Volumetric next-generation in situ sequencing

Abstract

A sequencing apparatus is provided for automated in situ sequencing of volumetric tissue samples. In particular, an automated volumetric in situ sequencing device capable of operating in parallel on multiple samples is provided. Methods of making and using the sequencing apparatus are also provided.

Description

[Background technology]

[0001] Biological samples contain complex and heterogeneous genetic information that spans the length scale of individual cells to whole tissues. Spatial patterns of nucleic acids within cells may reveal properties and abnormalities of cell function, cumulative distribution of RNA expression may define cell type or function, and systematic variations in the location of cell types within tissues may define tissue function. The combination of anatomical connectivity information encoded in nucleic acids and cell type distribution throughout tissues may span many tissue regions and sections. Thus, techniques for in situ nucleic acid sequencing must be able to bridge resolutions as small as individual molecules and as large as the entire brain. To efficiently collect and record this information across orders of magnitude of length, new inventions are needed to enhance the robustness, rapidity, automation, and high-throughput nature of in situ sequencing techniques. Summary of the Invention

[0002] A sequencing device is provided for automated in situ sequencing of volumetric tissue samples. In particular, an automated volumetric in situ sequencing device is provided that can operate in parallel at high resolution on multiple samples. The sequencing device combines automated immersion and automated in situ sequencing functions. The sequencing device is particularly useful for combinatorial sequencing that benefits from its high resolution capabilities. Methods of making and using the sequencing apparatus are also provided.

[0003] In one aspect, a sequencing device is provided, the device comprising: (a) a spinning disk confocal component including multiple laser lines for illumination with flat illumination correction, where the multiple laser lines are used to illuminate a sample with excitation light at one or more wavelengths; a bandpass emission filter; a longpass image splitter; a first camera that detects fluorescent emission in a first wavelength range; and a second camera that detects fluorescent emission in a second wavelength range, wherein the first camera and the second camera are capable of simultaneously detecting emission; (b) a microscope module comprising a motorized stage capable of multi-axis positioning along the x, y, and z axes, an objective Z drive, and an objective turret wheel comprising a plurality of objectives, each objective providing a different magnification, one or more of the objectives being immersion objectives, each immersion objective having an objective immersion collar and optics, the optics transmitting light from the objectives to the illumination and detection module; and (c) an automated immersion media module comprising: i) a container containing an immersion medium; and ii) an objective lens of the container and the microscope module. (iii) a series of pumps connected to the fluid lines and a microcontroller, the microcontroller controlling the pumping of immersion medium through the fluid lines, the automated immersion medium module providing a controlled volume of immersion medium to the objective immersion collar on top of the objective during imaging; (d) a multiwell plate, the motorized stage being capable of moving to position wells of the multiwell plate under the objective used for imaging; (e) a fluid connection tower, the fluid connection tower being on top of the motorized stage and for positioning the fluid lines within the wells of the multiwell plate; and (f) a fluid management module, the fluid connection tower comprising a symmetric rotary valve including a rotary valve mechanism and a pump, the pump connected to the fluid lines and an air bubble detector, the air bubble detector being configured toand a pump disposed on either side of the fluid line leading to the pump, the fluid management module enabling unidirectional or bidirectional movement of reagents, buffers, and waste through the fluid lines; (g) a reagent, buffer, and waste module comprising: i) a slide tray, where reagent cartridges and buffer cartridges may be disposed within the slide tray and coupled to the fluid management module; ii) a waste module comprising a waste container, where the waste container is coupled to a fluid line from the fluid management pump; and iii) a capping mechanism, where the capping mechanism enables the waste container to be removed for waste disposal. (h) an electrical module comprising: i) a first firmware board that controls media dispensed from the automated immersion media module; and ii) a second firmware board that controls the fluid management module and the reagent, buffer and waste module, the electrical module regulating power to other modules of the system; and (i) a processor programmed to provide a user interface and to operate the modules of the sequencing device.

[0004] In certain embodiments, the plurality of laser lines comprises at least 4 laser lines. In certain embodiments, the plurality of laser lines comprises at least 5 laser lines. In some embodiments, the bandpass emission filter is a penta-bandpass emission filter.

[0005] In one particular embodiment, the motorized stage has a piezo z-axis.

[0006] In one particular embodiment, the immersion medium is water.

[0007] In certain embodiments, the immersion medium is filtered and bubble-free.

[0008] In certain embodiments, the sequencing device further comprises an O-ring and a shrink-wrap coating on each objective lens.

[0009] In certain embodiments, the sequencing device further comprises a pressure monitor for monitoring the pressure in the fluid line, where an increase in pressure in the fluid line can be used to detect a potential blockage in the fluid line.

[0010] In certain embodiments, the sequencing device further comprises a plurality of light emitting diodes (LEDs), each capable of emitting light to provide a status indication for the system.

[0011] In certain embodiments, the sequencing device further comprises a display component for displaying information and providing a user interface.

[0012] In certain embodiments, the processor includes the steps of: (a) positioning selected samples in a multi-well plate; (b) detecting signals in an XY plane at low magnification from the selected samples using wide field imaging mode acquisition with camera binning; (c) segmenting an XY bounding box around the samples using the signals; (d) imaging the samples within the XY bounding box to generate an image, wherein the imaging is performed in confocal imaging mode at a higher magnification in Z than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint and across the XY range of the previously determined Z range; (e) displaying the image generated in step (d); (f) providing an interface for a user to probe a desired XY region of interest in the samples to be further imaged during sequencing of the selected samples; and (g) imaging the previously sampled samples in the XY bounding box to generate an image, wherein the imaging is performed in confocal imaging mode at a higher magnification than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint and across the XY range of the previously determined Z range. (h) imaging the sample in a selected XY region of interest over a framed Z range; (i) segmenting the image of the sample in the region of interest along the Z range; (j) providing an interface to the user for the user to adjust the Z range of the sample volume before starting sequencing, where the range of imaging derived from the region of interest defined by the user is automatically converted to an appropriate montaged field of view for a given imaging objective lens, and adjusting the microscope stage position, objective lens Z placement, and piezo boundaries for imaging of the region of interest along the XYZ axes during sequencing; and (k) repeating steps (a)-(j) multiple times to define a region of interest for each sample in the multiwell plate that the user intends to sequence.

[0013] In certain embodiments, the processor is further programmed to perform steps including providing an interface to the user for the user to select one or more samples for sequencing and a sequencing protocol, where the number of samples the user may select is limited depending on the amount of buffers and reagents available and the sequencing protocol selected; providing constraints on the total sequencing time across all samples to be sequenced and imaged, the total data acquired, the rate of acquisition, and the maximum total volume of regions of interest; and suggesting a protocol that maximizes sequencing of the desired regions of interest in the samples within the constraints.

[0014] In certain embodiments, the processor is further programmed to optimize sample sequencing parallelism as a function of the number of samples to be sequenced and the imaging type used for sequencing.

[0015] In one particular embodiment, the processor is further programmed to perform steps including performing a rapid confocal sweep in Z at a starting XY position of a given sample montage to determine a Z profile of the sample at the starting XY position; determining the top and bottom interfaces of the sample using a segmentation method; and setting the Z position of the objective at a fixed distance from the interfaces at the start of the sample montage, such that drift in Z of the sample relative to the stage and objective across rounds is reduced to below a selected tolerance to facilitate downstream sub-pixel registration across rounds during post-acquisition processing.

[0016] In certain embodiments, the sequencing is in situ sequencing of the target nucleic acid in a tissue sample. In some embodiments, the tissue sample is a thick tissue slice having a thickness of 50-200 μm. In other embodiments, the tissue sample is a thin tissue slice having a thickness of 5-20 μm. In some embodiments, the in situ sequencing is sequential or combined in situ sequencing.

[0017] In another aspect, a method of using the sequencing device described herein is provided, the method comprising loading samples into a multi-well plate, selecting which samples in the multi-well plate are to be sequenced, selecting a sequencing protocol, and sequencing the nucleic acid in the selected sample using the sequencing device described herein. In certain embodiments, the sequencing is in situ sequencing of a target nucleic acid in a tissue sample. In some embodiments, the tissue sample is a thick tissue slice having a thickness of 50-200 μm. In other embodiments, the tissue sample is a thin tissue slice having a thickness of 5-20 μm. In some embodiments, the in situ sequencing is sequential or combinatorial in situ sequencing.

[0018] In another aspect, a computer-implemented method is provided, the computer comprising: (a) positioning selected samples in a multi-well plate; (b) detecting signals in an XY plane at low magnification from the selected samples using wide field imaging mode acquisition with camera binning; (c) using the signals to segment an XY bounding box around the samples; (d) imaging the samples in the XY bounding box to generate an image, the imaging being performed in a confocal imaging mode at a higher magnification in Z than used in step (b) with camera binning to determine an approximate Z range of the samples, where a single Z plane is collected through the midpoint of the previously determined Z range and across the XY range; (e) displaying the image generated in step (d); and (f) providing an interface for a user to select a desired XY region of interest in the samples to be further imaged during sequencing of the selected samples. (g) imaging the sample in a selected XY region of interest over a previously sampled Z range; (h) calculating a sample volume of the region of interest and displaying the calculated sample volume of the region of interest to the user; (i) segmenting the image of the sample in the region of interest along the Z range; (j) providing an interface to the user for the user to adjust the Z range of the sample volume before starting sequencing, where the range of imaging derived from the region of interest defined by the user is automatically converted to an appropriate montaged field of view for a given imaging objective lens and adjusts the microscope stage position, objective lens Z placement, and piezo boundaries for imaging of the region of interest along the XYZ axes during sequencing; and (k) repeating steps (a)-(j) multiple times to define a region of interest for each sample in the multiwell plate that the user intends to sequence.

[0019] In another aspect, a computer-implemented method is provided, the computer performing steps including: providing an interface to a user for the user to select one or more samples for sequencing and a sequencing protocol, where the number of samples that the user may select is limited depending on the amount of buffers and reagents that are available and the sequencing protocol selected; providing constraints on total sequencing time across all samples to be sequenced and imaged, total data acquired, rate of acquisition, and maximum total volume of regions of interest; and proposing a protocol that maximizes sequencing of desired regions of interest in the samples within the constraints. In some embodiments, the computer is further programmed to optimize sample sequencing parallelism depending on the number of samples to be sequenced and the imaging type used for sequencing.

[0020] In another aspect, a computer-implemented method is provided, where the computer performs the steps including performing a rapid confocal sweep in Z at a starting XY position of a given sample montage to determine a Z profile of the sample at the starting XY position, determining top and bottom interfaces of the sample using a segmentation method, and setting the Z position of the objective at a fixed distance from the interfaces at the start of the sample montage, such that drift in Z of the sample relative to the stage and objective across rounds is reduced to below a selected tolerance to facilitate downstream sub-pixel registration across rounds during post-acquisition processing.

[0021] In another aspect, a non-transitory computer-readable medium is provided that includes program instructions that, when executed by a processor in a computer, cause the processor to perform any of the computer-implemented methods described herein.

[0022] In another aspect, an automated immersion medium module comprises: (a) a container containing immersion medium; (b) fluid lines connected to the container and to an objective immersion collar of an objective lens of a microscope module, the fluid lines carrying immersion medium to and from the objective immersion collar, the immersion collar capturing excess immersion medium; and (c) a series of pumps connected to the fluid lines and a microcontroller, the microcontroller controlling the pumping of immersion medium through the fluid lines, wherein the automated immersion medium module provides a controlled volume of immersion medium to the objective immersion collar on top of the objective lens during imaging.

[0023] In another aspect, a method of using an automated immersion medium module is provided, the method including using the automated immersion medium module to deliver immersion medium to an objective immersion collar attached to an immersion objective of a microscope.

[0024] In another aspect, a fluid management module is provided, the module comprising a symmetric rotary valve including a rotary valve mechanism and a pump, the pump connected to a fluid line and an air bubble detector, the air bubble detector being disposed on either side of the fluid line leading to the pump, the fluid management module enabling bidirectional or unidirectional movement of reagents, buffers and waste through the fluid lines.

[0025] In another aspect, a reagent, buffer, and waste module is provided that includes: (a) a slide tray, where reagent cartridges and buffer cartridges can be placed in the slide tray and coupled to a fluid management module; (b) a waste module including a waste container, where the waste container is coupled to a fluid line from a fluid management pump; and (c) a capping mechanism that closes the waste container when the waste container is removed from the system for waste disposal and opens the waste container when the waste container is returned to the system. [Brief description of the drawings]

[0026] [Figure 1] 1 shows a sequencing device comprising various modules. [Diagram 2] A dual camera capable of dual imaging in wide field and confocal modes is shown. [Diagram 3] Five laser lines covering 405 nm, 488 nm, 561 nm, 637 nm, and 730 nm are shown, as well as a beam conditioning unit. [Figure 4] 1 shows microscope components. [Diagram 5] Shown is the nosepiece in six positions with objectives having magnifications of 4x, 20x, 40x, and 60x. Watercolor is shown for the 40x and 60x objectives. [Figure 6] 1 shows a manual fluid connection system for a multiwell plate having 24 wells. [Figure 7] 1 shows a fluid connection system with a multiwell plate on top of an XY motorized stage with a nosepiece and piezo Z. [Figure 8] 1 illustrates an automated fluid delivery system. [Figure 9] 1 illustrates components of an automated fluid delivery system. [Figure 10] Shown is an enclosure for light-sensitive samples on top of a custom table that provides vibration isolation. Five laser lines with covers and beam conditioning units, as well as a workstation with a processor for high data throughput imaging, are shown on a shelf below the top of the table. Also shown is a 4K display component connected to the table. [Figure 11A] 1 shows a board port for an automated fluid delivery system. [Figure 11B] 1 shows a board port for an automated fluid delivery system. [Figure 12A] 1 shows the assembly of modular parts of a sequencing device. [Figure 12B]1 shows the assembly of modular parts of a sequencing device. [Figure 13] 1 shows a schematic of a stand-alone fluidic module that interfaces with an existing imaging setup including: 1) microscope and table, 2) multi-well plate, and 3) sequencing instrument, shown from various angles. [Figure 14] 1 shows a schematic of a multi-well plate and cover. [Figure 15] 1 shows a schematic of a multiwell plate having multiple fluid lines connected to it and inserted into several selected wells of the multiwell plate. [Figure 16] 1 shows a schematic of a multiwell plate having multiple fluid lines connected to it and inserted into all of the wells of the multiwell plate. [Figure 17] 1 shows a multiwell plate with a cover over the wells, where for each well of the multiwell plate the cover comprises a holder for a fluid line that guides the insertion of the fluid line into a hole in the cover over the well. [Figure 18] Schematics of the sequencing apparatus from various angles, showing 1) a microscope and table, 2) a covered multiwell plate, and 3) a multiwell plate either connected to the sequencing apparatus or removed from the sequencing apparatus. [Figure 19] 1 illustrates the design of a compact automated fluid delivery system. [Figure 20] 1 illustrates the design of a compact automated fluid delivery system. [Figure 21] The design for the buffer and reagent trays is shown. [Figure 22] 1 shows a buffer tray design that includes a carrier for sealed bottles of buffer and an RFID tag for tracking. [Diagram 23] 1 shows alternative designs for the buffer tray. At the top is a buffer tray with caps for the individual buffers. At the bottom is a buffer tray designed to hold sealed bottles of buffers. [Figure 24]1 shows a reagent tray design that includes carriers for Eppendorf tubes, seals, and RFID tags for tracking. [Diagram 25] Shown is a weighing station for reagent fill verification, a fixture for holding reagent consumables on a scale, and a fill manifold. [Figure 26] The buffer filling station is shown. [Figure 27] 40x and 60x objectives with color are shown. [Figure 28] 1 shows a fluid diagram for providing fluid to the collar in 40x and 60x objective lenses. [Figure 29] The supplied collar is shown. The 60x objective has one O-ring, the 40x objective has two O-rings. [Diagram 30] 1 shows a microscope with a connection for an immersion water dispenser. [Diagram 31] FIG. 1 shows an immersion water dispenser for use with a Nikon Ti2e microscope with connections for 40x and 60x objectives. [Diagram 32] 1 shows a schematic of an enclosure for light sensitive samples on top of a table with a shelf below for a workstation and a display component attached to the table. [Diagram 33] 1 shows a schematic of an enclosure for light sensitive samples on top of a table with a shelf below for a workstation and a display component above the table. [Diagram 34] FIG. 1 shows a fluid diagram for an automated fluid delivery system showing the fluidics line connections to the reagent tray, buffer tray, peristaltic pump, motor-driven rotary valves, pressure sensor, and air bubble detector. [Diagram 35] A fluidics diagram is shown with a series of pumps connected to an immersion medium module and fluidics lines with connections to 40x and 60x microscope objectives. [Diagram 36] A fluidics diagram with syringe pumps, motor-driven rotary valves, and connections to a multi-well plate is shown. [Figure 37] 1 shows a schematic diagram of a suction dual valve. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] A sequencing apparatus is provided for automated in situ sequencing of volumetric tissue samples. In particular, an automated volumetric in situ sequencing device capable of operating in parallel on multiple samples is provided. Methods of making and using the sequencing apparatus are also provided.

[0028] Before describing a sequencing apparatus for automated in situ sequencing of volumetric tissue samples, and methods of making and using such a sequencing apparatus, it is to be understood that this invention is not limited to the particular devices, methods, or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, since the scope of the invention will be limited only by the appended claims.

[0029] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are likewise included in the invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0031] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0032] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as oligopeptides or polypeptides.

[0033] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0034] definition The term "about" is meant to encompass a deviation of plus or minus 5 percent, particularly in relation to a given amount.

[0035] The terms "peptide," "oligopeptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as phosphorylation, glycosylation, acetylation, hydroxylation, oxidation, etc., as well as chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones. The term also includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, and the like. The term includes polypeptides that include one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.

[0036] As used herein, the term "target nucleic acid" refers to any polynucleotide nucleic acid molecule (e.g., DNA molecule, RNA molecule, modified nucleic acid, etc.) present within a single cell. In some embodiments, the target nucleic acid is a coding RNA (e.g., mRNA). In some embodiments, the target nucleic acid is a non-coding RNA (e.g., tRNA, rRNA, microRNA (miRNA), mature miRNA, immature miRNA, etc.). In some embodiments, the target nucleic acid is a splice variant of an RNA molecule (e.g., mRNA, pre-mRNA, etc.) in the context of a cell. Thus, suitable target nucleic acids can be unspliced ​​RNA (e.g., pre-mRNA, mRNA), partially spliced ​​RNA, or fully spliced ​​RNA, etc. Target nucleic acids of interest may be variably expressed, i.e., have different abundances, within a population of cells, and the methods of the present invention allow for profiling and comparison of expression levels of nucleic acids, including but not limited to RNA transcripts, in individual cells. Target nucleic acids may also be DNA molecules, such as modified genomes, viruses, plasmids, etc. For example, the methods may be used to detect copy number variants, e.g., in cancer cell populations where a target nucleic acid is present at different abundances in the genomes of cells in the population, or in virally infected cells to determine viral load and kinetics.

[0037] The terms "oligonucleotide", "polynucleotide" and "nucleic acid molecule", used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits, such as phosphoramidites and / or phosphorothioates, and thus may be oligodeoxynucleoside phosphoramidates or mixed phosphoramidate-phosphodiester oligomers. See Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848, Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may contain one or more L nucleosides. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be modified to contain N3'-P5' (NP) phosphoramidates, morpholino phosphorocyamidates (MF), locked nucleic acids (LNA), 2'-O-methoxyethyl (MOE), or 2'-fluoroarabinonucleic acid (FANA), which can enhance the resistance of the polynucleotide to nuclease degradation (see, e.g., Faria et al. (2001) Nature Biotechnol. 19:40-44; Toulme (2001) Nature Biotechnol. 19:17-18).Polynucleotides may be further modified after polymerization, for example, by conjugation with a labeling moiety. Other types of modifications included in this definition are capping, substitution with one or more analogs of naturally occurring nucleotides, and the introduction of a means for binding the polynucleotide to a protein, a metal ion, a labeling moiety, another polynucleotide, or a solid support. The immune modulatory nucleic acid molecule may be provided in various formulations, for example, in association with liposomes, microencapsulation, etc., as described in more detail herein. The polynucleotides used for amplification are generally single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the polynucleotide is first treated to separate its strands, which can then be used to prepare extension products. This denaturation step is typically affected by heat, but may alternatively be carried out using alkali, followed by neutralization.

[0038] "Isolated," when referring to a protein, polypeptide, or peptide, means that the indicated molecule is separated and separate from the whole organism in which it is found in nature, or exists in the substantial absence of other biological macromolecules of the same type. With reference to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or part of a sequence that is normally associated with it in nature, or a sequence that is naturally occurring but has heterologous sequences associated with it, or a molecule that is dissociated from the chromosome.

[0039] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to invertebrate and vertebrate animals, including, but not limited to, arthropods (e.g., insects, crustaceans, arachnids), cephalopods (e.g., octopus, squid), amphibians (e.g., frogs, salamanders, caecilians), fish, reptiles (e.g., turtles, crocodiles, snakes, earthworms, lizards, tuatara), mammals, including humans and non-human mammals, such as chimpanzees and other apes and non-human primates, including monkey species, laboratory animals, such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas, domestic animals, such as dogs and cats, farm animals, such as sheep, goats, pigs, horses, and cows, and birds, such as chickens, turkeys, and other pheasants, ducks, and geese, domestic, wild, and game birds. In some cases, the methods of the invention find use in laboratory animals, in veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents, including mice, rats, and hamsters, primates, and transgenic animals.

[0040] The term "user" as used herein refers to a person who interacts with the devices and / or systems disclosed herein to perform one or more steps of the methods disclosed herein. A user may be a subject who uses a sequencing device described herein.

[0041] Sequencing Device A sequencing apparatus is provided for automated in situ sequencing of volumetric tissue samples. In particular, an automated volumetric in situ sequencing device capable of operating in parallel on multiple samples is provided. In some embodiments, the sequencing device comprises an illumination and detection module, a microscope module, an automated immersion media module, a multi-well plate, a fluidic connection tower or a standalone fluidic module, a fluid management module, a reagent, buffer, and waste module, an electrical module, and a processor.

[0042] In some embodiments, the illumination and detection module comprises a spinning disk confocal component including multiple laser lines for illumination with flat illumination correction, where the multiple laser lines are used to illuminate the sample with excitation light at one or more wavelengths, a bandpass emission filter, a longpass image splitter, a first camera that detects fluorescent emission in a first wavelength range, and a second camera that detects fluorescent emission in a second wavelength range, where the first and second cameras can detect the emission simultaneously. In certain embodiments, the multiple laser lines include at least four laser lines. In some embodiments, the multiple laser lines include five laser lines used with a penta bandpass emission filter. In certain embodiments, the motorized stage has a piezo z-axis. In other embodiments, an objective z-axis drive is used and the motorized stage z is kept constant.

[0043] The microscope module includes a motorized stage that can be multi-axis positioned along the x, y, and z axes, an objective Z drive, and an objective turret wheel with multiple objectives, each objective providing a different magnification and optics, which transmits light from the objectives to the illumination and detection module. The objectives may include immersion objectives, each immersion objective having an objective immersion collar. In certain embodiments, the immersion objectives further include O-rings and shrink-wrap coating the multi-well plate, and the motorized stage prevents spills that could damage, for example, the optics or mechanical parts of the microscope. The objectives may also include dry objectives without immersion collars. In some embodiments, the microscope module includes a confocal microscope. In some embodiments, the microscope module includes an epifluorescence microscope.

[0044] The automated immersion medium module comprises: i) a container containing an immersion medium; ii) fluid lines connected to the container and to an objective immersion collar of an objective of the microscope module, the fluid lines carrying the immersion medium to and from the objective immersion collar, the immersion collar catching excess immersion medium; and iii) a series of pumps connected to the fluid lines and a microcontroller, the microcontroller controlling the pumping addition and removal of immersion medium through the fluid lines, the automated immersion medium module providing a controlled volume of immersion medium to the objective immersion collar on top of the objective during imaging. In certain embodiments, the immersion medium is water. In certain embodiments, the immersion medium is filtered and bubble-free.

[0045] The motorized stage can be moved to position the wells of the multiwell plate under the objective lens used for imaging. In some embodiments, a fluid connection tower is on top of the motorized stage and allows for the placement of fluid lines in the wells of the multiwell plate to add or remove samples from the wells using the fluid lines. In some embodiments, the fluid connection interface is not attached to the motorized stage. Instead, a stand-alone fluid connection interface module is used that is manually placed by the user above the sample plate and secured to the stage, and the fluid connection interface connects the fluid lines to the samples during sequencing.

[0046] The fluid management module includes a symmetrical rotary valve including a rotary valve mechanism and a pump connected to the fluid lines and a bubble detector disposed on either side of the fluid lines leading to the pump, the fluid management module allowing unidirectional or bidirectional movement of reagents, buffers, and waste through the fluid lines. A series of bubble detectors may be used to ensure that the immersion fluid lines are bubble-free. Additionally, air bubbles may be avoided by adding a volume of fluid, removing excess fluid, then adding more fluid, moving the stage to the edge of the well and back to the center of the sample to remove any additional bubbles that may have formed during the addition of immersion fluid.

[0047] The reagent, buffer, and waste module comprises: i) a slide tray, in which reagent cartridges and buffer cartridges can be placed and coupled to the fluid management module; ii) a waste module, in which the waste module includes a waste container coupled to a fluid line from the fluid management pump; and iii) a capping mechanism, in which the capping mechanism closes the waste container when it is removed from the system for waste disposal and opens the waste container when it is returned to the system.

[0048] The electrical module comprises i) a first firmware board that controls the media dispensed from the automated immersion media module, and ii) a second firmware board that controls the fluid management module and the reagent, buffer and waste modules, and the electrical module regulates power to the other modules of the system.

[0049] In certain embodiments, the sequencing device further comprises a pressure monitor for monitoring the pressure in the fluid line, where an increase in pressure in the fluid line can be used to detect a potential blockage in the fluid line.

[0050] In certain embodiments, the sequencing device further comprises a plurality of light emitting diodes (LEDs), each capable of emitting light to provide a status indication for the system.

[0051] In certain embodiments, the sequencing device comprises a processor programmed to provide a user interface and operate the modules of the sequencing device, hi some embodiments, the sequencing device further comprises a display component for displaying information and providing a user interface.

[0052] In certain embodiments, the processor includes the steps of: (a) positioning selected samples in a multi-well plate; (b) detecting signals in an XY plane at low magnification from the selected samples using wide field imaging mode acquisition with camera binning; (c) segmenting an XY bounding box around the samples using the signals; (d) imaging the samples within the XY bounding box to generate an image, wherein the imaging is performed in confocal imaging mode at a higher magnification in Z than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint and across the XY range of the previously determined Z range; (e) displaying the image generated in step (d); (f) providing an interface for a user to probe a desired XY region of interest in the samples to be further imaged during sequencing of the selected samples; and (g) imaging the previously sampled samples in the XY bounding box to generate an image, wherein the imaging is performed in confocal imaging mode at a higher magnification than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint and across the XY range of the previously determined Z range. (h) imaging the sample in a selected XY region of interest over a framed Z range; (i) segmenting the image of the sample in the region of interest along the Z range; (j) providing an interface to the user for the user to adjust the Z range of the sample volume before starting sequencing, where the range of imaging derived from the region of interest defined by the user is automatically converted to an appropriate montaged field of view for a given imaging objective lens, and adjusting the microscope stage position, objective lens Z placement, and piezo boundaries for imaging of the region of interest along the XYZ axes during sequencing; and (k) repeating steps (a)-(j) multiple times to define a region of interest for each sample in the multiwell plate that the user intends to sequence.

[0053] In certain embodiments, the processor is further programmed to perform steps including: providing an interface for the user to select one or more samples for sequencing and a sequencing protocol, where the number of samples that the user may select is limited depending on the amount of buffers and reagents available and the sequencing protocol selected; providing constraints on the total sequencing time, total data acquired, rate of acquisition, and maximum total volume of regions of interest across all samples to be sequenced and imaged; and proposing a protocol that maximizes sequencing of desired regions of interest in the samples within the constraints. In certain embodiments, the processor is further programmed to optimize sample sequencing parallelism depending on the number of samples to be sequenced and the imaging type used for sequencing.

[0054] In certain embodiments, the processor is further programmed to perform steps including performing a rapid confocal or epifluorescence sweep in Z at a starting XY position of a given sample montage to determine the Z profile of the sample at the starting XY position; determining the top and bottom interfaces of the sample using a segmentation method; and setting the Z position of the objective at a fixed distance from the interfaces at the start of the sample montage, such that drift in Z of the sample relative to the stage and objective across rounds is reduced to below a selected tolerance to facilitate downstream sub-pixel registration across rounds during post-acquisition processing.

[0055] In certain embodiments, the sequencing is in situ sequencing of the target nucleic acid in a tissue sample. In some embodiments, the tissue sample is a thick tissue slice having a thickness of 50-200 μm. In other embodiments, the tissue sample is a thin tissue slice having a thickness of 5-20 μm. In some embodiments, the in situ sequencing is sequential or combined in situ sequencing.

[0056] Modular Use of Sequencing Instrument Components In some embodiments, the fluidics components can function as a stand-alone sequencing module for use with any compatible imaging system. The fluid lines to the sample can be magnetically and / or mechanically coupled to the sample plate and microscope stage such that the coupling is easily attachable to and detachable from the engagement position, and such that the fluidic components are coupled to the sample wells. In one example of this coupling, the fluidic lines addressed to each sample well are bundled together and routed to each sample well via a removable plate lid (see schematic), which couples to the microscope stage via mechanical guides and magnetic fixtures. In another embodiment of the coupling, a modular coupling tower is provided that is fixed to the microscope stage. When used as a stand-alone sequencing module, the fluidic components facilitate the use of reagent and buffer kits and the automation of fluid exchange from multiple sample wells over multiple cycles of fluid addition and removal. When used as a device for in situ sequencing, the fluidic components can be coupled to existing microscopy setups that are compatible with the sample format, for example, an inverted microscope. For use with thin section samples (5-20 mm), the microscope can be an epifluorescence microscope with 3, 4, or 5 illumination or detection channels. For use with thin or thick section samples, the microscope can be an epifluorescence microscope, a confocal microscope (spinning disk or point scanning), a structured illumination microscope, or a light sheet or inclined plane light sheet microscope.

[0057] The immersion water distribution module (IWD) can be used as a submodule of an integrated fluidic system for a sequencing device. Alternatively, the IWD can be used as a standalone kit for automated immersion of a microscope immersion objective. In one example, the IWD is used in series with a reagent / buffer / consumable fluidic module to enable parallel and automated sequencing of samples on a separate and existing microscope setup. In this example, the immersion fluid reservoir and immersion fluid waste are external to the fluidic device so that the user can manually fill the immersion fluid reservoir and empty the waste reservoir. The immersion water distribution module is connected to the microscope objective via an immersion collar, which is designed to flow the immersion liquid across the imaging glass of the objective such that no air bubbles are introduced and the volume and flow rate of the liquid is accurate and consistent, while providing a tight seal against the objective body so that excess liquid can be removed. The exact dimensions of the immersion collar are adjusted to match a particular objective lens to ensure a proper fit, but the function of the other immersion water distribution module subcomponents is not relevant to the creation and manufacture of the imaging system.

[0058] The software that controls the sequencing instrument provides a layer of abstraction over the control of illumination, detection, microscope, and stage components, and can therefore be used modularly with a variety of imaging setups, provided that appropriate configuration files or other hardware plug-ins are provided. Thus, a particular imaging and microscopy setup is not privileged in the operation of the sequencing instrument and software, and the objective immersion module, sample, reagent and buffer fluidics, and consumables can be used modularly and reconfigured into one or more combinations of components.

[0059] In some embodiments, the reagent and buffer fluid components draw fluid from reusable reservoirs. In another embodiment of the sequencing device, the reagent and buffer fluid components draw fluid from consumable reservoirs. In one example, the consumable reservoirs are sealed after being filled, and the seals are punctured by sipper needles of the reagent / buffer fluid module. In one example, the seals are mechanically supported within the consumable assembly to ensure consistent puncture of the seals and to avoid excessive force on the sipper needle or forces not aligned with the parallel axis of the sipper needle. The consumable reservoirs are typically replaced at the beginning of each use of the sequencing device, and their use is tracked programmatically through detection of the identity of the consumables. In one example, the detection of the consumables is performed through the use of an RFID integrated into the consumables and an RFID reader in the sequencing device fluidics module. In another embodiment, the detection of the consumables is performed through the use of a barcode on the consumables and a barcode scanner integrated into the sequencing device fluidics module.

[0060] In another embodiment, the fluidic module draws from buffers and reagents used in the in situ sequencing cycle. In one example of a fluidic module, some or all of the buffers or reagents are cooled by a refrigeration component. In some embodiments of the fluidic module, some or all of the buffers or reagents are temperature sensitive, e.g., enzymes such as ligases, or molecules such as ATP involved in SCAL, SEDAL, or SEDAL2 sequencing chemistries. In another embodiment, the buffer and reagent fluidic module draws liquids used in other sequencing or cyclic labeling chemistries, e.g., oligos used to hybridize to sequences in the sample, or fluorescently labeled oligos used to detect hybridization events in the sample. In another embodiment, the buffer and reagent fluidic module draws liquids used to label the sample with dyes. In another embodiment, the buffer and reagent fluidic module draws liquids used in the CLICK chemistry with the sample. In another embodiment, the buffer and reagent fluidic module draws liquids to quench fluorescent signals in the sample. In another embodiment, the buffer and reagent fluidic module draws fluids containing enzyme components that add or remove signals from the sample.

[0061] Computer Implementation Method The present disclosure provides systems and computer-implemented methods that find use in the use of the sequencing devices described herein. In certain embodiments, the sequencing device comprises a processor programmed to provide a user interface and operate the modules of the sequencing device. In some embodiments, the sequencing device further comprises a display component for displaying information and providing a user interface. The system may also comprise one or more graphic boards for processing and outputting graphic information of tissue images to the display component.

[0062] In some embodiments, computer-implemented methods are used to provide an interface between a user and the sequencing device firmware and hardware, for example, to perform sequencing run setup, select sequencing run options, and select and define sample regions of interest (ROIs). Computer-implemented methods can be used to control the parallelization of sequencing across different modules and samples of the sequencing device, and to provide logging, error monitoring, data acquisition, management and transfer, and run progress monitoring.

[0063] In one embodiment, a computer-implemented method is provided, the computer comprising: (a) positioning selected samples in a multi-well plate; (b) detecting signals in an XY plane at low magnification from the selected samples using wide field imaging mode acquisition with camera binning; (c) segmenting an XY bounding box around the samples using the signals; (d) imaging the samples in the XY bounding box to generate an image, wherein the imaging is performed in a confocal imaging mode at a higher magnification in Z than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint of the previously determined Z range and across the XY range; (e) displaying the image generated in step (d); and (f) providing an interface for a user to select a desired XY region of interest in the samples to be further imaged during sequencing of the selected samples. (g) imaging the sample in a selected XY region of interest over a previously sampled Z range; (h) calculating a sample volume of the region of interest and displaying the calculated sample volume of the region of interest to the user; (i) segmenting the image of the sample in the region of interest along the Z range; (j) providing an interface to the user for the user to adjust the Z range of the sample volume before starting sequencing, where the range of imaging derived from the region of interest defined by the user is automatically converted to an appropriate montaged field of view for a given imaging objective lens and adjusts the microscope stage position, objective lens Z placement, and piezo boundaries for imaging of the region of interest along the XYZ axes during sequencing; and (k) repeating steps (a)-(j) multiple times to define a region of interest for each sample in the multi-well plate that the user intends to sequence.

[0064] In another embodiment, a computer-implemented method is provided, the computer performs steps including providing an interface to a user for the user to select one or more samples for sequencing and a sequencing protocol, where the number of samples that the user may select is limited depending on the amount of buffers and reagents available and the sequencing protocol selected, providing constraints on total sequencing time across all samples to be sequenced and imaged, total data acquired, rate of acquisition, and maximum total volume of regions of interest, and proposing a protocol that maximizes sequencing of desired regions of interest in the samples within the constraints. In some embodiments, the computer is further programmed to optimize sample sequencing parallelism depending on the number of samples to be sequenced and the imaging type used for sequencing.

[0065] In another embodiment, a computer-implemented method is provided, where the computer performs the steps including performing a rapid confocal sweep in Z at a starting XY position of a given sample montage to determine a Z profile of the sample at the starting XY position, determining top and bottom interfaces of the sample using a segmentation method, and setting the Z position of the objective at a fixed distance from the interfaces at the start of the sample montage, such that drift in Z of the sample relative to the stage and objective across rounds is reduced to below a selected tolerance to facilitate downstream sub-pixel registration across rounds during post-acquisition processing.

[0066] The methods can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware. The disclosed and other embodiments can be implemented as one or more computer program products, i.e., one or more modules of computer program instructions encoded on a computer-readable medium for execution by or for controlling the operation of a data processing apparatus. The computer-readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, a composition of matter affecting a machine-readable propagated signal, or any combination thereof.

[0067] A computer program (also known as a program, software, software application, script, or code) may be written in any form of programming language, including stored or interpreted languages, and it may be deployed as a stand-alone program or in any form including modules, components, subroutines, or other units suitable for use in a computing environment. A computer program does not necessarily correspond to a file in a file system. A program may be stored in part of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., a file that stores one or more modules, subprograms, or portions of code). A computer program may be deployed to be executed on one computer or multiple computers located at one site or distributed across multiple sites and interconnected by a communication network.

[0068] In further aspects, a system for performing the computer-implemented methods as described may include a processor, a storage component (i.e., memory), a display component, and other components typically present in a general purpose computer. The storage component stores information accessible by the processor, including instructions that may be executed by the processor and data that may be retrieved, manipulated, or stored by the processor.

[0069] The storage component includes instructions. For example, the storage component may include instructions for providing a user interface for the sequencing device, instructions for operating the sequencing device, and instructions for processing the in situ sequencing imaging data, as described herein. A computer processor is coupled to the storage component and configured to execute the instructions stored in the storage component to receive the in situ sequencing imaging data and analyze the data according to one or more algorithms, as described herein. The display component displays information and provides a user interface.

[0070] The storage component may be of any type capable of storing information accessible by the processor, such as a hard drive, memory card, ROM, RAM, DVD, CD-ROM, USB flash drive, writable memory, and read-only memory. The processor may be any well-known processor, such as a processor from Intel Corporation. Alternatively, the processor may be a dedicated controller, such as an ASIC.

[0071] In certain embodiments, the in situ sequencing imaging data is uploaded and stored in a cloud data storage system. In some embodiments, the cloud data storage system is a public cloud storage system. In other embodiments, the cloud data storage system is a private cloud storage system. The cloud data storage may be used to store raw images, intermediate processing files, and final data products. Processing may begin with uploading a dataset to the cloud storage by a data acquisition system. Configuration parameters such as encoding schemes, codebooks, image acquisition parameters, and sample metadata may be entered by a user using a data management web interface or may be automatically generated from a configuration file uploaded to the cloud storage along with the sequencing data. Each set of configuration parameters is stored in a cloud database. In some cases, multiple processing runs using different configuration parameters may be applied to a single dataset to optimize processing parameters.

[0072] Instructions may be any set of instructions that are executed directly (such as machine code) or indirectly (such as a script) by a processor. In that regard, the terms "instructions," "steps," and "program" may be used interchangeably herein. Instructions may be stored in object code format for direct processing by a processor, or in any other computer language, including a script or collection of independent source code modules that are interpreted on demand or pre-stored.

[0073] Data may be retrieved, stored, or modified by a processor in accordance with instructions. For example, the system is not limited by any particular data structure, but data may be stored in a computer register, a relational database, as a table with multiple different fields and records, an XML document, or a flat file. Data may also be formatted in any computer-readable format, such as, but not limited to, binary values, ASCII, or Unicode. Furthermore, data may include any information sufficient to identify related information, such as numbers, descriptive text, unique codes, pointers, references to data stored in other memory (including other network locations), or information used by a function to calculate related data.

[0074] In certain embodiments, the processor and storage components may comprise multiple processors and storage components that may or may not be stored in the same physical housing. For example, some of the instructions and data may be stored on a removable CD-ROM and others in a read-only computer chip. Some or all of the instructions and data may be stored in a location that is physically separate from the processor but still accessible by the processor. Similarly, a processor may comprise a collection of processors that may or may not be operated in parallel.

[0075] In some embodiments, the method may be performed using a cloud computing system. In some embodiments, image data files and programming for processing imaging data may be exported to a cloud computer that executes the program and returns the output to the user. The method may include optional compression of imaging data before transfer to reduce data size and increase transfer speed. During the data acquisition process, the acquired images are coupled with a metadata file detailing optical specifications, stage position, and sequencing information, optional compression of imaging data as a separate process from the imaging acquisition, and optional offloading of data from the acquisition to a remote cloud storage medium, a networked storage system, or a separate large-scale file system.

[0076] Components of systems for carrying out the methods disclosed herein are further described in the Examples below.

[0077] In situ gene sequencing The sequencing devices disclosed herein can be used for in situ gene sequencing of target nucleic acids in cells in intact tissue. In situ sequencing includes the steps of: (a) contacting fixed, permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow for specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first complementary region, a second complementary region sequence, and a third complementary region, the second oligonucleotide further comprising a barcode sequence, the first complementary region of the first oligonucleotide is complementary to a first portion of a target nucleic acid, the second complementary region of the first oligonucleotide is complementary to a first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to a third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and the first portion of the target nucleic acid is adjacent to the second portion of the target nucleic acid; and (b) adding a ligase to ligate the second oligonucleotide to the first complementary region of the second oligonucleotide. (c) performing rolling circle amplification in the presence of the nucleic acid molecule, comprising using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase to form one or more amplicons; (d) embedding the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons; and (e) contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a set of sequencing primers under conditions that allow for ligation, the set of sequencing primers comprising a third oligonucleotide configured to decode bases and a fourth oligonucleotide configured to convert the decoded bases into a signal, where ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide are complementary to adjacent sequences of the same amplicon.(f) repeating step (e) multiple times; and (g) imaging the amplicons embedded in one or more hydrogels using a sequencing device described herein to determine in situ the genetic sequence of the target nucleic acid in cells in the intact tissue.

[0078] In some embodiments, in situ sequencing is performed using sequencing with error correction by dynamic annealing and ligation (SEDAL) to determine the sequence of the target nucleic acid. The SEDAL method includes contacting one or more hydrogel-embedded amplicons with a pair of primers under conditions that allow ligation, the pair of primers including a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon. In some embodiments, SEDAL is used with STARmap. In such embodiments, the methods herein include operating at room temperature for best preservation of tissue morphology with low background noise and reduced errors. In other such embodiments, contacting one or more hydrogel-embedded amplicons includes eliminating error accumulation as sequencing proceeds.

[0079] In some embodiments, the contacting of one or more hydrogel-embedded amplicons occurs two or more times, including, but not limited to, for example, three or more, four or more, five or more, six or more, or seven or more times. In certain embodiments, the contacting of one or more hydrogel-embedded amplicons occurs four or more times for thin tissue specimens. In other embodiments, the contacting of one or more hydrogel-embedded amplicons occurs six or more times for thick tissue specimens. In some embodiments, the one or more amplicons may be contacted with a pair of primers for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less.

[0080] Specimens prepared using the present methods can be analyzed by any of several different types of microscopy, including optical microscopy (e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal, etc.), laser microscopy, electron microscopy, and scanning probe microscopy. In some embodiments, the non-transitory computer readable medium converts the raw images acquired through multiple rounds of in situ sequencing microscopy into decoded gene identities and spatial locations, and then analyzes the per-cell composition of gene expression.

[0081] SEDAL Oligonucleotide Primers In some embodiments, the disclosed method includes a third oligonucleotide and a fourth oligonucleotide. In certain aspects, the third oligonucleotide is configured to decode the bases, and the fourth oligonucleotide is configured to convert the decoded bases into a signal. In some aspects, the signal is a fluorescent signal. In an exemplary aspect, contacting one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation includes ligation of each of the third oligonucleotide and the fourth oligonucleotide to form a stable product for imaging only if a perfect match occurs. In certain aspects, the mismatch sensitivity of the ligase enzyme is used to determine the underlying sequence of the target nucleic acid molecule.

[0082] The term "perfectly matched", when used in reference to a double strand, means that the polynucleotides and / or oligonucleotide strands constituting the double strand form a double-stranded structure with each other, such that every nucleotide in each strand undergoes Watson-Crick base pairing with the nucleotide in the other strand. The term "double strand" includes, but is not limited to, pairings of nucleoside analogs such as deoxyinosine, nucleosides with 2-aminopurine bases, peptide nucleic acids (PNAs), etc., may be used. A "mismatch" in a double strand between two oligonucleotides means that a pair of nucleotides in the double strand cannot undergo Watson-Crick binding.

[0083] In some embodiments, the methods include a plurality of third oligonucleotides, including but not limited to, 5 or more third oligonucleotides, e.g., 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more, that hybridize to a target nucleotide sequence. In some embodiments, the methods of the disclosure include a plurality of third oligonucleotides, including but not limited to, 15 or more third oligonucleotides, e.g., 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences, that hybridize to a target nucleotide sequence. In some embodiments, the method includes a plurality of fourth oligonucleotides, including but not limited to 5 or more fourth oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure includes a plurality of fourth oligonucleotides, including but not limited to 15 or more fourth oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides, hybridizing to 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences. Multiple oligonucleotide pairs can be used in the reaction, with one or more pairs specifically binding to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability. It is also of interest to detect multiple different target nucleic acids within a cell, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids.

[0084] In certain embodiments, SEDAL involves a ligase whose activity is hindered by a base mismatch, a third oligonucleotide, and a fourth oligonucleotide. The term "hindered" in this context refers to the activity of the ligase being reduced by about 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 99% or more, such as 100%. In some embodiments, the third oligonucleotide has a length of 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the third oligonucleotide is m is room temperature (22-25° C.). In some embodiments, the third oligonucleotide is denatured or partially denatured. In some embodiments, the fourth oligonucleotide has a length of 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the fourth oligonucleotide is m is at room temperature (22-25°C). After each cycle of SEDAL corresponding to a base readout, the fourth oligonucleotide may be stripped off, thereby eliminating error accumulation as sequencing proceeds. In such an embodiment, the fourth oligonucleotide is stripped off by formamide.

[0085] In some embodiments, SEDAL involves washing the third and fourth oligonucleotides to remove unbound oligonucleotides, and then revealing the fluorescent product for imaging. In certain exemplary embodiments, detectable labels may be used to detect one or more nucleotides and / or oligonucleotides described herein. In certain embodiments, detectable labels may be used to detect one or more amplicons. Examples of detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs, and the like. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and the like. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle, and the like), luciferin, aequorin, and the like. Examples of enzyme systems with visually detectable signals include, but are not limited to, galactosidase, glucolimidase, phosphatase, peroxidase, cholinesterase, etc. Identifiable markers include: 125 I, 35 S, 14 C, or 3 Also included are radioactive compounds such as 3H. Identifiable markers are commercially available from a variety of sources.

[0086] Fluorescent labels and their attachment to nucleotides and / or oligonucleotides are described in many publications, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to the present invention are disclosed in the example references in U.S. Patent Nos. 4,757,141, 5,151,507, and 5,091,519. In one embodiment, one or more fluorescent dyes are used as labels for labeled target sequences, e.g., as disclosed by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes), U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes), U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes), U.S. Pat. No. 4,318,846 (ether-substituted fluorescein dyes), U.S. Pat. No. 5,800,996 (energy transfer dyes), Lee et al., U.S. Pat. No. 5,066,580 (xanthine dyes), U.S. Pat. No. 5,688,648 (energy transfer dyes), and the like. Labeling can be done with quantum dots, as disclosed in the following patents and patent publications: U.S. Patent Nos. 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, 2002 / 0045045, and 2003 / 0017264.As used herein, the term "fluorescent label" includes signaling moieties that transmit information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectral properties, energy transfer, and the like.

[0087] Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREEN™ 488-5 ...5-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-d RED(TM)-12-dUTP, BODIPY(TM) 630 / 650-14-dUTP, BODIPY(TM) 650 / 665-14-dUTP, ALEXA FLUOR(TM) 488-5-dUTP, ALEXA FLUOR(TM) 532-5-dUTP, ALEXA FLUOR(TM) 568-5-dUTP, ALEXA FLUOR(TM) 594-5-dUTP, ALEXA FLUOR(TM) 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED(TM)-5-UTP, mCherry, CASCADE BLUE(TM)-7-UTP, BODIPY(TM) FL-14-UTP, BODIPY These include, but are not limited to, TMR-14-UTP, BODIPY™ TR-14-UTP, RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.), etc. Protocols for the custom synthesis of nucleotides bearing other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0088] Other fluorophores available for post-synthetic conjugation include ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY Examples of fluorophores include, but are not limited to, 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), etc. FRET tandem fluorophores can also be used, including, but are not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, etc.

[0089] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0090] Biotin or its derivatives can also be used as a label on the nucleotide and / or oligonucleotide sequence, which can then be bound by detectably labeled avidin / streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or detectably labeled anti-biotin antibodies. Digoxigenin can be incorporated as a label, which can then be bound by detectably labeled anti-digoxigenin antibodies (e.g., fluoresceinated anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into the oligonucleotide sequence, which can then be bound to N-hydroxysuccinimide (NHS)-derivatized fluorescent dyes. In general, any member of the conjugate pair can be incorporated into the detection oligonucleotide, provided that the detectably labeled conjugate partner can be bound in a manner that allows detection. As used herein, the term antibody refers to any class of antibody molecule, or any subfragment thereof, such as Fab.

[0091] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phosphorescent amino acids (e.g., P-tyr, P-ser, P-thr), etc. In one embodiment, the following hapten / antibody pairs are used for detection, with each of the antibodies derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0092] In certain exemplary embodiments, the nucleotide and / or oligonucleotide sequences may be indirectly labeled with a hapten that is then specifically bound by a capture agent, as disclosed, for example, in U.S. Pat. Nos. 5,344,757, 5,702,888, 5,354,657, 5,198,537, and 4,849,336, PCT Publication No. 91 / 17160, and the like. Many different hapten capture agent pairs can be used. Exemplary haptens include, but are not limited to, biotin, desbiotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, and the like. In the case of biotin, the capture agent may be avidin, streptavidin, or an antibody. Antibodies can be used as capture agents for other haptens (many dye-antibody pairs are commercially available, e.g., Molecular Probes, Eugene, Oreg.).

[0093] In some embodiments, in situ sequencing is performed using sequencing by competitive annealing and ligation (SCAL) to determine a sequence of a target nucleic acid, the method comprising performing one or more sequencing cycles, each cycle comprising: (a) contacting the target nucleic acid with a readout oligonucleotide and a set of fluorescently labeled reading probes, where the readout oligonucleotide comprises a first region of complementarity that is complementary to a readout sequence on the target nucleic acid, and where each reading probe comprises a second region of complementarity that is complementary to a probe binding site on the target nucleic acid; and (b) ligating the readout oligonucleotide to one of the reading probes of the set of fluorescently labeled reading probes to generate a fluorescent ligation product, where the ligation is complementary to the readout oligonucleotide. (c) generating a ligation product in which the read oligonucleotide and the decoding probe bind to adjacent sequences on the target nucleic acid, the ligation product occurring only if both the read oligonucleotide and the decoding probe have sequences that are exactly complementary to a sequence of the target nucleic acid; (d) imaging the fluorescent ligation product to detect a fluorescent label of the decoding probe ligated to the read oligonucleotide, where the fluorescent label identifies a nucleotide of the sequence of the target nucleic acid; and (e) removing the fluorescent ligation product from the target nucleic acid by binding a competitor oligonucleotide to the target nucleic acid, where the competitor oligonucleotide comprises a third complementary region that includes a sequence that is complementary to the read sequence on the target nucleic acid, and removing the fluorescent ligation product.

[0094] In exemplary aspects, ligation involves ligation of each of the readout oligonucleotide and the fluorescently labeled decoding probe such that only if a perfect match occurs will a stable product be formed for imaging. In certain aspects, the mismatch sensitivity of the ligase enzyme is used to determine the underlying sequence of the target nucleic acid molecule. The inclusion of a polyethylene glycol (PEG) polymer in the sequencing ligation mixture substantially accelerates the addition of signal to the target nucleic acid. Exemplary PEG polymers have molecular weights ranging from 300 g / mol to 10,000,000 g / mol. In some embodiments, a PEG 6000 polymer is present during the ligation of the readout oligonucleotide and the fluorescently labeled decoding probe.

[0095] In certain embodiments, the set of fluorescently labeled decoding probes includes a first probe that encodes a guanine, where the first probe comprises a first fluorescent label, a second probe that encodes an adenine, where the second probe comprises a second fluorescent label, a third probe that encodes a cytosine, where the third probe comprises a third fluorescent label, and a fourth probe that encodes a thymine, where the fourth probe comprises a fourth fluorescent label.

[0096] In certain embodiments, each fluorescently labeled decoding probe encodes one to three bases adjacent to the ligation junction where the reader oligonucleotide is ligated to the fluorescently labeled decoding probe, and fluorescently labeled decoding probes encoding different sequences of bases comprise different fluorescent labels.

[0097] In certain embodiments, the sequence of the fluorescently labeled decoding probe for the current cycle of sequencing is optimized to minimize cross-hybridization with fluorescently labeled decoding probes for other sequencing cycles.

[0098] In certain embodiments, the reader oligonucleotide ranges in length from 8 to 11 nucleotides, including any length within this range, such as 8, 9, 10, or 11 nucleotides in length. In some embodiments, the reader oligonucleotide has a melting temperature range of 17°C to 20°C, including any melting temperature within this range, such as 17°C, 18°C, 19°C, or 20°C.

[0099] In certain embodiments, the competitor oligonucleotide further comprises a fourth region of complementarity comprising a sequence that is complementary to at least a portion of the probe binding site. In some embodiments, the fourth region of complementarity of the competitor oligonucleotide comprises a sequence that is completely complementary to the entire probe binding site on the target nucleic acid. In certain embodiments, the competitor oligonucleotide further comprises a fifth region of complementarity comprising a sequence that is complementary to a competitor-specific complementary site adjacent to the read sequence on the target nucleic acid. In some embodiments, the competitor-specific complementary site ranges in length from 2 nucleotides to 16 nucleotides, and may comprise any length within this range, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides. In certain embodiments, for sequencing cycles after an initial sequencing cycle, the competitor oligonucleotide used in the previous sequencing cycle is present in one or more subsequent sequencing cycles.

[0100] In certain embodiments, the reader oligonucleotide further comprises a competitor-specific complementary sequence. In some embodiments, the competitor-specific complementary sequence of the reader oligonucleotide ranges in length from 2 nucleotides to 16 nucleotides, including any length within this range, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides. In certain embodiments, the sequence of the reader oligonucleotide for the current cycle of sequencing is optimized to minimize cross-hybridization with the reader oligonucleotide for other sequencing cycles.

[0101] In certain embodiments, multiple readout oligonucleotides, sets of fluorescently labeled reading probes, and competitor oligonucleotides with specificity for different target nucleic acids are used to simultaneously or sequentially sequence multiple different target nucleic acids.

[0102] In certain embodiments, the competitor oligonucleotide removes ligation products from a previous sequencing round from a target nucleic acid different from the target nucleic acid currently undergoing step (a) or (b) of a sequencing cycle. In certain embodiments, the competitor oligonucleotide removes ligation products from a previous sequencing round from the same target nucleic acid currently undergoing step (a) or (b) of a sequencing cycle. In certain embodiments, the competitor oligonucleotide is a round-specific competitor oligonucleotide that includes a fourth region of complementarity that includes a sequence that is complementary to the read sequence for the next sequencing cycle.

[0103] The sequencing reads may be in the forward direction, 5' to 3', or in the reverse direction, 3' to 5'. For sequencing reads in the forward direction, each fluorescently labeled reading probe has a fluorophore modification at the 5' end and each reading oligonucleotide has a phosphate at the 5' end. For sequencing reads in the reverse direction, each fluorescently labeled reading probe has a phosphate at the 5' end and a fluorophore modification at the 3' end.

[0104] In certain embodiments, the sequencing is performed using continuous encoding. In some embodiments, each reader oligonucleotide comprises a unique continuous orthogonal readout sequence and a unique adjacent competitor-specific complementary sequence for each cycle of sequencing. In some embodiments, the unique continuous orthogonal readout sequence ranges from 8 nucleotides to 11 nucleotides in length, and includes any length within this range, for example, 8, 9, 10, or 11 nucleotides. In some embodiments, the unique adjacent competitor-specific complementary sequence ranges from 2 nucleotides to 16 nucleotides in length, and includes any length within this range, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides. In some embodiments, each competitor oligonucleotide comprises a unique continuous orthogonal readout sequence and a unique adjacent competitor-specific complementary sequence of the reader oligonucleotide for each cycle of sequencing, and a sequence that is complementary to at least a portion of the sequence of the fluorescently labeled decoding probe. In some embodiments, the sequence of the competitor oligonucleotide has partial or complete complementarity to the sequence of the fluorescently labeled decoding probe.

[0105] In certain embodiments, the sequencing is performed using combinatorial encoding. In some embodiments, multiple read oligonucleotides are used for sequencing, each read oligonucleotide comprises a first complementary region comprising a combined read sequence that is complementary to a read sequence at a distinct combined read position on the target nucleic acid, and the read sequence at each distinct position on the target nucleic acid is adjacent to a probe binding site. In some embodiments, each read oligonucleotide further comprises a competitor-specific complementary sequence adjacent to the read sequence. In some embodiments, the competitor-specific complementary sequence is not complementary to the fluorescently labeled decoding probe. In some embodiments, the read sequence ranges in length from 8 nucleotides to 11 nucleotides, including any length within this range, for example, 8, 9, 10, or 11 nucleotides. In some embodiments, the competitor-specific complementary sequence ranges in length from 2 nucleotides to 16 nucleotides, including any length within this range, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides. In some embodiments, for each distinct combination read position, the competitor oligonucleotide includes a sequence that is complementary to at least a portion of the combination read sequence and the competitor-specific complementary sequence of the read oligonucleotide and the sequence of the fluorescently labeled decoding probe for each cycle of sequencing. In some embodiments, the combination encoding uses a Hamming code.

[0106] The sequencing method described herein can be used for in situ genetic sequencing of target nucleic acid in cells in intact tissue. In some embodiments, the method of in situ genetic sequencing of target nucleic acid in cells in intact tissue includes: (a) contacting fixed and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first complementary region, a second complementary region sequence, and a third complementary region, the second oligonucleotide further comprising a barcode sequence, the first complementary region of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second complementary region of the first oligonucleotide being complementary to the first complementary region of the second oligonucleotide, and the third complementary region of the first oligonucleotide being complementary to the second oligonucleotide. The method includes contacting a nucleic acid molecule with a first oligonucleotide, the second oligonucleotide being complementary to a third complementary region of the first oligonucleotide, the second complementary region of the second oligonucleotide being complementary to a second portion of the target nucleic acid, the first portion of the target nucleic acid being adjacent to the second portion of the target nucleic acid; (b) adding a ligase to ligate the second oligonucleotide to generate a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of the nucleic acid molecule, comprising using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase to form one or more amplicons; (d) embedding the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons; and (e) sequencing the one or more amplicons according to the methods described herein. In certain embodiments, the sequencing is performed using sequential encoding. In other embodiments, the sequencing is performed using combinatorial encoding.

[0107] In some embodiments, the contacting of the one or more hydrogel-embedded amplicons occurs two or more times, including, but not limited to, for example, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more. In certain embodiments, the contacting of the one or more hydrogel-embedded amplicons occurs four or more times for thin tissue specimens. In other embodiments, the contacting of the one or more hydrogel-embedded amplicons occurs six or more times for thick tissue specimens. In some embodiments, the one or more amplicons may be contacted with a pair of primers for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. In some embodiments, 12 or more cycles of sequencing are performed, including 13 or more cycles, 14 or more cycles, 15 or more cycles, 16 or more cycles, 17 or more cycles, or 18 or more cycles of sequencing. In some embodiments, the method is performed at room temperature for preservation of tissue morphology, has low background noise and error reduction. In some embodiments, contacting one or more hydrogel-embedded amplicons includes eliminating error accumulation as sequencing proceeds.

[0108] Specimens prepared using the present methods can be analyzed by any of several different types of microscopy, including optical microscopy (e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal, etc.), laser microscopy, electron microscopy, and scanning probe microscopy. In some embodiments, the non-transitory computer readable medium converts the raw images acquired through multiple rounds of in situ sequencing microscopy into decoded gene identities and spatial locations, and then analyzes the per-cell composition of gene expression.

[0109] The term "perfectly matched", when used in reference to a double strand, means that the polynucleotides and / or oligonucleotide strands constituting the double strand form a double-stranded structure with each other, such that every nucleotide in each strand undergoes Watson-Crick base pairing with the nucleotide in the other strand. The term "double strand" includes, but is not limited to, pairings of nucleoside analogs such as deoxyinosine, nucleosides with 2-aminopurine bases, peptide nucleic acids (PNAs), etc., may be used. A "mismatch" in a double strand between two oligonucleotides means that a pair of nucleotides in the double strand cannot undergo Watson-Crick binding.

[0110] In some embodiments, the methods include a plurality of read oligonucleotides, including but not limited to, 5 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more, that hybridize to a target nucleotide sequence. In some embodiments, the methods of the present disclosure include a plurality of read oligonucleotides, including but not limited to, 15 or more, 15 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different read oligonucleotides that hybridize to 15 or more, such as, but not limited to, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences.

[0111] In some embodiments, the methods include a plurality of fluorescently labeled decoding probes, including but not limited to, 4 or more fluorescently labeled decoding probes, such as 8 or more, 10 or more, 12 or more, 16 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the methods of the present disclosure include a plurality of fluorescently labeled decoding probes, including but not limited to, 15 or more fluorescently labeled decoding probes, including but not limited to, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different fluorescently labeled decoding probes that hybridize to 15 or more, such as 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences.

[0112] Multiple pairs of oligonucleotide primers can be used in the reaction, with one or more pairs specifically binding to each target nucleic acid.For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability.It is also an object to detect multiple different target nucleic acids in a cell, for example, detect up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids.

[0113] In certain embodiments, sequencing is performed by a ligase whose activity is hindered by the base mismatch, the readout oligonucleotide, and the fluorescently labeled decoding probe. The term "hindered" in this context refers to the activity of the ligase being reduced by approximately 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 99% or more, such as 100%. In some embodiments, the third oligonucleotide has a length of 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the third oligonucleotide ism is at room temperature (22-25° C.). In some embodiments, the readout oligonucleotide is denatured or partially denatured. In some embodiments, the fluorescently labeled decoding probe oligonucleotide has a length of 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the fourth oligonucleotide is m is at room temperature (22°-25° C.). After each cycle of sequencing corresponding to a base readout, the fluorescent ligation product is removed from the target nucleic acid by binding a competitor oligonucleotide to the target nucleic acid, the competitor oligonucleotide containing a third region of complementarity that includes a sequence that is complementary to the readout sequence on the target nucleic acid, and the fluorescent ligation product dissociates from the target nucleic acid.

[0114] In some embodiments, sequencing involves washing to remove unbound oligonucleotides and unligated probes, and then reveals the fluorescent product for imaging. In certain exemplary embodiments, detectable fluorescent labels can be used to detect one or more nucleotides and / or oligonucleotides described herein. In certain embodiments, detectable fluorescent labels, such as fluorescent proteins, fluorescent dyes, or fluorescent quantum dots, are used to label the probes.

[0115] Fluorescent labels and their attachment to nucleotides and / or oligonucleotides are described in many publications, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to the present invention are disclosed in the example references in U.S. Patent Nos. 4,757,141, 5,151,507, and 5,091,519. In one embodiment, one or more fluorescent dyes are used as labels for labeled target sequences, e.g., as disclosed by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes), U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes), U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes), U.S. Pat. No. 4,318,846 (ether-substituted fluorescein dyes), U.S. Pat. No. 5,800,996 (energy transfer dyes), Lee et al., U.S. Pat. No. 5,066,580 (xanthine dyes), U.S. Pat. No. 5,688,648 (energy transfer dyes), and the like. Labeling can be done with quantum dots, as disclosed in the following patents and patent publications: U.S. Patent Nos. 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, 2002 / 0045045, and 2003 / 0017264.As used herein, the term "fluorescent label" includes signaling moieties that transmit information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectral properties, energy transfer, and the like.

[0116] Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREEN™ 488-5 ...5-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-d RED(TM)-12-dUTP, BODIPY(TM) 630 / 650-14-dUTP, BODIPY(TM) 650 / 665-14-dUTP, ALEXA FLUOR(TM) 488-5-dUTP, ALEXA FLUOR(TM) 532-5-dUTP, ALEXA FLUOR(TM) 568-5-dUTP, ALEXA FLUOR(TM) 594-5-dUTP, ALEXA FLUOR(TM) 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED(TM)-5-UTP, mCherry, CASCADE BLUE(TM)-7-UTP, BODIPY(TM) FL-14-UTP, BODIPY These include, but are not limited to, TMR-14-UTP, BODIPY™ TR-14-UTP, RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.), etc. Protocols for the custom synthesis of nucleotides bearing other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0117] Other fluorophores available for post-synthetic conjugation include ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY Examples of fluorophores include, but are not limited to, 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), etc. FRET tandem fluorophores can also be used, including, but are not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, etc.

[0118] Examples of fluorescent proteins include, but are not limited to, green fluorescent protein, superfolder green fluorescent protein, enhanced green fluorescent protein, Dronpa (photoswitchable green fluorescent protein), yellow-green fluorescent protein, yellow fluorescent protein, red fluorescent protein, orange fluorescent protein, blue fluorescent protein, cyan fluorescent protein, violet fluorescent protein, mApple, mNectarine, mNeptune, mCherry, mStrawberry, mPlum, mRaspberry, mCrimson3, mCarmine, mCardinal, mScarlet, mRuby2, FusionRed, mNeonGreen, TagRFP675, and mRFP1.

[0119] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0120] Biotin or its derivatives can also be used as a label on the nucleotide and / or oligonucleotide sequence, which can then be bound by detectably labeled avidin / streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or detectably labeled anti-biotin antibodies. Digoxigenin can be incorporated as a label, which can then be bound by detectably labeled anti-digoxigenin antibodies (e.g., fluoresceinated anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into the oligonucleotide sequence, which can then be bound to N-hydroxysuccinimide (NHS)-derivatized fluorescent dyes. In general, any member of the conjugate pair can be incorporated into the detection oligonucleotide, provided that the detectably labeled conjugate partner can be bound in a manner that allows detection. As used herein, the term antibody refers to any class of antibody molecule, or any subfragment thereof, such as Fab.

[0121] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phosphorescent amino acids (e.g., P-tyr, P-ser, P-thr), etc. In one embodiment, the following hapten / antibody pairs are used for detection, with each of the antibodies derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0122] In certain exemplary embodiments, the nucleotide and / or oligonucleotide sequences may be indirectly labeled with a hapten that is then specifically bound by a capture agent, as disclosed, for example, in U.S. Pat. Nos. 5,344,757, 5,702,888, 5,354,657, 5,198,537, and 4,849,336, PCT Publication No. 91 / 17160, and the like. Many different hapten capture agent pairs can be used. Exemplary haptens include, but are not limited to, biotin, desbiotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, and the like. In the case of biotin, the capture agent may be avidin, streptavidin, or an antibody. Antibodies can be used as capture agents for other haptens (many dye-antibody pairs are commercially available, e.g., Molecular Probes, Eugene, Oreg.).

[0123] In some embodiments, antioxidant compounds are included in the washing and imaging buffers (i.e., "anti-bleaching buffers") to reduce photobleaching during fluorescence imaging. Exemplary antioxidants include, but are not limited to, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and Trolox-quinone, propyl gallate, tertiary butyl hydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, ascorbic acid, and tocopherol. Such antioxidants have an anti-bleaching effect on fluorophores. That is, antioxidants reduce photobleaching during tiling and greatly enhance the signal-to-noise ratio (SNR) of sensitive fluorophores, allowing higher SNR imaging of thicker samples. For a fixed exposure time with antioxidants, the SNR is increased by increasing the concentration of fluorophores that are not bleached during exposure to light. The inclusion of antioxidants also removes the diminishing returns of longer exposure times (caused by limited fluorophore lifetime before photobleaching), providing an increase in SNR by allowing increased exposure times.

[0124] An exemplary sequencing cycle optionally begins with a short sample wash before proceeding to the first signal addition. Depending on whether sequential or combinatorial encoding is used for a particular round, a corresponding set of read oligonucleotides, fluorescently labeled decoding probes, and their round-specific competitors are added and ligated. In combinatorial encoding, a read oligonucleotide for a given position x is added, plus a set of fluorescently labeled dibase encoding oligonucleotides, plus a competitor oligonucleotide for the previous position that was labeled (unless it is the first round of labeling, in which case the competitor oligonucleotide is omitted). In sequential encoding, a read oligonucleotide for a given round x, a four-channel fluorophore mixture, and a round x-1 competitor oligonucleotide are added, unless it is the first round of labeling. The presence of PEG in the sequencing ligation mixture substantially accelerates signal addition to the target. After incubation of the sample in the imaging buffer, the sample is imaged and briefly rinsed before proceeding to the next sequencing cycle.

[0125] In addition, when multiple sequencing cycles are used, fluorophore cleavage or probe stripping from the probe can be used to eliminate signal carryover from one round to the next. For example, the fluorophore can be stripped away with formamide. Alternatively, thiol-linked dyes with a disulfide linkage between the fluorophore and the oligonucleotide probe can be used, which allows for cleavage of the fluorophore from the oligonucleotide probe under a reducing environment. Exemplary disulfide reducing agents that can be used to cleave disulfide bonds include, but are not limited to, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and b-mercaptoethanol (BME). After fluorescent imaging during a sequencing round, a stripping agent and / or reducing agent is added, followed by a washing step to remove diffusible fluorescent signal before performing another round of sequencing.

[0126] The methods disclosed herein also provide a method of screening a candidate agent to determine whether a candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue by performing the methods described herein to determine the gene sequence of a target nucleic acid in a cell in an intact tissue and detecting the level of gene expression of the target nucleic acid, where a change in the level of expression of the target nucleic acid in the presence of the candidate agent relative to the level of expression of the target nucleic acid in the absence of the candidate agent indicates that the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue.

[0127] In certain aspects, the methods disclosed herein provide faster processing times, higher multiplexing, higher efficiency, higher sensitivity, lower error rates, and more spatially resolved cell types compared to existing gene expression analysis tools. The methods provide improved sequencing by ligation techniques (SCAL and SEDAL2) for in situ sequencing with reduced errors. In some other aspects, the methods disclosed herein include spatial sequencing (e.g., reagents, chips, or services) for biomedical research and clinical diagnostics (e.g., cancer, bacterial infection, viral infection, etc.) with single cell and / or single molecule sensitivity.

[0128] Specific Amplification of Nucleic Acids via Intramolecular Ligation (SNAIL) An efficient approach for generating cDNA libraries in situ from cellular RNA may be utilized for specific amplification of nucleic acids via intramolecular ligation, referred to herein as SNAIL. In certain embodiments, the method comprises contacting a fixed, permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide.

[0129] More generally, the nucleic acid present in the cells of interest in the tissue serves as a scaffold for the assembly of a complex comprising a pair of primers, herein referred to as a first oligonucleotide and a second oligonucleotide. In some embodiments, contacting the fixed and permeabilized intact tissue comprises hybridizing a pair of primers to the same target nucleic acid. In some embodiments, the target nucleic acid is RNA. In such embodiments, the target nucleic acid may be mRNA. In other embodiments, the target nucleic acid is DNA.

[0130] As used herein, the terms "hybridize" and "hybridization" refer to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex through Watson-Crick base pairing. When a primer "hybridizes" with a target (template), such a complex (or hybrid) is sufficiently stable to perform the priming function required, for example, by DNA polymerase to initiate DNA synthesis. It is understood that hybridizing sequences do not need to have perfect complementarity to provide a stable hybrid. In many cases, stable hybrids are formed with less than about 10% mismatch of bases, ignoring loops of 4 or more nucleotides. Thus, as used herein, the term "complementary" refers to an oligonucleotide that forms a stable duplex with its "complement" under assay conditions where there is generally about 90% or more homology.

[0131] SNAIL oligonucleotide primers In the subject method, the SNAIL oligonucleotide primer comprises at least a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first region of complementarity, a second region of complementarity, and a third region of complementarity, the second oligonucleotide further comprising a barcode sequence, the first region of complementarity of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second region of complementarity of the first oligonucleotide being complementary to the first region of complementarity of the second oligonucleotide, the third region of complementarity of the first oligonucleotide being complementary to the third region of complementarity of the second oligonucleotide, the second region of complementarity of the second oligonucleotide being complementary to a second portion of the target nucleic acid, and the first region of complementarity of the first oligonucleotide being adjacent to the second region of complementarity of the second oligonucleotide. In an alternative embodiment, the second oligonucleotide is a closed circular molecule and the ligation step is omitted.

[0132] The present disclosure provides a method in which contacting fixed and permeabilized tissue comprises hybridizing a plurality of oligonucleotide primers having specificity for different target nucleic acids. In some embodiments, the method comprises a plurality of first oligonucleotides, including but not limited to, 5 or more first oligonucleotides hybridizing to target nucleotide sequences, such as 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure comprises a plurality of first oligonucleotides, including but not limited to, 15 or more first oligonucleotides hybridizing to 15 or more, such as 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences. In some embodiments, the method includes a plurality of second oligonucleotides, including but not limited to 5 or more second oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure includes a plurality of second oligonucleotides, including but not limited to 15 or more second oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides that hybridize to 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences. Multiple oligonucleotide pairs can be used in the reaction, with one or more pairs specifically binding to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability.It is also of interest to detect multiple different target nucleic acids in a cell, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids. Primers are typically denatured prior to use, typically by heating to a temperature of at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, and up to about 99°C, up to about 95°C, up to about 90°C.

[0133] In some embodiments, the primers are denatured by heating prior to contacting the sample. In certain aspects, the melting temperature (T m ) is chosen to minimize ligation in solution. The "melting temperature" or "T m " is defined as the temperature at which half of the helical structure of a nucleic acid is lost due to heating or other dissociation of the hydrogen bonds between base pairs, e.g., by acid or alkali treatment. m depends on its length and base composition. Nucleic acid molecules rich in GC base pairs have a higher T m The complementary strands of the separated nucleic acids have a temperature T m When the temperature drops below T, the nucleic acid will spontaneously reassociate or anneal to form a double-stranded nucleic acid. m This occurs approximately 25°C lower than T m can be estimated using the following relationship: T m =69.3+0.41(GC)% (Marmur et al. (1962) J. Mol. Biol. 5:109-118).

[0134] In certain embodiments, the plurality of second oligonucleotides comprises a padlock probe. In some embodiments, the probe comprises a detectable label that can be measured and quantified. The terms "label" and "detectable label" refer to a molecule that can be detected, including, but not limited to, a radioisotope, a fluorescer, a chemiluminescer, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (e.g., biotin or a hapten), and the like. The term "fluorescer" refers to a substance or a portion thereof that can exhibit fluorescence within the detectable range. Specific examples of labels that can be used with the present invention include, but are not limited to, phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acridine esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease.

[0135] In some embodiments, one or more of the first and second oligonucleotides bind to different regions of the target nucleic acid or target site. In a pair, each target site is different, and the target sites are adjacent sites on the target nucleic acid, e.g., typically no more than 15 nucleotides away from the other site, e.g., no more than 10, 8, 6, 4, or 2 nucleotides away, and may be contiguous sites. The target sites are typically on the same strand of the target nucleic acid in the same orientation. The target sites are also selected to provide unique binding sites for other nucleic acids present in the cell. Each target site is generally about 19 to about 25 nucleotides long, e.g., about 19 to 23 nucleotides, about 19 to 21 nucleotides, or about 19 to 20 nucleotides. The pair of first and second oligonucleotides is selected such that each oligonucleotide in the pair has a similar melting temperature for binding to its cognate target site, e.g., T m can be from about 50° C., from about 52° C., from about 55° C., from about 58° C., from about 62° C., from about 65° C., from about 70° C., or from about 72° C. The GC content of the target site generally can be selected to be about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less.

[0136] In some embodiments, the first oligonucleotide comprises a first, second, and third complementary region. The target site of the first oligonucleotide may refer to the first complementary region. As summarized above, the first complementary region of the first oligonucleotide may have a length of 19-25 nucleotides. In certain aspects, the second complementary region of the first oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In some aspects, the second complementary region of the first oligonucleotide has a length of 6 nucleotides. In some embodiments, the third complementary region of the first oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the first oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides.

[0137] In some embodiments, the second first oligonucleotide comprises a first, second and third complementary region. The target site of the second oligonucleotide may refer to the second complementary region. As summarized above, the second complementary region of the second oligonucleotide may have a length of 19-25 nucleotides. In certain aspects, the first complementary region of the first oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In some aspects, the first complementary region of the first oligonucleotide has a length of 6 nucleotides. In some aspects, the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. In some embodiments, the third complementary region of the second oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the second oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In further embodiments, the third region of complementarity of the second oligonucleotide comprises the 3' end of the second oligonucleotide. In some embodiments, the first region of complementarity of the second oligonucleotide is adjacent to the third region of complementarity of the second oligonucleotide.

[0138] In some embodiments, the second oligonucleotide comprises a barcode sequence, and the barcode sequence of the second oligonucleotide provides barcoded information for identifying the target nucleic acid. The term "barcode" refers to a nucleic acid sequence used to identify a single cell or a subpopulation of cells. The barcode sequence can be linked to the target nucleic acid of interest during amplification and can be used to trace the amplicon to the cell from which the target nucleic acid originated. The barcode sequence can be added to the target nucleic acid of interest during amplification by performing the amplification with an oligonucleotide that contains a region that includes the barcode sequence and a region that is complementary to the target nucleic acid such that the barcode sequence is ultimately incorporated into the amplified target nucleic acid product (i.e., amplicon).

[0139] organization As described herein, the disclosed methods include in situ sequencing techniques by contacting fixed and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization. Tissue specimens suitable for use with the methods described herein generally include any type of tissue specimen collected from a living or cadaveric subject, such as, for example, epithelial, muscle, connective tissue, and neural tissue, including but not limited to, those biopsy and autopsy specimens. The tissue specimen may be collected and processed using the methods described herein and subjected to microscopic analysis immediately after processing, or may be preserved and subjected to microscopic analysis in the future, for example after long-term storage. In some embodiments, the methods described herein may be used to preserve the tissue specimen in a stable, accessible, and completely intact form for future analysis. In some embodiments, the methods described herein may be used to analyze previously preserved or archived tissue specimens. In some embodiments, the intact tissue includes brain tissue, such as visual cortex slices. In some embodiments, the intact tissue is a thin slice having a thickness of 5-20 μm, for example, but not limited to, 5-18 μm, 5-15 μm, or 5-10 μm, while in other embodiments, the intact tissue is a thick slice having a thickness of 20-200 μm, for example, but not limited to, 20-150 μm, 50-100 μm, or 50-80 μm.

[0140] An embodiment of the present invention includes fixing intact tissue. The term "fixing" or "fixation" as used herein is a process of preserving biological material (e.g., tissues, cells, organelles, molecules, etc.) from decay and / or decomposition. Fixation can be accomplished using any convenient protocol. Fixation can include contacting the sample with a fixation reagent (i.e., a reagent containing at least one fixation agent). The sample can be contacted with the fixation reagent for a wide range of times that can depend on the temperature, the nature of the sample, and the fixation agent. For example, the sample can be contacted with the fixation reagent for 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less.

[0141] The sample can be contacted with the immobilization reagent for a period ranging from 5 minutes to 24 hours, e.g., 10 minutes to 20 hours, 10 minutes to 18 hours, 10 minutes to 12 hours, 10 minutes to 8 hours, 10 minutes to 6 hours, 10 minutes to 4 hours, 10 minutes to 2 hours, 15 minutes to 20 hours, 15 minutes to 18 hours, 15 minutes to 12 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1.5 hours, 15 minutes to 1 hour, 10 minutes to 30 minutes, 15 minutes to 30 minutes, 30 minutes to 2 hours, 45 minutes to 1.5 hours, or 55 minutes to 70 minutes.

[0142] The sample may be contacted by the immobilization reagent at a variety of temperatures, depending on the protocol and reagents used. For example, in some cases, the sample may be contacted by the immobilization reagent at a temperature ranging from -22°C to 55°C, with particular ranges of interest including, but not limited to, 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, 0-6°C, and -18-22°C. In some cases, the sample may be contacted by the immobilization reagent at temperatures of -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0143] Any convenient fixation reagent may be used. Common fixation reagents include cross-linking fixation agents, precipitation fixation agents, oxidation fixation agents, mercury, and the like. Cross-linking fixation agents chemically join two or more molecules by covalent bonds, and a wide range of cross-linking reagents may be used. Examples of suitable cross-linking fixation agents include, but are not limited to, aldehydes (e.g., formaldehyde, commonly also referred to as "paraformaldehyde" and "formalin", glutaraldehyde, and the like), imidoesters, NHS (N-hydroxysuccinimide) esters, and the like. Examples of suitable precipitation fixation agents include, but are not limited to, alcohols (e.g., methanol, ethanol, and the like), acetone, acetic acid, and the like. In some embodiments, the fixation agent is formaldehyde (i.e., paraformaldehyde or formalin). Suitable final concentrations of formaldehyde in the fixation reagent are 0.1-10%, 1-8%, 1-4%, 1-2%, 3-5%, or 3.5-4.5%, including about 1.6%, for 10 minutes. In some embodiments, the sample is fixed at a final concentration of 4% formaldehyde (diluted from a more concentrated stock solution, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%, etc.). In some embodiments, the sample is fixed at a final concentration of 10% formaldehyde. In some embodiments, the sample is fixed at a final concentration of 1% formaldehyde. In some embodiments, the fixative is glutaraldehyde. Suitable concentrations of glutaraldehyde in the fixation reagent are 0.1-1%. The fixation reagent may contain two or more fixatives in any combination. For example, in some embodiments, the sample is contacted with a fixation reagent that contains both formaldehyde and glutaraldehyde.

[0144] The term "permeabilization" or "permeabilize" as used herein refers to a process of making the cells (e.g., cell membranes) of a sample permeable to experimental reagents such as nucleic acid probes, antibodies, chemical substrates, etc. Any convenient method and / or reagent for permeabilization may be used. Suitable permeabilization reagents include detergents (e.g., saponin, Triton X-100, Tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), enzymes, etc. Detergents may be used in a wide range of concentrations. For example, 0.001% to 1% detergent, 0.05% to 0.5% detergent, or 0.1% to 0.3% detergent may be used for permeabilization (e.g., 0.1% saponin, 0.2% tween-20, 0.1 to 0.3% Triton X-100, etc.). In some embodiments, permeabilization is performed using methanol on ice for at least 10 minutes.

[0145] In some embodiments, the same solution may be used as the fixation reagent and the permeabilization reagent. For example, in some embodiments, the fixation reagent contains 0.1%-10% formaldehyde and 0.001%-1% saponin. In some embodiments, the fixation reagent contains 1% formaldehyde and 0.3% saponin.

[0146] The sample can be contacted with the permeabilization reagent for a wide range of times, which can depend on the temperature, the nature of the sample, and the permeabilization reagent. For example, the sample can be contacted with the permeabilization reagent for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample can be contacted with the permeabilization reagent at a variety of temperatures, depending on the protocol and reagents used. For example, in some examples, the sample can be contacted with the permeabilization reagent at a temperature ranging from -82°C to 55°C, with particular ranges of interest including 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, 0-6°C, -18-22°C, and -78-82°C. In some cases, the sample can be contacted by the permeabilization reagent at a temperature of -80°C, -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0147] In some embodiments, the sample is contacted with an enzyme permeabilization reagent. The enzyme permeabilization reagent permeabilizes the sample by partially degrading extracellular matrix or surface proteins that prevent permeabilization of the sample by the assay reagents. Contact with the enzyme permeabilization reagent can occur at any time after fixation and before target detection. In some cases, the enzyme permeabilization reagent is proteinase K, a commercially available enzyme. In such cases, the sample is contacted with proteinase K before contacting with the post-fixation reagent. Proteinase K treatment (i.e., contact with proteinase K, also commonly referred to as "proteinase K digestion") can be performed over a range of times, at a range of temperatures, and over a range of enzyme concentrations that are empirically determined for each cell or tissue type under investigation. For example, the sample can be contacted with proteinase K for 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample can be contacted with Proteinase K at 1 μg / ml or less, 2 μg / ml or less, 4 μg / ml or less, 8 μg / ml or less, 10 μg / ml or less, 20 μg / ml or less, 30 μg / ml or less, 50 μg / ml or less, or 100 μg / ml or less. The sample can be contacted with Proteinase K at a temperature ranging from 2° C. to 55° C., with specific ranges of interest including, but not limited to, 50-54° C., 40-44° C., 35-39° C., 28-32° C., 20-26° C., and 0-6° C. In some cases, the sample can be contacted with Proteinase K at a temperature of 4° C., room temperature (22-25° C.), 30° C., 37° C., 42° C., or 52° C. In some embodiments, the sample is not contacted with an enzyme permeabilization reagent. In some embodiments, the sample is not contacted with Proteinase K. Contacting the intact tissue with at least a fixation reagent and a permeabilization reagent results in the production of fixed and permeabilized tissue.

[0148] Ligase In some embodiments, the disclosed method comprises adding a ligase to ligate the second oligonucleotide and generate a closed nucleic acid circle. In some embodiments, the addition of the ligase comprises the addition of a DNA ligase. In alternative embodiments, the second oligonucleotide is provided as a closed nucleic acid circle, and the step of adding the ligase is omitted. In certain embodiments, the ligase is an enzyme that facilitates sequencing of the target nucleic acid molecule.

[0149] The term "ligase" as used herein refers to an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. Ligases include ATP-dependent double-stranded polynucleotide ligases, NAD-i-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, such as any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases such as E. coli DNA ligase and Taq DNA ligase, Ampligase® thermostable DNA ligase (Epicentre® Technologies Corp., part of Illumina®, Madison, Wis.), and phage ligases such as T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase, and variants thereof.

[0150] Rolling Circle Amplification In some embodiments, the method of the present invention comprises performing rolling circle amplification in the presence of a nucleic acid molecule, which comprises forming one or more amplicons using a second oligonucleotide as a template and a first oligonucleotide as a primer for a polymerase. In such an embodiment, a single-stranded circular polynucleotide template is formed by ligation of the second oligonucleotide, and the circular polynucleotide comprises a region complementary to the first oligonucleotide. Upon addition of DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the first oligonucleotide is extended by replication of multiple copies of the template. This amplification product can be easily detected by binding to a detection probe. In some embodiments, the polymerase is pre-incubated without dNTPs to allow the polymerase to uniformly permeate the sample before performing rolling circle amplification.

[0151] In some embodiments, only if the first and second oligonucleotides hybridize to the same target nucleic acid molecule, the second oligonucleotide may be circularized and rolled circle amplified to generate cDNA nanoballs (i.e., amplicons) containing multiple copies of the cDNA. The term "amplicon" refers to the amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process. In some embodiments, amine-modified nucleotides are spiked into the rolling circle amplification reaction.

[0152] Techniques for rolling circle amplification are known in the art (e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Schweitzer et al. Proc. Natl Acad. Sci. USA 97:101 13-1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el18, 2001; Dean et al. Genome Res. 11:1095-1099, 2001; Schweitzer et al, Nature (See, Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329, and 6,368,801.) In some embodiments, the polymerase is a phi29 DNA polymerase.

[0153] In certain aspects, the nucleic acid molecule comprises an amine-modified nucleotide. In such an embodiment, the amine-modified nucleotide comprises an acrylate N-hydroxysuccinimide partial modification. Other examples of amine-modified nucleotides include, but are not limited to, a 5-aminoallyl-dUTP partial modification, a 5-propargylamino-dCTP partial modification, an N6-6-aminohexyl-dATP partial modification, or a 7-deaza-7-propargylamino-dATP partial modification.

[0154] Amplicon embedding in tissue-hydrogel setup In some embodiments, the disclosed methods include embedding one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel-embedded amplicons. The described hydrogel histochemistry involves covalently attaching nucleic acids to in situ synthesized hydrogels for tissue clearing, enzyme diffusion, and multiple cycle sequencing, which existing hydrogel histochemistry methods are unable to do. In some embodiments, to enable amplicon embedding in a tissue-hydrogel setup, amine-modified nucleotides are spiked into a rolling circle amplification reaction, functionalized with acrylamide moieties using acrylic acid N-hydroxysuccinimide ester, and copolymerized with acrylamide monomers to form a hydrogel.

[0155] As used herein, the term "hydrogel" or "hydrogel network" refers to a network of water-insoluble polymer chains that are sometimes found as colloidal gels in which water is the dispersion medium. In other words, hydrogels are a class of polymeric materials that can absorb large amounts of water without dissolving. Hydrogels can contain more than 99% water and can include natural or synthetic polymers, or combinations thereof. Hydrogels also have a degree of flexibility that is very similar to natural tissues due to their significant water content. A detailed description of suitable hydrogels can be found in U.S. Patent Application Publication No. 2010 / 0055733, which is specifically incorporated herein by reference. As used herein, the term "hydrogel subunit" or "hydrogel precursor" refers to hydrophilic monomers, prepolymers, or polymers that can be crosslinked or "polymerized" to form a three-dimensional (3D) hydrogel network. Without being bound by any scientific theory, it is believed that this fixation of the biological specimen in the presence of the hydrogel subunits crosslinks components of the specimen to the hydrogel subunits, thereby fixing the molecular components in place and preserving tissue architecture and cellular morphology.

[0156] In some embodiments, embedding comprises copolymerizing one or more amplicons with acrylamide. As used herein, the term "copolymer" describes a polymer that contains two or more types of subunits. The term encompasses polymers that contain two, three, four, five, or six types of subunits.

[0157] In certain aspects, embedding comprises clarification of the one or more hydrogel-embedded amplicons, and the target nucleic acid is substantially retained in the one or more hydrogel-embedded amplicons. In such embodiments, clarification comprises substantially removing a plurality of cellular components from the one or more hydrogel-embedded amplicons. In some other embodiments, clarifying comprises substantially removing lipids and / or proteins from the one or more hydrogel-embedded amplicons. As used herein, the term "substantially" means that the original amount present in the sample prior to clarification is reduced by approximately 70% or more, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 99% or more, such as 100%.

[0158] In some embodiments, clarifying the amplicons embedded in the hydrogel comprises performing electrophoresis on the specimen. In some embodiments, the amplicons are electrophoresed using a buffer solution comprising an ionic detergent. In some embodiments, the ionic detergent is sodium dodecyl sulfate (SDS). In some embodiments, the specimen is electrophoresed using a voltage ranging from about 10 to about 60 volts. In some embodiments, the specimen is electrophoresed for a period ranging from about 15 minutes up to about 10 days. In some embodiments, the method further comprises incubating the cleared specimen in an encapsulation medium having a refractive index that matches the refractive index of the cleared tissue. In some embodiments, the encapsulation medium increases the optical clarity of the specimen. In some embodiments, the encapsulation medium comprises glycerol.

[0159] cell The methods disclosed herein include methods for in situ genetic sequencing of target nucleic acids in cells in intact tissues. In certain embodiments, the cells are present in a population of cells. In certain other embodiments, the population of cells includes multiple cell types, including, but not limited to, excitatory neurons, inhibitory neurons, and non-neuronal cells. The cells used in the assays of the present invention can be an organism, a single cell type from an organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells from transgenic animals, and the like. Virtually any cell type and size can be accommodated. Suitable cells include bacterial, fungal, plant, and animal cells. In one embodiment of the present invention, the cells are mammalian cells, e.g., complex cell populations of naturally occurring tissues, e.g., blood, liver, pancreas, neural tissue, bone marrow, skin, and the like. Some tissues can be disrupted into monodisperse suspensions. Alternatively, the cells can be cultured populations, e.g., cultures derived from complex populations, cultures derived from a single cell type in which the cells have differentiated into multiple lineages, or cells are differentially responsive to stimuli, and the like.

[0160] Cell types that may find use in the subject invention include stem and progenitor cells, e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc., endothelial cells, muscle cells, cardiac muscle, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells; hematopoietic cells, such as lymphocytes, including T cells, such as Th1 T cells, Th2 T cells, ThO T cells, cytotoxic T cells, etc.; B cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils; and macrophages; natural killer cells; mast cells, etc.; adipocytes, cells involved in specific organs, such as the thymus, endocrine glands, pancreas, brain, such as neurons, glia, astrocytes, dendritic cells, and genetically modified versions thereof. Hematopoietic cells may be associated with inflammatory processes, autoimmune diseases, etc., while endothelial cells, smooth muscle cells, cardiac muscle cells, etc. may be associated with cardiovascular diseases. Almost any type of cell may be associated with tumors, such as sarcomas, carcinomas, and lymphomas, liver diseases involving hepatocytes, kidney diseases involving renal cells, etc.

[0161] The cells may also be transformed or tumor cells of different types, e.g., carcinomas of different cellular origin, lymphomas of different cell types, etc. The American Type Culture Collection (Manassas, VA) has collected and made available over 4,000 cell lines from over 150 different species, over 950 cancer cell lines, including 700 human cancer cell lines. The National Cancer Institute has accumulated clinical, biochemical, and molecular data from a large panel of human tumor cell lines, which are available from the ATCC or NCI (Phelps et al. (1996) Journal of Cellular Biochemistry Supplement 24:32-91). Different cell lines that have been spontaneously derived or selected for desired growth or response characteristics from individual cell lines may include multiple cell lines derived from similar tumor types, but from different patients or sites.

[0162] The cells may be non-adherent, e.g., blood cells including monocytes, T cells, B cells, tumor cells, etc., or adherent cells, e.g., epithelial cells, endothelial cells, neuronal cells, etc. To profile adherent cells, they may be dissociated from the substrate to which they are adhered and from other cells in a manner that maintains the ability to recognize and bind to the probe molecule.

[0163] Such cells can be obtained from an individual, for example, by a variety of techniques known in the art, from a variety of tissues, e.g., blood, bone marrow, solid tissues (e.g., solid tumors), ascites, using withdrawal, lavage, wash, surgical dissection, etc. Cells can be obtained from fixed or unfixed, fresh or frozen, whole or disintegrated samples. Disintegration of tissues can occur either mechanically or enzymatically using known techniques.

[0164] Examples of Non-Limiting Aspects of the Disclosure The aspects (including embodiments) of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure, numbered 1-34, are provided below. As would be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding aspects or any of the aspects following the individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0165] 1. A sequencing device comprising: (a) an illumination and detection module comprising a spinning disk confocal component including multiple laser lines for illumination with flat illumination correction, where the multiple laser lines are used to illuminate a sample with excitation light at one or more wavelengths; a bandpass emission filter; a longpass image splitter; a first camera that detects fluorescent emission in a first wavelength range; and a second camera that detects fluorescent emission in a second wavelength range, where the first camera and the second camera are capable of simultaneously detecting emission; (b) a microscope module comprising a motorized stage capable of multi-axis positioning along the x, y, and z axes, an objective Z drive, and an objective turret wheel comprising a plurality of objective lenses, each objective lens providing a different magnification, one or more of the objective lenses being immersion objective lenses, each immersion objective lens having an objective immersion collar and optics, the optics transmitting light from the objective lens to an illumination and detection module; (c) an automated immersion medium module comprising: i) a container containing an immersion medium; ii) fluid lines connected to the container and to an objective immersion collar of an immersion objective of the microscope module, the fluid lines carrying immersion medium to and from the objective immersion collar, the immersion collar capturing excess immersion medium; and iii) a series of pumps connected to the fluid lines and a microcontroller, the microcontroller controlling the pumping addition and removal of immersion medium through the fluid lines, wherein the automated immersion medium module provides a controlled volume of immersion medium to the objective immersion collar on top of the immersion objective during imaging; (d) a multiwell plate, the motorized stage being capable of moving to position the wells of the multiwell plate under an objective lens used for imaging; (e) a fluid connection tower, the fluid connection tower being on top of the motorized stage and locating fluid lines into the wells of the multiwell plate; (f) a fluid management module comprising a symmetric rotary valve including a rotary valve mechanism and a pump connected to the fluid lines and an air bubble detector disposed on either side of the fluid lines leading to the pump, the fluid management module enabling unidirectional or bidirectional movement of reagents, buffers, and waste through the fluid lines; (g) a reagent, buffer, and waste module comprising: i) a slide tray, where reagent cartridges and buffer cartridges can be placed in the slide tray and coupled to the fluid management module; ii) a waste module comprising a waste container, where the waste container is coupled to a fluid line from the fluid management pump; and iii) a capping mechanism, where the capping mechanism closes the waste container when it is removed from the system for waste disposal and opens the waste container when it is returned to the system; (h) an electrical module comprising: i) a first firmware board that controls media dispensed from the automated immersion media module; and ii) a second firmware board that controls the fluid management module and the reagent, buffer and waste modules, the electrical module regulating power to the other modules of the system; (i) a processor programmed to provide a user interface and to operate the modules of the sequencing device; A sequencing device comprising: 2. The sequencing device of embodiment 1, wherein the plurality of laser lines comprises at least five laser lines. 3. The sequencing device of embodiment 2, wherein the bandpass emission filter is a penta-bandpass emission filter. 4. The sequencing device of any one of aspects 1-3, wherein the motorized stage has a piezo z-axis. 5. The sequencing device of any one of aspects 1 to 4, wherein the immersion medium is water.

[0166] 6. The sequencing device of any one of aspects 1-5, wherein the immersion medium is filtered and bubble-free. 7. The sequencing device of any one of aspects 1-6, further comprising an O-ring and shrink-wrap coating on each objective lens. 8. The sequencing device of any one of aspects 1 to 7, further comprising a pressure monitor for monitoring pressure in the fluid line, wherein an increase in pressure in the fluid line can be used to detect a potential blockage in the fluid line. 9. The sequencing device of any one of aspects 1-8, further comprising a plurality of light emitting diodes (LEDs), each LED capable of emitting light to provide a status indication for the system. 10. The sequencing device of any one of aspects 1-9, further comprising a display component for displaying information and providing a user interface.

[0167] 11. The processor: (a) positioning selected samples in a multiwell plate; (b) detecting signals in the XY plane at low magnification from a selected sample using wide-field imaging mode acquisition with camera binning; (c) using the signal to segment an XY bounding box around the sample; (d) imaging the sample within an XY bounding box to generate an image, where the imaging is performed in confocal imaging mode at a higher magnification in Z than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint of the previously determined Z range and across the XY range; (e) displaying the image generated in step (d); and (f) providing an interface for a user to select a desired XY region of interest in the sample to be further imaged during sequencing of the selected sample; (g) imaging the sample in a selected XY region of interest over the previously sampled Z range; (h) calculating the volume of a region of interest in the sample and displaying the calculated sample volume of the region of interest to the user; (i) segmenting an image of a sample within a region of interest along a Z range; (j) providing a user with an interface for adjusting the Z-range of the sample volume before starting sequencing, where the imaging range derived from the user-defined region of interest is automatically converted to the appropriate montaged field of view for a given imaging objective, and adjusting the microscope stage position, objective Z placement, and piezo boundaries for imaging of the region of interest along the XYZ axes during sequencing; (k) repeating steps (a)-(j) multiple times to define regions of interest for each sample in the multi-well plate that the user intends to sequence; The sequencing device according to any one of aspects 1 to 10, further programmed to perform a step comprising: 12. The processor: providing an interface for a user to select one or more samples for sequencing and a sequencing protocol, where the number of samples that the user may select is limited depending on the amount of buffers and reagents that are available and the sequencing protocol selected; providing constraints on total sequencing time across all samples to be sequenced and imaged, total data acquired, rate of acquisition, and maximum total volume of regions of interest; To propose a protocol that maximizes the sequencing of desired regions of interest in a sample within constraints; 12. The sequencing device according to any one of aspects 1 to 11, further programmed to perform a step comprising: 13. The sequencing device of any one of aspects 1 to 12, wherein the processor is further programmed to optimize sample sequencing parallelism depending on the number of samples to be sequenced and the imaging type used for sequencing. 14. The processor: performing a rapid confocal sweep in Z at a starting XY position of a given sample montage to determine a Z profile of the sample at the starting XY position; determining upper and lower interfaces of the specimen using a segmentation method; setting a Z position of the objective at a fixed distance from the interface at the start of a sample montage, such that drift in Z of the sample relative to the stage and objective across a round is reduced to below a selected tolerance to facilitate downstream sub-pixel registration across rounds during post-acquisition processing; 14. The sequencing device according to any one of aspects 1 to 13, further programmed to perform a step comprising: 15. The sequencing device according to any one of aspects 1 to 14, wherein the sequencing is in situ sequencing of a target nucleic acid in a tissue sample.

[0168] 16. The sequencing device of aspect 15, wherein the tissue sample is a tissue slice having a thickness of 20 μm to 200 μm. 17. The sequencing device according to any one of aspects 1 to 16, wherein the in situ sequencing is sequential or combinatorial in situ sequencing. 18. The sequencing device of any one of aspects 1-17, wherein the microscope module comprises a fluorescence microscope, a confocal microscope, a structured illumination microscope, or a light sheet or an inclined plane light sheet microscope. 19. The sequencing device according to aspect 18, wherein the confocal microscope is a spinning disk or point scanning confocal microscope.

[0169] 20. A method of using a sequencing device according to any one of aspects 1 to 19, comprising: Loading the samples into a multi-well plate; Selecting which samples in the multiwell plate are to be sequenced; selecting a sequencing protocol; Sequencing nucleic acids in a selected sample using a sequencing device according to any one of aspects 1 to 19. A method comprising: 21. The method of embodiment 20, wherein the sequencing is in situ volumetric sequencing of a tissue sample. 22. The method according to aspect 20 or 21, wherein the tissue sample is a tissue slice having a thickness of 20 to 200 μm. 23. The method according to any one of aspects 20 to 22, wherein the in situ sequencing is sequential or combined in situ sequencing.

[0170] 24. A computer-implemented method comprising: The computer (a) positioning selected samples in a multiwell plate; (b) detecting signals in the XY plane at low magnification from a selected sample using wide-field imaging mode acquisition with camera binning; (c) using the signal to segment an XY bounding box around the sample; (d) imaging the sample within an XY bounding box to generate an image, where the imaging is performed in confocal imaging mode at a higher magnification in Z than used in step (b) with camera binning to determine an approximate Z range of the sample, and a single Z plane is collected through the midpoint of the previously determined Z range and across the XY range; (e) displaying the image generated in step (d); and (f) providing an interface for a user to select a desired XY region of interest in the sample to be further imaged during sequencing of the selected sample; (g) imaging the sample in a selected XY region of interest over the previously sampled Z range; (h) calculating a sample volume of the region of interest and displaying the calculated sample volume of the region of interest to a user; (i) segmenting an image of a sample within a region of interest along a Z range; (j) providing a user with an interface for adjusting the Z-range of the sample volume before starting sequencing, where the imaging range derived from the user-defined region of interest is automatically converted to the appropriate montaged field of view for a given imaging objective, and adjusting the microscope stage position, objective Z placement, and piezo boundaries for imaging of the region of interest along the XYZ axes during sequencing; (k) repeating steps (a)-(j) multiple times to define regions of interest for each sample in the multi-well plate that the user intends to sequence; 23. A computer-implemented method comprising the steps of: 25. A non-transitory computer-readable medium comprising program instructions that, when executed by a processor in a computer, cause the processor to perform the method of embodiment 24. 26. A computer-implemented method comprising: The computer providing a user with an interface for selecting one or more samples for sequencing and a sequencing protocol, where the number of samples the user may select is limited depending on the amount of buffers and reagents available and the sequencing protocol selected; providing constraints on total sequencing time across all samples to be sequenced and imaged, total data acquired, rate of acquisition, and maximum total volume of regions of interest; To propose a protocol that maximizes the sequencing of desired regions of interest in a sample within constraints; 23. A computer-implemented method comprising the steps of: 27. The computer-implemented method of embodiment 26, wherein the computer is further programmed to optimize sample sequencing parallelism depending on the number of samples to be sequenced and the imaging type used for sequencing. 28. A non-transitory computer-readable medium comprising program instructions that, when executed by a processor in a computer, cause the processor to perform the method of aspect 26 or 27.

[0171] 29. A computer-implemented method comprising: The computer performing a rapid confocal sweep in Z at a starting XY position of a given sample montage to determine a Z profile of the sample at the starting XY position; determining upper and lower interfaces of the specimen using a segmentation method; setting a Z position of the objective at a fixed distance from the interface at the start of a sample montage, such that drift in Z of the sample relative to the stage and objective across a round is reduced to below a selected tolerance to facilitate downstream sub-pixel registration across rounds during post-acquisition processing; 23. A computer-implemented method comprising the steps of: 30. A non-transitory computer-readable medium comprising program instructions that, when executed by a processor in a computer, cause the processor to perform the method of embodiment 29.

[0172] 31. An automated immersion media module comprising: (a) a vessel containing an immersion medium; (b) a fluid line connected to the container and to an objective immersion collar of the objective of the microscope module, the fluid line conveying immersion medium to and from the objective immersion collar on the immersion objective, the immersion collar capturing excess immersion medium; (c) a series of pumps connected to the fluid lines and a microcontroller, the microcontroller controlling the pumping of the immersion medium through the fluid lines; and Equipped with an automated immersion media module provides a controlled volume of immersion media to an objective immersion collar above the objective during imaging; Automated immersion media module. 32. A method of using the automated immersion medium module described in embodiment 31, comprising using the automated immersion medium module described in embodiment 31 to deliver immersion medium to an objective immersion collar attached to an immersion objective of a microscope.

[0173] 33. A fluid management module comprising: a symmetric rotary valve including a rotary valve mechanism; and a pump connected to a fluid line and an air bubble detector, the air bubble detector being located on either side of the fluid line leading to the pump; A fluid management module that allows bidirectional or unidirectional movement of reagents, buffers, and waste through the fluid lines. 34. A reagent, buffer, and waste module comprising: (a) a slide tray, in which a reagent cartridge and a buffer cartridge can be placed and coupled to a fluid management module; (b) a waste module including a waste container, the waste container coupled to a fluid line from the fluid management pump; (c) a capping mechanism that closes the waste container when the waste container is removed from the system for waste disposal and that opens the waste container when the waste container is replaced in the system; and a reagent, buffer, and waste module comprising:

[0174] experiment The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0175] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0176] The present invention has been described with respect to specific embodiments found or provided by the inventors to include preferred modes for carrying out the invention. Those skilled in the art will understand in light of this disclosure that numerous modifications and changes can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, codon redundancy allows changes in the underlying DNA sequence to be made without affecting the protein sequence. Biological functional equivalence considerations allow changes in protein structure to be made without affecting biological action in type or amount. All such modifications are intended to be within the scope of the appended claims.

[0177] Example 1 Volumetric next-generation in situ sequencing overview 1. Integrated fluidics and rapid confocal platform, with up to 5 channel imaging 2. Automated Immersion Media Module Robust bubble prevention routine 3. Sample / Fluid Coupling Tower 4. Custom Waste Containers 5. Sequencing Machine Software a. GUI b. Rapid sample search algorithm (general for 3D sequencing samples) c. Joint optimization of sequencing time, parallelism, reagent consumption, and disk space (generalized for 3D sequencing samples) d. Closed-loop detection of sample interface and xyz position for minimal drift in data across multiple rounds.

[0178] Technical description The samples were sequenced on an automated integrated fluidics and imaging platform capable of performing multiple rounds of sequencing and imaging cycles on many samples in parallel. The sequencer has several important modules:

[0179] The illumination and detection module, which integrates components from Andor Technologies, consists of a spinning disk confocal component, five laser line illumination, Borealis flat illumination correction, a penta bandpass emission filter, a longpass image splitter, and two detection cameras for simultaneously detecting the emissions generated by the short and long wavelength illumination.

[0180] The custom microscope module combines a motorized XYZ stage (with piezo Z), objective Z drive, turret wheels, multiple objectives ranging from low to high magnification, and optics that deliver light from the objectives to the illumination and detection module.

[0181] A new automated immersion media module, which interfaces with objectives that require immersion media between the objective and the cover glass, provides a precisely controlled volume of filtered, bubble-free media (such as water) to the top of the objective and manages both inflow and outflow. This module enables parallel sequencing of samples with high magnification immersion objectives that would otherwise require manual addition of immersion media. Addition and removal of immersion media is managed by a series of pumps and a microcontroller such that surface tension on the cover glass is minimized (minimizing shifts in the Z dimension of the sample), air bubbles are avoided, liquid overflow is prevented, and sufficient volume is present above the objective for long-term imaging. This involves algorithmic adjustment of liquid flow rate, x, y, and z position of the objective relative to the sample, liquid exchange steps to remove air bubbles, and fluid exchange timing. For example, after delivering a calibrated volume of immersion media (typically water) through the collar and onto the objective, an angled stage movement is performed that prevents the occurrence of air bubble formation. The immersion collar captures excess immersion medium and prevents the medium from accessing the objective lens through multiple O-rings and shrink-wrap coatings on the objective lens. The fluid lines bringing the immersion medium to and from the objective lens immersion collar are managed by a central tower that is integrated into the objective turret.

[0182] A novel fluid connection tower located on the XYZ stage positions the fluid lines in the multi-well sample plate. The connection allows easy addition and removal of samples from the microscope stage, as well as mating of fluid lines to the samples, while minimizing structural load on the automated microscope stage (excessive weight on the stage would inhibit accurate placement and rapid movement). A beam break detects if the sample is fully connected to the system, and structural elements prevent damage to the fluid lines or harm to the user during operation.

[0183] The fluid management module allows bidirectional movement of reagents, buffers, and waste through symmetrical rotary valves, pumps, and rotary valve mechanisms. Pump movement is controlled and line pressure is monitored by custom firmware. Liquid moving towards the pump is primed for accurate volumetric movement through the use of air bubble detectors on either side of the lines leading to the pump (before the rotary valve). Pressure in the lines is monitored for nominal fluid flow to detect potential blockages in the lines.

[0184] The reagent, buffer, and waste module orchestrates the addition of sequencing reagents and buffers to the system, as well as the removal of waste from the system. It is composed of a slide tray into which custom reagent and buffer cartridges are placed, which is then coupled to the fluid management module in an automated fashion. The waste module receives an outlet line from the fluid management pump via a capping mechanism design that closes the waste container when removed from the system for waste disposal, but ensures that it is open when placed back into the system.

[0185] The electrical module regulates power for the various components of the system. One firmware board controls the automated immersion media dispenser, and a second firmware board controls the fluid management module, and the reagent, buffer and waste modules, in addition to LEDs that display status indications related to the system.

[0186] A custom computer software program, including both back-end and GUI modules, provides an interface between the user and the sequencer firmware and hardware, including sequencing run setup, sequencing run options, sample region of interest (ROI) definition, high level operations required for sequencing and imaging, high level control of the automated immersion system, parallelization of sequencing across samples, logging, error monitoring, data acquisition, management and transfer, and run progress monitoring. The computer software program includes several novel algorithms for run setup and consistent imaging of sequencing rounds. An automated, 3D sample search and ROI specification algorithm rapidly detects signal in the XY plane from the sample via low magnification widefield imaging mode acquisition with maximal camera binning. This signal is used to segment an XY bounding box around the sample. This XY boundary is then rapidly subsampled in Z in confocal imaging mode with higher magnification but maximal camera binning to determine the approximate Z range of the sample. A single Z plane is rapidly collected across the XY range through the midpoint of the previously determined Z range and displayed to the user. At this point, the user can select via the interface the desired XY ROI to be imaged during sequencing for a given sample. A more detailed acquisition of the XY ROI is then acquired across the previously sampled Z range. This volume is displayed to the user and is additionally used to segment in Z the sample range at the given ROI. The user may further fine-tune the Z range of the volume via the interface before sequencing begins. The range of imaging derived from this ROI definition is automatically translated into the appropriate montaged field of view for a given imaging objective, and further automatically translated into the microscope stage position, objective Z placement, and piezo boundaries for optimal imaging of the XYZ ROI during sequencing.Using this ROI definition algorithm provides rapid semi-automatic ROI definition in 3D and minimizes the amount of user interaction (e.g., any manual control and searching of wells for sample and montage definition and testing, which can be excessively time-consuming and difficult for inexperienced users). This automated ROI procedure is performed for each sample well that the user intends to sequence.

[0187] The second algorithm guides the user in the sequencing run setup to ensure optimal sample parallelism and sequencer time efficiency while preventing the collection (storage or transfer) of excessive amounts of data or the use of more buffers and reagents than are available. This algorithm is essential for spatial sequencing approaches, especially volumetric sequencing approaches, since the user is interested in ROIs of varying XYZ extent across different sample types and excess imaging volume beyond the ROI is irrelevant to the user. Furthermore, a single sample acquisition may generate many terabytes of raw data that need to be stored and / or transferred. Furthermore, since sequencing kits have maximum buffer and reagent volumes, the user is limited in the number of samples that can be defined for use, especially depending on the exact sequencing protocol and the number of rounds required. Thus, an optimization algorithm is needed that balances multiple sample sequencing time, buffer / reagent availability, sequencing protocol, total data collection and transfer capacity, and ROI extent in XYZ. The algorithm may impose strict constraints on total sequencing time (e.g., 3 days), total data acquired and acquisition speed (related to available data transfer / offloading speed), maximum ROI budget across all samples, and maximum available buffers / reagents, while suggesting combinations of ROI and sample protocols that pack the maximum amount of desired sequencing into these constraints. This optimization also involves a subroutine optimizer for sample sequencing parallelization, as different numbers of samples and imaging types may yield optimal parallelization solutions for varying times.

[0188] Another algorithm applied during sample sequencing utilizes angled infrared laser light reflected from the cover slip to find the Z position set point in a closed loop, eliminating the need for so-called Perfect Focus system hardware. At the starting XY position of a given sample montage, the algorithm uses a rapid confocal sweep in Z to determine the characteristic Z profile of the sample at that position. A segmentation method is used to determine the top and bottom interface of the sample, and the Z position of the objective is set a fixed distance from this interface at the start of the montage. This ensures that even if there is drift in Z of the sample relative to the stage and objective across rounds, the sample Z drift is reduced to below some tolerance, facilitating downstream sub-pixel registration across rounds during post-acquisition processing. This algorithm is especially important in the absence of a perfect focus hardware system for combinatorial sequencing, where the exact position of the diffraction limited spot must be aligned across rounds, and even more so when the sample thickness is small, where drift in Z that might otherwise occur in any round is large relative to the total sample range in Z, resulting in a larger percentage of data loss in each round at the edges of the sample range. Such an algorithm may also be performed for alignment in XY, although due to the aspect ratio of the acquisition, the percentage of data loss from unexpected sample movement in XY is generally minimal.

Claims

1. A method for automatically maintaining an amount of immersion fluid on an optical immersion lens, comprising: (a) providing a chamber having said optical immersion lens and fluidly coupled to a fluid delivery line; (b) dispensing an immersion fluid into the chamber via the fluid delivery line, the chamber having at least a predetermined amount of immersion fluid contained therein after dispensing; (c) maintaining at least the predetermined amount of immersion fluid within the chamber; and A method comprising:

2. The method of claim 1, further comprising, between (a) and (b), determining that the optical immersion lens does not have the predetermined amount of immersion fluid on the optical immersion lens.

3. The method described in claim 2, wherein the determining is an automated determination.

4. The method of claim 2, wherein the determining includes the use of a sensor configured to determine the amount of immersion fluid on the optical immersion lens.

5. The method of claim 2, wherein the determining includes using a timer to measure the time since the last addition of immersion fluid.

6. A method according to any one of claims 1 to 5, wherein the optical immersion lens is part of a system configured to autonomously image a sample over a period of at least two days using at least in part an objective lens.

7. The method of claim 1, further comprising using the optical immersion lens to image at least a portion of an in situ biological sample.

8. A method according to any one of claims 1 to 5, wherein the chamber is configured to capture excess immersion fluid.

9. A method according to any one of claims 1 to 5, further comprising using a fluid removal line to remove immersion fluid from the chamber.

10. The method of claim 9, wherein the fluid delivery line and the fluid removal line operate in series.

11. An automated fluidic module for an optical immersion lens, comprising: a pump coupled to a fluid delivery line configured to distribute immersion fluid into an immersion fluid collar provided on said optical immersion lens; a processor configured to, in response to determining that the optical immersion lens does not have a predetermined level of immersion fluid, drive the pump to deliver the immersion fluid to the optical immersion lens until the optical immersion lens has at least the predetermined level of immersion fluid; An automated fluidic module comprising:

12. The automated fluid module of claim 11, wherein the immersion fluid is water.

13. The automated fluid module of claim 11, wherein the immersion fluid collar is configured to retain the immersion fluid on the optical immersion lens.

14. The automated fluid module of claim 11, wherein substantially no air bubbles are introduced into the immersion fluid in the fluid delivery line.

15. An automated fluid module as described in any one of claims 11 to 14, further comprising a further optical immersion lens and a further fluid delivery line configured to distribute the immersion fluid onto the further optical immersion lens.

16. The automated fluid module of claim 15, configured to maintain the predetermined level of the immersion fluid on both the optical immersion lens and the further optical immersion lens.

17. An automated fluidic module as described in any one of claims 11 to 14, which maintains the optical immersion lens under the immersion fluid for at least about five days.

18. An automated fluid module as described in any one of claims 11 to 14, further comprising an additional optical immersion lens coupled to an additional immersion fluid collar.

19. An automated fluid module as described in any one of claims 11 to 14, wherein the immersion fluid collar is configured to capture excess immersion fluid.

20. An automated fluid module as described in any one of claims 11 to 14, further comprising an additional pump configured to remove the immersion fluid from the immersion fluid collar.

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