deaggregated samples on a spatially barcoded array prior to analyte capture. "Cell immobilizer" may refer to an antibody attached to the substrate that can bind to cell surface markers. In some embodiments, the distribution of multiple cells on the substrate follows Poisson statistics.

[0343] In some embodiments, cells from multiple cell types are fixed to a substrate. In some embodiments, the cells are fixed to prevent lateral diffusion, for example by adding hydrogel and / or by applying an electric field.

[0344] (12) Suspension and adherent cells

[0345] In some embodiments, the biological sample may be obtained from an in vitro grown cell culture. The sample from the cell culture may include one or more suspension cells that are anchored independently within the cell culture. Examples of such cells include, but are not limited to, hematopoietic cells and cell lines derived from the following cell lines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI-H322M, and MALME-3M.

[0346] Samples from cell cultures may include one or more adherent cells grown on the surface of a container containing culture medium. Non-limiting examples of adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical carcinoma) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myeloid leukemia) cells, LNCaP (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells, and SH-SY5Y (neuroblastoma, cloned from myeloma) cells. Cells, including T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, National Cancer Institute 60 Cancer Cell Line Group (NCI60), Vero (African green monkey kidney epithelial cell line) cells, MC3T3 (embryonic skull) cells, GH3 (pituitary adenoma) cells, PC12 (pheochromocytoma) cells, canine MDCK kidney epithelial cells, Xenopus A6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insect epithelial cells.

[0347] Other examples of adherent cells are shown in Table 1 and are categorized in, for example, “A Catalog of in Vitro Cell Lines, Transplantable Animal and Human Tumors and Yeast”, Division of Diagnostic and Therapeutic Use of Cancer, National Cancer Institute (DCTD) (2013) and “The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology”, Cancer Research 73(14):4372-82, 2013, the full contents of which are incorporated herein by reference.

[0348] Table 1: Examples of adherent cells

[0349]

[0350]

[0351] In some embodiments, the adherent cells are cells corresponding to one or more of the following cell lines: BT549, HS578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31, A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOX. IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1, U87, vero, MC3T3, GH3, PC12, canine MDCK renal epithelium, Xenopus A6 renal epithelium, zebrafish AB9, and Sf9 insect epithelial cell lines.

[0352] (13) Tissue permeability

[0353] In some implementations, biological samples may be permeabilized to facilitate the transfer of analytes from the sample and / or to facilitate the transfer of substances (e.g., capture probes) into the sample. If the sample is not permeabilized sufficiently, the amount of analyte captured from the sample may be too low for adequate analysis. Conversely, if the tissue sample is too permeabilized, the relative spatial relationships of analytes within the tissue sample may be lost. Therefore, a balance is ideal between adequately permeating the tissue sample to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution within the sample.

[0354] Generally, biological samples can be permeated by exposing them to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), crosslinking agents (e.g., paraformaldehyde), and detergents (e.g., saponins, Triton X-100). TMOr Tween-20 TM The detergent is an anionic detergent (e.g., SDS or sodium lauryl sarcosinate solution). In some embodiments, the detergent is permeabilized using any of the methods described herein (e.g., using any detergent described herein, such as SDS and / or sodium lauryl sarcosinate solution) before or after enzyme treatment (e.g., treatment with any enzyme described herein, such as trypsin, protease (e.g., pepsin and / or proteinase K)).

[0355] In some embodiments, biological samples can be permeated by exposing the sample to greater than about 1.0 w / v% (e.g., greater than about 2.0 w / v%, greater than about 3.0 w / v%, greater than about 4.0 w / v%, greater than about 5.0 w / v%, greater than about 6.0 w / v%, greater than about 7.0 w / v%, greater than about 8.0 w / v%, greater than about 9.0 w / v%, greater than about 10.0 w / v%, greater than about 11.0 w / v%, greater than about 12.0 w / v%, or greater than about 13.0 w / v%) of sodium dodecyl sulfate (SDS) and / or N-lauroyl sarcosine or sodium N-lauroyl sarcosine. In some embodiments, the biological sample may be permeated in such a manner as to expose the sample to about 1.0 w / v% to about 14.0 w / v% (e.g., about 2.0 w / v% to about 14.0 w / v%, about 2.0 w / v% to about 12.0 w / v%, about 2.0 w / v% to about 10.0 w / v%, about 4.0 w / v% to about 14.0 w / v%, about 4.0 w / v% to about 14.0 w / v%, about 4.0 w / v% to about 12.0 w / v%, about 2.0 w / v% to about 10.0 w / v%, about 4.0 w / v% to about 14.0 w / v%, about 4.0 w / v% to about 12.0 w / v%). SDS and / or N-lauroyl sarcosinate solution and / or proteinase K (e.g., for about 5 minutes to about 1 hour, about 5 minutes to about 40 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, or about 5 minutes to about 10 minutes) of about 4.0 w / v% to about 10.0 w / v%, about 6.0 w / v% to about 14.0 w / v%, about 6.0 w / v% to about 12.0 w / v%, about 6.0 w / v% to about 10.0 w / v%, about 8.0 w / v% to about 14.0 w / v%, about 8.0 w / v% to about 12.0 w / v%, about 8.0 w / v% to about 10.0 w / v%, about 10.0% w / v% to about 14.0 w / v%, about 10.0% w / v% to about 12.0 w / v%, or about 12.0 w / v% to about 14.0 w / v%).

[0356] In some implementations, biological samples may be incubated with a permeabilizing agent to promote sample permeation. For example, other methods of sample permeation are described in Jamur et al., Method Mol. Biol. 588: 63-66, 2010, the entire contents of which are incorporated herein by reference.

[0357] lysis reagent

[0358] In some implementations, biological samples can be permeated by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive reagents such as lysins for lysing different cell types, such as Gram-positive or Gram-negative bacteria, plants, yeast, mammals, such as lysozyme, colorless peptidase, lysostaphin, Labiase, kitalase, cytolysin, and a variety of other commercially available lysins.

[0359] Other lysis agents may be added to biological samples, either additionally or alternatively, to facilitate permeation. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions may include ionic surfactants such as creatine amide and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents may include, but are not limited to, organic solvents, chelating agents, detergents, surfactants, and dissociation agents.

[0360] In some embodiments, biological samples can be permeated using non-chemical permeation methods. Non-chemical permeation methods are known in the art. For example, usable non-chemical permeation methods include, but are not limited to, physical dissolution techniques such as electroporation, mechanical permeation methods (e.g., bead milling to mechanically disrupt the tissue structure of the sample using a homogenizer and grinding balls), acoustic permeation (e.g., ultrasound), and thermal decomposition techniques, such as heating to induce thermal permeation of the sample.

[0361] protease

[0362] In some embodiments, the culture medium, solution, or permeation solution may contain one or more proteases. In some embodiments, treatment of biological samples with a histone-degrading protease may result in the production of fragmented genomic DNA. The same capture domain used for capturing mRNA (e.g., a capture domain having a poly(T) sequence) can be used to capture the fragmented genomic DNA. In some embodiments, biological samples are treated with a histone-degrading protease and an RNA protectant prior to spatial profiling analysis to facilitate the capture of genomic DNA and mRNA.

[0363] In some embodiments, a biological sample is permeated by exposing it to a protease capable of degrading histones. As used herein, the term "histone" generally refers to adaptor histones (e.g., H1) and / or core histones (e.g., H2A, H2B, H3, and H4). In some embodiments, the protease can degrade adaptor histones, core histones, or both. Any suitable protease capable of degrading histones in a biological sample can be used. Non-limiting examples of proteases capable of degrading histones include proteases inhibited by leuprorein and TLCK (toluenesulfonyl-L-lysyl-chloromethane hydrochloride), proteases encoded by the EUO gene from Chlamydia trachomatis serovar A, granzyme A, serine proteases (e.g., trypsin or trypsin-like proteases, neutral serine proteases, elastase, cathepsin G), aspartic proteases (e.g., cathepsin D), peptidase family C1 enzymes (e.g., cathepsin L), pepsin, proteinase K, proteases inhibited by the diazomethane inhibitor Z-Phe-Phe-CHN(2) or the epoxide inhibitor E-64, lysosomal proteases or azophiles (e.g., cathepsin G, elastase, proteinase 3, neutral serine proteases).In some embodiments, the serine protease is trypsin, trypsin-like enzyme, or a functional variant or derivative thereof (e.g., P00761; C0HK48; Q8IYP2; Q8BW11; Q6IE06; P35035; P00760; P06871; Q90627; P16049; P07477; P00762; P35031; P19799; P350). 36; Q29463; P06872; Q90628; P07478; P07146; P00763; P35032; P70059; P29786; P3503 7; Q90629; P35030; P08426; P35033; P35038; P12788; P29787; P35039; P35040; Q8NHM4; P35041;P35043;P35044;P54624;P04814;P35045;P32821;P54625;P35004;P35046;P 32822;P35047;C0HKA5;C0HKA2;P54627;P35005;C0HKA6;C0HKA3;P52905;P83348;P00 765; P35042; P81071; P35049; P51588; P35050; P35034; P35051; P24664; P35048; P00764; P00775; P54628; P42278; P54629; P42279; Q91041; P54630; P42280; COHKA4), or combinations thereof. In some embodiments, the trypsin is P00761, P00760, Q29463, or combinations thereof. In some embodiments, the protease capable of degrading one or more histones comprises an amino acid sequence having at least 80% sequence identity with P00761, P00760, or Q29463. In some embodiments, the protease capable of degrading one or more histones comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with P00761, P00760, or Q29463. A protease is considered a functional variant if it has at least 50% of the activity of a protease under optimal conditions, for example, at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of that of a protease under optimal conditions.In some embodiments, enzymatic treatment with pepsin or pepsin-like enzymes may include: P03954 / PEPA1_MACFU; P28712 / PEPA1_RABIT; P27677 / PEPA2_MACFU; P27821 / PEPA2_RABIT; P0DJD8 / PEPA3_HUMAN; P27822 / PEPA3_RABIT; P0DJD7 / PEPA4_HUMAN; P27678 / PEPA4_MACFU; P28713 / PEPA4_RABIT; P0DJD9 / PEPA5_HUMAN; Q9D106 / PEPA5_MOUSE; P27823 / PEPAF_RABIT; P00792 / PEPA_BOVIN; Q9N2D4 / PEPA_CALJA; Q9GMY6 / PEPA_CANLF; P00793 / PEPA_CHICK; P11489 / PEPA_MACMU; P00791 / PEPA_PIG; Q9GMY7 / PEPA_RHIFE; Q9GMY8 / PEPA_SORUN; P81497 / PEPA_SUNMU; P13636 / PEPA_URSTH and their functional variants and derivatives, or combinations thereof. In some embodiments, pepsin may include: P00791 / PEPA_PIG; P00792 / PEPA_BOVIN, functional variants, derivatives, or combinations thereof.

[0364] In addition, the protease may be included in the reaction mixture (solution), which also includes other components (e.g., buffers, salts, chelating agents (e.g., EDTA), and / or detergents (e.g., SDS, N-lauroyl sarcosinate sodium solution)). The reaction mixture may be buffered and have a pH of about 6.5-8.5, for example, about 7.0-8.0. Furthermore, the reaction mixture can be used at any suitable temperature, for example, about 10 to 50°C, such as about 10 to 44°C, 11 to 43°C, 12 to 42°C, 13 to 41°C, 14 to 40°C, 15 to 39°C, 16 to 38°C, 17 to 37°C, such as about 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 33°C, 35°C, or 37°C, preferably about 35 to 45°C, for example, about 37°C.

[0365] Other reagents

[0366] In some embodiments, the permeation solution may contain other reagents or the biological sample may be treated with other reagents to optimize permeation of the biological sample. In some embodiments, the other reagents are RNA protectants. As used herein, the term "RNA protectant" generally refers to a reagent that protects RNA from RNA nucleases (e.g., RNases). Any suitable RNA protectant that protects RNA from degradation can be used. Non-limiting examples of RNA protectants include organic solvents (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% v / v organic solvents), including but not limited to ethanol, methanol, propanol, acetone, trichloroacetic acid, propanol, polyethylene glycol, acetic acid, or combinations thereof. In some embodiments, the RNA protectant includes ethanol, methanol, and / or propanol, or combinations thereof. In some embodiments, the RNA protectant includes RNAlater ICE (Thermo Fisher Scientific). In some embodiments, the RNA protectant contains at least about 60% ethanol. In some embodiments, the RNA protectant comprises about 60-95% ethanol, about 0-35% methanol, and about 0-35% propan-2-ol, wherein the total amount of organic solvents in the culture medium does not exceed about 95%. In some embodiments, the RNA protectant comprises about 60-95% ethanol, about 5-20% methanol, and about 5-20% propan-2-ol, wherein the total amount of organic solvents in the culture medium does not exceed about 95%.

[0367] In some embodiments, the RNA protectant comprises a salt. The salt may include ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron(II) sulfate, chromium(III) sulfate, cobalt(II) sulfate, copper(II) sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, potassium chloride, potassium sulfate, sodium chloride, sodium acetate, sodium sulfate, zinc chloride, zinc acetate, and zinc sulfate. In some embodiments, the salt is a sulfate, such as ammonium sulfate, ammonium bisulfate, cesium sulfate, cadmium sulfate, cesium iron(II) sulfate, chromium(III) sulfate, cobalt(II) sulfate, copper(II) sulfate, lithium sulfate, magnesium sulfate, manganese sulfate, potassium sulfate, sodium sulfate, or zinc sulfate. In some embodiments, the salt is ammonium sulfate. Salt may be present at a concentration of about 20 g / 100 mL of culture medium or lower, such as about 15 g / 100 mL, 10 g / 100 mL, 9 g / 100 mL, 8 g / 100 mL, 7 g / 100 mL, 6 g / 100 mL, 5 g / 100 mL or lower, such as about 4 g, 3 g, 2 g or 1 g / 100 mL.

[0368] In addition, the RNA protectant may be included in a culture medium that also contains a chelating agent (e.g., EDTA), a buffer (e.g., sodium citrate, sodium acetate, potassium citrate or potassium acetate, preferably sodium acetate) and / or buffered to a pH between about 4 and 8 (e.g., about 5).

[0369] In some embodiments, the biological sample is treated with one or more RNA protectants before, during, or after permeabilization. For example, the biological sample is treated with one or more RNA protectants before treatment with one or more permeabilization agents (e.g., one or more proteases). In another example, the biological sample is treated with a solution containing one or more RNA protectants and one or more permeabilization agents (e.g., one or more proteases). In yet another example, the biological sample is treated with one or more RNA protectants after it has been treated with one or more permeabilization agents (e.g., one or more proteases). In some embodiments, the biological sample is treated with one or more RNA protectants before fixation.

[0370] In some embodiments, identifying the location of a captured analyte in a biological sample includes a nucleic acid extension reaction. In some embodiments where the capture probe captures fragmented genomic DNA molecules, the nucleic acid extension reaction includes a DNA polymerase. For example, the nucleic acid extension reaction includes using a DNA polymerase to extend a capture probe that hybridizes to the captured analyte (e.g., fragmented genomic DNA) using the captured analyte (e.g., fragmented genomic DNA) as a template. The product of the extension reaction includes a spatially barcoded analyte (e.g., spatially barcoded fragmented genomic DNA). The spatially barcoded analyte (e.g., spatially barcoded fragmented genomic DNA) can be used to identify the spatial location of the analyte in a biological sample. Any DNA polymerase capable of using a captured analyte as a template to extend a capture probe can be used in the methods described herein. Non-limiting examples of DNA polymerases include T7 DNA polymerase; Bsu DNA polymerase; and E. coli DNA polymerase pol I.

[0371] Anti-diffusion medium

[0372] In some embodiments, the antidiffusion medium, typically used to limit analyte diffusion, may include at least one permeation reagent. For example, the antidiffusion medium (e.g., a hydrogel) may include pores (e.g., micropores, nanopores, or lenticels or pores) containing a permeation buffer or reagent. In some embodiments, the antidiffusion medium (e.g., the hydrogel) is soaked in a permeation buffer before the hydrogel is contacted with a sample. In some embodiments, when the antidiffusion medium is applied to a biological sample, the hydrogel or other antidiffusion medium may contain a drying reagent or monomer to deliver the permeation reagent. In some embodiments, the antidiffusion medium (e.g., the hydrogel) is covalently attached to a solid substrate (e.g., an acrylic glass slide).

[0373] In some embodiments, the hydrogel can be modified to both deliver a permeation reagent and contain a trap. For example, the hydrogel membrane can be modified to include a spatially barcoded trap probe. The spatially barcoded hydrogel membrane is then immersed in a permeation buffer before being contacted with the sample. In another example, the hydrogel can be modified to include a spatially barcoded trap probe and is designed to function as a porous membrane (e.g., a permeable hydrogel) when exposed to a permeation buffer or any other biological sample preparation reagent. The permeation reagent diffuses through the spatially barcoded permeable hydrogel and permeates the biological sample on the other side of the hydrogel. After exposure to the permeation reagent, the analyte subsequently diffuses into the spatially barcoded hydrogel. In this case, the spatially barcoded hydrogel (e.g., a porous membrane) facilitates the diffusion of bioanalytes from the biological sample into the hydrogel. In some embodiments, the bioanalyte diffuses into the hydrogel first and is then exposed to the permeation reagent (e.g., when secreted analytes are present outside the biological sample, or when the biological sample has been otherwise dissolved or permeated before the permeation reagent is added). In some embodiments, the permeation reagent flows through the hydrogel at a variable flow rate (e.g., any flow rate that promotes diffusion of the permeation reagent through the spatially barcoded hydrogel). In some embodiments, the permeation reagent flows through microfluidic chambers or channels on the spatially barcoded hydrogel. In some embodiments, after introducing the permeation reagent into the biological sample using flow, a biological sample preparation reagent can be flowed through the hydrogel to further promote the diffusion of the bioanalyte into the spatially barcoded hydrogel. Thus, the spatially barcoded hydrogel membrane delivers the permeation reagent to the sample surface in contact with the spatially barcoded hydrogel, enhancing analyte migration and capture. In some embodiments, the spatially barcoded hydrogel is applied to the sample and placed in a large volume of permeation solution. In some embodiments, a hydrogel membrane immersed in the permeation reagent is sandwiched between the sample and the spatially barcoded array. In some embodiments, the target analyte is capable of diffusing through the permeation reagent-immersed hydrogel and hybridizing or binding a capture probe on the other side of the hydrogel. In some embodiments, the thickness of the hydrogel is proportional to the resolution loss. In some embodiments, the pores (e.g., micropores, nanopores, or picometer pores) may contain spatially barcoded capture probes and permeation reagents and / or buffers. In some embodiments, the spatially barcoded capture probes and permeation reagents are held between spacers. In some embodiments, the sample is punched, cut, or transferred into the pores, wherein the target analyte diffuses through the permeation reagent / buffer to the spatially barcoded capture probes. In some embodiments, the resolution loss may be proportional to the gap thickness (e.g., the amount of permeation buffer between the sample and the capture probe). In some embodiments, the thickness of the anti-diffusion medium (e.g., hydrogel) is between about 50 and 500 micrometers, including thicknesses of 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 micrometers, or any thickness within 50 and 500 micrometers.

[0374] In some embodiments, the biological sample is exposed to a porous membrane (e.g., a permeable hydrogel) to aid permeation and limit diffusion loss of analytes while allowing permeation reagents to reach the sample. Membrane chemistry and pore volume can be controlled to minimize analyte loss. In some embodiments, the porous membrane may be made of glass, silicon, paper, hydrogel, polymer monolith, or other materials. In some embodiments, the material may be naturally porous. In some embodiments, the material may have pores or holes etched into a solid material. In some embodiments, the permeation reagent flows through microfluidic chambers or channels on the porous membrane. In some embodiments, flow control measures the sample's accessibility to the permeation reagent. In some embodiments, the porous membrane is a permeable hydrogel. For example, the hydrogel is permeable when the permeation reagent and / or biological sample preparation reagent can diffuse through the hydrogel. Any suitable permeation reagent and / or biological sample preparation reagent described herein can be used under conditions sufficient to release analytes (e.g., nucleic acids, proteins, metabolites, lipids, etc.) from the biological sample. In some embodiments, the hydrogel is exposed to the biological sample on one side and to the permeation reagent on the other side. The permeabilizing agent diffuses through a permeable hydrogel and permeates the biological sample on the other side of the hydrogel. In some embodiments, the permeabilizing agent is allowed to flow through the hydrogel at a variable flow rate (e.g., any flow rate that promotes diffusion of the permeabilizing agent through the hydrogel). In some embodiments, the permeabilizing agent flows through a microfluidic chamber or channel above the hydrogel. The operation of allowing the permeabilizing agent to flow through the hydrogel allows control over the concentration of the agent. In some embodiments, the hydrogel chemistry and pore volume can be adjusted to enhance permeation and limit the loss of diffused analytes.

[0375] In some embodiments, a porous membrane is sandwiched between a spatial barcode array and a sample, wherein a permeation solution is applied to the porous membrane. The permeation reagent diffuses through the membrane pores into the biological sample. In some embodiments, the biological sample may be placed on a substrate (e.g., a glass slide). The bioanalyte then diffuses through the porous membrane and into the space containing the capture probe. In some embodiments, the porous membrane is modified to include the capture probe. For example, the capture probe can be attached to the surface of the porous membrane using any of the methods described herein. In another example, the capture probe may be embedded in the porous membrane at any depth that allows interaction with the bioanalyte. In some embodiments, the porous membrane is placed on the biological sample in a configuration that allows interaction between the capture probe on the porous membrane and the bioanalyte from the biological sample. For example, the capture probe is located on the side of the porous membrane closer to the biological sample. In this case, the permeation reagent on the other side of the porous membrane diffuses through the porous membrane to the location containing the biological sample and the capture probe to facilitate permeation of the biological sample (e.g., also facilitates capture of the bioanalyte by the capture probe). In some embodiments, the porous membrane is located between the sample and the capture probe. In some implementations, the permeation reagent flows through microfluidic chambers or channels on the porous membrane.

[0376] Selective permeation / selective lysis

[0377] In some embodiments, biological samples may be processed according to established methods to selectively release analytes from subcellular regions of cells. In some embodiments, the methods provided herein may include detecting at least one bioanalyte present in subcellular regions of cells within a biological sample. As used herein, "subcellular region" may refer to any subcellular region. For example, a subcellular region may refer to the cytoplasm, mitochondria, nucleus, nucleolus, endoplasmic reticulum, lysosomes, vesicles, Golgi apparatus, plastids, vacuoles, ribosomes, cytoskeleton, or combinations thereof. In some embodiments, a subcellular region comprises at least one of cytosol, nucleus, mitochondria, and microsomes. In some embodiments, the subcellular region is the cytosol. In some embodiments, the subcellular region is the nucleus. In some embodiments, the subcellular region is the mitochondria. In some embodiments, the subcellular region is the microsome.

[0378] For example, bioanalytes can be selectively released from subcellular regions of a cell through selective permeabilization or selective lysis. In some embodiments, "selective permeabilization" can refer to a permeabilization method that can permeate a membrane into a subcellular region while maintaining the different subcellular regions substantially intact (e.g., the bioanalyte is not released from the subcellular region due to the applied permeabilization method). Non-limiting examples of selective permeabilization methods include the use of electrophoresis and / or the application of permeabilization reagents. In some embodiments, "selective lysis" can refer to a lysis method that can lyse a membrane into a subcellular region while maintaining the different subcellular regions substantially intact (e.g., the bioanalyte is not released from the subcellular region due to the applied lysis method). Several methods for selective permeation or lysis are known to those skilled in the art, including those described below: Lu et al. Lab Chip. Jan 2005; 5(1): 23-9; Niklas et al. Anal Biochem. Sep 15 2011; 416(2): 218-27; Cox and Emili. NatProtoc. 2006; 1(4): 1872-8; Chiang et al. J Biochem. Biophys. Methods. Nov 20 2000; 46(1-2): 53-68; and Yamauchi and Herr et al. Microsyst. Nanoeng. 2017; 3. pii: 16079; each of which is incorporated herein by reference in its entirety.

[0379] In some implementations, "selective permeabilization" or "selective lysis" refers to the selective permeabilization or selective lysis of a specific cell type. For example, "selective permeabilization" or "selective lysis" can refer to lysing one cell type while maintaining the different cell types substantially intact (e.g., the bioanalyte is not released from the cells due to the applied permeabilization or lysis method). Cells of a "different cell type" from another cell can refer to cells from different taxa, prokaryotic cells versus eukaryotic cells, cells from different tissue types, etc. Many methods for selectively permeabilizing or lysing different cell types are known to those skilled in the art. Non-limiting examples include the application of permeabilizing agents, electroporation, and / or sonication. See, for example, International Application No. WO 2012 / 168003; Han et al., Microsyst Nanoeng. June 17, 2019; 5:30; Gould et al. Oncotarget. March 20, 2018; 9(21):15606-15615; Oren and Shai. Biochemistry. February 18, 1997; 36(7):1826-35; Algayer et al. Molecules. May 31, 2019; 24(11).pii:E2079; Hipp et al. Leukemia. October 2017; 31(10):2278; International Application No. WO 2012 / 168003; and U.S. Patent No. 7,785,869; all of which are incorporated herein by reference in their entirety.

[0380] In some implementations, applying a selective permeabilizing or lysis reagent involves contacting a biological sample with a hydrogel containing the permeabilizing or lysis reagent.

[0381] In some implementations, the biological sample is contacted with two or more arrays (e.g., flexible arrays as described herein). For example, after the subcellular region is permeated and bioanalytes from the subcellular region are captured by the first array, the first array can be removed, and bioanalytes from different subcellular regions can be captured on a second array.

[0382] (14) Selective enrichment of RNA substances

[0383] In some embodiments where RNA is the analyte, one or more RNA analyte species of interest can be selectively enriched (e.g., Adiconis et al., Comparative analysis of RNA sequencing methods for degraded and low-input samples, Nature, Vol. 10, July 2013, 623-632, incorporated herein by reference in its full text). For example, one or more RNAs can be selected by adding one or more oligonucleotides to the sample. In some embodiments, the other oligonucleotides are sequences used to initiate a reaction via a polymerase. For example, one or more primer sequences that are sequence complementary to one or more RNAs of interest can be used to amplify one or more RNAs of interest, thereby selectively enriching these RNAs. In some embodiments, oligonucleotides that are sequence complementary to the complementary strand of the captured RNA (e.g., cDNA) can bind to the cDNA. For example, biotinylated oligonucleotides having sequences complementary to one or more cDNAs of interest can bind to the cDNA and can be selected using any of the various methods known in the art (e.g., streptavidin beads) utilizing biotinylation-streptavidin affinity.

[0384] Alternatively, any of a variety of methods can be used to downselect (e.g., remove, consume) one or more RNAs (e.g., ribosomal and / or mitochondrial RNA). Non-limiting examples of hybridization and capture methods for ribosomal RNA consumption include RiboMinus. TM RiboCop TM and Ribo-Zero TM Another non-limiting RNA consumption method involves hybridizing complementary DNA oligonucleotides with unwanted RNA, followed by degradation of the RNA / DNA hybrid using RNase H. Non-limiting examples of hybridization and degradation methods include... Consumption, NuGEN AnyDeplete, TruSeq TM Another non-restrictive ribosomal RNA consumption method includes ZapR. TMThe ribosomal RNA is digested, for example, by SMARTer. In the SMARTer method, a random nucleic acid conjugate hybridizes with RNA for first-strand synthesis and tailing via reverse transcriptase, followed by template conversion and extension via reverse transcriptase. Additionally, a full-length Illumina sequencing adapter (e.g., Illumina index) is added during the first round of PCR amplification. The ribosomal RNA is lysed by ZapRv2 and R probe v2. A second round of PCR is performed to amplify non-rRNA molecules (e.g., cDNA). Some or all steps of these ribosomal consumption protocols / kits can be further combined with the methods described herein to optimize protocols for specific biological samples.

[0385] In consumption protocols, probes can be applied to samples that selectively hybridize with ribosomal RNA (rRNA), thereby reducing the pooling and concentration of rRNA in the sample. Probes can be applied to biological samples that selectively hybridize with mitochondrial RNA (mtRNA), thereby reducing the pooling and concentration of mtRNA in the sample. In some embodiments, probes complementary to mitochondrial RNA can be added during cDNA synthesis, or probes complementary to both ribosomal and mitochondrial RNA can be added during cDNA synthesis. Subsequent application of capture probes to the sample may result in improved capture of other types of RNA due to a reduction in nonspecific RNA (e.g., downselective RNA) present in the sample. Alternatively, double-stranded specific nuclease (DSN) treatment can remove rRNA (see, for example, Archer et al., Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage, BMC Genomics, 15 401, (2014), the entire contents of which are incorporated herein by reference). In addition, hydroxyapatite chromatography can remove abundant substances (e.g., rRNA) (see, for example, Vandernoot, VA, cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6)373-80, (2012), the entire contents of which are incorporated herein by reference).

[0386] (15) Other reagents

[0387] Before analyzing a sample, other reagents can be added to the biological sample for various functions. In some embodiments, nuclease inhibitors, such as DNase and RNase inactivators, or protease inhibitors and / or chelating agents, such as EDTA, can be added to the biological sample. In other embodiments, nucleases, such as DNase or RNase, or proteases, such as pepsin or proteinase K, are added to the sample. In some embodiments, other reagents can be dissolved in solution or applied to the sample as a medium. In some embodiments, other reagents (e.g., pepsin) can be dissolved in HCl before application to the sample.

[0388] In some implementations, the biological sample may be treated with one or more enzymes. For example, one or more endonucleases for fragmenting DNA, DNA polymerases, and dNTPs for amplifying nucleic acids may be added. Other enzymes that may also be added to the biological sample include, but are not limited to, polymerases, transposases, ligases, DNases, and RNases.

[0389] In some embodiments, reverse transcriptase may be added to the sample, including an enzyme with terminal transferase activity, primers, and template-changing oligonucleotides (TSOs). Template changing can be used to increase the length of cDNA, for example, by appending a predefined nucleic acid sequence to the cDNA. In some embodiments, the appended nucleic acid sequence comprises one or more ribonucleotides.

[0390] In some embodiments, other reagents may be added to improve the recovery rate of one or more target molecules (e.g., eDNA molecules, mRNA transcripts). For example, adding vector RNA in an RNA sample workflow can increase the yield of RNA / DNA hybrids extracted from biological samples. In some embodiments, vector molecules are useful when the concentration of the input or target molecule is low compared to the remaining molecules. Typically, a single target molecule cannot form a precipitate, and adding a vector molecule helps to form a precipitate. Some target molecule recovery protocols use vector RNA to prevent irreversible binding of small amounts of target nucleic acids present in the sample. In some embodiments, vector RNA may be added immediately before the second-strand synthesis step. In some embodiments, vector RNA may be added immediately before the synthesis of second-strand eDNA on oligonucleotides released from the array. In some embodiments, vector RNA may be added immediately before the in vitro post-transcriptional purification step. In some embodiments, vector RNA may be added before the purification and quantification of amplified RNA. In some embodiments, vector RNA may be added before RNA quantification. In some embodiments, vector RNA may be added immediately before the second-strand eDNA synthesis and in vitro post-transcriptional purification steps.

[0391] (16) Preprocessing for capturing probe interactions

[0392] In some embodiments, the analyte in the biological sample may be pretreated before interacting with the capture probe. For example, a polymerization reaction catalyzed by a polymerase (e.g., DNA polymerase or reverse transcriptase) may be performed in the biological sample before interaction with the capture probe. In some embodiments, the primers used for the polymerization reaction include functional groups that enhance hybridization with the capture probe. The capture probe may include appropriate capture domains to capture the bioanalyte of interest (e.g., a poly(dT) sequence to capture poly(A) mRNA).

[0393] In some embodiments, the bioanalyte is pretreated using next-generation sequencing to generate a library. For example, the analyte can be pretreated by adding a modifier (e.g., ligating a sequence that interacts with a capture probe). In some embodiments, the analyte (e.g., DNA or RNA) is fragmented using fragmentation techniques (e.g., using transposases and / or fragmentation buffers).

[0394] After fragmentation, the analyte can be modified. For example, modification can be added by ligating an adaptor sequence that hybridizes with the capture probe. In some embodiments, where the analyte of interest is RNA, poly(A) tailing is performed. Adding a poly(A) tail to RNA that does not contain a poly(A) tail promotes hybridization with a capture probe containing a capture domain with a functional amount of poly(dT) sequence.

[0395] In some embodiments, a ligation reaction catalyzed by a ligase is performed in the biological sample prior to interaction with the capture probe. In some embodiments, ligation can be performed by chemical ligation. In some embodiments, click chemistry can be used for ligation, as described below. In some embodiments, the capture domain includes a DNA sequence complementary to an RNA molecule, wherein the RNA molecule is complementary to a second DNA sequence, and wherein the RNA-DNA sequence complementarity is used to ligate the second DNA sequence to the DNA sequence in the capture domain. In these embodiments, the RNA molecule can be detected directly.

[0396] In some embodiments, a target-specific reaction is performed in the biological sample prior to interaction with the capture probe. Examples of target-specific reactions include, but are not limited to, the ligation of target-specific adaptors, probes, and / or other oligonucleotides; target-specific amplification using primers specific to one or more analytes; and target-specific detection using in situ hybridization, DNA microscopy, and / or antibody detection. In some embodiments, the capture probe includes a capture domain (e.g., amplified or ligated) that is targeted to a target-specific product.

[0397] II. General Spatial Array-Based Analysis Methods

[0398] This article provides methods, apparatus, systems, and compositions for spatial array-based analysis of biological samples.

[0399] (a) Spatial analysis methods

[0400] Array-based spatial analysis methods involve transferring one or more analytes from a biological sample onto a feature array on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the analyte identity and the spatial location of each analyte within the biological sample. The spatial location of each analyte within the biological sample is determined based on the features bound to each analyte in the array and the relative spatial positions of those features on the array.

[0401] There are at least two general methods for associating spatial barcodes with one or more neighboring cells, such that the spatial barcode identifies one or more cells and / or the contents of one or more cells as associated with a specific spatial location. One general method facilitates analysis of cells and their orientation towards a spatially barcoded array. Figure 1 Exemplary implementations of this general method are described. Figure 1 In this process, a spatially barcoded array containing capture probes (as further described herein) is brought into contact with a biological sample 101, and the biological sample is permeated, allowing analytes to migrate from the sample to the array. The analytes interact with the capture probes on the spatially barcoded array 102. Once the analytes hybridize / bind to the capture probes, the sample is optionally removed from the array and the capture probes are analyzed to obtain spatially resolved analyte information 103.

[0402] Another common approach is to capture probes by spatial barcoding from array fragmentation and to facilitate the spatial barcoding capture probes toward and / or onto biological samples. Figure 2 An exemplary implementation of this general method is depicted, enabling a spatially barcoded array of capture probes (as further described herein) to contact sample 201. The spatially barcoded capture probes are cleaved and then interact with cells within the provided biological sample 202. This interaction can be a covalent or non-covalent cell surface interaction. The interaction can be an intracellular interaction facilitated by a delivery system or cell-penetrating peptides. Once the spatially barcoded capture probes are associated with specific cells, the sample can optionally be removed for analysis. The sample can be selectively segregated prior to analysis. Once the labeled cells are associated with the spatially barcoded capture probes, the capture probes can be analyzed to obtain spatial resolution information about the labeled cells 203.

[0403] Figure 3An exemplary workflow is illustrated, including the preparation of a biological sample on a spatially barcoded array 301. Sample preparation may include placing the sample on a glass slide, fixing the sample, and / or staining the biological sample for imaging. The stained sample is then imaged on array 302 using bright-field (imaging for hematoxylin and eosin staining of the sample) and fluorescence (imaging for features) modes. Optionally, the sample may be destained before permeation. In some embodiments, the analyte is then released from the sample, and the capture probes forming the spatially barcoded array hybridize or bind to the released analyte 303. The sample is then removed from array 304, and the capture probes are lysed from array 305. The biological sample and array are then optionally second-imaged in two modes 305B, while the analyte is reverse-transcribed into cDNA, and an amplicon library 306 is prepared and sequenced 307. The two sets of images are then spatially overlaid to correlate spatially identified biological sample information 308. When the sample and array are not second-imaged 305B, a point coordinate file is provided instead. The point coordinate file replaces the second imaging step 305B. Additionally, amplicon library preparation 306 can be performed using a unique PCR adaptor and sequencing 307.

[0404] Figure 4 Another exemplary workflow is shown, which utilizes a spatially barcoded array on a substrate, where spatially barcoded capture probes are clustered in regions called features. The spatially barcoded capture probes may include cleavage domains, one or more functional domains, spatial barcodes, unique molecular identifiers, and capture domains. The spatially barcoded capture probes may also include 5′ end modifications for reversible attachment to the substrate. The spatially barcoded array contacts a biological sample 401 and permeates the sample by applying a permeation reagent 402. The permeation reagent can be administered by placing the array / sample assembly in a bulk solution. Alternatively, the permeation reagent can be administered to the sample via an antidiffusion medium and / or a physical barrier (e.g., a cap), where the sample is sandwiched between the antidiffusion medium and / or barrier and the substrate containing the array. Analytes are migrated to the spatially barcoded capture array using any number of techniques disclosed herein. For example, analyte migration can be performed using an antidiffusion medium cap and passive migration. As another example, analyte migration can be active migration, for example, using an electrophoretic transfer system. Once the analyte is near the spatially barcoded capture probe, the capture probe may hybridize or otherwise bind to the target analyte 403. Biological samples can be optionally removed from array 404.

[0405] The capture probe can optionally be cleaved from array 405, and the captured analyte can be spatially barcoded by performing a reverse transcriptase first-strand cDNA reaction. The first-strand cDNA reaction can optionally be performed using template-converting oligonucleotides. For example, the template-converting oligonucleotides can hybridize in a template-independent manner with a poly(C) tail added to the 3′ end of the cDNA via reverse transcriptase. The original mRNA template and template-converting oligonucleotides can be denatured from the cDNA, and then the spatially barcoded capture probe can hybridize with the cDNA and generate a cDNA complement. The first-strand cDNA can then be purified and collected for downstream amplification steps. The first-strand cDNA can be amplified using PCR 406, where forward and reverse primers are side-attached to the spatial barcode of interest and the analyte region, producing a library associated with a specific spatial barcode. In some embodiments, the cDNA contains sequencing-by-synthesis (SBS) primer sequences. The library amplicon is sequenced and analyzed to decode the spatial information 407.

[0406] Using the methods, compositions, systems, kits, and devices described herein, RNA transcripts present in biological samples (e.g., tissue samples) can be used for spatial transcriptome analysis. Specifically, in some cases, barcoded oligonucleotides can be configured to initiate, replicate, and thus produce barcoded extension products from an RNA template or a derivative thereof. For example, in some cases, the barcoded oligonucleotides may include mRNA-specific initiating sequences, such as poly-T primer fragments or other targeted initiating sequences that allow initiation and replication of mRNA in a reverse transcription reaction. Alternatively or additionally, random RNA initiation can be performed using random N-mer (N-mer) primer segments of the barcoded oligonucleotides. Reverse transcriptase (RT) can use an RNA template and primers complementary to the 3′ end of the RNA template to direct the synthesis of first-strand complementary DNA (cDNA). Many RTs can be used for this reverse transcription reaction, including, for example, avian myeloblastomavirus (AMV) reverse transcriptase, Moloney murine leukemia virus (M-MuLV or MMLV), and other variants. Some recombinant M-MuLV reverse transcriptases, such as… Reverse transcriptases, compared to their wild-type counterparts, may exhibit reduced RNase H activity and increased thermostability, and provide higher specificity, higher cDNA yield, and more complete cDNA products up to 12 kilobases (kb) in length. In some embodiments, the reverse transcriptase is a mutant reverse transcriptase, such as, but not limited to, a mutant MMLV reverse transcriptase. In another embodiment, the reverse transcriptase is a mutant MMLV reverse transcriptase, such as, but not limited to, one or more variants described in U.S. Patent Publication No. 20180312822, which is incorporated herein by reference in its entirety.

[0407] Figure 5An exemplary workflow is described in which a biological sample is removed from a spatially barcoded array, and a spatially barcoded capture probe is removed from the array for barcoded analyte amplification and library preparation. Another embodiment includes first-strand synthesis using template-converting oligonucleotides on the spatially barcoded array without cleaving the capture probe. In this embodiment, sample preparation 501 and permeabilization 502 are performed as described elsewhere herein. Once the capture probe captures the analyte, the first-strand eDNA generated via template conversion and reverse transcriptase 503 is subsequently denatured, and the second strand is subsequently extended 504. The second-strand eDNA is then denatured, neutralized, and transferred from the first-strand eDNA to tube 505. eDNA quantification and amplification can be performed using the standard techniques discussed herein. The eDNA can then undergo library preparation 506 and indexing 507, including fragmentation, end repair and A-tailing, and indexing PCR steps.

[0408] In a non-limiting example of the above workflow, biological samples (e.g., tissue sections) can be fixed with methanol, stained with hematoxylin and eosin, and imaged. Optionally, the sample can be destained before permeabilization. These images can be used to map spatial gene expression patterns back to the biological sample. Permeabilizing enzymes can be used to permeabilize biological samples directly on a slide. Analytes (e.g., polyadenylated mRNA) released from overlying cells of the biological sample can be captured by capture probes in capture regions on the substrate. Reverse transcription (RT) reagents can be added to the permeabilized biological sample. Incubation with RT reagents can generate spatially barcoded full-length cDNA from the captured analytes (e.g., polyadenylated mRNA). Second-strand reagents (e.g., second-strand primers, enzymes) can be added to the biological sample on the slide to initiate second-strand synthesis. The resulting cDNA can be denatured from the capture probe template and transferred (e.g., to a clean tube) for amplification and / or library construction. The spatially barcoded full-length cDNA can be amplified by PCR before library construction. Then, cDNA can be enzymatically fragmented and size-selected to optimize the size of the cDNA amplicon. P5, P7, i7, and i5 can be used as sample indices, and TruSeq Read 2 can be added using methods such as end repair, A-tailing, adapter ligation, and PCR. TruSeq Read 1 and TruSeq Read 2 can then be used as sequencing primer sites, and paired-ended sequencing can be used to sequence the cDNA fragments.

[0409] In some implementations, correlation analysis of the data generated by this workflow and other workflows described herein can produce a correlation of more than 95% (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher) across genes expressed in two capture regions. When the workflow is performed using single-cell RNA sequencing of the cell nucleus, in some implementations, correlation analysis of the data can produce a correlation of more than 90% (e.g., more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) across genes expressed in two capture regions.

[0410] In some embodiments, after cDNA is generated (e.g., via reverse transcription), the cDNA can be amplified directly on the substrate surface. Generating multiple copies of cDNA (e.g., cDNA synthesized from captured analytes) by direct amplification on the substrate surface can increase the complexity of the final sequencing library. Therefore, in some embodiments, cDNA can be amplified directly on the substrate surface by isothermal nucleic acid amplification. In some embodiments, isothermal nucleic acid amplification can amplify either RNA or DNA.

[0411] In some embodiments, isothermal amplification can be faster than a standard PCR reaction. In some embodiments, isothermal amplification can be linear amplification (e.g., asymmetric with a single primer) or exponential amplification (e.g., with two primers). In some embodiments, isothermal nucleic acid amplification can be performed using template-converting oligonucleotide primers. In some embodiments, template-converting oligonucleotides add a common sequence to the 5′ end of RNA being reverse transcribed. For example, after a capture probe interacts with an analyte (e.g., mRNA) and undergoes reverse transcription, additional nucleotides are added to the ends of cDNA, producing 3′ overhangs as described herein. In some embodiments, template-converting oligonucleotides hybridize with untemplated poly(C) nucleotides added by reverse transcriptase to continue replication to the 5′ end of the template-converting oligonucleotide, thereby generating full-length cDNA ready for further amplification. In some embodiments, template-converting oligonucleotides add a common 5′ sequence (e.g., the reverse complement of the template-converting oligonucleotide) to the full-length cDNA used for cDNA amplification.

[0412] In some embodiments, once a full-length cDNA molecule is generated, template-changing oligonucleotides can be used as primers in a cDNA amplification reaction (e.g., using DNA polymerase). In some embodiments, double-stranded cDNA (e.g., first-strand cDNA and second-strand reverse complementary cDNA) can be isothermally amplified using a helicase or recombinase, followed by strand displacement DNA polymerase. The strand displacement DNA polymerase generates a substituted second strand, thereby producing the amplification product.

[0413] In any of the isothermal amplification methods described herein, barcode exchange (e.g., spatial barcodes) may occur after the first amplification cycle, where unused capture probes remain on the substrate surface. In some embodiments, the free 3′OH ends of the unused capture probes can be blocked by any suitable 3′OH blocking method. In some embodiments, the 3′OH can be blocked by hairpin connection.

[0414] Isothermal nucleic acid amplification can be used as a supplement to or alternative to standard PCR reactions (e.g., PCR reactions that require heating to approximately 95°C to denature double-stranded DNA). Isothermal nucleic acid amplification typically does not require the use of a thermal cycler; however, in some embodiments, isothermal amplification can be performed in a thermal cycler. In some embodiments, isothermal amplification can be performed at approximately 35°C to 75°C. In some embodiments, isothermal amplification can be performed at approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, approximately 60°C, approximately 65°C, or approximately 70°C, or any temperature in between, depending on the polymerase and coenzyme used.

[0415] Isothermal nucleic acid amplification techniques are known in the art and can be used alone or in combination with any spatial method described herein. For example, non-limiting examples of suitable isothermal nucleic acid amplification techniques include: transcription-mediated amplification, nucleic acid sequence-based amplification, signal-mediated RNA amplification, strand displacement amplification, rolling circle amplification, loop-mediated isothermal DNA amplification (LAMP), isothermal multiple displacement amplification, recombinase polymerase amplification, helicase-dependent amplification, single-primer isothermal amplification, and loop helicase-dependent amplification (see, for example, Gill and Ghaemi, Nucleic acid isothermal amplification technologies: a review), Nucleosides, Nucleotides, & Nucleic Acids, 27(3), 224-43, doi: 10.1080 / 15257770701845204 (2008), which is incorporated herein by reference in its entirety.

[0416] In some embodiments, isothermal nucleic acid amplification is helicase-dependent nucleic acid amplification. Helicase-dependent isothermal nucleic acid amplification is described in Vincent et al., Helicase-dependent isothermal DNA amplification, EMBO Rep., 795-800 (2004) and U.S. Patent No. 7,282,328, both of which are incorporated herein by reference in their entirety. Helicase-dependent nucleic acid amplification on a matrix (e.g., on a chip) is described in Andresen et al., Helicase-dependent amplification: use in OnChip amplification and potential for point-of-care diagnostics, Expert Rev Mol Diagn., 9, 645-650, doi: 10.1586 / erm.09.46 (2009), which is incorporated herein by reference in its entirety. In some embodiments, isothermal nucleic acid amplification is recombinase polymerase nucleic acid amplification. The description of recombinase polymerase amplification is found in Piepenburg et al., DNA Detection Using Recombinant Proteins, PLoS Biol., 4, 7 e204 (2006), and Li et al., Review: a comprehensive summary of adecade development of the recombinase polymerase amplification, Analyst, 144, 31-67, doi: 10.1039 / C8AN01621F (2019). Both are included in this paper by citing their full text.

[0417] Typically, isothermal amplification techniques use standard PCR reagents known in the art (e.g., buffers, dNTPs, etc.). Some isothermal amplification techniques may require additional reagents. For example, helicase-dependent nucleic acid amplification uses single-strand binding proteins and accessory proteins. In another example, recombinase polymerase nucleic acid amplification uses recombinases (e.g., T4 UvsX), recombinase loading factors (e.g., TF UvsY), single-strand binding proteins (e.g., T4 gp32), congesting agents (e.g., PEG-35K), and ATP.

[0418] Following isothermal nucleic acid amplification of full-length cDNA using any of the methods described herein, the isothermally amplified cDNA (e.g., single-stranded or double-stranded) can be recovered from the substrate and optionally subsequently amplified using typical cDNA PCR in a microcentrifuge tube. The sample can then be used with any of the spatial methods described herein.

[0419] (i) Immunohistochemistry and immunofluorescence

[0420] In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) may be performed as part or an additional part of the exemplary spatial workflows presented herein. For example, tissue sections may be fixed according to the methods described herein. Biological samples may be transferred to an array (e.g., a capture probe array) where an analyte (e.g., a protein) is detected using an immunofluorescence protocol. For example, the sample may be rehydrated, blocked, and permeabilized (3X SSC, 2% BSA, 0.1% Triton X, 1 U / μl RNase inhibitor, 4°C for 10 min), followed by staining with a fluorescent primary antibody (1:100 in 3X SSC, 2% BSA, 0.1% Triton X, 1 U / μl RNase inhibitor, 4°C for 30 min). Biological samples may be washed, covered with coverslips (in glycerol + 1 U / μl RNase inhibitor), imaged (e.g., using a confocal microscope or other device capable of fluorescence detection), washed, and processed (according to the analyte capture or spatial workflows described herein).

[0421] As used herein, “antigen retrieval buffer” can improve antibody capture in IF / IHC protocols. An exemplary protocol for antigen retrieval may be to preheat the antigen retrieval buffer (e.g., to 95°C), immerse the biological sample in the heated antigen retrieval buffer for a predetermined time, and then remove and wash the biological sample from the antigen retrieval buffer.

[0422] In some implementations, optimizing permeabilization can be useful for identifying intracellular analytes. Permeabilization optimization may include selecting the permeabilizing agent, its concentration, and the duration of permeabilization. Tissue permeabilization is discussed elsewhere in this document.

[0423] In some embodiments, blocking the array and / or biological sample during the preparation of the labeled biological sample can reduce nonspecific binding of the antibody to the array and / or biological sample (reduce background). Some embodiments provide a blocking buffer / blocking solution that can be applied before and / or during the application of the label, wherein the blocking buffer may include a blocking agent and optionally a surfactant and / or a salt solution. In some embodiments, the blocking agent may be bovine serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk (e.g., skim milk powder), casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP), biotin blocking agent, peroxidase blocking agent, levamisole, Carnoy's solution, glycine, lysine, sodium borohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blocking agent, and / or acetic acid. Blocking buffer / blocking solution can be applied to the array and / or biological sample before and / or during labeling of biological sample (e.g., application of antibody conjugated with a fluorophore).

[0424] In some implementations, additional steps or optimizations may be included in the IF / IHC scheme using a combined space array. Additional steps or optimizations may also be included in the space-spiked analyte capture workflow discussed herein.

[0425] In some embodiments, this document provides a method for spatially detecting an analyte (e.g., a detection analyte, such as a bioanalyte) from a biological sample (e.g., an analyte present in a biological sample, such as a tissue section), the method comprising: (a) providing the biological sample on a substrate; (b) staining the biological sample on the substrate, imaging the stained biological sample, and selecting the biological sample or a subsection of the biological sample (e.g., a region of interest) for analysis; (c) providing an array on the substrate comprising one or more sets of multiple capture probes; (d) contacting the biological sample with the array to allow one or more sets of multiple capture probes to capture the analyte of interest; and (e) analyzing the captured analyte to perform spatial analysis of the analyte of interest. Any kind of staining and imaging technique described herein or known in the art may be used according to the methods described herein. In some embodiments, staining includes optical markers as described herein, including but not limited to fluorescent markers, radiolabels, chemiluminescent markers, calorimetric or colorimetric detectable markers. In some embodiments, staining includes fluorescent antibodies against a target analyte (e.g., a protein on the cell surface or inside the cell) in the biological sample. In some embodiments, staining includes immunohistochemical staining for a target analyte in the biological sample (e.g., cell surface or intracellular proteins). In some embodiments, staining includes chemical stains such as hematoxylin and eosin (H&E) or periodic acid Schiff (PAS). In some embodiments, a considerable time interval (e.g., days, months, or years) may exist between staining and / or imaging of the biological sample and performing analysis. In some embodiments, reagents for analysis are added to the biological sample before, simultaneously with, or after the array contacts the biological sample. In some embodiments, step (d) includes placing the array on the biological sample. In some embodiments, the array is a flexible array in which multiple spatially barcoded features (e.g., a substrate with capture probes, beads with capture probes) are attached to a flexible substrate. In some embodiments, measures are taken to slow the reaction before the array contacts the biological sample (e.g., lowering the temperature of the biological sample or using enzymes that preferentially perform their primary function at temperatures lower or higher than their optimal functional temperature). In some embodiments, step (e) is performed without removing the biological sample from the array. In some embodiments, step (e) is performed after the biological sample is no longer in contact with the array. In some embodiments, the biological sample is spiked with an analyte trapping agent before, simultaneously with, or after staining and / or imaging. In this case, a considerable amount of time may separate staining and / or imaging from the analysis (e.g., days, months, or years). In some embodiments, the array is adapted to facilitate the migration of bioanalytes from the stained and / or imaged biological sample to the array (e.g., using any of the materials or methods described herein).In some embodiments, the biological sample is permeabilized before contact with the array. In some embodiments, the permeabilization rate is slowed before contact with the array (e.g., to limit diffusion of the analyte from its original location in the biological sample). In some embodiments, modulating the permeabilization rate (e.g., modulating the activity of the permeabilizing agent) can be achieved by modulating the conditions to which the biological sample is exposed (e.g., adjusting temperature, pH, and / or light). In some embodiments, modulating the permeabilization rate involves using an external stimulus (e.g., a small molecule, enzyme, and / or activating agent) to modulate the permeabilization rate. For example, a permeabilizing agent that is inactive before contact with the array can be provided to the biological sample.

[0426] In some embodiments, this document provides a method for spatial detection of an analyte (e.g., a detection analyte, such as a bioanalyte) from a biological sample (e.g., an analyte present in a biological sample, such as a tissue section), the method comprising: (a) providing a biological sample on a substrate; (b) staining the biological sample on the substrate, imaging the stained biological sample, and selecting the biological sample or a subsection of the biological sample (e.g., a region of interest) for spatial transcriptomics analysis; (c) providing an array on the substrate comprising one or more sets of multiple capture probes; (d) contacting the biological sample with the array to allow one or more sets of multiple capture probes to capture the bioanalyte of interest; and (e) analyzing the captured bioanalyte to perform spatial analysis of the bioanalyte of interest.

[0427] (b) Capture probe

[0428] A “capture probe” is any molecule capable of capturing (directly or indirectly) and / or labeling an analyte (e.g., the analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or peptide. In some embodiments, the capture probe is a conjugate (e.g., an oligonucleotide-antibody conjugate). In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI)) and a capture domain.

[0429] Figure 6This is a schematic diagram illustrating an example of a capture probe as described herein. As shown, capture probe 602 is optionally coupled to feature 601 via a cleavage domain 603, such as a disulfide linker. The capture probe may include functional sequences useful for subsequent processing, such as functional sequence 604, which may include sequencer-specific flow cell attachment sequences, such as the P5 sequence, and functional sequence 606, which may include sequencing primer sequences, such as the R1 primer binding site. In some embodiments, sequence 604 is the P7 sequence and sequence 606 is the R2 primer binding site. Spatial barcode 605 may be included within the capture probe for barcoding of the target analyte. Functional sequences are typically selected for compatibility with any of a variety of different sequencing systems, such as 454 sequencing, Ion Torrent Proton or PGM, Illumina X10, PacBio, Nanopore, etc., and their requirements. In some embodiments, functional sequences may be selected for compatibility with non-commercial sequencing systems. Examples of such sequencing systems and techniques that can use appropriate functional sequences include (but are not limited to) Roche 454 sequencing, ion-fluid proton or PGM sequencing, Illumina X10 sequencing, PacBio SMRT sequencing, and Oxford nanopore sequencing. Furthermore, in some implementations, functional sequences can be selected for compatibility with other sequencing systems, including non-commercial sequencing systems.

[0430] In some implementations, the spatial barcode 605, functional sequence 604 (e.g., flow cell attachment sequence), and 606 (e.g., sequencing primer sequence) may be shared by all probes attached to a given feature. The spatial barcode may also include a capture field 607 to facilitate the capture of the target analyte.

[0431] (i) Capture domain

[0432] As described above, each capture probe includes at least one capture domain. The "capture domain" can be an oligonucleotide, peptide, small molecule, or any combination thereof, which specifically binds to the desired analyte. In some embodiments, the capture domain can be used to capture or detect the desired analyte.

[0433] In some embodiments, the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes, such as one or more different types of nucleic acids (e.g., RNA molecules and DNA molecules). In some embodiments, the functional nucleic acid sequence may include an N-mer sequence (e.g., a random N-mer sequence) configured to interact with multiple DNA molecules. In some embodiments, the functional sequence may include a poly(T) sequence configured to interact with messenger RNA (mRNA) molecules via a poly(A) tail of an mRNA transcript. In some embodiments, the functional nucleic acid sequence is a binding target for a protein (e.g., a transcription factor, a DNA-binding protein, or an RNA-binding protein), wherein the analyte of interest is a protein.

[0434] The capture probe may include ribonucleotides and / or deoxyribonucleotides, as well as synthetic nucleotide residues capable of participating in Watson-Crick-type or similar base-pair interactions. In some embodiments, the capture domain is capable of initiating a reverse transcription reaction to produce cDNA complementary to the captured RNA molecule. In some embodiments, the capture domain of the capture probe may initiate a DNA extension (polymerase) reaction to produce DNA complementary to the captured DNA molecule. In some embodiments, the capture domain may serve as a template for a ligation reaction between the captured DNA molecule and a surface probe directly or indirectly immobilized on a substrate. In some embodiments, the capture domain may be ligated to one strand of the captured DNA molecule. For example, SplintR ligase, along with an RNA or DNA sequence (e.g., degenerate RNA), may be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, ligases with RNA template ligase activity (e.g., SplintR ligase, T4 RNA ligase 2, or KOD ligase) may be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, the capture domain comprises a splint oligonucleotide. In some embodiments, the capture domain captures the splint oligonucleotide.

[0435] In some embodiments, the capture domain is located at the 3′ end of the capture probe and includes a free 3′ end that can be extended, for example, by template-dependent polymerization to form an extended capture probe as described herein. In some embodiments, the capture domain includes a nucleotide sequence capable of hybridizing with nucleic acids (e.g., RNA or other analytes) present in cells of a biological sample in contact with the array. In some embodiments, the capture domain may be selected or designed to bind selectively or specifically to a target nucleic acid. For example, the capture domain may be selected or designed to capture mRNA by hybridizing with the poly(A) tail of mRNA. Thus, in some embodiments, the capture domain includes a poly(T) DNA oligonucleotide, for example, a series of consecutive deoxythymidine residues linked by phosphodiester bonds, capable of hybridizing with the poly(a) tail of mRNA. In some embodiments, the capture domain may include a nucleotide that is functionally or structurally similar to a poly(T) tail. For example, a poly(U) oligonucleotide or an oligonucleotide containing a deoxythymidine analogue. In some embodiments, the capture domain comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capture domain comprises at least 25, 30, or 35 nucleotides.

[0436] In some embodiments, the capture probe includes a capture domain having a sequence capable of binding to mRNA and / or genomic DNA. For example, the capture probe may include a capture domain comprising a nucleic acid sequence (e.g., a poly(T) sequence) capable of binding to a poly(A) tail of mRNA and / or a poly(A) homopolymeric sequence present in genomic DNA. In some embodiments, a homopolymeric sequence is added to an mRNA molecule or a genomic DNA molecule using a terminal transferase to produce an analyte having a poly(A) or poly(T) sequence. For example, a poly(A) sequence may be added to an analyte (e.g., a fragment of genomic DNA) to enable the analyte to be captured by a poly(T) capture domain.

[0437] In some embodiments, a random sequence (e.g., a random hexamer or similar sequence) may be used to form all or part of the capture domain. For example, the random sequence may be used in conjunction with a poly(T) (or poly(T) analog) sequence. Therefore, in cases where the capture domain comprises a poly(T) (or “poly(T)-like”) oligonucleotide, it may also comprise a random oligonucleotide sequence (e.g., a “poly(T)-random sequence” probe). This may be located, for example, at the 5′ or 3′ end of the poly(T) sequence, such as at the 3′ end of the capture domain. The poly(T)-random sequence probe facilitates the capture of the poly(A) tail of the mRNA. In some embodiments, the capture domain may be a completely random sequence. In some embodiments, a degenerate capture domain may be used.

[0438] In some embodiments, sets of two or more capture probes form a mixture, wherein the capture domain of one or more capture probes comprises a poly(T) sequence, and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, sets of two or more capture probes form a mixture, wherein the capture domain of one or more capture probes comprises a poly(T) sequence, and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, sets of two or more capture probes form a mixture, wherein the capture domain of one or more capture probes comprises a poly(T) random sequence, and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, a probe having a degenerate capture domain may be added to any of the foregoing combinations listed herein. In some embodiments, a probe having a degenerate capture domain may replace one of the probes in each pair described herein.

[0439] The capture domain may be based on a specific gene sequence or motif sequence or common / conserved sequence that it is designed to capture (i.e., a sequence-specific capture domain). Therefore, in some embodiments, the capture domain is capable of selectively binding to a desired nucleic acid subtype or subset, such as a specific type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tRNA, snRNA, snRNA, SmY RNA, sARNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA, or other types of RNA. In a non-limiting example, the capture domain is capable of selectively binding to a desired subset of ribonucleic acid, such as microbiome RNA, such as 16S rRNA.

[0440] In some embodiments, the capture domain includes an "anchor" or "anchoring sequence," which is a nucleotide sequence designed to ensure hybridization of the capture domain with the intended bioanalyte. In some embodiments, the anchoring sequence includes a nucleotide sequence, including a 1-mer, 2-mer, 3-mer, or longer sequence. In some embodiments, the short sequence is random. For example, a capture domain including a poly(T) sequence can be designed to capture mRNA. In such embodiments, the anchoring sequence may include a random 3-mer (e.g., GGG) that helps ensure hybridization of the poly(T) capture domain with mRNA. In some embodiments, the anchoring sequence may be VN, N, or NN. Alternatively, a specific nucleotide sequence can be used to design the sequence. In some embodiments, the anchoring sequence is located at the 3′ end of the capture domain. In some embodiments, the anchoring sequence is located at the 5′ end of the capture domain.

[0441] In some embodiments, the capture domain of the capture probe is blocked before the biological sample comes into contact with the array, and blocking probes are used when nucleic acids in the biological sample are modified before they are captured onto the array. In some embodiments, blocking probes are used to block or modify the free 3′ end of the capture domain. In some embodiments, blocking probes may hybridize with capture probes to mask the free 3′ end of the capture domain, such as hairpin probes, partially double-stranded probes, or complementary sequences. In some embodiments, the free 3′ end of the capture domain can be blocked by chemical modification, for example, by adding an azidomethyl group as a chemically reversible capping moiety, such that the capture probe does not include a free 3′ end. Blocking or modifying the capture probe, particularly the free 3′ end of the capture domain, before the biological sample comes into contact with the array prevents modification of the capture probe, for example, preventing the addition of a poly(A) tail to the free 3′ end of the capture probe.

[0442] Non-limiting examples of 3′ modifications include dideoxyC-3′ (3′-ddC), 3′ inverted dT, 3′C3 spacers, 3′ amino groups, and 3′ phosphorylation. In some embodiments, nucleic acids in a biological sample can be modified such that they can be captured by a capture domain. For example, an adaptor sequence (including a binding domain capable of binding to the capture domain of a capture probe) can be added to the end of a nucleic acid (e.g., fragmented genomic DNA). In some embodiments, this is achieved by ligating the adaptor sequence or by nucleic acid extension. In some embodiments, an enzyme is used to incorporate other nucleotides, such as poly(A) tails, at the ends of the nucleic acid sequence. In some embodiments, the capture probe can be reversibly masked or modified such that the capture domain of the capture probe does not include a free 3′ end. In some embodiments, the 3′ end is removed, modified, or rendered inaccessible such that the capture domain is not susceptible to the effects of processes (e.g., ligation or extension) used to modify the nucleic acid in the biological sample.

[0443] In some embodiments, the capture domain of the capture probe is modified to remove any modifications to the capture probe that occur during the modification of nucleic acid molecules in the biological sample. In some embodiments, the capture probe may include an additional sequence downstream of the capture domain, such as the 3′ of the capture domain, i.e., the blocking domain.

[0444] In some implementations, the capture domain of the capture probe may be a non-nucleic acid domain. Examples of suitable capture domains, including but not limited to proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any capture domain described herein, are not limited to these examples.

[0445] (ii) Fracturing domain

[0446] Each capture probe may optionally include at least one cleavage domain. A cleavage domain represents a portion of the probe used to reversibly attach the probe to the array feature, as described herein. Furthermore, one or more segments or regions of the capture probe may optionally be released from the array feature by cleaving the cleavage domain. For example, spatial barcodes and / or universal molecular identifiers (UMIs) may be released by cleaving the cleavage domain.

[0447] Figure 7 This is a schematic diagram illustrating a cleavable capture probe, where the cleaved capture probe can enter non-permeable cells and bind to analytes within the sample. Capture probe 701 includes a cleavage domain 702, a cell-penetrating peptide 703, a reporter molecule 704, and a disulfide bond (-SS-). 705 represents all other parts of the capture probe, such as spatial barcodes and the capture domain.

[0448] In some embodiments, the cleavage domain that attaches the capture probe to the feature is a disulfide bond. A reducing agent can be added to break the disulfide bond, thereby releasing the capture probe from the feature. As another example, heating can also cause degradation of the cleavage domain and release of the attached capture probe from the array feature. In some embodiments, laser radiation is used to heat and degrade the cleavage domain of the capture probe at specific locations. In some embodiments, the cleavage domain is a photosensitive chemical bond (e.g., a chemical bond that dissociates when exposed to light, such as ultraviolet light).

[0449] Oligonucleotides with photosensitive chemical bonds (e.g., photolytically cleavable linkers) offer several advantages. They can be cleaved efficiently and rapidly (e.g., in nanoseconds and milliseconds). In some cases, photomasks can be used to expose only specific regions of an array to the cleavable stimulus (e.g., exposure to ultraviolet light, light, or laser-induced heat). When using photolytically cleavable linkers, the cleavage reaction is light-triggered and highly selective for the linker, resulting in a bisorthogonal reaction. Typically, the wavelength absorption of photolytically cleavable linkers lies in the near-ultraviolet range of the spectrum. In some embodiments, the λ of the photolytically cleavable linker... 最大 The wavelength is approximately 300 nm to approximately 400 nm, or approximately 310 nm to approximately 365 nm. In some embodiments, the λ of the optically spliable connector... 最大 The wavelengths are approximately 300nm, 312nm, 325nm, 330nm, 340nm, 345nm, 355nm, 365nm, or 400nm.

[0450] Non-limiting examples of photosensitive chemical bonds that can be used in the cleavage domain include those described in Leriche et al., Bioorg Med Chem., Jan 15, 2012; 20(2): 571-82 and U.S. Publication No. 2017 / 0275669, both of which are incorporated herein by reference in their entirety. For example, linkers containing photosensitive chemical bonds include: 3-amino-3-(2-nitrophenyl)propionic acid (ANP), benzoyl ester derivatives, 8-quinolinebenzenesulfonate, biscoumarin, 6-bromo-7-alkylcoumarin-4-ylmethoxycarbonyl, bimane-based linkers, and bisarylhydrazone-based linkers. In some embodiments, the photosensitive bond is part of a cleavable linker, such as the following o-nitrobenzyl (ONB) linker:

[0451]

[0452] in:

[0453] X is selected from O and NH;

[0454] R 1 Selected from H and C 1-3 alkyl;

[0455] R 2 Selected from H and C 1-3 Alkoxy;

[0456] n is 1, 2, or 3; and

[0457] a and b represent the connection point between the connector and the substrate, or the connection point between the connector and the capture probe, respectively.

[0458] In some embodiments, at least one spacer is included between the substrate and the o-nitrobenzyl (ONB) connector, and at least one spacer is included between the o-nitrobenzyl (ONB) connector and the capture probe. In some aspects of these embodiments, the spacer comprises at least one group selected from: C1-6 alkylene, C2-6 alkenylene, C2-6 ynylene, C=O, O, S, NH, -(C=O)O-, -(C=O)NH-, -SS-, ethylene glycol, polyethylene glycol, propylene glycol, and polypropylene glycol, or any combination thereof. In some embodiments, X is O. In some embodiments, X is NH. In some embodiments, R... 1 For H. In some implementations, R 1 C 1-3 Alkyl group. In some embodiments, R 1 It is a methyl group. In some embodiments, R 2 It is H. In some implementations, R 2 It is C 1-3 Alkyloxy group. In some embodiments, R2 It is a methoxy group. In some embodiments, R 1 For H and R 2 For H. In some implementations, R 1 For H and R 2 It is methoxylated. In some embodiments, R 1 It is methyl and R 2 For H. In some implementations, R 1 It is methyl and R 2 It is a methoxy group.

[0459] In some embodiments, the photodegradable connector has the following formula:

[0460]

[0461] In some embodiments, the photodegradable connector has the following formula:

[0462]

[0463] In some embodiments, the photodegradable connector has the following formula:

[0464]

[0465] In some embodiments, the photodegradable connector has the following formula:

[0466]

[0467] In some embodiments, the photodegradable connector has the following formula:

[0468]

[0469] Unbound by any particular theory, it is believed that the excitation of the ortho-nitrobenzyl (ONB) linker leads to Norrish-type hydrogen abstraction at the γ-position, subsequently forming a highly reactive azazinic acid that rearranges into a nitroso compound, resulting in complete cleavage of the linker, as shown in the figure below:

[0470]

[0471] In some embodiments, the photolytically cleavable linker is a 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker:

[0472]

[0473] Among them, X and R 2 n, a, and b are as described in this paper for the ortho-nitrobenzyl (ONB) connector.

[0474] In some embodiments, the photodegradable connector has the following formula:

[0475]

[0476] In some implementations, the photolytically degradable connector is a benzoyl ester connector:

[0477]

[0478] Where a and b are as described in this paper for the ortho-nitrobenzyl (ONB) connector.

[0479] Other examples of photosensitive chemical bonds that can be used in the cleavage domain include halogenated nucleosides, such as bromodeoxyuridine (BrdU). BrdU is an analogue of thymidine and can be readily incorporated into oligonucleotides (e.g., in the cleavage domain of a capture probe) and is sensitive to UVB light (280–320 nm range). Upon exposure to UVB light, photocleavage occurs (e.g., at the nucleoside immediately adjacent to the 5′ BrdU incorporation site (Doddridge et al. Chem. Comm., 1998, 18: 1997–1998 and Cook et al. Chemistry and Biology. 1999, 6: 451–459)), resulting in the release of the capture probe from the characteristic region.

[0480] Other examples of cleavage domains include unstable chemical bonds, such as, but not limited to, ester bonds (e.g., cleavable with acids, bases, or hydroxylamine), vicinal diol bonds (e.g., cleavable with sodium periodate), Diels-Alder bonds (e.g., cleavable by heat), sulfone bonds (e.g., cleavable with bases), silyl ether bonds (e.g., cleavable with acids), glycosidic bonds (e.g., cleavable with amylases), peptide bonds (e.g., cleavable with proteases), base-free or purine / pyrimidine (AP)-free sites (e.g., cleavable with bases or AP endonucleases), or phosphodiester bonds (e.g., cleavable with nucleases (e.g., DNases)).

[0481] In some embodiments, the cleavage domain includes a sequence recognized by one or more enzymes capable of cleaving nucleic acid molecules, such as those capable of breaking phosphodiester bonds between two or more nucleotides. The bonds can be cleaved by other nucleic acid molecule-targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases). For example, the cleavage domain may include a restriction endonuclease (restriction enzyme) recognition sequence. Restriction endonucleases cleave double-stranded or single-stranded DNA at a specific recognition nucleotide sequence known as a restriction site. In some embodiments, rare-cleavage restriction enzymes are used, such as enzymes with long recognition sites (at least 8 base pairs in length), to reduce the likelihood of cleavage at other locations within the capture probe.

[0482] In some embodiments, the cleavage domain includes a poly(U) sequence, which may be generated by uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER). TM The mixture of enzymes is cleaved. Once released, the releasable capture probe can be used for the reaction. Therefore, for example, the activatable capture probe can be activated by releasing the capture probe from the feature.

[0483] In some embodiments, when the capture probe is indirectly attached to the substrate, for example via a surface probe, the cleavage domain comprises one or more mismatched nucleotides such that the complementary portions of the surface probe and the capture probe are not 100% complementary (e.g., the number of mismatched base pairs can be one, two, or three base pairs). Such mismatches are recognized, for example, by MutY and T7 endonuclease I enzymes, leading to cleavage of the nucleic acid molecule at the mismatch site. As described herein, a “surface probe” can be any portion present on the surface of the substrate capable of being attached to a reagent (e.g., a capture probe). In some embodiments, the surface probe is an oligonucleotide. In some embodiments, the surface probe is a portion of the capture probe.

[0484] In some embodiments where the capture probe is indirectly (e.g., via a surface probe) attached (e.g., immobilized) to the feature, the cleavage domain includes a nickase recognition site or sequence. A nickase is a nuclease that can only cleave single strands of a DNA duplex. Therefore, the cleavage domain may include a nickase recognition site near the 5′ end of the surface probe (and / or the 5′ end of the capture probe), such that cleavage of the surface probe or the capture probe destabilizes the duplex between the surface probe and the capture probe, thereby releasing the capture probe from the feature.

[0485] Cleavage enzymes can also be used in some embodiments where the capture probe is directly attached (e.g., immobilized) to the feature. For example, the substrate can be contacted with a nucleic acid molecule hybridized to the cleavage domain of the capture probe to provide or reconstitute a cleavage enzyme recognition site, such as a cleavage helper probe. Thus, contact with the cleavage enzyme will result in cleavage of the cleavage domain, thereby releasing the capture probe from the feature. Such cleavage helper probes can also be used to provide or reconstitute cleavage recognition sites for other cleavage enzymes, such as restriction endonucleases.

[0486] Some nickases introduce single-strand cleavage only at specific sites on the DNA molecule by binding to and recognizing specific nucleotide recognition sequences. Many natural nickases have been discovered, and the sequence recognition characteristics of at least four nickases have been identified. Nickases are described in U.S. Patent No. 6,867,028, which is incorporated herein by reference in its entirety. Generally, any suitable nickase can be used to bind to a complementary nickase recognition site of the cleavage domain. After use, the nickase can be removed from the analysis or inactivated after the release of the capture probe to prevent unnecessary cleavage of the capture probe.

[0487] Examples of suitable capture domains based on nucleic acids include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any capture domain described herein.

[0488] In some implementations, the capture probe does not contain a cleavage domain. For example, an example of a substrate with an attachment capture probe lacking a cleavage domain is described in Macosko et al., (2015) Cell 161, 1202-1214, the entire contents of which are incorporated herein by reference.

[0489] In some embodiments, the capture probe region corresponding to the cleavage domain can be used for certain other functions. For example, other regions for nucleic acid extension or amplification may be included at the location where the cleavage domain would normally be located. In such embodiments, the region may complement the functional domain or even exist as another functional domain. In some embodiments, a cleavage domain is present, but its use is optional.

[0490] (iii) Functional domains

[0491] Each capture probe may optionally include at least one functional domain. Each functional domain typically includes a functional nucleotide sequence for downstream analytical steps throughout the analytical procedure.

[0492] In some implementations, the capture probe may include a functional domain for attaching to the sequencing flow cell, for example, for... The sequenced P5 sequence. In some embodiments, the capture probe or a derivative thereof may include another functional domain, for example, for attachment to... The P7 sequence of the sequencing flow cell. Selectable functional domains allow for compatibility with any of the various sequencing systems, such as 454 sequencing, ion-fluid proton or PGM, Illumina X10, and their requirements.

[0493] In some implementations, the functional domain includes a primer. The primer may include components for use with... The R1 primer sequence for sequencing, and in some embodiments, including those for... The R2 primer sequence for sequencing. Examples of such capture probes and their uses are described in U.S. Patent Publications 2014 / 0378345 and 2015 / 0376609, the entire contents of which are incorporated herein by reference.

[0494] (iv) Spatial barcode

[0495] As described above, the capture probe may include one or more spatial barcodes (e.g., two or more, three or more, four or more, five or more). A “spatial barcode” is a continuous nucleic acid segment or two or more non-contiguous nucleic acid segments used as a tag or identifier that conveys or is capable of conveying spatial information. In some embodiments, the capture probe includes spatial barcodes with spatial aspects, wherein the barcodes are associated with a specific location within an array or a specific location on a substrate.

[0496] Spatial barcodes can be part of the analyte or independent of it (e.g., part of a capture probe). A spatial barcode can be a tag attached to the analyte (e.g., a nucleic acid molecule) or a combination of tags other than endogenous characteristics of the analyte (e.g., the size or terminal sequence of the analyte). Spatial barcodes can be unique. In some implementations where the spatial barcode is unique, it functions both as a spatial barcode and as a unique molecular identifier (UMI) associated with a specific capture probe.

[0497] Spatial barcodes can have a variety of different formats. For example, spatial barcodes may include polynucleotide spatial barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. In some embodiments, spatial barcodes are attached to analytes in a reversible or irreversible manner. In some embodiments, spatial barcodes are added to fragments of, for example, DNA or RNA samples before, during, and / or after sample sequencing. In some embodiments, spatial barcodes enable the identification and / or quantification of individual sequence readouts. In some embodiments, spatial barcodes are used as fluorescent barcodes, wherein fluorescently labeled oligonucleotide probes hybridize with the spatial barcode.

[0498] In some embodiments, the spatial barcode is a nucleic acid sequence that substantially does not hybridize with the analyte nucleic acid molecule in the biological sample. In some embodiments, the spatial barcode has less than 80% sequence identity to the nucleic acid sequence on a substantial portion (e.g., 80% or more) of the nucleic acid molecule in the biological sample (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity).

[0499] The spatial barcode sequence may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the spatial barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0500] These nucleotides can be completely continuous, such as in a single sequence segment of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by one or more nucleotides. The length of the separated spatial barcode subsequences is approximately 4 to 16 nucleotides. In some embodiments, the spatial barcode subsequences can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0501] For multiple capture probes attached to a common array feature, one or more spatial barcode sequences of the multiple capture probes may include the same sequence for all capture probes coupled to the feature and / or different sequences among all capture probes coupled to the feature.

[0502] Figure 8 This is a schematic diagram illustrating an example of multi-spatial barcode features. Figure 8In this design, feature 801 can be coupled to a spatially barcoded capture probe, wherein the spatially barcoded probe for a specific feature may have the same spatial barcode but different capture domains designed to associate the spatial barcode of the feature with multiple target analytes. For example, the feature can be coupled to four different types of spatially barcoded capture probes, each type having a spatial barcode 802. One type of capture probe associated with this feature includes a combination of spatial barcode 802 and a poly(T) capture domain 803, designed to capture mRNA target analytes. A second type of capture probe associated with this feature includes a combination of spatial barcode 802 and a random N-mer capture domain 804 for gDNA analysis. A third type of capture probe associated with this feature includes a combination of spatial barcode 802 and a capture domain complementary to a capture domain on the analyte capture agent capture barcode structural domain 805. A fourth type of capture probe associated with this feature includes a combination of a spatial barcode 802 and a capture probe that specifically binds to nucleic acid molecule 806, which can function in CRISPR analysis (e.g., CRISPR / Cas9). Although Figure 8 Only four different capture probe barcoded constructs are shown, but capture probe barcoded constructs can be customized for the analysis of any given analyte related to nucleic acids and can be bound to such constructs. For example, Figure 8 The protocols illustrated can also be used for the simultaneous analysis of other analytes disclosed herein, including but not limited to: (a) mRNA, lineage-tracing constructs, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, available chromatin (e.g., ATAC-seq, DNase-seq, and / or MNAase-seq), cell surface or intracellular proteins and metabolites, and perturbants (e.g., CRISPR-crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein); and (c) V(D)J sequences of mRNA, cell surface or intracellular proteins and / or metabolites, barcode markers (e.g., MHC multimers as described herein), and immune cell receptors (e.g., T cell receptors). In some embodiments, the perturbant may be a small molecule, antibody, drug, aptamer, miRNA, physical environment (e.g., temperature change), or any other known perturbant.

[0503] Capture probes attached to a single array feature may include the same (or common) spatial barcode sequence, different spatial barcode sequences, or a combination of both. Capture probes attached to a feature may include multiple sets of capture probes. A given set of capture probes may include the same spatial barcode sequence. The same spatial barcode sequence may differ from the spatial barcode sequence of another set of capture probes.

[0504] Multiple capture probes may include spatial barcode sequences (e.g., nucleic acid barcode sequences) associated with specific locations on a spatial array. For example, a first plurality of capture probes may be associated with a first region based on a spatial barcode sequence common to capture probes within a first region, and a second plurality of capture probes may be associated with a second region based on a spatial barcode sequence common to capture probes within a second region. The second region may or may not be associated with the first region. Other plurality of capture probes may be associated with spatial barcode sequences common to capture probes within other regions. In some embodiments, the spatial barcode sequence may be identical across the plurality of capture probe molecules.

[0505] In some implementations, multiple distinct spatial barcodes are combined into a single array capture probe. For example, a mixed but known set of spatial barcode sequences can provide stronger addressing or attributes of the spatial barcode to a given point or location by offering repeated or independent verification of location characteristics. In some implementations, multiple spatial barcodes represent an increased specificity of the location of a particular array point.

[0506] (v) Unique molecular identifier

[0507] Capture probes may include one or more (e.g., two or more, three or more, four or more, five or more) unique molecular identifiers (UMIs). A unique molecular identifier is a continuous nucleic acid segment or two or more non-continuous nucleic acid segments that serve as a tag or identifier for a particular analyte or a capture probe bound to a particular analyte (e.g., by a capture domain).

[0508] UMIs can be unique. A UMI may include one or more specific polynucleotide sequences, one or more random nucleic acid and / or amino acid sequences, and / or one or more synthetic nucleic acid and / or amino acid sequences.

[0509] In some embodiments, the UMI is a nucleic acid sequence that substantially does not hybridize with the analyte nucleic acid molecule in the biological sample. In some embodiments, the UMI has less than 80% sequence identity to the nucleic acid sequence in a substantial portion (e.g., 80% or more) of the nucleic acid molecule in the biological sample (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity).

[0510] The UMI may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the UMI may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the UMI may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the UMI may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0511] These nucleotides can be completely continuous, i.e., within a single sequence segment of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by one or more nucleotides. The length of the separated UMI subsequence is approximately 4 to 16 nucleotides. In some embodiments, the UMI subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0512] In some embodiments, the UMI is attached to the analyte in a reversible or irreversible manner. In some embodiments, the UMI is added to fragments of, for example, DNA or RNA samples before, during, and / or after sequencing the analyte. In some embodiments, the UMI enables the identification and / or quantification of individual sequence reads. In some embodiments, the UMI is used as a fluorescent barcode, wherein a fluorescently labeled oligonucleotide probe hybridizes with the UMI.

[0513] (vi) Other aspects of the capture probe

[0514] For capture probes attached to an array feature, an individual array feature may include one or more capture probes. In some embodiments, an individual array feature includes hundreds or thousands of capture probes. In some embodiments, capture probes are associated with a specific individual feature, wherein the individual feature contains capture probes, which include spatial barcodes unique to a defined area or location on the array.

[0515] In some implementations, a particular feature may include capture probes comprising more than one spatial barcode (e.g., one capture probe at a particular feature may include a spatial barcode different from the spatial barcode included in another capture probe at the same particular feature, while both capture probes include a second common spatial barcode), wherein each spatial barcode corresponds to a specific defined region or location on the array. For example, multiple spatial barcode sequences associated with a particular feature on the array can provide a stronger address or attribute to a given location by providing repeating or independent confirmation of the location. In some implementations, multiple spatial barcodes represent an increased specificity of the location of a particular array point. In a non-limiting example, a particular array point may be encoded using two different spatial barcodes, wherein each spatial barcode identifies a specific defined region within the array, and an array point having both spatial barcodes identifies a sub-region where the two defined regions overlap, such as, for example, the overlapping portion of a Venn diagram.

[0516] In another non-limiting example, a particular array point can be represented by three different spatial barcodes, wherein a first spatial barcode identifies a first region within the array, a second spatial barcode identifies a second region, wherein the second region is a sub-region entirely within the first region, and a third spatial barcode identifies a third region, wherein the third region is a sub-region entirely within the first and second sub-regions.

[0517] In some embodiments, the capture probe attached to the array feature is released from the array feature for sequencing. Alternatively, in some embodiments, the capture probe remains attached to the array feature and is sequenced while remaining attached to the array feature (e.g., by in situ sequencing). Other aspects of sequencing of the capture probe are described in subsequent sections of the invention.

[0518] In some implementations, an array feature may include different types of capture probes attached to the feature. For example, an array feature may include a first type of capture probe and a second type of capture probe, the first type of capture probe having a capture domain designed to bind to a class of analytes, and the second type of capture probe having a capture domain designed to bind to a second type of analytes. Typically, an array feature may include one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more) different types of capture probes attached to a single array feature.

[0519] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is attached to an array feature via its 5′ end. In some embodiments, the capture probe from its 5′ end to its 3′ end includes: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe from its 5′ end to its 3′ end includes: a barcode (e.g., a spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe from its 5′ end to its 3′ end includes: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, the capture probe from its 5′ end to its 3′ end includes: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain, and a capture domain. In some embodiments, the capture probe from its 5′ end to its 3′ end includes: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probe does not include a spatial barcode. In some embodiments, the capture probe does not include a UMI. In some implementations, the capture probe includes a sequence used to initiate the sequencing reaction.

[0520] In some embodiments, the capture probe is fixed to the feature via its 3' end. In some embodiments, the capture probe from its 3' end to its 5' end includes: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture fields. In some embodiments, the capture probe from its 3' end to its 5' end includes: a barcode (e.g., a spatial barcode or a UMI) and a capture field. In some embodiments, the capture probe from its 3' end to its 5' end includes: a fragmentation field, a functional field, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture field. In some embodiments, the capture probe from its 3' end to its 5' end includes: a fragmentation field, a functional field, a spatial barcode, a UMI, and a capture field.

[0521] In some embodiments, the capture probe comprises an in-situ synthesized oligonucleotide. The in-situ synthesized oligonucleotide may be attached to a substrate or to a feature on the substrate. In some embodiments, the in-situ synthesized oligonucleotide comprises one or more constant sequences, one or more of which serve as initiating sequences (e.g., primers for amplifying target nucleic acids). The in-situ synthesized oligonucleotide may, for example, comprise a constant sequence located at the 3′ end, which is attached to the substrate or to a feature on the substrate. Alternatively, the in-situ synthesized oligonucleotide may include a constant sequence at the free 5′ end. In some embodiments, the one or more constant sequences may be cleavable sequences. In some embodiments, the in-situ synthesized oligonucleotide comprises a barcode sequence, such as a variable barcode sequence. The barcode can be any barcode described herein. The length of the barcode can be about 8 to 16 nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides). The length of the in-situ synthesized oligonucleotide can be less than 100 nucleotides (e.g., less than 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20 nucleotides). In some cases, the length of the in-situ synthesized oligonucleotide is about 20 to about 40 nucleotides. Exemplary in-situ synthesized oligonucleotides are produced by Affymetrix. In some embodiments, the in-situ synthesized oligonucleotide is attached to the features of the array.

[0522] Other oligonucleotides can be ligated to an in-situ synthesized oligonucleotide to generate a capture probe. For example, a primer complementary to a portion of the in-situ synthesized oligonucleotide (e.g., a constant sequence within the oligonucleotide) can be used to hybridize other oligonucleotides and extend them (using the in-situ synthesized oligonucleotide as a template, e.g., a primer extension reaction) to form a double-stranded oligonucleotide and further generate a 3′ overhang. In some embodiments, the 3′ overhang can be generated by a template-independent ligase (e.g., terminal deoxynucleotidyl transferase (TdT) or polymerase). Additional oligonucleotides containing one or more capture domains can be ligated to the 3′ overhang using suitable enzymes (e.g., ligases) and splice oligonucleotides to generate a capture probe. Thus, in some embodiments, the capture probe is the product of two or more oligonucleotide sequences linked together (e.g., an in-situ synthesized oligonucleotide and other oligonucleotides). In some embodiments, one of the oligonucleotide sequences is an in-situ synthesized oligonucleotide.

[0523] In some embodiments, a splice oligonucleotide (e.g., any splice oligonucleotide described herein) can be used to prepare the capture probe. Two or more oligonucleotides can be ligated together using a splice oligonucleotide and any type of ligase known in the art or described herein (e.g., splice ligase).

[0524] One of the oligonucleotides may include, for example, a constant sequence (e.g., a sequence partially complementary to a splice oligonucleotide), a degenerate sequence, and / or a capture domain (e.g., as described herein). One of the oligonucleotides may also include a sequence compatible with linking or hybridizing to an analyte of interest in a biological sample. The analyte of interest (e.g., mRNA) may also be used as a splice oligonucleotide to link additional oligonucleotides to the capture probe. In some embodiments, the capture probe is generated by causing an enzyme to add a polynucleotide to the end of the oligonucleotide sequence. The capture probe may include a degenerate sequence that can be used as a unique molecular identifier.

[0525] A degenerate sequence is a sequence in which certain positions of a nucleotide sequence contain a number of possible bases. A degenerate sequence can be a degenerate nucleotide sequence comprising about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In some embodiments, the nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more degenerate positions within the nucleotide sequence. In some embodiments, the degenerate sequence is used as a UMI (Unique Minute Injection).

[0526] In some embodiments, the capture probe comprises a restriction endonuclease recognition sequence or a nucleotide sequence that can be cleaved by a specific enzyme activity. For example, a uracil DNA glycosylase (UDG) or a uracil-specific excision reagent (USER) can be used to enzymatically cleave the uracil sequence from the nucleotide sequence. As another example, other modified bases (e.g., methylated) can be recognized and cleaved by a specific endonuclease. The capture probe can be subjected to enzymatic cleavage, which removes the blocking domain and any other nucleotides added to the 3′ end of the capture probe during modification. Removal of the blocking domain will expose and / or restore the free 3′ end of the capture domain of the capture probe. In some embodiments, other nucleotides may be removed to expose and / or restore the 3′ end of the capture domain of the capture probe.

[0527] In some embodiments, the blocking domain may be incorporated into the capture probe during or after its synthesis. The terminal nucleotide of the capture domain is a reversible terminator nucleotide (e.g., a 3′-O-blocking reversible terminator and a 3′-unblocking reversible terminator) and may be included in the capture probe during or after probe synthesis.

[0528] (vii) Extended capture probe

[0529] An "extended capture probe" is a capture probe with an expanded nucleic acid sequence. For example, in cases where the capture probe comprises nucleic acids, an extended "3' end" means that additional nucleotides are added to the terminal 3' nucleotide of the capture probe to extend its length, for example, by extending the nucleic acid molecule through a standard polymerization reaction, including template polymerization catalyzed by a polymerase (e.g., DNA polymerase or reverse transcriptase).

[0530] In some implementations, extending the capture probe includes generating cDNA from the captured (hybridized) RNA. This process involves synthesizing a complementary strand of the hybridized nucleic acid, for example, generating cDNA based on a captured RNA template (RNA hybridized to the capture domain of the capture probe). Thus, in the initial step of extending the capture probe (e.g., cDNA generation), the captured (hybridized) nucleic acid (e.g., RNA) serves as a template for extension (e.g., a reverse transcription step).

[0531] In some embodiments, the capture probe uses reverse transcription extension. For example, reverse transcription includes the synthesis of cDNA (complementary or copy DNA) from RNA (e.g., messenger RNA) using a reverse transcriptase. In some embodiments, reverse transcription is performed while the tissue is still in situ, producing an analyte library, wherein the analyte library includes spatial barcodes from adjacent capture probes. In some embodiments, the capture probe uses one or more DNA polymerases for extension.

[0532] In some embodiments, the capture domain of the capture probe includes primers for generating a complementary strand of a nucleic acid that hybridizes to the capture probe, such as primers for DNA polymerase and / or reverse transcription. The nucleic acid (e.g., DNA and / or cDNA) molecule produced by the extension reaction contains the sequence of the capture probe. The extension of the capture probe, such as by DNA polymerase and / or reverse transcription, can be performed using a variety of suitable enzymes and protocols.

[0533] In some embodiments, a full-length DNA (e.g., cDNA) molecule is generated. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if the nucleic acid (e.g., RNA) is partially degraded in the tissue sample, the captured nucleic acid molecule will differ in length from the initial RNA in the tissue sample. In some embodiments, the 3' end of the extended probe (e.g., a first-strand cDNA molecule) is modified. For example, a adapter or adaptor can be attached to the 3' end of the extended probe. This can be achieved by using a single-stranded ligase such as T4 RNA ligase or CircleGase. TM(Available from Epicentre Biotechnologies, Madison, Wisconsin). In some embodiments, template-converting oligonucleotides are used to extend the cDNA to produce full-length cDNA (or cDNA as close to full-length as possible). In some embodiments, a second-strand synthetic helper probe (a partial double-stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe) can be ligated to the 3′ end of the extended probe, such as the first-stranded cDNA molecule, using a double-stranded ligase (e.g., T4 DNA ligase). Other enzymes suitable for the ligation step are known in the art and include, for example, Tth DNA ligase, Taq DNA ligase, and Thermococcus spp. (strain 9°N) DNA ligase (9°N). TM DNA ligase, New England Biolabs, Ampligase TM (Available from Epicentre Biotechnologies, Madison, Wisconsin) and SplintR (available from New England Biolabs, Ipswich, Massachusetts). In some embodiments, a polynucleotide tail (e.g., a poly(A) tail) is incorporated into the 3′ end of the extended probe molecule. In some embodiments, a terminal transferase-active enzyme is used to incorporate the polynucleotide tail.

[0534] In some implementations, the double-stranded extended capture probe is treated to remove any unextended capture probes prior to amplification and / or analysis (e.g., sequence analysis). This can be achieved through various methods, such as enzymatic degradation of unextended probes, like exonucleases or purification columns.

[0535] In some embodiments, the extended capture probe is amplified to produce a sufficient quantity for analysis, such as by DNA sequencing. In some embodiments, the first strand of the extended capture probe (e.g., DNA and / or cDNA molecules) is used as a template for an amplification reaction (e.g., polymerase chain reaction).

[0536] In some embodiments, the amplification reaction uses primers comprising an affinity group to incorporate the affinity group onto an extended capture probe (e.g., RNA-cDNA hybridization). In some embodiments, the primers comprise an affinity group, and the extended capture probe comprises an affinity group. The affinity group may correspond to any affinity group described above.

[0537] In some embodiments, the extended capture probe, including an affinity group, can be coupled to an affinity group-specific array feature. In some embodiments, the substrate may include an antibody or antibody fragment. In some embodiments, the array feature includes avidin or streptavidin, and the affinity group includes biotin. In some embodiments, the array feature includes maltose, and the affinity group includes a maltose-binding protein. In some embodiments, the array feature includes a maltose-binding protein, and the affinity group includes maltose. In some embodiments, the amplified extended capture probe can serve to release the amplified probe from the array feature, provided that a copy of the extended probe is not attached to the array feature.

[0538] In some embodiments, a capture probe or its complement or amplicon extended from the array feature is released. The step of releasing the capture probe or its complement or amplicon extended from the array feature can be implemented in a variety of ways. In some embodiments, the capture probe or its complement is released from the feature by nucleic acid lysis and / or denaturation (e.g., by heating to denature double-stranded molecules).

[0539] In some embodiments, extended capture probes or their complements or amplicones are released from an array feature by physical means. For example, methods for inducing physical release include denaturing double-stranded nucleic acid molecules. Another method for releasing extended capture probes is using a solution that interferes with the hydrogen bonds of the double-stranded molecules. In some embodiments, extended capture probes are released by applying heated water (e.g., water at least 85°C or a buffer solution, such as water at least 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or 99°C). In some embodiments, solutions including salts, surfactants, etc., are added to further destabilize the interactions between nucleic acid molecules to release extended capture probes from the array feature. In some embodiments, formamide solutions can be used to destabilize the interactions between nucleic acid molecules to release extended capture probes from the array feature.

[0540] (viii) Analyte trapping agent

[0541] This invention also provides methods and materials for spatial profiling of bioanalytes (e.g., mRNA, genomic DNA, proteins and / or metabolites accessible to chromatin and cell surface or intracellular) using analyte capture agents. As used herein, an analyte capture agent (formerly sometimes also referred to as a "cell spike") is a substance that interacts with an analyte (e.g., an analyte in a sample) and a capture probe (e.g., a capture probe attached to a substrate) to identify the analyte. In some embodiments, the analyte capture agent includes an analyte-binding portion and a capture agent barcode domain.

[0542] Figure 9This is a schematic diagram of an exemplary analyte trapping agent 902, comprising an analyte binding portion 904 and a trapping agent barcode domain 908. The analyte binding portion 904 is a molecule capable of binding to an analyte 906 and interacting with a spatially barcode-coded trapping probe. The analyte binding portion can bind to the analyte 906 with high affinity and / or high specificity. The analyte trapping agent may include the trapping agent barcode domain 908, a nucleotide sequence (e.g., an oligonucleotide), which can hybridize to at least part or all of the trapping domain of the trapping probe. The analyte binding portion 904 may include a polypeptide and / or aptamer (e.g., an oligonucleotide or peptide molecule bound to a specific target analyte). The analyte binding portion 904 may include an antibody or antibody fragment (e.g., an antigen-binding fragment).

[0543] As used herein, the term "analyte-binding moiety" refers to a molecule or portion capable of binding to a macromolecular component (e.g., an analyte, such as a bioanalyte). In some embodiments of any spatial profile analysis method described herein, the analyte-binding moiety of an analyte trap bound to a bioanalyte may include, but is not limited to, antibodies or epitope-binding fragments thereof, cell surface receptor-binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T-cell conjugates, T-cell receptor conjugates, B-cell receptor conjugates, precursors, aptamers, monomers, adhesins, DARPin, and protein scaffolds, or any combination thereof. The analyte-binding moiety may bind to a macromolecular component (e.g., an analyte) with high affinity and / or high specificity. The analyte-binding moiety may include a nucleotide sequence (e.g., an oligonucleotide) that may correspond to at least part or all of the analyte-binding moiety. The analyte-binding moiety may include peptides and / or aptamers (e.g., peptides and / or aptamers bound to a specific target molecule, such as an analyte). The analyte-binding moiety may include an antibody or antibody fragment (e.g., an antigen-binding fragment) bound to a specific analyte (e.g., a peptide).

[0544] In some embodiments, the analyte-binding portion of an analyte capture agent comprises one or more antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment including the analyte-binding portion can specifically bind to a target analyte. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, multiple analyte capture agents comprising multiple analyte-binding portions bind multiple analytes present in a biological sample. In some embodiments, the multiple analytes comprise a single class of analytes (e.g., a single class of peptides). In some embodiments where the multiple analytes comprise a single class of analytes, the analyte-binding portions of the multiple analyte capture agents are identical. In some embodiments where the multiple analytes comprise a single class of analytes, the analyte-binding portions of the multiple analyte capture agents are different (e.g., a member of the multiple analyte capture agents may have two or more analyte-binding portions, each of which binds a single class of analyte (e.g., at a different binding site)). In some embodiments, the multiple analytes comprise multiple different classes of analytes (e.g., multiple different classes of peptides).

[0545] An analyte trap may include an analyte-binding moiety. The analyte-binding moiety may be an antibody. Exemplary non-limiting antibodies that can be used as the analyte-binding moiety in an analyte trap or for the IHC / IF applications disclosed herein include any of the following, including variants thereof: A-ACT, A-AT, ACTH, actin-muscle-specific, actin-smooth muscle (SMA), AE1, AE1 / AE3, AE3, AFP, AKT phosphate, ALK-1, amyloid A, androgen receptor, annexin A1, B72.3, BCA-225, BCL-1 (cyclin D1), BCL-1 / CD20, BCL-2, BCL-2 / BCL-6, BCL-6, Ber-EP4, β-amyloid, β-catenin, BG8 (Lewis Y), BOB-1, CA 19.9, CA125, CAIX, calcitonin, calmodulin-binding protein, calmodulin, calreticulin, CAM 5.2, CAM 5.2 / AE1, CD1a, CD2, CD3(M), CD3(P), CD3 / CD20, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD20, CD21, CD22, CD 23, CD25, CD30, CD31, CD33, CD34, CD35, CD43, CD45(LCA), CD45RA, CD56, CD57, CD61, CD68, CD71, CD74, CD79a, CD99, CD117(c-KIT), CD123, CD138, CD163, CDX-2, CDX-2 / CK-7, CEA(M), CEA(P), Chromogranin A, Chymotrypsin, CK-5, CK-5 / 6, CK-7, CK-7 / TTF-1, CK-14, CK-17, CK-18, CK-19, CK-20, CK-H MW, CK-LMW, CMV-IH, COLL-IV, COX-2, D2-40, DBA44, myodermal dermin, DOG1, EBER-ISH, EBV (LMP1), E-cadherin, EGFR, EMA, ER, ERCC1, Factor VIII (vWF), Factor XIIIa, myofascitis, FLI-1, FHS, galactagogue-3, gastrin, GCDFP-15, GFAP, glucagon, blood group glycoprotein A, phosphatidylinositol glycan-3, granzyme B, growth hormone (GH), GST, HAM56, HMBE-1, HBP, HCAg, HCG, hemoglobin A, HEP B CORE (HBcAg), HEP B SURF (HBsAg), HepPar1, HER2, Herpes I, Herpes II, HHV-8, HLA-DR, HMB 45, HPL, HPV-IHCHPV(6 / 11)-ISH, HPV(16 / 18)-ISH, HPV(31 / 33)-ISH, HPV WSS-ISH, High HPV-ISH, Low HPV-ISH, High and Low HPV-ISH, IgA, IgD, IgG, IgG4, IgM, Inhibin, Insulin, JC Virus-ISH, κ-ISH, KERPAN, Ki-67, λ-IHC, λ-ISH, LH, Lipase, Lysozyme (MURA), Globin, MART-1, MBP, M-Cellular Trypsin, MEL-5, Melan-A, Melan-A / Ki-67, Mesothelin, MiTF, MLH-1, MOC-31, MPO, MSH-2, MSH-6, MUC1, MUC2, MUC4, MUC5AC, MUM-1, MYO D1, myopoietin, myoglobin, myoin heavy chain, neoaspartate protease A, NB84a, NEW-N, NF, NK1-C3, NPM, NSE, OCT-2, OCT-3 / 4, OSCAR, p16, p21, p27 / Kip1, p53, p57, p63, p120, P504S, pan-melanoma, PANC.POLY, parvovirus B19, PAX-2, PAX-5, PAX-5 / CD43, PAX=5 / CD5, PAX-8, PC, PD1, perforin, PGP 9.5, PLAP, PMS-2, PR, prolactin, PSA, PSAP, PSMA, PTEN, PTH, PTS, RB, RCC, S6, S100, serotonin, somatostatin, surfactant (SP-A), synaptic vesicle protein, synuclein, TAU, TCL-1, TCRβ, TdT, thrombomodulin, thyroglobulin, TIA-1, TOXO, TRAP, TriView. TM Chest, TriView TM Prostate, trypsin, TS, TSH, TTF-1, tyrosinase, ubiquitin, urinary protein, VEGF, chorionic villi, vimentin (VIM), VIP, VZV, WT1(M)N-terminus, WT1(P)C-terminus, ZAP-70.

[0546] In addition, exemplary non-limiting antibodies that can be used as the analyte binding portion in an analyte trap or for the IHC / IF applications disclosed herein include any of the following antibodies (and their variants): cell surface proteins, intracellular proteins, kinases (e.g., the AGC kinase family (e.g., AKT1, AKT2, PDK1, protein kinase C, ROCK1, ROCK2, SGK3), the CAMK kinase family (e.g., AMPK1, AMPK2, CAMK, Chk1, Chk2, Zip), the CK1 kinase family, the TK kinase family (e.g., Abl2, AXL, CD167, CD246 / ALK, c-Met, CSK, c-Src, EGFR, ErbB2 (HER2 / neu), ErbB3, ErbB4, FAK, Fyn, LCK, Lyn, PKT7, Syk, Zap70), and the STE kinase family (e.g., ASK1, MAPK, MEK1, MEK2, MEK3). MEK4, MEK5, PAK1, PAK2, PAK4, PAK6), CMGC kinase family (e.g., Cdk2, Cdk4, Cdk5, Cdk6, Cdk7, Cdk9, Erk1, GSK3, Jnk / MAPK8, Jnk2 / MAPK9, JNK3 / MAPK10, p38 / MAPK), and TKL kinase family (e.g., ALK1, ILK1, IRAK1, IRAK2, IRAK3, IRAK4, LIMK1, LIMK2, M3K11, RAF1, RIP1, RIP3, VEGFR1, VEGFR2, VEGFR3), Aurora A kinase, Aurora B kinase, IKK, Nemo-like kinase, PINK, PLK3, ULK2, WEE1, transcription factors (e.g., FOXP3, ATF3, BACH1, EGR, ELF3, FOXA1, FOXA2, FOX01, GATA), growth factor receptors, tumor suppressors (e.g., anti-p53, anti-BLM, anti-Cdk2, anti-Chk2, anti-BRCA-1, anti-NBS1, anti-BRCA-2, anti-WRN, anti-PTEN, anti-WT1, anti-p38).

[0547] In some embodiments, the analyte trapping agent is capable of binding analytes present within the cell. In some embodiments, the analyte trapping agent is capable of binding cell surface analytes, which may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, and post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) states, gap junctions, and adhesion junctions of cell surface proteins. In some embodiments, the analyte trapping agent is capable of binding to post-translational modified cell surface analytes. In such embodiments, the analyte trapping agent may be specific to cell surface analytes based on a given state of post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation), such that the cell surface analyte profile may include post-translational modification information of one or more analytes.

[0548] In some embodiments, the analyte capture agent includes a capture agent barcode domain coupled to or otherwise attached to the analyte binding moiety. In some embodiments, the capture agent barcode domain is covalently linked to the analyte binding moiety. In some embodiments, the capture agent barcode domain is a nucleic acid sequence. In some embodiments, the capture agent barcode domain includes an analyte binding moiety barcode and an analyte capture sequence.

[0549] As used herein, the term "analyte binding portion barcode" refers to a barcode associated with or otherwise identifying an analyte binding portion. In some embodiments, an analyte bound to an analyte binding portion can also be identified by recognizing the analyte binding portion and the associated analyte binding portion barcode. An analyte binding portion barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the analyte binding portion. An analyte binding portion barcode can generally include any of the various aspects of barcodes described herein. For example, an analyte-specific analyte capture agent of one type may have a first capture agent barcode domain coupled thereto (e.g., which includes a first analyte binding portion barcode), while analyte capture agents of different analyte specificities may have different capture agent barcode domains coupled thereto (e.g., which include a second barcode analyte binding portion barcode). In some aspects, such a capture agent barcode domain may include an analyte binding portion barcode that identifies the analyte binding portion to which the capture agent barcode domain is coupled. The selection of capture agent barcode domains allows for significant sequence diversity, while also enabling easy attachment to most analyte binding sites (e.g., antibodies or aptamers) and easy detection (e.g., using sequencing or array technologies).

[0550] In some embodiments, the capture agent barcode domain of the analyte capture agent includes an analyte capture sequence. As used herein, the term "analyte capture sequence" refers to a region or portion configured to hybridize to, bind to, couple to, or otherwise interact with the capture domain of a capture probe. In some embodiments, the analyte capture sequence includes a nucleic acid sequence complementary or substantially complementary to the capture domain of a capture probe, such that the analyte capture sequence hybridizes with the capture domain of the capture probe. In some embodiments, the analyte capture sequence comprises a poly(A) nucleic acid sequence that hybridizes with a capture domain containing a poly(T) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a poly(T) nucleic acid sequence that hybridizes with a capture domain containing a poly(A) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a non-homogeneous nucleic acid sequence that hybridizes with a capture domain containing a non-homogeneous nucleic acid sequence complementary (or substantially complementary) to a non-homogeneous nucleic acid sequence in the analyte capture region.

[0551] In some embodiments of any spatial analysis method described herein using analyte trapping agents, trapping agent barcode domains may be directly coupled to the analyte binding portion, or they may be attached to beads, molecular lattices, such as linear, spherical, cross-linked, or other polymers, or other frameworks attached to or otherwise associated with the analyte binding portion, enabling t...

Claims

1. A method for preparing biological samples for space analysis, the method comprising: The first substrate and the second substrate are fixed to the support device, the support device comprising: A first component includes a first holding mechanism configured to hold a first substrate comprising a biological sample; A second component includes a second holding mechanism configured to hold a second substrate comprising an array, the array including a plurality of capture probes, wherein the capture probes include (i) spatial barcodes and (ii) capture fields; and An alignment mechanism, the alignment mechanism including a hinge connecting a first component and a second component; A reagent medium comprising a permeation reagent is added to the first substrate and / or the second substrate; and The first and second components are aligned such that, when the first and second components are aligned, the biological sample is aligned with the array, and thus the reagent medium contacts the biological sample and the array, wherein the alignment includes rotating the first or second component about the axis of the hinge.

2. The method of claim 1, wherein alignment includes maintaining the interval between the first and second substrates when the first and second substrates are aligned.

3. The method of claim 1, wherein alignment includes maintaining the first and second substrates in an approximately parallel relationship when the first and second substrates are aligned.

4. The method of claim 3, wherein the angle between the first and second substrates is two degrees or less.

5. The method of claim 1, wherein the support device further comprises one or more spacer members configured to maintain the spacing or parallel arrangement of the first and second substrates.

6. The method of claim 1, wherein the array comprises a plurality of features, wherein: The feature includes multiple sets of capture probes, each of which includes the same spatial barcode, and other features of the multiple features include multiple sets of capture probes that include different spatial barcodes; and / or the feature includes embossed dots, gel beads, gel pads, or any combination thereof.

7. The method of claim 1, further comprising obtaining an image of a biological sample at least partially aligned with the array through one or more apertures defined by the first and / or second member.

8. The method of claim 1, further comprising capturing a first analyte, a second analyte, and / or a third analyte from the biological sample, wherein: The capture domain of the capture probe specifically binds to the first analyte; The second capture probe of the plurality of capture probes includes a second spatial barcode and a second capture domain, wherein the second capture domain specifically binds to the second analyte; and / or The third capture probe of the plurality of capture probes includes a third spatial barcode and a third capture domain, wherein the third capture domain specifically binds to the analyte capture sequence of the analyte capture agent, wherein the analyte capture agent includes an analyte binding portion that specifically binds to the third analyte, an analyte binding portion barcode, and an analyte capture sequence.

9. The method of claim 8, wherein the spatial barcode, the second spatial barcode, and the third spatial barcode comprise the same sequence.

10. The method of claim 8, wherein the spatial barcode, the second spatial barcode, and the third spatial barcode comprise different sequences.

11. The method of claim 8, wherein the first analyte and the second analyte comprise nucleic acids.

12. The method of claim 11, wherein the nucleic acid is RNA.

13. The method of claim 11, wherein the nucleic acid is DNA.

14. The method of claim 8, wherein the analyte binding portion comprises an antibody or an antigen-binding fragment thereof.

15. The method of claim 14, wherein the third analyte comprises a protein.

16. The method of claim 8, further comprising the steps of using the first analyte as a template to extend the 3' end of the capture probe, using the second analyte as a template to extend the 3' end of the second capture probe, or using the analyte combined with a portion of a barcode as a template to extend the 3' end of the third capture probe.

17. The method of claim 8, wherein the second analyte is a ligation product generated by ligating a first probe and a second probe that hybridize with a nucleic acid analyte from a biological sample, and wherein the method further comprises ligating a first probe and a second probe that hybridize with the nucleic acid analyte.

18. The method of claim 8, wherein capturing comprises contacting the biological sample and the array with the reagent medium.

19. The method of claim 18, wherein the reagent medium comprises a permeabilizing enzyme, a nuclease, a buffer solution, a detergent, or any combination thereof.

20. The method of claim 18, wherein the permeabilizing agent comprises a solid, liquid, gel, or dry permeabilizing agent.

21. The method of claim 20, wherein the permeation agent promotes the release of the analyte from the biological sample.

22. The method of claim 18, wherein the reagent medium comprises one or more of the following: dodecyl sulfate (SDS), proteinase K, pepsin, N-lauroyl sarcosine, RNase and its sodium salt.

23. The method of claim 18, further comprising adding a reagent medium to the second substrate.

24. The method of claim 23, wherein the reagent medium is added before aligning the first and second components.

25. The method of claim 1, wherein alignment further includes observing or imaging through one or more apertures defined by the first and / or second member and adjusting the position of the biological sample relative to the array.

26. The method of claim 1, wherein the first and / or second substrate includes one or more reference markers, and wherein alignment includes adjusting the position of the first and / or second substrate such that the one or more reference markers are aligned with each other.

27. The method of claim 1, wherein the first and / or second component comprises one or more reference markers, and wherein alignment comprises adjusting the position of the first and / or second component such that the biological sample is aligned with one or more reference markers of the first and / or second component.

28. A method for capturing analytes from biological samples for space analysis, the method comprising: A support device is provided that holds a first substrate, a second substrate, and a reagent medium, the first substrate being on a first member of the support device, the second substrate being on a second member of the support device, the reagent medium comprising a permeation reagent, wherein the first substrate comprises a biological sample fixed thereon, and the second substrate comprises an array comprising a plurality of capture probes, wherein the capture probes of the plurality of capture probes comprise (i) spatial barcodes and (ii) capture domains, and wherein the biological sample comprises an analyte; Align the first and second substrates such that when the first and second components are aligned, the biological sample is at least partially aligned with the array, and the reagent medium is in contact with the biological sample and the array, wherein the support device includes a hinge connecting the first and second components, and the alignment includes rotating the first or second component about an axis of the hinge. The analyte is passively or actively migrated from the biological sample to the array; and The analytes are captured by the array.

29. A method comprising: A first substrate containing a biological sample is fixed to a first member of a support device, such that a first holding mechanism on the first member holds the first substrate on the first member. The second substrate is fixed to the second member of the support device such that the second holding mechanism on the second member holds the second substrate on the second member, the second substrate comprising a capture probe array, wherein the capture probes of the capture probe array include (i) spatial barcodes and (ii) capture fields; A reagent solution is added to the first substrate and / or the second substrate, the reagent solution comprising a permeation reagent; and A hinge connected to the first and second components of the support device is operated to move the first component and / or the second component such that the biological sample comes into at least partial contact with the capture probe array through the reagent solution, wherein the operation includes rotating the first component or the second component about the axis of the hinge.

30. The method of claim 29, further comprising maintaining a gap between the first substrate and the second substrate when the biological sample comes into contact with at least a portion of the capture probe array via a reagent solution.

31. The method of claim 29, further comprising maintaining an angle of 2 degrees or less between the first substrate and the second substrate when the biological sample comes into contact with at least a portion of the capture probe array via a reagent solution.

32. The method of claim 29, further comprising maintaining the first and second substrates spaced apart or arranged in parallel as the biological sample comes into contact with at least a portion of the capture probe array via a reagent solution.

33. The method of claim 29, wherein the capture probe array comprises a plurality of features, wherein: The plurality of features include multiple sets of capture probes, wherein each capture probe in the plurality of sets of capture probes includes the same spatial barcode, and other features of the plurality of features include multiple sets of capture probes that include different spatial barcodes; and / or The features include printing dots, gel beads, gel pads, or any combination thereof.

34. The method of claim 29, further comprising obtaining an image of at least a portion of a biological sample in contact with at least a portion of the capture probe array via a reagent solution, wherein obtaining an image of at least a portion of the biological sample comprises obtaining an image of at least a portion of the biological sample through one or more orifices defined by the first and / or second member.

35. The method of claim 29, further comprising capturing a first analyte, a second analyte, and / or a third analyte from the biological sample, wherein: The capture domain of the capture probe specifically binds to the first analyte; The second capture probe of the capture probe array includes a second spatial barcode and a second capture domain, wherein the second capture domain specifically binds to the second analyte; and / or The third capture probe of the capture probe array includes a third spatial barcode and a third capture domain, wherein the third capture domain specifically binds to the analyte capture sequence of the analyte capture agent, wherein the analyte capture agent includes an analyte binding portion, an analyte binding portion barcode, and an analyte capture sequence, wherein the analyte binding portion specifically binds to a third analyte.

36. The method of claim 35, wherein the spatial barcode, the second spatial barcode, and the third spatial barcode comprise the same sequence.

37. The method of claim 35, wherein the spatial barcode, the second spatial barcode, and the third spatial barcode comprise different sequences.

38. The method of claim 35, wherein the first analyte and the second analyte comprise nucleic acids.

39. The method of claim 38, wherein the nucleic acid is RNA.

40. The method of claim 38, wherein the nucleic acid is DNA.

41. The method of claim 35, wherein the analyte binding portion comprises an antibody or an antigen-binding fragment thereof.

42. The method of claim 41, wherein the third analyte comprises a protein.

43. The method of claim 35, further comprising the steps of using the first analyte as a template to extend the 3' end of the capture probe, using the second analyte as a template to extend the 3' end of the second capture probe, or using the analyte combined with a portion of a barcode as a template to extend the 3' end of the third capture probe.

44. The method of claim 29, wherein the reagent solution comprises a permeabilizing enzyme.

45. The method of claim 29, wherein the reagent solution is added prior to the operation.

46. ​​The method of claim 29, further comprising observing or imaging at least a portion of the first and / or second substrate through one or more apertures defined by the first and / or second member and adjusting the position of the biological sample relative to the array.

47. The method of claim 29, wherein the first and / or second substrate includes one or more reference markers, and wherein the operation includes moving the first and / or second substrate such that the one or more reference markers are aligned with each other.

48. The method of claim 29, wherein the first and / or second component includes one or more reference markers, and wherein the operation includes moving the first substrate and / or the second substrate such that the biological sample is aligned with one or more reference markers of the first and / or second component.

49. The method of claim 29, wherein the operation includes operating the alignment mechanism to move the first member toward the second member and / or move the second member toward the first member.

50. The method of claim 29, wherein the operation includes operating the actuator to move the first member and / or the second member.

51. The method of claim 50, wherein the actuator is a rotary actuator, and the operation includes operating the alignment mechanism to rotate the first member and / or the second member.

52. The method of claim 29, further comprising operating an adjustment mechanism to adjust the distance between the first member and / or the second member in a direction orthogonal to the surface of the first substrate on which the biological sample is located.

53. The method of claim 52, wherein the adjustment mechanism includes a linear actuator to translate the first member and / or the second member in a direction orthogonal to the surface of the first substrate on which the biological sample is located.

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