Compositions and methods for in situ sequencing

Abstract

Methods and compositions for in situ base-by-base sequencing in a cell or tissue sample are described that minimize optical congestion. In some embodiments, a sequencing primer hybridizes to a priming site at 3'of an identifier sequence (e.g., a barcode sequence) in a sample, such that the sequencing primer can be extended in a per-base manner by a polymerase using the identifier sequence as a template. In some embodiments, a sample may be contacted with a nucleotide in a cyclic series of nucleotide incorporation or binding steps, and a signal indicative of an incorporation or binding event may be detected to generate a signal code sequence comprising a series of signal codes (corresponding to a signal (ON signal), a signal loss (OFF signal), or a combination thereof) detected in successive cycles. A corresponding analyte may be detected and localized using decoding of the sequence of identifiers based at least in part on the sequence of signal codes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 340,730, filed on May 11, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to methods and compositions for in situ detection of analytes in a sample. Background Art

[0004] Genomic, transcriptomic and proteomic analysis of cell and tissue samples using microscopic imaging can simultaneously resolve multiple analytes of interest, thereby providing valuable information about analyte abundance and location in situ. Therefore, in situ assays are important tools, for example, for understanding the molecular basis of cell identity and developing disease treatment methods. New and improved in situ assay methods are needed. Methods and compositions that address these and other needs are provided herein. Summary of the invention

[0005] The multiple scalability of the in-situ detection method may be limited by optical crowding. For example, the signal associated with large-size features (for example, rolling circle amplification (RCA) products, nucleic acid probes or nucleic acid complexes) and / or high intensity may overlap with other signals and / or cover other signals, especially when the signal comes from the feature next to each other, and the signal associated with small-size features and / or weak intensity may not reach the detection threshold. In either case, the quality of signal detection and decoding of identifier sequences associated with target analytes in multiple determinations, for example, may be impaired. In addition, some existing methods require complex oligonucleotide probe pools, which increase the cost and time of detection and decoding.

[0006] The present disclosure relates in some aspects to the method and composition for carrying out in situ base-by-base sequencing in a cell or tissue sample.In some embodiments, a sequencing primer is hybridized with the priming site at 3' of an identifier sequence (e.g., a barcode sequence) in a sample so that the sequencing primer can be extended by a polymerase in a base-by-base manner using the sequence of the identifier sequence as a template.Cells or tissue samples can be contacted with nucleotides in the nucleotide incorporation or nucleotide binding step of a cycle series, and the signal indicating incorporation or binding events can be detected in each cycle to generate a signal code sequence, which includes a series of signal codes corresponding to the signal (ON signal) detected in a continuous cycle, a signal absence (OFF signal) or a combination of ON and OFF signals.Signal code sequences of a variety of different identifier sequences can be generated at multiple positions in a sample and used to decode corresponding analytes.

[0007] In some embodiments, provided herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: the biological sample is a cell or tissue sample, the biological sample comprises a first analyte and a second analyte at a first position and a second position in the biological sample, respectively, the first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively, the first probe or a product thereof comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and the second probe or a product thereof comprises i) a second priming site of a second sequencing primer and ii) a second identifier sequence associated with the second analyte; b) using the first sequencing primer and a second sequencing primer to perform base-by-base sequencing of the first identifier sequence and the second identifier sequence, such as sequencing by synthesis (SBS) or sequencing by binding (SBB), thereby generating a first signal code sequence and a second signal code sequence, each signal code sequence comprising a signal code corresponding to a signal (ON signal), a signal absence (OFF signal) or a combination thereof detected at a first position and a second position, respectively, in consecutive cycles, wherein in one or more of the consecutive cycles, an ON signal is detected at the first position and an OFF signal is detected at the second position; and c) detecting the first identifier sequence and the second identifier sequence in a biological sample based at least in part on the first signal code sequence and the second signal code sequence.

[0008] In some embodiments, the first analyte and the second analyte are the same. In some embodiments, the first analyte and the second analyte are different. In any embodiments herein, the first identifier sequence and the second identifier sequence may be different. In any embodiments herein, the first identifier sequence and the second identifier sequence may comprise an analyte sequence or its complementary sequence.

[0009] In any embodiment herein, the first identifier sequence and the second identifier sequence may comprise a barcode sequence or its complementary sequence. In any embodiment herein, the barcode sequence may be assigned to the first analyte and the second analyte, respectively. In some embodiments, the barcode sequence is assigned based on a decision rule designed to minimize the maximum predicted density of the ON signal detected in each of one or more continuous cycles. In some embodiments, the decision rule for assigning the first barcode sequence to the first analyte and the second barcode sequence to the second analyte includes an assignment based on the expression data of the first analyte and the second analyte. In some embodiments, the first barcode sequence is assigned to the first analyte and the second barcode sequence is assigned to the second analyte including an assignment based on the expression data of the first analyte and the second analyte in the clustered cell type. In some embodiments, the clustered cell type represents the distribution of the cell types present in the biological sample. In some embodiments, the expression data of the first analyte and the second analyte overlap at least partially. In some embodiments, the expression data of the first analyte and the second analyte include bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single cell gene expression data, single cell protein expression data, or any combination thereof.

[0010] In any of the embodiments herein, the method may include assigning a first barcode sequence to a first analyte and assigning a second barcode sequence to a second analyte. In some embodiments, the nucleotides in the first barcode sequence detected in a particular cycle correspond to a signal code comprising an ON signal, and the corresponding nucleotides in the second barcode sequence detected in the particular cycle correspond to a signal code comprising an OFF signal. In some embodiments, the nucleotides in the first barcode sequence detected in a particular cycle correspond only to ON signals, and the corresponding nucleotides in the second barcode sequence detected in the particular cycle correspond only to OFF signals. In any of the embodiments herein, one or more pairs of corresponding nucleotides in the first barcode sequence and the second barcode sequence to be detected in the same cycle can be selected to reduce optical crowding of the signals detected in the cycle.

[0011] In any of the embodiments herein, base-by-base sequencing can be performed by contacting a biological sample with nucleotides in continuous cycles, wherein in each cycle a complex is formed, the complex comprising i) a first sequencing primer or a second sequencing primer hybridized to a first priming site or a second priming site, respectively, or an extension product thereof, ii) a polymerase, and iii) a homologous nucleotide that base pairs with a nucleotide in the first identifier sequence or the second identifier sequence, and a signal (ON signal) and / or a lack of signal (OFF signal) associated with the homologous nucleotide and / or the polymerase in the complex is detected at a specific position in the biological sample, wherein the ON signal, the OFF signal, or a combination thereof corresponds to a base in the homologous nucleotide and the corresponding nucleotide in the first identifier sequence or the second identifier sequence.

[0012] In any embodiment herein, 25% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, the first identifier sequence and / or the second identifier sequence can be designed so that more than 25% of the nucleotides correspond to the OFF signal. In any embodiment herein, 30% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, the first identifier sequence and / or the second identifier sequence can be designed so that more than 30% of the nucleotides correspond to the OFF signal. In any embodiment herein, the first identifier sequence and / or the second identifier sequence can be designed so that more than 35% of the nucleotides correspond to the OFF signal. In any embodiment herein, 40% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, the first identifier sequence and / or the second identifier sequence can be designed so that more than 45% of the nucleotides correspond to the OFF signal. In any embodiment herein, 50% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, the first identifier sequence and / or the second identifier sequence can be designed so that more than 55% of the nucleotides correspond to the OFF signal. In any embodiment herein, 60% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, the first identifier sequence and / or the second identifier sequence can be designed so that more than 65% of the nucleotides correspond to the OFF signal. In any embodiment herein, 70% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, 75% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, 80% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, 85% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any embodiment herein, 90% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence can be assigned to correspond to the OFF signal. In any of the embodiments herein, 95% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence may be assigned to correspond to an OFF signal.

[0013] In any embodiment herein, a variety of different identifier sequences can be detected in a biological sample, and each different identifier sequence can be detected at one or more positions in a biological sample. In some embodiments, the multiple different identifier sequences are multiple unique identifier sequences, for example, each uniquely corresponding to an analyte. In any embodiment herein, 50% or more of the different identifier sequences can each contain 50% or more of the nucleotides corresponding to an OFF signal in the identifier sequence. In any embodiment herein, 80% or more of the different identifier sequences can each contain 80% or more of the nucleotides corresponding to an OFF signal in the identifier sequence.

[0014] In any embodiment herein, the signal code can each correspond to a signal of the first color, a signal of the second color, a signal of the third color, or a signal absence, and wherein the first, second, and third colors are different. In any embodiment herein, the signal code can each correspond to a signal of the first color, a signal of the second color, a combination of signals of the first color and the second color, or a signal absence, wherein the first color and the second color are different. In any embodiment herein, the signal code can each correspond to a combination of signals (ON signals) and / or signal absence (OFF signals), wherein a combination of ON and / or OFF signals is detected in two or more imaging steps.

[0015] In any embodiments herein, the method may further include detecting a first analyte and a second analyte in a biological sample based on detecting a first identifier sequence and a second identifier sequence. In any embodiments herein, the first identifier sequence may be a sequence of the first analyte or its complement. In any embodiments herein, the second identifier sequence may be a sequence of the second analyte or its complement. In any embodiments herein, the first identifier sequence may be a first barcode sequence corresponding to, associated with, and / or identifying the first analyte, or its complement. In any embodiments herein, the second identifier sequence may be a second barcode sequence corresponding to, associated with, and / or identifying the second analyte, or its complement.

[0016] In any of the embodiments herein, a first barcode sequence can be used to identify a first analyte. In any of the embodiments herein, a second barcode sequence can be used to identify a second analyte.

[0017] In any embodiments herein, the first probe can be provided as a first plurality of probes that bind directly or indirectly to a first analyte. In some embodiments, the first plurality of probes collectively comprise a first combination of barcode sequences, and the first combination of barcode sequences identifies the first analyte. In any embodiments herein, the second probe can be provided as a second plurality of probes that bind directly or indirectly to a second analyte. In some embodiments, the second plurality of probes collectively comprise a second combination of barcode sequences, and the second combination of barcode sequences identifies the second analyte.

[0018] In any embodiments herein, the first analyte and the second analyte may comprise nucleic acid sequences.In any embodiments herein, the first identifier sequence may comprise the sequence of the first analyte or its complement, and the second identifier sequence may comprise the sequence of the second analyte or its complement.

[0019] In any embodiment herein, base-by-base sequencing can include the use of a fluorescently labeled polymerase and one or more non-fluorescently labeled nucleotides. In any embodiment herein, base-by-base sequencing can include the use of a polymerase-nucleotide conjugate comprising a fluorescently labeled polymerase attached to a non-fluorescently labeled nucleotide moiety. In any embodiment herein, base-by-base sequencing can include the use of a multivalent polymer-nucleotide conjugate comprising a polymer core, a plurality of nucleotide moieties, and one or more fluorescent labels.

[0020] In some embodiments, during base-by-base sequencing, the polymerase does not incorporate homologous nucleotides into the first sequencing primer or the second sequencing primer or an extension product thereof. In some embodiments, the incorporation of homologous nucleotides into the first sequencing primer or the second sequencing primer or an extension product thereof by the polymerase is weakened or inhibited.

[0021] In any of the embodiments herein, base-by-base sequencing can include contacting the biological sample with a mixture of nucleotides comprising fluorescently labeled nucleotides and non-fluorescently labeled nucleotides. In some embodiments, during base-by-base sequencing, a polymerase incorporates homologous nucleotides into a first sequencing primer or a second sequencing primer or an extension product thereof, and the homologous nucleotides are either fluorescently labeled or non-fluorescently labeled.

[0022] In any of the embodiments herein, base-by-base sequencing can include: contacting the biological sample with a first nucleotide mixture, wherein the nucleotides comprising the first base are not detectably labeled, and the nucleotides comprising bases other than the first base are each labeled with one or more detectable labels, and contacting the biological sample with a subsequent nucleotide mixture, wherein the nucleotides comprising the subsequent base are not detectably labeled, and the nucleotides comprising bases other than the subsequent base are each labeled with one or more detectable labels, wherein the subsequent base is the same as the first base, optionally wherein the first base and the subsequent base are A, T, C or G.

[0023] In any of the embodiments herein, base-by-base sequencing may include contacting the biological sample with a first nucleotide mixture, wherein nucleotides comprising the first base are not detectably labeled, and nucleotides comprising bases other than the first base are each labeled with one or more detectable labels, and contacting the biological sample with a subsequent nucleotide mixture, wherein nucleotides comprising the subsequent base are not detectably labeled, and nucleotides comprising bases other than the subsequent base are each labeled with one or more detectable labels, wherein the subsequent base is different from the first base.

[0024] In any of the embodiments herein, a biological sample can be contacted with two or more of the following nucleotide mixtures in any order in consecutive cycles: nucleotide mixture 1, in which nucleotides comprising G are not detectably labeled, and nucleotides comprising A, C, or T are detectably labeled; nucleotide mixture 2, in which nucleotides comprising T are not detectably labeled, and nucleotides comprising A, C, or G are detectably labeled; nucleotide mixture 3, in which nucleotides comprising C are not detectably labeled, and nucleotides comprising A, G, or T are detectably labeled; and nucleotide mixture 4, in which nucleotides comprising A are not detectably labeled, and nucleotides comprising G, C, or T are detectably labeled.

[0025] In any embodiment herein, each nucleotide mixture can be contacted with a biological sample in one or more cycles independently of each other, wherein the cycles are continuous or non-continuous. In any embodiment herein, independently of each other, each nucleotide mixture can include: detectably labeled nucleotides with three different colors of fluorescent labels, one color for each of the three bases, for example, A is red, G is blue, T is green, and C has no detectable label; detectably labeled nucleotides with two different colors of fluorescent labels, one color for each of two of the three bases, wherein the nucleotides containing the remaining one base are labeled with two colors, for example, A is red and green, G is red, T is green, and C has no detectable label; or detectably labeled nucleotides with fluorescent labels of the same color, wherein the fluorescent label on the nucleotide containing one of the three bases is configured to be cut, and the nucleotide containing another of the three bases is configured to be labeled with a fluorescent label.

[0026] In any of the embodiments herein, the biological sample can be contacted with two or more of the following nucleotide mixtures in any order in consecutive cycles: nucleotide mixture 1, in which nucleotides comprising G or A are not detectably labeled, and nucleotides comprising C or T are detectably labeled; nucleotide mixture 2, in which nucleotides comprising G or T are not detectably labeled, and nucleotides comprising C or A are detectably labeled; nucleotide mixture 3, in which nucleotides comprising G or C are not detectably labeled, and nucleotides comprising A or T are detectably labeled; and nucleotide mixture 4, in which nucleotides comprising C or A are not detectably labeled, and nucleotides comprising G or T are detectably labeled; nucleotide mixture 5, in which nucleotides comprising C or T are not detectably labeled, and nucleotides comprising G or A are detectably labeled; and nucleotide mixture 6, in which nucleotides comprising A or T are not detectably labeled, and nucleotides comprising G or C are detectably labeled.

[0027] In any embodiment herein, the first priming site and the second priming site may be different. In any embodiment herein, the method may include: b1) hybridizing a first sequencing primer to the first priming site and performing base-by-base sequencing to generate an extension product of the first sequencing primer and a first signal code sequence; b2) removing, cutting or blocking the extension product of the first sequencing primer in b1); and b3) hybridizing a second sequencing primer to the second priming site and performing base-by-base sequencing (e.g., SBS or sBB) to generate an extension product of the second sequencing primer and a second signal code sequence.

[0028] In any embodiments herein, probes for a first plurality of analytes or their products can share a common first priming site, and probes for a second plurality of analytes or their products can share a common second priming site. In any embodiments herein, the second plurality of analytes can comprise two or more different analytes that are different from two or more different analytes in the first plurality of analytes.

[0029] In any embodiment herein, biological sample can be contacted with a plurality of probes each configured to be directly or indirectly combined with different analytes, and every kind of probe or its product can comprise the combination of different initiation sites.In any embodiment herein, the first probe or its product can comprise the first combination of different initiation sites comprising the first initiation site.In any embodiment herein, the second probe or its product can comprise the second combination of different initiation sites comprising the second initiation site.

[0030] In any embodiment herein, biological sample can be contacted with the third probe that directly or indirectly binds to the third analyte. In any embodiment herein, the third probe or its product can include the third combination of different priming sites including the first priming site, the second priming site and / or the third priming site.

[0031] In any of the embodiments herein, any two or more of the first combination, the second combination, and the third combination may share one or more common priming sites.

[0032] In any of the embodiments herein, the method may include: b') contacting the biological sample with a first sequencing primer for base-by-base sequencing, thereby hybridizing the first sequencing primer to a first priming site in a first probe or its product and in one or more other probes or their products, and generating an extension product of the first sequencing primer; b") removing, cutting or blocking the extension product of the first sequencing primer in b'); and b'") contacting the biological sample with a second sequencing primer for base-by-base sequencing, thereby hybridizing the second sequencing primer to a second priming site in a second probe or its product and in one or more other probes or their products, and generating an extension product of the second sequencing primer.

[0033] In any of the embodiments herein, the base-by-base sequencing in b') can be performed by contacting the biological sample with nucleotides in successive cycles, detecting signals associated with nucleotide incorporation or binding of each successive cycle, and generating signal code sequences for the first plurality of analytes.

[0034] In any of the embodiments herein, the base-by-base sequencing in b″′) can be performed by contacting the biological sample with nucleotides in successive cycles, detecting a signal associated with each successive cycle of nucleotide incorporation or binding, and generating a signal code sequence for a second plurality of analytes. In some embodiments, the first plurality of analytes and the second plurality of analytes comprise one or more common analytes. In some embodiments, the first plurality of analytes and the second plurality of analytes do not comprise a common analyte.

[0035] In any embodiment herein, each analyte can be independently a nucleic acid analyte or a non-nucleic acid analyte. In any embodiment herein, each probe can be independently i) a primary probe directly bound to its corresponding analyte, or ii) a probe directly or indirectly bound to the primary probe. In some embodiments, the primary probe and the probe directly or indirectly bound to the primary probe are independently selected from: a probe comprising a 3' or 5' protrusion, optionally wherein the 3' or 5' protrusion comprises one or more barcode sequences; a probe comprising a 3' protrusion and a 5' protrusion, optionally wherein the 3' protrusion and the 5' protrusion each independently comprise one or more barcode sequences; a circular probe; a circularizable probe or probe set; a probe or probe set comprising a split hybridization region configured to hybridize with a splint, optionally wherein the split hybridization region comprises one or more barcode sequences; and combinations thereof.

[0036] In any of the embodiments herein, the product of each probe may include a rolling circle amplification (RCA) product generated in situ in the biological sample. In any of the embodiments herein, base-by-base sequencing may be performed in situ in the biological sample.

[0037] In some aspects, disclosed herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a first sequencing primer and a second sequencing primer, wherein: the biological sample is a cell or tissue sample, the biological sample comprises a first nucleic acid and a second nucleic acid at a first position and a second position in the biological sample, respectively, the first nucleic acid comprising i) a first priming site complementary to the first sequencing primer and ii) a first identifier sequence, and the second nucleic acid comprising i) a second priming site complementary to the second sequencing primer and ii) a second identifier sequence; b) performing base-by-base sequencing of the first identifier sequence using the first sequencing primer that hybridizes to the first priming site to generate a first signal code sequence, the first signal code sequence comprising a sequence detected at the first position in a continuous cycle; a signal code, wherein the signal code corresponds to a signal (ON signal), a signal absence (OFF signal) or a combination thereof; c) subsequently performing base-by-base sequencing of a second identifier sequence using a second sequencing primer that hybridizes to a second priming site to generate a second signal code sequence, wherein the second signal code sequence comprises a signal code detected at a second position in an additional continuous cycle; wherein in at least one or more of the continuous cycles in b) and the additional continuous cycles in c), an OFF signal is detected at one or both of the first position and the second position; and d) detecting the first identifier sequence and the second identifier sequence at the first position and the second position in the biological sample, respectively, based at least in part on the first signal code sequence and the second signal code sequence.

[0038] In some aspects, disclosed herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: the biological sample is a cell or tissue sample, the biological sample comprises a first analyte and a second analyte at a first location and a second location in the biological sample, respectively, the first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively, the first probe or a product thereof comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and the second probe or a product thereof comprises i) a second priming site of a second sequencing primer and ii) a second identifier sequence associated with the second analyte; b) performing base-by-base sequencing of the first identifier sequence using the first sequencing primer (e.g., using at least one dark base (dark base)). base)) to generate a first signal code sequence, the first signal code sequence comprising a signal code detected at a first position in a continuous cycle, wherein the signal code corresponds to a signal (ON signal), a signal absence (OFF signal), or a combination thereof; c) subsequently performing base-by-base sequencing of a second identifier sequence using a second sequencing primer (e.g., using at least one dark base) to generate a second signal code sequence, the second signal code sequence comprising a signal code detected at a second position in an additional continuous cycle; wherein in at least one or more of the continuous cycles in b) and the additional continuous cycles in c), an OFF signal is detected; and d) detecting the first identifier sequence and the second identifier sequence at a first position and a second position in the biological sample, respectively, based at least in part on the first signal code sequence and the second signal code sequence. In some embodiments, in at least one or more of the continuous cycles in b) and the additional continuous cycles in c), an OFF signal is detected at one or both of the first position and the second position.

[0039] In some embodiments, in at least one or more of the continuous cycles using the first sequencing primer and the other continuous cycles using the second sequencing primer, no ON signal is detected at the first position and the second position in the same base-by-base sequencing cycle. In some embodiments, in at least one or more of the continuous cycles using the first sequencing primer and at least one or more of the other continuous cycles using the second sequencing primer, no ON signal is detected at the first position and the second position in the same base-by-base sequencing cycle. In some embodiments, in two or more of the continuous cycles using the first sequencing primer and two or more of the other continuous cycles using the second sequencing primer, no ON signal is detected at the first position and the second position in the same base-by-base sequencing cycle. In some embodiments, in three or more of the continuous cycles using the first sequencing primer and three or more of the other continuous cycles using the second sequencing primer, no ON signal is detected at the first position and the second position in the same base-by-base sequencing cycle. In the case where the first position and the second position are adjacent to each other, the method disclosed herein reduces optical crowding during in situ base-by-base sequencing in a biological sample.

[0040] In any embodiment herein, OFF signal can be generated by base sequencing using one, two or three nucleotides (e.g., any one, two or three of A, T / U, C and G) that are not detectably labeled, while other one or more nucleotides are detectably labeled. Different nucleotides or different combinations of nucleotides can be used in a specific cycle compared to one or more other cycles. In some embodiments, the nucleotides that are not detectably labeled can be natural nucleotides or derivatives thereof, such as naturally occurring nucleotides, which do not include exogenous labels (e.g., fluorescent dyes or any other labels) or chemical modifications. One or more nucleotides that are detectably labeled can be covalently (e.g., via a bond or a joint) or non-covalently (e.g., via a binding pair such as biotin or a derivative or analog thereof and streptavidin or a derivative or analog thereof) combined with a detectable label (e.g., a fluorescent dye or any other label). In other examples, one or more detectably labeled nucleotides can be conjugated to a moiety (e.g., an antigen or antigen binding molecule such as an antibody) that allows detection of the nucleotide with an agent that specifically binds to the moiety and is capable of producing a detectable signal, and the detectable signal produced by the agent can be used to detect incorporation of the nucleotide. The moiety can be conjugated to the nucleotide via a cleavable linker.

[0041] In any embodiment herein, an OFF signal can be generated by omitting one, two or three nucleotides (e.g., any one, two or three of A, T / U, C and G) from the nucleotide mixture contacted with the sample in a specific sequencing cycle, and the nucleotide mixture contains only other one or more nucleotides that are detectably labeled. Different nucleotides or different combinations of nucleotides can be used (or omitted from the nucleotide mixture) in a specific cycle compared to one or more other cycles.

[0042] In any embodiment herein, an OFF signal can be generated by performing base-by-base sequencing without detecting one, two or three nucleotides (e.g., any one, two or three of A, T / U, C and G) in contact with the sample in a specific sequencing cycle, while only detecting other one or more nucleotides. Different nucleotides or different combinations of nucleotides can be detected (or not detected) in a specific cycle compared to one or more other cycles. For example, in the case of using an antigen or antibody-labeled nucleotide in a sequencing cycle, not detecting a signal associated with the nucleotide can be achieved by omitting the corresponding antibody or antigen labeled with a detectable label from the signal detection in the sequencing cycle.

[0043] In any embodiment herein, the number of "ON" cycles / bits that each code word (e.g., corresponding to a signal code sequence) must have can be selected, and the code words can be designed accordingly. In any embodiment herein, the number of "ON" cycles / bits in a code word can be between about 3 and about 8. In any embodiment herein, the number of "ON" cycles / bits in a code word can be 4, 5, 6, or 7. In any embodiment herein, constraints on code words can be used to facilitate identification of the correct identifier sequence (e.g., a barcode sequence, such as a barcode sequence corresponding to an RCP). In any embodiment herein, each code word can have "ON" bits of at least X colors, where X is 3 or 4, to distinguish from background fluorescent sources that tend to remain a single color.

[0044] In any embodiment herein, the first sequencing primer and the second sequencing primer can be contacted with the sample simultaneously or in any order. In any embodiment herein, the first sequencing primer and the second sequencing primer can be contacted with the sample simultaneously or in any order. In any embodiment herein, the first identifier sequence and the second identifier sequence can be in a DNA (e.g., genomic DNA), RNA (e.g., mRNA), a probe or product directly or indirectly bound to DNA (e.g., genomic DNA) or RNA (e.g., mRNA) such as a rolling circle amplification product of a DNA (e.g., genomic DNA), RNA (e.g., mRNA) or a probe.

[0045] In any embodiments herein, the first identifier sequence and the second identifier sequence may be the same. In any embodiments herein, the first identifier sequence and the second identifier sequence may be different.

[0046] In any embodiment herein, the first identifier sequence and the second identifier sequence may include barcode sequences or their complementary sequences assigned to the first analyte and the second analyte, respectively. In some embodiments, based on a decision rule designed to minimize the maximum predicted density of ON signals detected in each of one or more consecutive cycles, the first priming site and / or the first barcode sequence may be assigned to the first analyte, and the second priming site and / or the second barcode sequence is assigned to the second analyte. In some embodiments, the allocation may include the allocation based on the expression data of the first analyte and the second analyte. In some embodiments, the allocation may include the allocation based on the expression data of the first analyte and the second analyte in the clustered cell type. In some embodiments, the clustered cell type may represent the distribution of cell types present in the biological sample. In some embodiments, the expression data of the first analyte and the second analyte may overlap at least partially. In some embodiments, the expression data of the first analyte and the second analyte include bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single cell gene expression data, single cell protein expression data, or any combination thereof.

[0047] In any of the embodiments herein, the nucleotides in the first barcode sequence or the second barcode sequence detected in a particular cycle may correspond to a signal code comprising an ON signal. In any of the embodiments herein, the nucleotides in the first barcode sequence or the second barcode sequence detected in a particular cycle may correspond to a signal code comprising an OFF signal. In some embodiments, the nucleotides in the first barcode sequence or the second barcode sequence detected in a particular cycle correspond only to an ON signal. In some embodiments, the nucleotides in the first barcode sequence or the second barcode sequence detected in a particular cycle may correspond only to an OFF signal.

[0048] Also disclosed herein is a method for decoding a barcode sequence in a biological sample while minimizing optical crowding, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: the biological sample is a cell or tissue sample, the biological sample comprises a first analyte and a second analyte at a first position and a second position in the biological sample, respectively, the first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively, the first probe or a product thereof comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and the second probe or a product thereof comprises i) a second priming site of a second sequencing primer and ii) a second identifier sequence associated with the second analyte ; b) performing base-by-base sequencing of the first identifier sequence and the second identifier sequence using a first sequencing primer and a second sequencing primer to generate a first signal code sequence and a second signal code sequence, each signal code sequence comprising a signal code corresponding to a signal (ON signal), a signal absence (OFF signal), or a combination thereof detected at a first position and a second position, respectively, in consecutive sequencing cycles, wherein the base-by-base sequencing comprises contacting the biological sample with a polymerase and a nucleotide mixture comprising at least one nucleotide that is not detectably labeled in each consecutive cycle; and c) detecting the first identifier sequence and the second identifier sequence in the biological sample based at least in part on the first signal code sequence and the second signal code sequence.

[0049] In some embodiments, the first analyte and the second analyte can be the same or different.In some embodiments, the first identifier sequence and the second identifier sequence are different.

[0050] In any embodiment herein, the first identifier sequence and the second identifier sequence may comprise an analyte sequence or its complement. In any embodiment herein, the first identifier sequence and the second identifier sequence comprise barcode sequences or their complements assigned to the first analyte and the second analyte, respectively. In any embodiment herein, the method may include assigning the first barcode sequence to the first analyte and assigning the second barcode sequence to the second analyte. In some embodiments, assigning the barcode sequence may be based on a decision rule designed to minimize the maximum predicted density of ON signals detected in each of one or more consecutive cycles. In some embodiments, assigning the first barcode sequence to the first analyte and assigning the second barcode sequence to the second analyte includes assigning based on the expression data of the first analyte and the second analyte. In some embodiments, assigning the first barcode sequence to the first analyte and assigning the second barcode sequence to the second analyte may include assigning based on the expression data of the first analyte and the second analyte in the clustered cell type. In some embodiments, the clustered cell type represents the distribution of cell types present in the biological sample. In some embodiments, the expression data of the first analyte and the second analyte overlap at least partially. In some embodiments, the expression data for the first analyte and the second analyte comprises bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single cell gene expression data, single cell protein expression data, or any combination thereof.

[0051] In any embodiment herein, the nucleotide mixture may include at least two nucleotides that are not detectably labeled. In some embodiments, the biological sample is contacted with two or more of the following nucleotide mixtures in any order in consecutive sequencing cycles: nucleotide mixture 1, wherein the nucleotides comprising G are not detectably labeled, and the nucleotides comprising A, C or T are detectably labeled; nucleotide mixture 2, wherein the nucleotides comprising T are not detectably labeled, and the nucleotides comprising A, C or G are detectably labeled; nucleotide mixture 3, wherein the nucleotides comprising C are not detectably labeled, and the nucleotides comprising A, G or T are detectably labeled; and nucleotide mixture 4, wherein the nucleotides comprising A are not detectably labeled, and the nucleotides comprising G, C or T are detectably labeled. In some embodiments, independently of each other, each nucleotide mixture is contacted with the biological sample in one or more cycles, wherein the cycles are continuous or non-continuous. In some embodiments, independently of each other, in each nucleotide mixture, the detectably labeled nucleotides include: i) fluorescent labels of three different colors, one color for each of the three bases; ii) fluorescent labels of two different colors, one color for each of two of the three bases, wherein the nucleotides containing the remaining one base are labeled with both colors; or iii) fluorescent labels of the same color, wherein the fluorescent label on the nucleotide containing one of the three bases is configured to be cleaved, and the nucleotide containing another of the three bases is configured to be labeled with the fluorescent label.

[0052] In some embodiments, the biological sample can be contacted with two or more of the following nucleotide mixtures in any order in consecutive cycles: nucleotide mixture 1, in which nucleotides comprising G or A are not detectably labeled, and nucleotides comprising C or T are detectably labeled; nucleotide mixture 2, in which nucleotides comprising G or T are not detectably labeled, and nucleotides comprising C or A are detectably labeled; nucleotide mixture 3, in which nucleotides comprising G or C are not detectably labeled, and nucleotides comprising A or T are detectably labeled; and nucleotide mixture 4, in which nucleotides comprising C or A are not detectably labeled, and nucleotides comprising G or T are detectably labeled; nucleotide mixture 5, in which nucleotides comprising C or T are not detectably labeled, and nucleotides comprising G or A are detectably labeled; and nucleotide mixture 6, in which nucleotides comprising A or T are not detectably labeled, and nucleotides comprising G or C are detectably labeled. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The accompanying drawings illustrate certain features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0054] Figure 1An exemplary workflow is shown that includes analyzing a first analyte and a second analyte in an in situ assay.

[0055] Figure 2 Depicted are exemplary molecules and complexes comprising a priming site and a downstream identifier sequence (e.g., a barcode sequence) that can be analyzed using in situ base-by-base sequencing (e.g., SBS or SBB). Rolling circle amplification products, probes tiled on analytes, hybridization chain reaction complexes, and branched structures are shown as examples (bottom panels, from left to right). Each molecule or complex can contain multiple copies of an identifier sequence and a 3' priming site for sequencing primer binding.

[0056] Figure 3 Depicted is a method for in situ detection of multiple analytes (eg, genes) by sequentially decoding blocks (eg, subsets) of analytes. The probes for a given block of different genes contain a common priming site.

[0057] Figure 4 A method for detecting multiple analytes (eg, genes) in parallel is depicted. Each probe for a particular gene may contain two or more different priming sites, and probes for any two different genes may share one or more common priming sites.

[0058] Figure 5 2 is an example workflow for analyzing a biological sample (eg, a cell or tissue sample) using an optofluidic instrument according to various embodiments. DETAILED DESCRIPTION

[0059] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or contradictory to definitions set forth in patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0060] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0061] I. Overview

[0062] The multiple scalability of in situ detection methods may be limited by optical crowding. For example, signals with large size and / or high intensity may overlap and / or mask other signals, especially when the signals are close together, while signals with small size and / or weak intensity may not reach the detection threshold. In either case, the quality of signal detection and decoding may be compromised. In addition, some existing methods require complex oligonucleotide probe pools, which increase the cost and time of detection and decoding.

[0063] For example, in some aspects, single nucleotide sequencing chemistries (e.g., sequencing by synthesis (SBS), sequencing by binding (SBB), etc.) can provide fast reaction times and avoid the need for complex oligonucleotide probe pools and sequential hybridization probe pools to decode identifier sequences associated with target analytes in multiplex assays, for example. However, due to optical crowding during signal detection and decoding steps, these base-by-base sequencing methods may be limited in terms of multiplex scalability for in situ analysis. In particular, in situ parallel detection of a large number of analytes (e.g., genes and / or their transcripts) in a cell or tissue sample may be challenging.

[0064] When there are a large number of signals to be detected, signal crowding may occur.Using a conventional base-by-base sequencing method in which each nucleotide generates an optical signal (e.g., spots in an image obtained using fluorescence microscopy) in a sequencing cycle, a sample may be crowded with (e.g., overlapped to a certain extent) signal spots next to each other, making it difficult to resolve a single spot.Therefore, spatial overlap may limit the ability to realize multiplex in the nucleic acid sequencing assay based on microscopy.In some aspects, when one or more detected signals are significantly stronger than (e.g., having significantly greater amplitude or intensity) other signals, signal crowding may occur.For example, in the same microscope field of view, one or more fluorescent spots may be significantly stronger than other spots including adjacent spots.When there are too many signal spots in a sample, or when the amplitude of a signal is significantly greater than the amplitude of another signal, it may be difficult to accurately and reliably detect all signals in the same field of view and / or the same detection channel (e.g., the same fluorescence channel).In some cases, signal crowding may cause weaker (e.g., lower amplitude) or overlapping signals to be covered and / or leaked, which ultimately results in the loss of information from analytes in the sample.In such a case, the effective dynamic range of detection may be reduced.

[0065] In some embodiments, provided herein are methods and compositions that can be used to prevent and / or resolve problems associated with optical crowding during in situ base-by-base sequence determination.

[0066] In some embodiments, a method for analyzing a cell or tissue sample is provided herein, wherein the identifier sequence (e.g., sequence of nucleic acid analyte, or barcode sequence in analyte targeting probe) of various analytes is sequenced in situ, so as to detect the corresponding analyte at one or more positions in a cell or tissue sample. In some embodiments, in order to detect the identifier sequence of a plurality of analytes sequenced in situ, the signal corresponding to at least two different identifier sequences is staggered between cycles. In some embodiments, the identifier sequence is decoded in a base-by-base sequencing cycle using SBS or SBB or any other base-by-base sequencing chemistry (e.g., affinity sequencing). In some embodiments, for many (e.g., most) base-by-base sequencing cycles, only the signal associated with a limited number of analytes is detected in a specific cycle, and the signal associated with many (e.g., most) other analytes is dark in the specific cycle. For example, in a specific cycle, when the incorporation (e.g., in SBS) or the combination (e.g., in SBB) of the homologous nucleotides paired with the nucleotide bases in the identifier sequence of the analyte does not generate a detectable signal, then the analyte may be dark in the cycle. In some embodiments, homologous nucleotides comprise a base and are not detectably labeled (eg, generate an OFF signal), while nucleotides comprising one or more other different bases are detectably labeled (eg, each generate an ON signal).

[0067] In some embodiments, using the methods disclosed herein, an ON signal is detected at a first position in a cell or tissue sample, and an OFF signal is detected at a second position in a cell or tissue sample. In some embodiments, the first position and the second position do not overlap. In some embodiments, the first position and the second position overlap at least partially. In some embodiments, the identifier sequence is such that only a portion of the nucleotides in the identifier sequence to be detected in the same base-by-base sequencing cycle produce a detectable signal, thereby limiting the optical crowding of the signal detected in the cycle.

[0068] Provided herein is a method for analyzing one or more analytes (e.g., one or more messenger RNAs) present in a cell or biological sample such as a tissue sample using one or more polynucleotides (e.g., cyclizable probes such as padlock probes). Also provided are probes, probe groups, compositions, test kits, systems and devices used according to the provided method. In some respects, the provided method and system can be applied to sequencing, detection, imaging, quantitative and / or determining the presence of one or more analytes (e.g., target nucleic acids) or parts thereof. In some respects, the provided method and system can be applied to sequencing multiple analytes or identifier sequences associated therewith in situ in parallel, while reducing the optical crowding of the signal generated in the sample.

[0069] In some embodiments, an exemplary workflow for analyzing a biological sample includes contacting the biological sample with a first probe and a second probe, wherein the biological sample is a cell or tissue sample, and the biological sample comprises a first analyte and a second analyte at a first position and a second position in the biological sample, respectively, and wherein the first probe and the second probe are directly or indirectly bound to the first analyte and the second analyte, respectively. In some embodiments, the first probe and the second probe undergo connection, circularization and amplification to form a first product and a second product. In some aspects, the amplification is a rolling circle amplification and the product formed is a first rolling circle amplification product (RCP) and a second rolling circle amplification product. In some embodiments, the product of each probe is a rolling circle amplification (RCA) product generated in situ in a biological sample. In some embodiments, the first probe or its product comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with a first analyte, and the second probe or its product comprises i) a second priming site of a second sequencing primer and ii) a second identifier sequence associated with a second analyte. In some embodiments, the first identifier sequence is a first barcode sequence or its complementary sequence, and the second identifier sequence is a second barcode sequence or its complementary sequence.

[0070] In some embodiments, an exemplary workflow for analyzing a biological sample includes base-by-base sequencing of a first identifier sequence and a second identifier sequence using a first sequencing primer and a second sequencing primer and a cyclic series of nucleotide incorporation or nucleotide binding steps, respectively, to generate a first signal code sequence and a second signal code sequence, each signal code sequence comprising a series of signal codes corresponding to a signal (ON signal), a signal absence (OFF signal), or a combination thereof, detected at a first position and a second position, respectively, in a continuous cycle, wherein in one or more of the continuous cycles, an ON signal is detected at the first position and an OFF signal is detected at the second position. In some embodiments, the method includes detecting a first identifier sequence and a second identifier sequence in a biological sample based on the first signal code sequence and the second signal code sequence. In some embodiments, the method includes detecting an amplification product or a portion thereof (e.g., an RCA product) based on the first signal code sequence and the second signal code sequence. In some aspects, the first identifier sequence (e.g., a barcode sequence) identifies the first analyte, and / or the second barcode sequence identifies the second analyte.

[0071] In some embodiments, an exemplary workflow for analyzing a biological sample includes hybridizing a first sequencing primer to a first priming site and performing base-by-base sequencing to generate an extension product of the first sequencing primer and a first signal code sequence, removing (e.g., stripping), cutting or blocking (e.g., by using terminator nucleotides for the last cycle) the extension product of the first sequencing primer so that base-by-base sequencing is prevented, and hybridizing a second sequencing primer to a second priming site and performing base-by-base sequencing to generate an extension product of the second sequencing primer and a second signal code sequence. In some embodiments, the first priming site and the second priming site are different. In some embodiments, probes for a first plurality of analytes or their amplified products share a common first priming site, and probes for a second plurality of analytes or their amplified products share a common second priming site. The second plurality of analytes may include two or more different analytes that are different from two or more different analytes in the first plurality of analytes.

[0072] In some embodiments, the exemplary workflow for analyzing biological samples includes contacting the biological sample with a variety of probes, each of which is configured to directly or indirectly bind to different analytes. In some embodiments, each probe or its rolling circle amplification product comprises a combination of two or more different priming sites. In some embodiments, the first probe or its product comprises a first combination of two or more different priming sites comprising a first priming site, and / or the second probe or its product comprises a second combination of two or more different priming sites comprising a second priming site. In some embodiments, the first combination of two or more different priming sites and the second combination of two or more different priming sites can share one or more common priming sites. In some embodiments, an exemplary workflow for analyzing a biological sample includes contacting the biological sample with a first sequencing primer and performing base-by-base sequencing using a cyclic series of nucleotide incorporation or binding, respectively, to generate extension products of the first sequencing primer and detectable signal code sequences for a first plurality of analytes, removing, cleaving, or blocking the extension products of the first sequencing primer, and contacting the biological sample with a second sequencing primer and performing base-by-base sequencing using a cyclic series of nucleotide incorporation or binding, respectively, to generate extension products of the second sequencing primer and detectable signal code sequences for a second plurality of analytes different from the first plurality of analytes.

[0073] In some embodiments, an exemplary workflow for analyzing a biological sample includes contacting the biological sample with a first probe and a second probe, wherein: the biological sample is a cell or tissue sample, the biological sample comprises a first analyte and a second analyte at a first location and a second location in the biological sample, respectively, the first probe is provided as a first plurality of probes that bind directly or indirectly to the first analyte, the second probe is provided as a second plurality of probes that bind directly or indirectly to the second analyte, and wherein the first plurality of probes collectively comprise a first combination of barcode sequences, and the second plurality of probes collectively comprise a second combination of barcode sequences. In some embodiments, the first plurality of probes and the second plurality of probes comprise a first combination and a second combination of priming sites for binding to a plurality of first sequencing primers and a second sequencing primer. In some embodiments, the exemplary method includes performing base-by-base sequencing of a first combination and a second combination of barcode sequences using a plurality of first sequencing primers and a second sequencing primer and a cyclic series of nucleotide incorporation or nucleotide binding steps, respectively, thereby generating a first signal code sequence and a second signal code sequence, each signal code sequence comprising a series of signal codes corresponding to a signal (ON signal), a signal absence (OFF signal), or a combination thereof detected at a first position and a second position, respectively, in a continuous cycle, wherein in one or more of the continuous cycles, an ON signal is detected at the first position and an OFF signal is detected at the second position. In some embodiments, the exemplary method includes detecting a first combination and a second combination of barcode sequences in a biological sample based on the first signal code sequence and the second signal code sequence. In some embodiments, the first combination of barcode sequences identifies a first analyte and the second combination of barcode sequences identifies a second analyte.

[0074] II. Identifier Sequence

[0075] In some embodiments, provided herein are methods and compositions for analyzing multiple analytes in a sample by detecting the identifier sequence of an analyte in a sample. In some embodiments, the identifier sequence is present in an analyte in a sample such as a DNA or RNA analyte or is derived from an analyte in a sample such as a DNA or RNA analyte. For example, the identifier sequence can be a part of a DNA or RNA analyte sequence. In some embodiments, the identifier sequence can be an analyte sequence (such as the arm of a padlock probe, or a gap filling sequence) or its complementary sequence. In some embodiments, the analyte can include two or more different identifier sequences. In some embodiments, the analyte can include two or more copies of the same identifier sequence. In some embodiments, different identifier sequences and / or copies of the same identifier sequence can be directly connected by a phosphodiester bond. In some embodiments, different identifier sequences and / or copies of the same identifier sequence can be separated from each other by one or more nucleotide residues. In some embodiments, different identifier sequences and / or copies of the same identifier sequence can partially overlap.

[0076] In some embodiments, the identifier sequence is present in a labeling agent, such as in a nucleic acid probe comprising an identifier sequence, or in an antibody conjugated to a reporter oligonucleotide comprising an identifier sequence. In some embodiments, the labeling agent may include a binding portion that interacts (e.g., binds and / or reacts) directly or indirectly with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labeling agent may include a reporter oligonucleotide indicating an analyte or a portion thereof that interacts with a binding portion. For example, the reporter oligonucleotide may include a barcode sequence (as an identifier sequence) that allows identification of a binding portion and a corresponding analyte. In some cases, the sample contacted with the labeling agent may be further contacted with a nucleic acid probe hybridized with the reporter oligonucleotide of the labeling agent. In some embodiments, the labeling agent includes one or more barcode sequences, such as barcode sequences corresponding to analyte binding portions and / or analytes. In some embodiments, the barcode sequence is associated with the analyte binding portion or otherwise identifies the analyte binding portion. In some embodiments, the analyte binding portion is identified by identifying its associated barcode sequence, and the analyte bound by the analyte binding portion can be identified. In some embodiments, the barcode sequence can be a nucleic acid sequence of a given length and / or a sequence associated with, corresponding to, and / or identifying an analyte binding moiety. The barcode sequence can generally include any of the various aspects of the barcode sequence described herein (e.g., in Section II-B).

[0077] In some embodiments, the identifier sequence is present in a product of a DNA or RNA analyte in a sample. For example, the identifier sequence can be present in a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product such as a rolling circle amplification (RCA) product of a DNA or RNA analyte in a sample.

[0078] In some embodiments, the identifier sequence is present in the product of the labeling agent. In some embodiments, the identifier sequence is present in the product of the nucleic acid probe. In some embodiments, the identifier sequence is present in the product of the reporter oligonucleotide of the labeling agent. For example, the identifier sequence can be present in the hybridization product of the nucleic acid probe or the reporter oligonucleotide in the sample, the connection product, the extension product (for example, by DNA or RNA polymerase), the replication product, the transcription / reverse transcription product and / or the amplification product such as rolling circle amplification (RCA) product.

[0079] In some embodiments, the product of the DNA or RNA analyte, nucleic acid probe and / or reporter oligonucleotide in the sample can include two or more different identifier sequences. In some embodiments, the product can include two or more copies of the same identifier sequence. In some embodiments, different identifier sequences and / or copies of the same identifier sequence can be in the same molecule or in different molecules (for example, molecules such as branched structures formed via hybridization). In some embodiments, different identifier sequences and / or copies of the same identifier sequence can be directly connected by phosphodiester bonds. In some embodiments, different identifier sequences and / or copies of the same identifier sequence can be separated from each other by one or more nucleotide residues. In some embodiments, different identifier sequences and / or copies of the same identifier sequence can partially overlap. The product can be generated in a sample (for example, in situ), or at least a portion of the product can be generated outside the sample and then contacted with the sample. The product can be generated by enzymatic and / or non-enzymatic means. Exemplary products include, but are not limited to, RCA products, hybridization chain reaction (HCR) products, linear oligonucleotide hybridization chain reaction (LO-HCR) products, branched DNA reaction (bDNA) products, primer exchange reaction (PER) products, or products generated using any combination of these enzymatic and / or non-enzymatic reactions, for example, as described in Section II-C.

[0080] The identifier sequence herein may be a continuous sequence or may comprise two or more sequences in the same molecule or in separate molecules. When in the same molecule, the two or more sequences may be separated by one or more nucleotide residues.

[0081] The identifier sequence herein can have any suitable length.In some embodiments, the length of the identifier sequence is between about 1 and about 500 nucleotides.In some embodiments, the length of the identifier sequence is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195 or about 200 nucleotides or the nucleotide of any integer (or integer range) between the indicated value.

[0082] In some embodiments, at a specific position of a cell or tissue sample, a molecule or complex comprises multiple copies of an identifier sequence associated with an analyte at the specific position in the sample, corresponding to an analyte at the specific position in the sample and / or identifying an analyte at the specific position in the sample. In some embodiments, multiple copies of the identifier sequence are detected using base-by-base sequencing, and the signal is used to identify the analyte. In some embodiments, a molecule or complex comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25 or more different identifier sequences associated with an analyte, corresponding to an analyte and / or identifying an analyte. In some embodiments, a molecule or complex comprises at least 2, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1,000, at least 2,500, at least 5,000 or more copies of each of the one or more identifier sequences. Exemplary molecules and complexes comprising one or more different identifier sequences are described in Section II-C.

[0083] In some embodiments, multiple analytes are detected in a cell or tissue sample, and each analyte can be identified using an identifier sequence or a combination of identifier sequences. In some embodiments, the number of different analytes to be detected in a sample is at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1,000, at least 2,500, at least 5,000 or more. In some embodiments, the number of different identifier sequences used to identify multiple analytes in a sample is at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1,000, at least 2,500, at least 5,000 or more.

[0084] A. Identifier sequence from analyte

[0085] In some embodiments, the identifier sequence herein comprises the analyte sequence, a sequence from which the analyte is derived, or a complementary sequence thereof. In some embodiments, the analyte comprises a nucleic acid sequence, and the identifier sequence comprises a nucleic acid sequence in the analyte or a complementary sequence of a nucleic acid sequence.

[0086] In some embodiments, the identifier sequence includes the sequence of a virus or cell nucleic acid. In some embodiments, the identifier sequence includes the sequence of a viral DNA or RNA. In some embodiments, the identifier sequence is in a virus or a viral particle (for example, in a cell or tissue sample) or from a virus or a viral particle. In some embodiments, the identifier sequence includes the sequence of a cell DNA or RNA. In some embodiments, the cell DNA or RNA is in a prokaryotic cell (for example, in a tissue sample) or from a prokaryotic cell. In some embodiments, the cell DNA or RNA is in a eukaryotic cell (for example, in a tissue sample) or from a eukaryotic cell. In some embodiments, the identifier sequence includes the sequence of a nucleic acid molecule in or from a nucleus, mitochondria or chloroplast. In some embodiments, the identifier sequence includes the sequence of genomic DNA, cell RNA or cDNA. In some embodiments, the identifier sequence includes the sequence of coding RNA and / or non-coding RNA. In some embodiments, the identifier sequence comprises the sequence of a messenger RNA (mRNA), including nascent RNA, pre-mRNA, primary transcribed RNA, and processed RNA (such as capped mRNA (e.g., with a 5′ 7-methylguanosine cap), polyadenylated mRNA (poly A tail at the 3′ end), and spliced ​​mRNA from which one or more introns have been removed). In some embodiments, the identifier sequence comprises the sequence of a non-capped mRNA, non-polyadenylated mRNA, or non-spliced ​​mRNA. In some embodiments, the identifier sequence comprises the sequence of a transcript of another nucleic acid molecule (e.g., DNA or RNA, such as viral RNA) present in a cell or tissue sample. In some embodiments, the identifier sequence includes the sequence of non-coding RNA, and examples of non-coding RNA (ncRNA) that is not translated into protein include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNA, such as microRNA (miRNA), small interfering RNA (siRNA), Piwi interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body (Cajal body) specific RNA (scaRNA) and long ncRNA (such as Xist and HOTAIR). In some embodiments, the identifier sequence includes the sequence of small RNA (e.g., less than 200 nucleic acid bases in length) or large RNA (e.g., RNA with a length greater than 200 nucleic acid bases). Examples of small RNA include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNA, piRNA, tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). RNA can include double-stranded RNA or single-stranded RNA.RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16s rRNA or 23s rRNA). In some embodiments, the identifier sequence includes a sequence spanning an exon-exon junction, such as in a spliced ​​RNA. In some embodiments, the identifier sequence includes a sequence spanning an intron-exon or exon-intron junction, such as in a DNA or non-splicing RNA. In some embodiments, the identifier sequence may include the sequence of a reverse transcription product of any RNA disclosed herein. In some embodiments, the identifier sequence may include a cDNA of any RNA disclosed herein or a complementary sequence of a cDNA.

[0087] In some embodiments, the nucleic acid analyte or its complementary sequence is circularized, for example, using template-independent ligation, and used as a template for rolling circle amplification (RCA). For example, cDNA generated by reverse transcription of mRNA in a cell or tissue sample can be ligated using a single-stranded DNA ligase (e.g., CircLigase TM ) is directly circularized and used as a template for RCA. In some cases, no prior knowledge of the mRNA is required, and a direct ligation method can be used to sample the entire transcriptome in situ. The RCA product contains the complementary sequence of the cDNA (i.e., the sequence of the mRNA that was reverse transcribed to generate the cDNA), which can be an identifier sequence for the corresponding mRNA that is sequenced using the methods disclosed herein to detect a location in a cell or tissue sample.

[0088] In some embodiments, circularized probe or probe group can be hybridized with nucleic acid analyte (for example, cDNA or mRNA), and nucleic acid analyte is used as template to be connected to form circularized probe, and gap filling is performed or not performed before connection.In some embodiments, circularized probe or probe group can include 3' hybridization zone and 5' hybridization zone (for example, 3' arm and 5' arm) hybridized with nucleic acid analyte (for example, cDNA or mRNA).In some embodiments, after hybridization with nucleic acid analyte, 3' terminal nucleotide and 5' terminal nucleotide of circularized probe or probe group can be configured to be connected after gap filling by polymerase (for example, enzyme with DNA polymerase or reverse transcriptase activity).In some embodiments, 3' hybridization zone and 5' hybridization zone of circularized probe or probe group are complementary to sequences that are not directly connected by phosphodiester bond in nucleic acid analyte (for example, cDNA or mRNA), and the gap of one or more nucleotides is formed between two hybridization zones.In some embodiments, the length of the gap is between about 1 to about 500 nucleotides. In some embodiments, the length of the gap is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 nucleotides, or any integer (or range of integers) between the indicated values. In some embodiments, the length of the gap is between about 200 to about 300 nucleotides, between about 300 to about 400 nucleotides, or between about 400 to about 500 nucleotides. In some embodiments, after hybridization with a nucleic acid analyte (e.g., cDNA or mRNA), one or more gaps are formed between the 3' and 5' ends of the circularizable probe or probe set, for example, two, three or more gaps can be filled using the nucleic acid analyte as a template.

[0089] In some embodiments, nucleic acid analyte is DNA (for example, cDNA) and the gap is filled by an enzyme with DNA polymerase activity. In some embodiments, nucleic acid analyte is RNA (for example, mRNA) and the gap is filled by an enzyme with reverse transcriptase activity. In some embodiments, the gap filling of enzyme copies the sequence of nucleic acid analyte (for example, cDNA or mRNA) into the circularization probe formed by circularization probe or probe group. In some embodiments, because the gap side joint known sequence, it is possible to read nucleic acid analyte sequence or its complementary sequence (for example, " cell barcode ") as the identifier sequence of nucleic acid analyte using method disclosed herein. For example, the known sequence at 3' of the identifier sequence of nucleic acid analyte can provide the priming site for combining sequencing primer disclosed herein, and the identifier sequence can be sequenced by base base.

[0090] In some embodiments, one or more barcode sequences can be built into the backbone of a circularizable probe or probe set, for example, to distinguish between circularizable probes (e.g., padlock probes) that target the same nucleic acid analyte or different nucleic acid analytes (e.g., cDNA or mRNA). In some embodiments, in addition to an identifier sequence (e.g., a "cell barcode"), one or more barcode sequences from a probe or probe set can be read using a base-by-base sequencing method disclosed herein. In some embodiments, in a circularized probe, a barcode sequence is adjacent to an identifier sequence (e.g., a sequence complementary to the sequence of a nucleic acid analyte) generated using gap filling, and both the barcode sequence and the identifier sequence can be sequenced in situ, for example, by sequencing the corresponding sequence in the RCA product of the circularized probe base-by-base. Adjacent barcode sequences and identifier sequences can be sequenced using the same sequencing primer or using separate sequencing primers. In some embodiments, in circularized probes, the 3' or 5' hybridizing region sequence of the circularizable probe is between adjacent barcode sequences and identifier sequences, and the 3' or 5' hybridizing region sequence (known sequence) can be used as a priming site or can be sequenced as a control.

[0091] Exemplary methods for generating circularized probes and RCA products thereof comprising identifier sequences derived from nucleic acid analytes (e.g., genomic DNA, mRNA, or cDNA) include, but are not limited to, those described in Chen et al., Efficient in situ barcode sequencing using padlock probe-based BaristaSeq, Nucleic Acid Research (2018) 46(4): e22; Lee et al., Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues, Nature Protocols (2015) 10: 442-458; Lee et al., Highly multiplexed subcellular RNA sequencing in situ, Science (2014) 343: 1360-1363; US 10,138,509; US 10,179,932; US 10,494,662; US 11,078,520; and US 11,085,072, all of which are incorporated herein by reference.

[0092] B. Barcode sequence as identifier sequence

[0093] In some embodiments, a method is provided herein, which includes contacting a cell or tissue sample with a plurality of probes, each probe directly or indirectly binding to a different analyte in the sample. In some embodiments, each probe may include: i) a hybridization sequence (or analyte binding portion, such as an antibody or antibody fragment) for direct or indirect binding to its corresponding analyte and ii) one or more identifier sequences associated with the corresponding analyte, corresponding to the corresponding analyte and / or identifying the corresponding analyte. In some embodiments, the one or more identifier sequences are not derived from the corresponding analyte, but are assigned to the barcode sequence of the corresponding analyte. In some cases, assigning an identifier sequence to the corresponding analyte provides certain advantages over the sequence of the endogenous analyte, because it allows the design of an identifier sequence containing the desired number and / or type (e.g., A, T, C or G) of nucleotides, most of which (e.g., 70% or more) can be associated with an OFF signal. In some cases, using a barcode sequence in place of the sequence of an endogenous analyte allows the design of multiple barcode sequences that collectively reduce optical crowding of signals detected in consecutive decoding cycles (e.g., by having a sufficient number of "OFF" bases among the bases analyzed in the same cycle between the multiple barcode sequences) and facilitate unambiguous analyte identification.

[0094] In some embodiments, in a barcode sequencing method, a barcode sequence is detected to identify other molecules that contain a nucleic acid molecule (DNA or RNA) that is longer than the barcode sequence itself, as opposed to directly sequencing a longer nucleic acid molecule. In some embodiments, given a sequenced read of N bases, an N-mer barcode sequence contains 4 N The complexity of barcode sequencing is greatly increased, and molecular identification may require much shorter sequencing reads compared to non-barcode sequencing methods such as direct sequencing. For example, 1024 molecular species can be identified using a 5-nucleotide barcode sequence (4 5 =1024), and an 8-nucleotide barcode can be used to identify up to 65,536 molecular species, a number greater than the total number of different genes in the human genome. In some embodiments, the barcode sequence (e.g., contained in the probe or RCA product) is detected instead of the endogenous sequence, which can be an effective read segment for the information of each sequencing cycle. In some embodiments, because the barcode sequence is predetermined, they can also be designed to characterize error detection and correction mechanisms, see, for example, U.S. Patent Publication 20190055594 and U.S. Patent Publication 20210164039, which are hereby incorporated by reference in their entirety.

[0095] In some aspects, an analyte can be associated with, correspond to, and / or identified using one or more barcode sequences (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more different barcode sequences). Barcode sequences can be assigned to analytes as described in Section III.

[0096] In some embodiments, a single barcode sequence can uniquely identify one analyte in a plurality of different analytes. In some embodiments, two or more different barcode sequences can be assigned to the same analyte, and any barcode sequence can uniquely identify one analyte in a plurality of different analytes. In some embodiments, a single barcode sequence is provided with a combination of barcode sequences, wherein the combination can uniquely identify one analyte in a plurality of different analytes. The combination of barcode sequences can be provided in two or more probe molecules. In some embodiments, the number of different barcode sequences in the nucleic acid probe population is less than the number of different target analytes (e.g., nucleic acid analytes and / or protein analytes) of the nucleic acid probe, but different target analytes can still be uniquely identified from each other, for example, by encoding probes with different combinations of barcode sequences. However, it is not necessary to use all possible combinations of a given set of barcode sequences. For example, each probe can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more barcode sequences. In some embodiments, for example, a population of nucleic acid probes may each contain the same number of barcode sequences, but in other cases, different numbers of barcode sequences may be present on various probes.

[0097] As an illustrative example, a first probe can contain a first target binding sequence (or first target binding portion), a first barcode sequence, and a second barcode sequence, while a second, different probe can contain a second target binding sequence or target binding portion that is different from the first target binding sequence (or target binding portion) in the first probe, the same first barcode sequence as in the first probe, but a third barcode sequence instead of the second barcode sequence. Such probes can thus be distinguished by determining the various barcode sequence combinations present at a given location in a sample or associated with a given probe.

[0098] The barcode sequence can be attached to an analyte or another moiety or structure in a reversible or irreversible manner. In some aspects, the barcode sequence can comprise about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more 30 nucleotides.

[0099] In some embodiments, probes such as nucleic acid probes (or combinations of nucleic acid probes configured to target the same analyte) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 32 or more, 40 or more, or 50 or more different barcode sequences. In some embodiments, nucleic acid probes or combinations of nucleic acid probes may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 32 or more, 40 or more, or 50 or more copies of a specific barcode sequence. Different barcode sequences or copies of the same barcode sequence can be positioned anywhere within or between nucleic acid probes. If there are more than one barcode sequence or more than one copy, the barcode sequences or copies can be positioned adjacent to each other and / or interspersed with other sequences. In some embodiments, two or more of the barcode sequences or copies may also at least partially overlap. In some embodiments, two or more barcode sequences or copies in the same probe do not overlap. In some embodiments, any two or more or all barcode sequences or copies in the same probe may be separated from each other by at least one phosphodiester bond (e.g., they may be immediately adjacent to each other but not overlapping), such as separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0100] The barcode sequence can be of any length. In some embodiments, the barcode sequences can independently have the same or different lengths, such as at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50 nucleotides. In some embodiments, the length of a single barcode sequence can be no more than 24, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 10, no more than 9, or no more than 8 nucleotides. Any combination of these is also possible, for example, the barcode sequence can be between 5 and 10 nucleotides, between 8 and 15 nucleotides, etc.

[0101] In some embodiments, a barcode sequence may comprise two or more sub-barcode sequences that together act as a single barcode sequence. For example, a polynucleotide may comprise two or more polynucleotide sequences (e.g., sub-barcode sequences) separated by one or more non-barcode sequences. In some embodiments, a barcode sequence may also provide a platform for targeting functions, such as oligonucleotides, oligonucleotide-antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functions, and / or enzymes for detecting and identifying polynucleotides comprising a barcode sequence or directly or indirectly bound to a barcode sequence.

[0102] The barcode sequence can be arbitrary or random. In some cases, the barcode sequence is selected to reduce or minimize homology with other components in the sample, for example, so that the barcode sequence itself does not bind or hybridize with other nucleic acids suspected to be in the cell or other sample. In some embodiments, the homology between a specific barcode sequence and another sequence (e.g., a cell nucleic acid sequence in the sample or other barcode sequences in a probe added to the sample) may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%. In some embodiments, the homology may be less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 bases, and in some embodiments, the bases are continuous bases.

[0103] In some embodiments, the nucleic acid probes and barcode sequences in the probes disclosed herein can be prepared using only 2 or only 3 of the 4 bases, such as excluding all "G"s and / or excluding all "C"s within the probe. Sequences lacking "G"s or "C"s can form very little secondary structure and can contribute to more uniform and faster hybridization in certain embodiments.

[0104] In some embodiments, the signal group detected during the cyclic in-situ decoding process can be used to generate a signal code sequence corresponding to a barcode sequence assigned to the analyte, and the signal code sequence can be compared with the code words in the code book to identify a match. In some cases, the code words in the code book can correspond to a physical barcode, which includes a series of ON and / or OFF bits corresponding to a series of ON and OFF signals detected in images acquired in continuous cycles during the decoding process. Then, the presence and position of one or more target analytes can be inferred from the signal code sequence detected at each specific position in a series of images of the sample and the detection position of the corresponding ON and OFF signals of each signal code sequence. In some aspects, the signal code of the signal code sequence can include, for example, a signal (ON signal), a signal absence (OFF signal) or a combination thereof.

[0105] A codebook containing codewords (corresponding to identifier sequences or "physical" nucleic acid barcode sequences) can be designed to meet a particular set of design criteria. For example, a codebook can be designed to ensure that it contains a specified number (or minimum number) of unique codewords / barcode sequences (e.g., corresponding to a specified number (or minimum number) of target analytes to be identified). In some cases, for example, the codebook may include at least 2, at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, at least 1,000, at least 2,000, at least 4,000, at least 6,000, at least 8,000, at least 10,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, at least 1,000,000, at least 2x10 6 , at least 3x10 6 , at least 4x10 6 , at least 5x10 6 , at least 6x10 6 , at least 7x10 6 , at least 8x10 6 , at least 9x10 6 , at least 10 7 , at least 10 8 , at least 10 9 or more than 10 9 In some cases, the codebook may include any number of unique codewords / barcode sequences within the range of values ​​in this paragraph.

[0106] In some cases, the codewords / barcode sequences in a given codebook may be designed to satisfy a specified pairwise edit distance (e.g., a specified minimum pairwise edit distance) to enable barcode error detection and correction. "Edit distance" is a numerical value that quantifies how different two strings (e.g., text strings) are from each other by counting the minimum number of edit operations required to transform one string into another. Examples of edit distance metrics include, but are not limited to, Hamming distance, Levenshtein distance, longest common subsequence (LCS) distance, and the like. For example, the Levenshtein distance between two strings is the minimum number of single-character edits (e.g., insertions, deletions, or substitutions) required to transform one string into another. The longest common subsequence (LCS) distance is the edit distance for which the only allowed edit operations are insertions and deletions (with a unit cost assigned to each). The Hamming distance between two strings of equal length (i.e., substitution is the only edit operation allowed) is the number of positions where the corresponding symbols in the two strings are different.

[0107] The Hamming distance and / or the Levenshtein distance (where the error penalty assigned to the difference between two strings is an integer value) allow a natural interpretation of error correction, where the minimum pairwise barcode distance of 2k+1 allows correction of at most k errors. In some cases, the designed barcodes for a given codebook may need to have a minimum pairwise edit distance (e.g., minimum pairwise Hamming distance, minimum pairwise Levenshtein distance, or minimum pairwise LCS distance) such that they guarantee an error correction capability of correcting at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 barcode errors (e.g., errors arising during the decoding process).

[0108] In some cases, it is desirable to design a codebook so as to minimize optical crowding in the image used for decoding. For example, in some cases, a set of codewords / barcodes may be designed so that the ON signals (or combined weights) of the set of codewords / barcodes are more or less evenly distributed over multiple decoding cycles. In some cases, the codewords / barcodes in a codebook may be designed so that at most one bit is ON during any given decoding cycle. For example, a codeword / barcode in a codebook designed for a four "color" (e.g., four detection channels) decoding instrument may contain 4-bit blocks per decoding cycle (one for each detection channel), where at most one bit is ON in any given cycle. The "colors" described herein may include any color used in fluorescence microscopy, as well as a dark or OFF state (e.g., no detectable signal).

[0109] In some embodiments, nucleotides within the barcode sequence corresponding to the codeword can be selected to reduce optical crowding of signals detected in the cycle. For example, among multiple barcode sequences, corresponding nucleotides to be detected in the same base-by-base sequencing cycle can be selected to reduce optical crowding of signals detected in the cycle, and multiple barcode sequences can be designed so that optical crowding in multiple (e.g., most) cycles is limited. In some embodiments, for each cycle, the signal code representing the nucleotide in the first barcode sequence (e.g., a signal of a first color, a signal of a second color, a signal of a third color, or a signal absence) is an ON signal, and the signal code representing the corresponding nucleotide in the second barcode sequence is an OFF signal. In some embodiments, a dark or OFF state can be included in one or more codewords / barcode sequences, and a nucleotide mixture for cyclic base-by-base sequencing is used to detect the dark or OFF state. In some embodiments, a barcode sequence is assigned to each of the multiple analytes using any suitable scheme (e.g., as described in Section III.A).

[0110] C. Molecule or complex containing the identifier sequence

[0111] Identifier sequences, including those from the analyte and the barcode sequence assigned to the analyte, can be present in various molecules or complexes thereof and detected using in situ base-by-base sequencing.Exemplary molecules and complexes are described herein.

[0112] (i) Labeling agent

[0113] In the methods and systems described herein, one or more labeling agents that can be combined with one or more features or otherwise coupled can be used to characterize analyte, cell and / or cell feature. In some cases, cell feature includes cell surface feature. Analyte can include but is not limited to protein, receptor, antigen, surface protein, transmembrane protein, differentiation protein cluster, protein channel, protein pump, carrier protein, phospholipid, glycoprotein, glycolipid, cell-cell interaction protein complex, antigen presentation complex, major histocompatibility complex, engineered T cell receptor, T cell receptor, B cell receptor, chimeric antigen receptor, gap connection, adhesion connection or any combination thereof. In some cases, cell feature can include intracellular analyte, such as protein, protein modification (for example, phosphorylation state or other post-translational modifications), nucleoprotein, nuclear membrane protein or any combination thereof. In some embodiments, described method is included in one or more post-fixation (post-fixing, also referred to as post-fixation) steps after making sample contact with one or more labeling agents.

[0114] In some embodiments, the labeling agent comprises an analyte binding portion that can be bound to an analyte (e.g., a biological analyte, e.g., a macromolecular component). The binding portion may include, but is not limited to, proteins, peptides, antibodies (or their epitope binding fragments), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell adapters, T cell receptor adapters, B cell receptor adapters, antibody prodrugs, aptamers, monoclonal antibodies, affimers, darpins, and protein scaffolds or any combination thereof. The binding portion may be directly or indirectly attached to a reporter oligonucleotide indicating the analyte or feature bound by the binding portion. For example, the reporter oligonucleotide may include a barcode sequence that allows identification of the binding portion and the corresponding analyte. For example, a labeling agent specific for a type of analyte or cell feature may have a first reporter oligonucleotide coupled thereto, while a labeling agent specific for different analytes or cell features may have different reporter oligonucleotides coupled thereto. For a description of exemplary labeling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Patent 10,550,429; U.S. Patent Publication 20190177800; and U.S. Patent Publication 20190367969, each of which is incorporated herein by reference in its entirety.

[0115] In some embodiments, the analyte binding moiety comprises one or more nucleic acid moieties. The one or more nucleic acid moieties can specifically bind to a target analyte (e.g., a target nucleic acid) via nucleic acid hybridization. In some embodiments, the analyte binding moiety comprises one or more antibodies or epitope binding fragments thereof. The antibody or epitope binding fragment comprising the analyte binding moiety can specifically bind to a target analyte. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein).

[0116] In some embodiments, a plurality of analyte labeling agents comprising a plurality of analyte binding moieties are combined with a plurality of analytes present in a biological sample. In some embodiments, a plurality of analytes include a single species of analyte (e.g., a single species of polynucleotides or polypeptides). In some embodiments in which a plurality of analytes include a single species of analyte, the analyte binding moieties of a plurality of analyte labeling agents are identical. In some embodiments in which a plurality of analytes include a single species of analyte, the analyte binding moieties of a plurality of analyte labeling agents are different (e.g., a member of a plurality of analyte labeling agents can have two or more species of analyte binding moieties, wherein each species in two or more species of analyte binding moieties is combined with a single species of analyte, such as in different binding sites). In some embodiments, a plurality of analytes include a plurality of different species of analyte (e.g., a plurality of different species of polynucleotides or polypeptides).

[0117] In other cases, for example, to facilitate sample multiplexing, different subsets of labeling agents specific for particular analytes or cell characteristics can be used. For example, a first subset of labeling agents comprises a binding moiety (e.g., a nucleic acid or antibody or a lipophilic moiety) coupled to a first reporter oligonucleotide, and a second subset of labeling agents comprises a binding moiety coupled to a second reporter oligonucleotide different from the first reporter oligonucleotide.

[0118] In some aspects, the reporter oligonucleotides may comprise nucleic acid barcode sequences that allow identification of the binding moiety to which the reporter oligonucleotide is coupled. Selecting an oligonucleotide as a reporter can provide the advantages of being able to generate significant diversity in sequence while also being able to attach to most biomolecules, e.g., nucleic acids and antibodies, etc., and being detectable, e.g., using the in situ detection techniques described herein.

[0119] Attachment (coupling) of the reporter oligonucleotide to the binding moiety can be achieved by any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, the oligonucleotide can be conjugated using chemical conjugation techniques (e.g., Lightning- Antibody labeling kit) is covalently attached to a portion of a binding moiety (such as a protein, such as an antibody or antibody fragment), as well as using other non-covalent attachment mechanisms, such as using a biotinylated antibody and an oligonucleotide with an avidin or streptavidin linker (or a bead comprising one or more biotinylated linkers coupled to an oligonucleotide). Antibody and oligonucleotide biotinylation techniques are available. See, for example, Fang et al., "Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labelling and Affinity Purification of Synthetic Oligonucleotides," Nucleic Acids Res. 2003 Jan 15; 31(2):708-715, which is incorporated herein by reference in its entirety for all purposes. Similarly, protein and peptide biotinylation techniques can be used. See, for example, U.S. Pat. No. 6,265,552, which is incorporated herein by reference for all purposes. In addition, click reaction chemistry can be used to couple the reporter oligonucleotide to the binding moiety. The reporter oligonucleotide can be coupled to a binding moiety using a commercially available kit (such as those from Thunderlink and Abcam) when appropriate. In another example, a binding moiety is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide, which includes a barcode sequence for identifying a binding moiety. For example, a binding moiety can be directly coupled (e.g., covalently bound) to a hybrid oligonucleotide, which includes a sequence hybridized with a sequence of a reporter oligonucleotide. The hybridization of a hybrid oligonucleotide and a reporter oligonucleotide couples a binding moiety to a reporter oligonucleotide. In some embodiments, such as when applying stimulation, the reporter oligonucleotide can be released from a binding moiety. For example, a reporter oligonucleotide can be attached to a binding moiety by an unstable bond (e.g., chemically unstable, light unstable, thermally unstable, etc.).

[0120] In some embodiments, a variety of different species of analytes (e.g., polynucleotides or polypeptides) from a biological sample can be subsequently associated with one or more physical properties of the biological sample. For example, a variety of different species of analytes can be associated with the position of the analyte in the biological sample. Such information (e.g., proteome information when the analyte binding moiety recognizes a polypeptide) can be used in combination with other spatial information (e.g., genetic information from a biological sample, such as DNA sequence information, transcriptome information (e.g., transcript sequence) or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., the shape, size, activity or type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte labeling agent, the analyte labeling agent comprising an analyte binding moiety that binds to a cell surface protein and an analyte binding moiety barcode that identifies the analyte binding moiety. The protein analysis results in a sample (e.g., a tissue sample or a cell) can be associated with DNA and / or RNA analysis in the sample.

[0121] (ii) Nucleic Acid Molecules and Complexes

[0122] In some respects, the combination of one or more nucleic acid molecules (for example, probe) and the analyte in the biological sample can be direct or indirect.In some embodiments, one or more nucleic acid molecules comprise the primary probe directly combined with its corresponding analyte.In other embodiments, one or more nucleic acid molecules comprise one or more probes directly or indirectly combined with the primary probe.The identifier sequence can be present in nucleic acid molecules and the complex comprising one or more nucleic acid analytes and / or one or more nucleic acid probes, and the one or more nucleic acid probes are directly or indirectly combined with the nucleic acid analyte.One or more identifier sequences in nucleic acid molecules and the complex can be located at one or more downstream (5') of the priming site for combining one or more sequencing primers.One or more identifier sequences can be associated with the analyte combined with the primary probe, and the sequencing of the identifier sequence can make it possible to identify the analyte.

[0123] For example, single molecule fluorescence in situ hybridization (smFISH) can be used to determine expression levels by detecting cell nucleic acid analytes such as mRNA. In smFISH, a group of conventional 30-50 oligonucleotides (length is each about 20 nucleotides) can be hybridized with complementary mRNA targets. Then a single transcript is visualized as a diffraction-limited spot using wide-field epifluorescence microscopy, and quantified. In some embodiments, smFISH probes can carry a 10-30 nucleotide long protruding sequence (rather than a directly conjugated fluorescent label), which is not hybridized with the mRNA target, and can be detected by hybridizing with it detectable (e.g., fluorescently detectable) probes labeled. The protruding sequence in the smFISH probe can include one or more identifier sequences, such as barcode sequences. Therefore, smFISH probes and mRNA targets can form a nucleic acid complex comprising a variety of barcode sequences, and methods disclosed herein can be used to perform in situ base sequencing.

[0124] In some aspects, the present invention discloses a probe (e.g., a first nucleic acid probe and / or a second nucleic acid probe) that is introduced into a cell or used to contact a biological sample, such as a tissue sample, in other ways. The probe may include any of a variety of entities that can hybridize with nucleic acids by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc. The probe typically contains a targeting sequence or hybridization region that can be directly or indirectly bound to at least a portion of a nucleic acid (e.g., a target nucleic acid or a probe). For example, the probe described herein may be capable of binding to a specific target nucleic acid (e.g., mRNA or other nucleic acids as discussed herein). In some embodiments, the probe includes one or more priming sites for binding to a sequencing primer and one or more downstream identifier sequences that can be sequenced using a sequencing primer.

[0125] Any probe described herein can be a linear probe. In some embodiments, a linear probe can be a probe comprising an analyte binding sequence (also sometimes referred to as a target recognition sequence). In some embodiments, a linear probe may include a sequence that is not hybridized with a target nucleic acid or a target probe, such as a 5' protrusion and / or a 3' protrusion. In some embodiments, a sequence (e.g., a 5' protrusion, a 3' protrusion) is not hybridized with a target nucleic acid or a target probe, but can hybridize with each other and / or with one or more other probes to form a hybridization complex, e.g., those in a hybridization chain reaction (HCR), a branched DNA reaction, etc. The hybridization complex can include one or more priming sites for binding to a sequencing primer and one or more downstream identifier sequences that can be sequenced using a sequencing primer.

[0126] The analyte binding sequence of the probe can be positioned at any position within the probe. For example, the analyte binding sequence of the probe that binds to the analyte can be 5' or 3' of an identifier sequence (e.g., a barcode sequence) in the probe. In some embodiments, the analyte binding sequence of the probe can be 5' or 3' of a priming site or a portion thereof in the probe. In some embodiments, the analyte binding sequence may include a sequence that is substantially complementary to a portion of the analyte. In some embodiments, the portion may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary. The analyte binding sequence of the probe can be determined with reference to a target analyte (e.g., cellular RNA) present or suspected in a sample. In some embodiments, more than one analyte binding sequence can be used to identify a specific analyte that contains a target nucleic acid or is associated with a target nucleic acid. The more than one analyte binding sequence can be in the same probe or in different probes. For example, multiple probes can be used sequentially and / or simultaneously, which can be combined (e.g., hybridized) with different regions of the same target analyte or with different target analytes. In some embodiments, the first probe is provided with a first multiple probe that is directly or indirectly combined with the first analyte. In some embodiments, the second probe is provided with a second multiple probe that is directly or indirectly combined with the second analyte. The first analyte and the second analyte can be the same or different, and the first multiple probe can be contacted with the sample, and the signal associated with the first multiple probe is subsequently detected, and the sample is contacted with the second multiple probe and the signal associated with it is detected.

[0127] In some embodiments, the identifier sequence (e.g., barcode sequence) can be on a single protrusion of a probe provided herein (e.g., on a protrusion of an L-shaped nucleic acid probe, or on a protrusion of a U-shaped probe). In other embodiments, the barcode sequence can be located on two protrusions of the probe (e.g., both 5' and 3' protrusions of a U-shaped probe). In some embodiments, the barcode sequence of a single probe is associated with an analyte and uniquely identifies the analyte. In other embodiments, a combination of barcode sequences of multiple probes hybridized with the same target analyte uniquely identifies the target analyte. For example, a first probe is provided with a first multiple probe that is directly or indirectly bound to a first analyte. In some aspects, a first multiple probe comprises a first combination of barcode sequences together, and the first combination of barcode sequences identifies the first analyte. In some embodiments, a second probe is provided with a second multiple probe that is directly or indirectly bound to a second analyte. In some aspects, a second multiple probe comprises a second combination of barcode sequences together, and the second combination of barcode sequences identifies the second analyte. In some embodiments, dividing the barcode sequence identifying the target analyte among multiple probes (eg, multiple primary probes) can reduce the length requirement for the probe (eg, the first probe).

[0128] In some embodiments, the identifier sequence (e.g., barcode sequence) may be in a primary probe, such as a primary probe that directly hybridizes with a cell nucleic acid molecule such as mRNA, or in a product of the primary probe (e.g., RCA product). In some embodiments, when hybridizing with a cell nucleic acid molecule, the primary probe may include a 3' or 5' protrusion. In some embodiments, the 3' or 5' protrusion includes one or more barcode sequences. In some embodiments, when hybridizing with a cell nucleic acid molecule, the primary probe may include a 3' protrusion and a 5' protrusion. In some embodiments, the 3' protrusion and the 5' protrusion each independently include one or more barcode sequences. In some embodiments, the primary probe may be a circular primary probe. In some embodiments, the circular primary probe includes one or more barcode sequences outside the analyte binding sequence of the probe. In some embodiments, the primary probe may be a circularizable primary probe or probe set. In some embodiments, the circularizable primary probe or probe set includes one or more barcode sequences outside the analyte binding sequence of the probe or probe set. In some embodiments, the primary probe may include a segmented hybridization zone configured to hybridize with a splint. In some embodiments, the partitioned hybridization regions comprise one or more barcode sequences. In some embodiments, the primary probes comprise one or more barcode sequences outside of the analyte binding sequence and the partitioned hybridization regions of the primary probes.

[0129] In some embodiments, the identifier sequence (e.g., barcode sequence) can be in a probe that directly binds to a cell nucleic acid molecule such as mRNA, or in a product of the probe (e.g., RCA product). In some embodiments, the probe can be an intermediate probe that bridges the binding of a probe (e.g., a primary probe) or its product to another probe or its product. In some embodiments, the probe can be a detection probe that hybridizes with a primary probe or an intermediate probe or its product but does not bind directly or indirectly to any other probe. The detection probe can also include a priming site for sequencing the identifier sequence in the detection probe using the methods disclosed herein.

[0130] In some embodiments, when hybridized with another probe, the intermediate or detection probe may include a 3' or 5' overhang. In some embodiments, the 3' or 5' overhang includes one or more barcode sequences. In some embodiments, when hybridized with another probe, the intermediate or detection probe may include a 3' overhang and a 5' overhang. In some embodiments, the 3' overhang and the 5' overhang each independently include one or more barcode sequences. In some embodiments, the intermediate or detection probe may be a circular probe. In some embodiments, the circular probe includes one or more barcode sequences outside the target binding sequence of the probe. In some embodiments, the intermediate or detection probe may be a circularizable probe or probe set. In some embodiments, the circularizable probe or probe set includes one or more barcode sequences outside the target binding sequence of the probe or probe set. In some embodiments, the intermediate or detection probe may include a split hybridization region configured to hybridize with a splint. In some embodiments, the split hybridization region may include a priming site, and the splint or a portion thereof may be used as a sequencing primer to sequence the identifier sequence in the intermediate or detection probe using the methods disclosed herein. Exemplary probes comprising split hybridization zones include, but are not limited to, those described in US 2022 / 0049302, which is incorporated herein by reference in its entirety. In some embodiments, the split hybridization zones comprise one or more barcode sequences. In some embodiments, the intermediate or detection probe comprises one or more barcode sequences outside the target binding sequence and split hybridization zones of the probe.

[0131] In some embodiments, the identifier sequence (e.g., barcode sequence) can be in a rolling circle amplification (RCA) product molecule, a complex comprising an initiator and amplifier for hybridization chain reaction (HCR), a complex comprising an initiator and amplifier for linear oligonucleotide hybridization chain reaction (LO-HCR), a primer exchange reaction (PER) product molecule, a complex comprising a pre-amplifier and amplifier for branched DNA (bDNA), or a complex comprising any two or more of the aforementioned molecules and complexes. For example, bDNA complexes or HCR complexes can be assembled on RCA products. See, for example, US 2021 / 0198727, which is incorporated herein by reference in its entirety. A priming site can be provided at 3' of each identifier sequence in a molecule or complex to sequence the identifier sequence in situ using SBS, SBB or any other base-by-base sequencing method.

[0132] In some embodiments, a molecule or complex comprising an identifier sequence (e.g., a barcode sequence) and a priming site for sequencing the identifier sequence can be generated using methods such as targeted assembly of branched structures (e.g., bDNA or branched assays using locked nucleic acids (LNA)), programmed in situ growth of concatemers by enzymatic rolling circle amplification (RCA) (e.g., as described in US 2019 / 0055594, which is incorporated herein by reference), HCR, etc., assembly of topologically linked DNA structures using successive rounds of chemical ligation (clampFISH), hairpin-mediated concatemerization (e.g., as described in US 2020 / 0362398, which is incorporated herein by reference), such as primer exchange reactions, such as signal amplification by exchange reaction (SABER) or SABER coupled with DNA-exchange (Exchange-SABER). In some embodiments, non-enzymatic methods can be used.

[0133] In some embodiments, the complex comprising an identifier sequence (e.g., a barcode sequence) and a priming site comprises an amplifier that hybridizes directly or indirectly (via one or more oligonucleotides) to a sequence of a nucleic acid analyte, a probe that directly or indirectly targets a nucleic acid analyte, or a product of a nucleic acid analyte or a probe, e.g., Figure 2 In some embodiments, the assembly includes one or more amplifiers, each amplifier including an amplifier repeating sequence. In some aspects, the one or more amplifiers are labeled. In some aspects, the one or more amplifiers are not labeled. For exemplary complexes, see, for example, US 2020 / 0399689 and US 2022 / 0064697, which are fully incorporated herein by reference.

[0134] In some embodiments, the complex comprising an identifier sequence (e.g., a barcode sequence) and a priming site comprises an HCR complex, e.g., Figure 2HCR is an enzyme-free nucleic acid amplification based on a hybridization-triggered chain of nucleic acid molecules, starting with HCR monomers, which hybridize to each other to form a nicked nucleic acid polymer. The polymer is the product of the HCR reaction, which is ultimately detected to indicate the presence of the target analyte. HCR is described in detail in the following literature: Dirks and Pierce, 2004, PNAS, 101 (43), 15275-15278; and US 7,632,641 and US 7,721,721 (see also US 2006 / 00234261; Chemeris et al., 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al., 2010, 46, 3089-3091; Choi et al., 2010, Nat. Biotechnol. 28 (11), 1208-1212; and Song et al., 2012, Analyst, 137, 1396-1401). HCR monomers typically contain hairpins or other metastable nucleic acid structures. In the simplest HCR form, when introducing " initiator " nucleic acid molecule, two different types of stable hairpin monomers (here referred to as the first and second HCR monomers) experience hybridization chain reaction events, forming a double-stranded DNA molecule with long nicks.HCR monomer has a hairpin structure, which comprises a double-stranded stem region, a loop region connecting two chains in the stem region and a single-stranded region at one end of the double-stranded stem region.When monomer is in a hairpin structure, the single-stranded region exposed (and therefore can be used for hybridization with another molecule such as initiator or other HCR monomers) can be referred to as "foothold region" (or "input domain").The first HCR monomer also includes a sequence complementary to the sequence in the foothold region exposed with the second HCR monomer.This complementary sequence in the first HCR monomer can be referred to as "interaction region" (or "output domain").Similarly, the second HCR monomer includes an interaction region (output domain) each, such as a sequence complementary to the foothold region (input domain) exposed with the first HCR monomer. In the absence of HCR initiators, these interaction regions are protected by secondary structures (e.g., they are not exposed), so hairpin monomers are stable or dynamically trapped (also referred to as "metastable"), and remain as monomers (e.g., to prevent rapid system equilibrium), because the first and second groups of HCR monomers cannot hybridize with each other. However, once an initiator is introduced, it is able to hybridize with the exposed foothold region of the first HCR monomer, and invade it, causing it to open. This will expose the interaction region of the first HCR monomer (e.g., a sequence complementary to the foothold region of the second HCR monomer), thereby allowing it to hybridize with the second HCR monomer at the foothold region and invade the second HCR monomer.This hybridization and invasion and then open the second HCR monomer, expose its interaction zone (it is complementary to the foothold zone of the first HCR monomer), and allow it to hybridize with another first HCR monomer and invade another first HCR monomer.Reaction continues in this way, until all HCR monomers are exhausted (for example, all HCR monomers are incorporated into polymer chain).Finally, this chain reaction causes the notched chain of the alternating unit forming the first and second monomer species.Therefore, it is necessary to have the presence of HCR initiator in order to trigger HCR reaction by hybridizing with the first HCR monomer and invading the first HCR monomer.The first and second HCR monomers are designed to hybridize each other and therefore can be defined as homologous to each other.They are also homologous to given HCR initiator sequence.The HCR monomer interacting with each other (hybridization) can be described as a group of HCR monomer or HCR monomer or hairpin system.

[0135] The HCR reaction can be carried out with more than two kinds or types of HCR monomers. For example, a system involving three HCR monomers can be used. In such a system, each first HCR monomer can include an interaction zone combined with the foothold area of ​​the second HCR monomer; each second HCR can include an interaction zone combined with the foothold area of ​​the 3rd HCR monomer; and each 3rd HCR monomer can include an interaction zone combined with the foothold area of ​​the first HCR monomer. Then, the HCR polymerization reaction will be carried out as described above, except that the product obtained will be a polymer of a repeating unit with the first, second and third monomers continuously. A corresponding system with a larger number of HCR monomer groups can be used.

[0136] In some embodiments, HCR product (for example, the notched chain of the alternating unit of monomer species) can include multiple protrusions, and each protrusion includes one or more identifier sequences (for example, barcode sequence) and a priming site for sequencing the identifier sequence in situ. Therefore, as used herein, HCR does not need to use detectably labeled HCR monomers; On the contrary, one or more HCR monomers can include one or more identifier sequences (for example, barcode sequence), and the detection of HCR product includes sequencing the identifier sequence in situ.

[0137] In some embodiments, similar to the HCR reaction using hairpin monomers, a linear oligonucleotide hybridization chain reaction (LO-HCR) can also be used to generate a complex comprising an identifier sequence (e.g., a barcode sequence) and a priming site for sequencing the identifier sequence. In some embodiments, a method for detecting an analyte in a sample is provided herein, the method comprising: (i) performing a linear oligonucleotide hybridization chain reaction (LO-HCR), wherein an initiator is contacted with a plurality of LO-HCR monomers of at least a first species and a second species to generate a polymerized LO-HCR product hybridized with a target nucleic acid molecule, wherein the first species comprises a first hybridization region complementary to the initiator and a second hybridization region complementary to the second species, wherein the first species and the second species are linear single-stranded nucleic acid molecules; wherein the initiator is provided in one or more parts and is directly or indirectly hybridized with the target nucleic acid molecule or is contained in the target nucleic acid molecule; and (ii) detecting the polymer product, thereby detecting the analyte. In some embodiments, the first species and / or the second species may not contain a hairpin structure. In some embodiments, the plurality of LO-HCR monomers may not contain a metastable secondary structure. In some embodiments, the LO-HCR polymer may not contain a branched structure. In some embodiments, performing a linear oligonucleotide hybridization chain reaction comprises contacting the target nucleic acid molecule with an initiator to provide an initiator that hybridizes to the target nucleic acid molecule. Exemplary methods and compositions for LO-HCR are described in US 2021 / 0198723, which is incorporated herein by reference in its entirety.

[0138] In some embodiments, the polymer product in LO-HCR can include multiple protrusions, each protrusion including one or more identifier sequences (e.g., barcode sequences) and a priming site for in situ sequencing of the identifier sequences. Thus, as used herein, LO-HCR does not require the use of detectably labeled LO-HCR monomers; instead, one or more LO-HCR monomers can include one or more identifier sequences (e.g., barcode sequences), and detection of the LO-HCR product includes in situ sequencing of the identifier sequences.

[0139] In some embodiments, a molecule (e.g., a concatemer molecule) comprising an identifier sequence (e.g., a barcode sequence) and a priming site is generated by a primer exchange reaction (PER). In various embodiments, a primer having a domain at its 3' end is bound to a catalytic hairpin and a new domain is extended by a strand displacement polymerase. In various embodiments, the strand displacement polymerase is a Bst polymerase. In various embodiments, the catalytic hairpin includes a terminator that releases a strand displacement polymerase. In various embodiments, the branch migration displaces the extended primer, and then the extended primer can dissociate. In various embodiments, the primer undergoes repeated cycles to form a concatemer molecule, see, for example, US 2019 / 0106733 (which is incorporated herein by reference), for exemplary molecules and PER reaction components. In various embodiments, the concatemer molecule comprises multiple copies of one or more identifier sequences (e.g., barcode sequences) and a priming site for sequencing the identifier sequence. Therefore, instead of hybridizing a variety of labeled oligonucleotide probes to the concatemer molecule, the concatemer molecule can be detected by sequencing the identifier sequence in situ.

[0140] (iii) Rolling circle amplification (RCA) products

[0141] In some embodiments, molecules (e.g., concatemer molecules) comprising an identifier sequence (e.g., a barcode sequence) and a priming site are generated by RCA of circular nucleic acid molecules, e.g., as Figure 2 As shown in . As described in Section II-A, a nucleic acid analyte (e.g., cDNA) can be cyclized to generate a circular nucleic acid molecule and the RCA product comprises an identifier sequence derived from the nucleic acid analyte. In some embodiments, any suitable cyclizable probe or probe set disclosed herein can be cyclized, for example, using a nucleic acid analyte or probe as a template, with or without gap filling prior to cyclization. The RCA product may comprise a barcode sequence or its complementary sequence that can be sequenced in situ.

[0142] In some aspects, probes disclosed herein (e.g., primary probes, intermediate probes, detection probes, etc.) are connected to form a circular construct (e.g., a circular probe). In some embodiments, a template primer is used to extend and then connect to form a circular construct. In some embodiments, an analyte is used as a template to generate a connected probe. In some embodiments, a circular construct is formed by providing an insert between the ends to be connected. In some embodiments, a combination of any of the foregoing is used to form a circular construct. In some embodiments, the connection is a DNA templated connection. In some embodiments, the connection is an RNA templated connection. In some embodiments, a splint is provided as a template for connection.

[0143] The property of ligation reaction depends on the structural component of the probe used. In some embodiments, analyte (for example, RNA) can be used as template to connect the 3' end and 5' end of circularizable probe or probe group. In some embodiments, 3' end and 5' end are connected without filling the gap before connection. In some embodiments, before the connection of 3' end and 5' end, the gap is filled first. The gap can be 1,2,3,4,5 or more nucleotides. In some embodiments, connection may include enzymatic connection, chemical connection, template-dependent connection and / or non-template-dependent connection. In any one of the embodiments herein, connection may include the use of a ligase with RNA-templated DNA ligase activity and / or RNA-templated RNA ligase activity. In some embodiments, enzymatic connection relates to the use of a ligase (for example, RNA ligase, DNA ligase). Ligase includes ATP-dependent double-stranded polynucleotide ligase, NAD-i-dependent double-stranded DNA or RNA ligase and single-stranded polynucleotide ligase, such as any one of the ligases described in EC 6.5.1.1 (ATP-dependent ligase), EC 6.5.1.2 (NAD+-dependent ligase), EC 6.5.1.3 (RNA ligase). Specific examples of ligases include bacterial ligases (such as Escherichia coli DNA ligase), Tth DNA ligase, Thermococcus species (strain 9°N) DNA ligase (9°N TM DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase TM (Epicentre Biotechnologies) and bacteriophage ligases (such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase) and their mutants. In any one of the embodiments herein, the connection can include the use of a ligase selected from Chlorella virus DNA ligase (PBCV DNA ligase), T4 RNA ligase, T4 DNA ligase and single-stranded DNA (ssDNA) ligase. In any one of the embodiments herein, the connection can include the use of a PBCV-1 DNA ligase or its variant or derivative and / or T4 RNA ligase 2 (T4 Rnl2) or its variant or derivative. In some embodiments, the ligase is T4 RNA ligase. In some embodiments, the ligase comprises splintR ligase. In some embodiments, the ligase is a single-stranded DNA ligase. In some embodiments, the ligase is T4 DNA ligase. In some embodiments, the ligase is a ligase with DNA splinting DNA ligase activity. In some embodiments, the ligase is a ligase with RNA splinting DNA ligase activity.

[0144] In some aspects, a high-fidelity ligase is used, such as a thermostable DNA ligase (e.g., Taq DNA ligase). The thermostable DNA ligase is active at elevated temperatures, allowing further differentiation by incubating the ligation at a temperature close to the melting temperature of the DNA strands. High-fidelity ligation can be achieved by a combination of inherent selectivity of the ligase active site and equilibrium conditions to reduce the incidence of annealing mismatched dsDNA.

[0145] In some embodiments, a removal step is performed to remove molecules that are not specifically hybridized. In some embodiments, a removal step is performed to remove unconnected probes. In some embodiments, the removal step is performed after connection and before amplification. A washing step can be performed at any time point during the process to remove non-specifically bound probes, unconnected probes, etc. In some embodiments, after the removal step, the circularized probe remains specifically hybridized with the analyte.

[0146] In some cases, primer oligonucleotides are added for amplification. In some cases, primer oligonucleotides are added together with circularizable probes or probe groups. In some cases, primer oligonucleotides are added before or after circularizable probes or probe groups are contacted with a sample. In some cases, primer oligonucleotides for amplifying circular nucleic acid molecules may include sequences complementary to nucleic acid (e.g., cDNA or mRNA), and sequences complementary to circularizable probes with nucleic acid hybridization. In some embodiments, washing steps are carried out to remove any unbound probes, primers, etc. In some embodiments, washing is stringent washing.

[0147] The primer oligonucleotide for amplifying circular nucleic acid molecules can include a single-stranded nucleic acid sequence with a 3' end, and the 3' end can be used as a substrate for the nucleic acid polymerase in the nucleic acid extension reaction. The primer oligonucleotide can include both RNA nucleotides and DNA nucleotides (for example, in a random or designed pattern). The primer oligonucleotide can also include other natural or synthetic nucleotides that can have additional functions as described herein. The primer oligonucleotide can be about 6 bases to about 100 bases, such as about 25 bases.

[0148] In some cases, after adding DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the amplification primer is extended by the replication of multiple copies of the template. The amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after forming a hybridization complex and any subsequent cyclization (such as the connection of a padlock probe), the circular nucleic acid molecule rolling circle amplification is performed to generate RCA products (e.g., amplicons) containing multiple copies of the sequence of the circular nucleic acid molecule. See, e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 Nov 15;49(11):2540-2550; Schweitzer et al., Proc. Natl Acad. Sci. USA 97:10113-119, 2000; Faruqi et al., BMC Genomics 2:4, 2000; Nallur et al., Nucl. Acids Res. 29:e118, 2001; Dean et al., Genome Res. 11:1095-1099, 2001; Schweitzer et al., Nature 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, all of which are incorporated herein by reference.

[0149] In some embodiments, the rolling circle amplification product is generated using a polymerase selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and variants or derivatives thereof. In some embodiments, the polymerase is Phi29 DNA polymerase.

[0150] In some embodiments, the polymerase comprises a modified recombinant Phi29-type polymerase. In some embodiments, the polymerase comprises a modified recombinant Phi29, B103, GA-1, PZA, Phi15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In some embodiments, the polymerase comprises a modified recombinant DNA polymerase having at least one amino acid substitution or combination of substitutions compared to a wild-type Phi29 polymerase. Exemplary polymerases are described in U.S. Pat. Nos. 8,257,954; 8,133,672; 8,343,746; 8,658,365; 8,921,086; and 9,279,155, all of which are incorporated herein by reference. In some embodiments, the polymerase is not directly or indirectly immobilized to a substrate, such as a bead or a planar substrate (eg, a glass slide), prior to contacting the sample, but the sample may be immobilized on a substrate.

[0151] In some embodiments, amplification is performed at a temperature equal to or approximately between 20° C. and 60° C. In some embodiments, amplification is performed at a temperature equal to or approximately between 30° C. and 40° C. In some aspects, amplification is performed at a temperature between at or approximately between 25° C. and at or approximately 50° C., such as at or approximately 25° C., 27° C., 29° C., 31° C., 33° C., 35° C., 37° C., 39° C., 41° C., 43° C., 45° C., 47° C., or 49° C.

[0152] In any of the embodiments herein, the RCA product may be generated in situ in a biological sample.In any of the embodiments herein, the product may be generated using linear RCA, branched RCA, dendritic RCA or any combination thereof.

[0153] In some embodiments, the RCA product comprises multiple copies of one or more identifier sequences or their complements. In some embodiments, the RCA product comprises multiple copies of one or more priming sites or their complements. In some aspects, the one or more identifier sequences or their complements are located downstream of the one or more priming sequences or their complements.

[0154] In some aspects, during the amplification step, modified nucleotides can be added to the reaction to incorporate the modified nucleotides into the amplification product (e.g., nanospheres). Examples of modified nucleotides include amine-modified nucleotides. In some aspects of the method, for example, the generated amplification product (e.g., nanospheres) is anchored or cross-linked to a scaffold, a cell structure, and / or other amplification products (e.g., other nanospheres). In some aspects, the amplification product comprises modified nucleotides, such as amine-modified nucleotides. In some embodiments, amine-modified nucleotides react with acrylic acid N-hydroxysuccinimide moieties. Examples of other amine-modified nucleotides include, but are not limited to, 5-aminoallyl-dUTP moiety modifications, 5-propargylamino-dCTP moieties, N 6 -6-aminohexyl-dATP partial modification or 7-deaza-7-propargylamino-dATP partial modification. In some embodiments, the modified nucleotide comprises a base modification, such as azide and / or alkynyl base modification, dibenzylcyclooctyl (DBCO) modification, vinyl modification, trans-cyclooctene (TCO) and the like.

[0155] In some embodiments, the primer extension reaction mixture may include deoxynucleoside triphosphates (dNTPs) or derivatives, variants, or analogs thereof. In some embodiments, the primer extension reaction mixture may include a catalytic cofactor of a polymerase. In any of the foregoing embodiments, the primer extension reaction mixture may include a catalytic divalent cation, such as Mg. 2+ and / or Mn 2+ .

[0156] In some respects, the amplified product (e.g., RCA product) can be anchored to a polymer matrix. The amplified product can be fixed in the matrix, usually in the position where the nucleic acid is amplified, thereby producing a local colony of an amplicon. The amplified product can be fixed in the matrix by spatial factors. The amplified product can also be fixed in the matrix by covalent bonds or non-covalent bonds. In this way, it can be considered that the amplified product is connected to the matrix. By being fixed on the matrix, such as by covalent bonds or crosslinking, the size and spatial relationship of the original amplicon are maintained. By being fixed on the matrix, such as by covalent bonds or crosslinking, the amplified product is resistant to moving or spreading out under mechanical stress.

[0157] In some aspects, the amplification products (e.g., RCA products) are copolymerized and / or covalently attached to the surrounding matrix, thereby retaining their spatial relationship and any inherent information thereof. In some embodiments, the RCA products are generated by DNA or RNA embedded in the cells in the matrix. In some embodiments, the RCA products can also be functionalized to form covalent attachments to the matrix, retaining their spatial information in the cell, thereby providing a subcellular localization distribution pattern. In some embodiments, the provided method involves embedding the RCA products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the described hydrogel-histochemistry includes covalently attaching nucleic acids to hydrogels synthesized in situ for tissue clearance, enzyme diffusion, and multi-cycle sequencing or probe hybridization, while existing hydrogel-histochemistry methods cannot. In some embodiments, in order to enable the amplification products to be embedded in the tissue-hydrogel setting, amine-modified nucleotides are included in the amplification step (e.g., RCA), functionalized to acrylamide moieties using acrylic acid N-hydroxysuccinimide esters, and copolymerized with acrylamide monomers to form a hydrogel.

[0158] III. In situ sequencing of identifier sequences

[0159] In some embodiments, a method for analyzing a cell or tissue sample is provided herein, the method comprising contacting the sample with a probe that binds directly or indirectly to an analyte at a position in the sample, wherein the probe or its product (e.g., an amplification product, such as an RCA product) comprises a priming site and an identifier sequence present in, associated with, corresponding to, and / or identifying the analyte. In some embodiments, the method further comprises contacting the sample with a sequencing primer that is configured to hybridize to the priming site and be bound by a polymerase and nucleotides to sequence the identifier sequence or a portion thereof in the probe or its product, base by base.

[0160] In some embodiments, the method includes contacting the biological sample with nucleotides in continuous cycles (e.g., for each cycle, the nucleotide mixture includes nucleotides containing different bases), wherein a complex is formed in each cycle and the complex includes: i) a sequencing primer or an extension product thereof hybridized with a probe or its product, ii) a polymerase, and iii) a homologous nucleotide that is base-paired with a nucleotide in an identifier sequence. In some embodiments, in each cycle, the method includes detecting a signal (ON signal) and / or a signal absence (OFF signal) associated with a homologous nucleotide at the position and / or detecting a polymerase, wherein the ON signal, the OFF signal, or a combination thereof corresponds to a base in a homologous nucleotide and a corresponding nucleotide in an identifier sequence. In some embodiments, the method includes generating a signal code sequence, the signal code sequence comprising a signal code corresponding to an ON signal, an OFF signal, or a combination thereof at the position in a continuous cycle, thereby detecting an identifier sequence at the position in a biological sample. Since the identifier sequence can be present in an analyte, associated with an analyte, corresponding to an analyte, and / or identifying an analyte, detecting the identifier sequence at the position can be used to identify the analyte at the position. For example, where the identifier sequence uniquely identifies an analyte from a plurality of analytes, detecting the identifier sequence at that position will identify the analyte at that position. In some embodiments, a combination of identifier sequences in the analyte and / or in the probe or its product identifies the analyte, and each identifier sequence can be sequenced to decode the combination and identify the analyte.

[0161] The present disclosure provides methods for in situ detection of multiple analytes in a cell sample or a tissue sample. Provided herein are probes (e.g., a first probe and a second probe) designed to reduce optical crowding of signals in a biological sample. In some embodiments, the first probe and the second probe are contacted with a first analyte and a second analyte (e.g., a nucleic acid molecule, such as mRNA) in a biological sample. Figure 1(101)). In some embodiments, the first probe and the second probe directly or indirectly bind to the first analyte and the second analyte at the first position and the second position in the sample, respectively. In some aspects, the first probe and the second probe are primary probes that directly bind to their corresponding analytes at the position in the sample. In some aspects, the first probe and the second probe directly or indirectly bind to the first primary probe and the second primary probe in the sample. In some embodiments, the first probe and the second probe can be circularized probes or probe groups. In some embodiments, the first probe and the second probe can be linear probes or probe groups. In some embodiments, the first probe and the second probe are amplified in situ to generate the first product and the second product of the first probe and the second probe (e.g., the first and second rolling circle amplification (RCA products)). In some embodiments, the first probe and the second probe or their products each contain i) a priming site of a sequencing primer and ii) an identifier sequence associated with the corresponding analyte in the sample. For example, the first probe or its product may contain i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte at, for example, the first position in the sample. The priming site of a sequencing primer is a site for starting a sequencing reaction, such as sequencing by synthesis (SBS) or sequencing by binding (SBB) reactions. In some embodiments, the first identifier sequence and the second identifier sequence are associated with the first analyte and the second analyte in the sample. The decoding of the first identifier sequence and the second identifier sequence can enable the identification of the first analyte and the second analyte in the sample and their corresponding positions. In some embodiments, the identifier sequence is a barcode sequence associated with the analyte or corresponding to the identity of the analyte, and the barcode sequence decoding enables the identification of the analyte in addition to revealing the spatial position of the analyte in the biological sample.

[0162] In some embodiments, the method comprises performing SBS or SBB on the first identifier sequence and the second identifier sequence. Figure 1 (102)). In some embodiments, a SBS or SBB reaction is performed using a first sequencing primer and a second sequencing primer. The first sequencing primer and the second sequencing primer bind to the first priming site and the second priming site on the first probe and the second probe or their products, respectively. In some embodiments, the method includes performing a cyclic series of nucleotide incorporation steps, such as incorporating A, T, C, or G in SBS. In some embodiments, the method includes performing a cyclic series of nucleotide incorporation steps, such as incorporating A, T, C, or G in SBB.

[0163] In some embodiments, the method includes detecting a signal such as an ON signal or a lack of a signal such as an OFF signal during the sequencing step. Figure 1(103)). In some embodiments, the method includes detecting first and second ON signals and / or OFF signals at a first position and a second position in the sample. In some embodiments, the signal detected in a particular cycle corresponds to a signal code for that cycle. In some embodiments, the signal code corresponds to a signal of a first color, a signal of a second color, a signal of a third color, or a lack of signal. In some aspects, the first, second, and third colors are different. In some aspects, the first, second, and third colors are the same. In some aspects, the SBS or SBB reaction is repeated in one or more consecutive cycles to generate a series of signal codes, each signal code corresponding to a signal (ON signal) or a lack of signal (OFF signal) or a combination of ON signals and / or OFF signals ( Figure 1 (104)).

[0164] In some embodiments, a series of signal codes are detected at different positions (e.g., a first position and a second position) in a sample. In some embodiments, a nucleotide in a first barcode sequence detected in a particular cycle corresponds to a signal code comprising an ON signal, and a corresponding nucleotide in a second barcode sequence detected in a particular cycle corresponds to a signal code comprising an OFF signal. In some embodiments, one or more pairs of corresponding nucleotides in a first barcode sequence and a second barcode sequence to be detected in the same cycle are selected to reduce optical crowding of signals detected in that cycle.

[0165] In some aspects, each series of signal codes comprises a signal code sequence. For example, the signal code of the series detected at the first position in the continuous cycle comprises a first signal code sequence. The signal code of the series detected at the second position in the continuous cycle comprises a second signal code sequence. In some aspects, the first signal code sequence and the second signal code sequence are used to decode the identifier sequence on the first probe and the second probe or their products. In some embodiments, the method includes detecting the first identifier sequence and the second identifier sequence based on the first signal code sequence and the second signal code sequence ( Figure 1 (105)). In some aspects, the identifier sequence on the probe or its product is a barcode sequence. In some aspects, the barcode sequence on the probe or its product is assigned to the analyte bound to the probe. In some aspects, in situ detection of the barcode sequence by SBS or SBB enables parallel identification of analytes while reducing optical crowding of the signal during multiple cycles of decoding.

[0166] Figure 2 An exemplary schematic diagram of a sequencing-by-synthesis reaction of an identifier sequence on a probe or its product (eg, an RCA product) is depicted. An exemplary hybridization complex comprising the identifier sequence is also shown.

[0167] The probe or product may comprise i) a priming site for a sequencing primer and ii) an identifier sequence. In some embodiments, the priming site is a site for hybridizing a sequencing primer to begin sequencing. In some embodiments, the identifier sequence is a barcode sequence. In some embodiments, the identifier sequence or barcode sequence is associated with a corresponding analyte in the sample. Decoding of the identifier sequence or barcode sequence on the probe or its product can enable identification of the analyte bound to the probe. In some embodiments, the SBS reaction is performed by contacting the biological sample with a mixture of nucleotides in a continuous cycle ( Figure 2 ). In some embodiments, a complex is formed in each cycle, the complex comprising i) a sequencing primer or an extension product thereof that hybridizes to the priming site, ii) a polymerase, and iii) a homologous nucleotide that base pairs with a nucleotide in the identifier sequence. In some aspects, the sequencing primer binds to the priming site on the probe or its product and generates an extension product as the sequencing reaction proceeds. In some embodiments, the sequencing reaction comprises a cyclic series of nucleotide incorporation steps. Homologous nucleotides A, T, C, or G bind to their corresponding nucleotides in the identifier sequence and are incorporated into the sequencing primer or the extension product. In some embodiments, the polymerase incorporates homologous nucleotides into the sequencing primer or the extension product. In some embodiments, the nucleotide mixture contacted with the biological sample comprises a mixture of fluorescently labeled and non-fluorescently labeled nucleotides. In some aspects, the nucleotide mixture comprises three fluorescently labeled nucleotides and one non-fluorescently labeled nucleotide. In some aspects, the nucleotide mixture comprises two fluorescently labeled nucleotides and two non-fluorescently labeled nucleotides. For example, as Figure 2 As shown in , nucleotides T, A, and G are labeled with fluorescent moieties, while nucleotide C is unlabeled. In some embodiments, a signal (ON signal) and / or a signal absence (OFF signal) associated with a homologous nucleotide is detected at a specific position in a biological sample. In some embodiments, the ON signal, the OFF signal, or a combination thereof corresponds to a base in the homologous nucleotide and the corresponding nucleotide in the identifier sequence. For example, an ON signal may be detected due to incorporation of labeled nucleotides T, A, or G into a sequencing primer or extension product, while an OFF signal may be detected due to incorporation of unlabeled nucleotide C into a sequencing primer or extension product. In some aspects, the identifier sequence is detected based on a signal code sequence generated by SBS. In some aspects, as Figure 2 Detection of the identifier sequence shown in enables identification of the analyte while reducing optical crowding of the signal.

[0168] A. Dark base and nucleotide mixture

[0169] In some embodiments, a method is provided herein that includes determining the sequence of an identifier sequence in situ in a cell or tissue sample, wherein the identifier sequence comprises bases that do not generate a detectable signal in a corresponding base-by-base sequencing cycle, for example, these bases are "dark". In any particular identifier sequence, the dark bases can be the same (for example, all G) or different (the first dark base is G and the second dark base is C). In any particular identifier sequence, any two or more dark bases can be continuous (the nucleotide residues are directly connected by a phosphodiester bond) or non-continuous (for example, the nucleotide residues are separated by one or more nucleotide residues, at least one of which contains a non-dark base).

[0170] In some embodiments, multiple different identifier sequences are sequenced in situ in a cell or tissue sample using a base-by-base sequencing method (e.g., SBS or SBB), and each of the multiple different identifier sequences comprises multiple dark bases, such that optical crowding in many (e.g., most) base-by-base sequencing cycles is limited to facilitate accurate and efficient signal detection and decoding.

[0171] In some embodiments, the present invention provides a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: the biological sample is a cell or tissue sample, the biological sample comprises a first analyte and a second analyte at a first position and a second position in the biological sample, respectively, the first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively, the first probe or its product comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and the second probe or its product comprises i) a second priming site of a second sequencing primer and ii) a second identifier sequence associated with the second analyte; b) performing base-by-base sequencing of the first identifier sequence and the second identifier sequence using the first sequencing primer and the second sequencing primer, thereby generating a first signal code sequence and a second signal code sequence, each signal code sequence comprising a signal code corresponding to a signal (ON signal), a signal absence (OFF signal), or a combination thereof detected at the first position and the second position, respectively, in consecutive cycles, wherein in one or more of the consecutive cycles, an ON signal is detected at the first position and an OFF signal is detected at the second position; and c) detecting the first identifier sequence and the second identifier sequence in the biological sample based on the first signal code sequence and the second signal code sequence.

[0172] In some embodiments, the first identifier sequence and the second identifier sequence are different and are associated with different first and second analytes, respectively. In some embodiments, the first identifier sequence and the second identifier sequence are different and are associated with the same analyte. In some embodiments, the first identifier sequence and the second identifier sequence can comprise a barcode sequence or a complement thereof. In some embodiments, the first barcode sequence and the second barcode sequence can be assigned to the first analyte and the second analyte, respectively.

[0173] In some embodiments, a code word (corresponding to a physical barcode or identifier sequence) is assigned to each of a plurality of analytes using any suitable scheme that minimizes optical crowding in a fluorescent image for decoding the identifier sequence. Methods for designing code books and assigning code words to analytes to minimize optical crowding are described in US 63 / 317,842, filed on March 8, 2022, entitled "in situ Code Design Methods for Minimizing Optical Crowding," and International Patent Application No. PCT / US2023 / 063866, filed on March 7, 2023, entitled "in situ Code Design Methods forMinimizing Optical Crowding," which are incorporated herein by reference in their entirety. In some aspects, an identifier sequence (corresponding to a code word) is assigned to each of a plurality of analytes for optimization of each of the analytes. In some embodiments, for example, the assignment of identifier sequences to each of a plurality of analytes is performed based on expression data of a plurality of analytes. For example, expression data may include full transcriptome, single cell reference gene expression data that have been clustered according to cell type. In some embodiments, the code word / identifier sequence containing the maximum number of OFF bits can be assigned to the analyte (e.g., gene) in descending order of the number of OFF bits in the code word and the highest expression level of the analyte (e.g., gene) in all cell types. In some embodiments, assigning a code word (corresponding to an identifier sequence) to a gene includes calculating a metric (e.g., worst case density or maximum predicted density) that will be obtained by assigning any still available code word to the current gene. Then, the code word that produces the lowest worst case density is assigned to a specific analyte. In some cases, the worst case density is the expression density of the (cell type, code bit) pair with the highest total expression density of the gene that is ON in the code bit.

[0174] In some cases, codewords (corresponding to identifier sequences) can be assigned to multiple analytes according to a decision rule (e.g., a minimax decision rule) designed to minimize the maximum predicted density of ON signals on a series of images acquired on one or more detection channels (e.g., 1, 2, 3, 4, or more than 4 detection channels) during multiple sequencing cycles. For example, in some cases, codewords of a codebook can be assigned according to a minimax decision rule that minimizes the maximum predicted density of ON signals (corresponding to ON bits of target analyte-associated codewords) detected in each image in a series of images acquired during multiple sequencing cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 sequencing cycles) for sequencing multiple analytes (e.g., where each of one, two, three, four, or more than four detection channels acquires or receives a different image of the biological sample in each sequencing cycle). For multiple analytes (e.g., at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, at least 1,000, at least 2,000, at least 4,000, at least 6,000, at least 8,000, at least 10,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, at least 1,000,000, at least 2 x 10 6 , at least 3x10 6 , at least 4x10 6 , at least 5x10 6 , at least 6x10 6 , at least 7x10 6 , at least 8x10 6 , at least 9x10 6 , at least 10 7 , at least 10 8 , at least 10 9 10 or more 9 analyte).

[0175] In some cases, codewords in a codebook can be assigned to multiple analytes based on a decision rule that will ensure that the ON bits are more or less evenly distributed across multiple sequencing cycles and detection channels used for sequencing. For example, in some cases, codewords in a codebook can be assigned to multiple analytes based on a decision rule that will ensure that the total number of ON signals detected in a given image for a given sequencing cycle is within ±5%, ±10%, ±15%, ±20%, or ±25% of the average number of ON signals detected in each image in a series of images acquired during multiple sequencing cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 sequencing cycles) for sequencing multiple analytes (e.g., where each of one, two, three, four, or more than four detection channels acquires or receives a different image of the biological sample in each sequencing cycle). The policy rules assign code words of the code book to a plurality of analytes (e.g., at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, at least 1,000, at least 2,000, at least 4,000, at least 6,000, at least 8,000, at least 10,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, at least 1,000,000, at least 2x10 6 , at least 3x10 6 , at least 4x10 6 , at least 5x10 6 , at least 6x10 6 , at least 7x10 6 , at least 8x10 6 , at least 9x10 6 , at least 10 7 , at least 10 8 , at least 10 9 10 or more 9 analyte).

[0176] In some cases, code words in a codebook may be assigned to multiple analytes based on decision rules that will ensure that the number of target analytes visible in a given image (e.g., the number of target analytes whose corresponding code words have an ON position in a given image) is more or less evenly distributed across multiple sequencing cycles and detection channels used for sequencing. For example, in some cases, code words of a codebook can be assigned to a plurality of analytes (e.g., at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, at least 1,000, at least 2,000, at least 4,000, at least 6,000, at least 8,000, at least 10,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, at least 1,000,000, at least 2 x 10 6 , at least 3x10 6 , at least 4x10 6 , at least 5x10 6 , at least 6x10 6 , at least 7x10 6 , at least 8x10 6 , at least 9x10 6 , at least 10 7 , at least 10 8 , at least 10 9 10 or more 9 The method further comprises: ensuring that the number of target analytes visible in a given image (e.g., target analytes whose corresponding codewords have an ON position) for a given sequencing cycle is within ±5%, ±10%, ±15%, ±20% or ±25% of the average number of target analytes detected in each image in a series of images acquired during multiple sequencing cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 sequencing cycles) for sequencing the multiple analytes (e.g., wherein each of one, two, three, four or more detection channels acquires or receives a different image of the biological sample in each cycle).

[0177] In some cases, a minimax decision rule (e.g., designed to minimize the maximum predicted density of ON signals across a series of images), an average ON signal decision rule (e.g., designed to ensure that the total number of ON signals detected in a given image for a given sequencing cycle is within ±5%, ±10%, ±15%, ±20%, or ±25% of the average number of ON signals detected in each image), an average target number decision rule (e.g., designed to ensure that the number of target analytes visible in a given image (e.g., target analytes whose corresponding codewords have ON bits) for a given sequencing cycle is within ±5%, ±10%, ±15%, ±20%, or ±25% of the average number of target analytes detected in each image), or any combination thereof may be used.

[0178] In some cases, the decision rule (or decision process) can be implemented in an iterative manner. For example, in some cases, one or more code words can be sorted according to code word weights (e.g., the total number of ON bits in a given code word), one or more analytes can be sorted according to predicted density, and one or more sorted code words can be assigned to one or more sorted analytes in descending order of maximum predicted density using an iterative process repeated for each of the one or more analytes, the iterative process comprising: calculating the predicted density of ON signals for each combination of the remaining unassigned code words and the analytes on the series of images; selecting a code word from the remaining unassigned code words that will minimize the predicted density of ON signals on the series of images; and assigning the selected code word to the analyte. In some cases, the iterative process may also include reviewing code words previously assigned to the analyte, and changing the code word selected for the current analyte to minimize the predicted density of ON signals on the series of images for the analyte to which the code word was previously assigned.

[0179] In some aspects, optimizing the codewords assigned to analytes is a method in which codewords are assigned to corresponding analytes according to a decision rule based on a priori knowledge of the abundance or distribution of target analytes in a given biological sample (e.g., expression data of the analyte) to reduce the weight of codewords corresponding to highly expressed analytes. In some cases, codewords may be assigned to corresponding analytes according to a decision rule based on, for example, single-cell expression data of target analytes in clustered cell types to reduce the weight of codewords corresponding to highly expressed target analytes, wherein the clustered cell types represent the distribution of cell types present in the biological sample. In some cases, the expression data of one or more target analytes include bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single-cell gene expression data, single-cell protein expression data, or any combination thereof.

[0180] For example, assume that single-cell expression data (e.g., single-cell gene expression data or single-cell protein expression data) are available for a biological sample of interest (e.g., a tissue sample of interest), wherein the expression data has been clustered according to cell type clusters, each cluster having an average gene or protein expression profile, and wherein the clustered cell types represent the distribution of cell types present in the biological sample. In some cases, the clustered single-cell expression data may provide the best prior information for the expression profile that may be observed in an in situ experiment, wherein the density of the marked features may simulate the expression profile.

[0181] Given a codebook and the assignment of analytes (e.g., gene transcripts) to codewords in the codebook, single cell type expression data can be used to determine the expected density of marker spots that will be observed for each cell type in each sequencing cycle and detection channel of the sequencing process.

[0182] In some aspects, the codewords for each gene transcript are designed to specifically avoid this and reduce the incidence of overcrowded cycles by distributing the number (or density) of ON features across multiple sequencing cycles and detection channels and including signal loss (OFF signals) as described herein. As noted above, in some cases, codewords may be selected / assigned according to a decision rule that minimizes the maximum predicted density of ON signals in images acquired during the cycle sequencing process (e.g., a minimize-maximum decision rule). In some cases, codewords may be selected / assigned according to a decision rule that will ensure that the total number of ON signals detected in an image for a given sequencing cycle is within ±5%, ±10%, ±15%, ±20%, or ±25% of the average number of ON signals detected per image (e.g., an average ON signal decision rule). In some cases, code words may be selected / assigned according to a decision rule (e.g., an average target quantity decision rule) that will ensure that the number of target analytes visible in a given image (e.g., target analytes for which the corresponding code word has an ON position) for a given sequencing cycle is within ±5%, ±10%, ±15%, ±20%, or ±25% of the average number of target analytes detected per image. In any of these cases, the decision rule may also include assignments based on expression data.

[0183] In some cases, for example, the codewords may be ranked according to codeword weights, the analytes may be ranked according to maximum expression levels on clustered cell types, and the ranked codewords may be assigned to the ranked analytes in descending order of maximum expression level using an iterative process repeated for each of the analytes, the iterative process comprising: calculating the predicted density of ON signals for each combination of the remaining unassigned codewords and the analytes on the series of images; selecting from the remaining unassigned codewords a codeword that will minimize the predicted density of ON signals on the series of images; and assigning the selected codeword to the analyte. In some cases, the iterative process may also include reviewing codewords previously assigned to the analyte, and changing the codeword selected for the current analyte to minimize the predicted density of ON signals on the series of images for the analyte to which the codeword was previously assigned.

[0184] In some cases, for example, codewords can be ranked according to codeword weights (i.e., the total number of ON bits in a given codeword), and the analytes to be detected can be ranked according to their corresponding single cell expression data or predicted density in the sample. In some cases, the lowest ranked codeword can then be assigned to the highest ranked analyte. In some cases, an algorithm can be developed to assign codewords to, for example, gene transcripts, where the assignment algorithm is optimized to minimize optical crowding and to distribute the total number or density of ON bits over multiple sequencing cycles and detection channels.

[0185] In some embodiments, the nucleotides in the first identifier sequence (e.g., barcode sequence) detected in a particular cycle correspond to a signal code comprising an ON signal, and the corresponding nucleotides in the second identifier sequence (e.g., barcode sequence) detected in a particular cycle correspond to a signal code comprising an OFF signal. In some embodiments, one or more pairs of corresponding nucleotides in the first barcode sequence and the second barcode sequence to be detected in the same cycle can be selected to reduce optical crowding of the signals detected in that cycle.

[0186] In some embodiments, the first identifier sequence and / or the second identifier sequence, such as a barcode sequence (or its corresponding codeword) can be designed such that about or at least 30% of the nucleotides therein correspond to an OFF signal, e.g., they are dark bases. In some embodiments, about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence (e.g., a barcode sequence) are dark bases.

[0187] In some aspects, the signal detected in each cycle of base-by-base sequencing corresponds to a block of bits (e.g., a signal code) in a codeword corresponding to an identifier sequence (e.g., a first identifier sequence and / or a second identifier sequence). The identifier sequence (e.g., a barcode sequence) may be assigned based on the codeword design and the base-dye labeling scheme in use (where the latter may be the same for each decoding (imaging) cycle, or may be different for different decoding cycles (e.g., a different dark base (corresponding to an OFF signal) may be used in each cycle). For example, the signal detected in each cycle of base-by-base sequencing may correspond to 3 bits in a codeword (corresponding to the identifier sequence), or a portion thereof, where each group of 3 bits may be mapped to a specific nucleotide. The barcode sequence may be derived from the codeword design, and the base / dye assignment scheme in use may be the same in each cycle, or the assignment of dark bases may be adjusted in each cycle. Table 1 provides examples of groups of 3 bits in a codeword (corresponding to the identifier sequence), which may be mapped to nucleotides using the exemplary patterns provided:

[0188] Table 1

[0189] Nucleotides 3-bit block dye / signal A 001 Blue Dye C 010 Green Dye G 100 Red Dye T 000 No dye (dark)

[0190] In some cases, the identifier is made of a 3-bit block extracted from four options. The design of the code book may include generating candidate code words (e.g., identifier sequences) with an appropriate number of ON bits (e.g., 4, 5, or 6), and then continuously identifying new valid code words that are also fully spaced apart from the existing code words generated in the code book. As long as the code word has the correct 3-bit valid block and has the correct number of ON bits (corresponding to dye / signal), the ON bit can appear anywhere in the identifier. In some aspects, it is determined that each code word must have a plurality of "ON" cycles / bits, and the code word can be designed accordingly. For example, each code word uses 3, 4, 5, 6, 7, or 8 "ON" cycles / bits. In some instances, each code word uses 5 "ON" cycles / bits. In some cases, there may be certain constraints on the code word that make it easier to identify "real" features, for example, by requiring each code word to have an "ON" bit lit with at least a certain number of colors. For example, each code word may be required to have an "ON" bit lit with at least 2, 3, or 4 colors. In some cases, this allows the signal to be distinguished from background fluorescent sources that tend to remain a single color. In some cases, the barcode sequence is derived from a codeword assignment scheme (e.g., an optimized codeword assignment), and a nucleotide is designated as "dark" using a base and corresponding dye assignment scheme. In some cases, for a given cycle, a nucleotide can be designated as dark, or the dark nucleotide can be moved around.

[0191] In some cases, the codeword is made of 3-bit blocks extracted from four dye labeling options (e.g., as shown in Table 1), where each bit in the block corresponds to a different detection (color) channel. The design of the codebook can include generating candidate codewords (e.g., identifier sequences) with an appropriate number of ON bits (e.g., 2, 3, 4, 5, or 6), and then continuously identifying new valid codewords that are also fully spaced apart from the existing codewords generated in the codebook (e.g., according to edit distance, such as Hamming distance). As long as the codeword has the correct number of 3-bit blocks (or codeword bits) and has the correct number of ON codeword bits (corresponding to the detection of ON signals in a given sequencing cycle), the ON bit can appear anywhere in the codeword. In some aspects, multiple ON codeword bits can be specified so that each codeword must have a specified number of ON codeword bits (corresponding to the number of sequencing cycles in which ON signals are detected), and the codeword can be designed accordingly. For example, in some cases, each codeword can use 1, 2, 3, 4, 5, 6, 7, or 8 ON bits. In some instances, each codeword uses 5 ON bits. In some cases, there may be certain constraints on the codeword design that make it easier to identify "real" image features, for example, by requiring each codeword to contain ON bits that light up in at least a certain number of colors. For example, each codeword may be required to have ON bits that light up in at least 2, 3, or 4 colors. In some cases, this allows the signal to be distinguished from background fluorescent sources that tend to remain a single color.

[0192] In some cases, an identifier sequence, such as a barcode sequence, is derived from a codeword assignment scheme (e.g., by optimizing the codeword assignment to minimize optical crowding), and a given nucleotide can be designated as "dark" using a base and corresponding dye labeling scheme. In some cases, a nucleotide can be designated as dark for a given sequencing cycle, or the designated dark nucleotide can be different for different cycles.

[0193] In some embodiments, a variety of different identifier sequences (or barcode sequences) can be detected in a biological sample, and each different identifier sequence can be detected at one or more positions in the biological sample. In some embodiments, 30% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of nucleotides that are dark bases. In some embodiments, 40% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of nucleotides that are dark bases. In some embodiments, 50% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of nucleotides that are dark bases. In some embodiments, 60% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the nucleotides that are dark bases. In some embodiments, 70% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the nucleotides that are dark bases. In some embodiments, 80% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the nucleotides that are dark bases. In some embodiments, 90% or more of the different identifier sequences can each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the nucleotides that are dark bases. In some embodiments, 95% or more of the different identifier sequences may each contain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more nucleotides that are dark bases.

[0194] In some embodiments, the signal code can correspond to the signal of the first color, the signal of the second color, the signal of the third color, or the signal absence (for example, corresponding to a dark base), wherein the first, second and third colors are different. The dark base can be any one or both of A, T, C and G. The dark base in the identifier sequence can be "detected" by using a nucleotide mixture in which the homologous nucleotides of the base complementary to the dark base are not detectably labeled. In some embodiments, the biological sample is contacted with the first nucleotide mixture, wherein the nucleotides comprising the first base are not detectably labeled, and the nucleotides comprising the base except the first base are each labeled with one or more detectable labels, and the biological sample is contacted with a subsequent nucleotide mixture, wherein the nucleotides comprising the subsequent bases are not detectably labeled, and the nucleotides comprising the base except the subsequent bases are each labeled with one or more detectable labels.

[0195] In some embodiments, the same nucleotide mixture can be used in multiple sequencing cycles, for example, the subsequent base can be the same as the first base. Any two or more of the multiple sequencing cycles can be continuous or non-continuous. For example, the two or more cycles can be separated by one or more cycles, and the one or more cycles may or may not use the same nucleotide mixture as in the two or more cycles.

[0196] In some cases, for example, a G in an identifier sequence (e.g., a barcode sequence) can be designated as a dark base, and a nucleotide mixture containing unlabeled C nucleotides can be used in two or more or all sequencing cycles, while nucleotides containing A, T, or G in the nucleotide mixture are detectably labeled. In some aspects, nucleotides assigned as dark bases can alternate in different cycles. In some cases, assigning different nucleotides as dark bases can avoid long homopolymer runs of the same base in the identifier sequence. For example, a G in an identifier sequence can be designated as a dark base in cycles 1, 5, and 9; a T in an identifier sequence can be designated as a dark base in cycles 2, 6, and 10; a C in an identifier sequence can be designated as a dark base in cycles 3, 7, and 11; and an A in an identifier sequence can be designated as a dark base in cycles 4, 8, and 12. In some aspects, two nucleotides assigned as dark bases can alternate in different cycles. In some embodiments, long homopolymer stretches of the same base in the barcode sequence can be avoided by assigning different dark bases in different cycles (e.g., the dark base can be G in cycles 1, 5, 9, T in 2, 6, 10, C in 3, 7, 11, and A in 4, 8, 12).

[0197] In some cases, the nucleotide mixture comprises unlabeled C nucleotides and A, T, and G nucleotides labeled with three different colored fluorophores, e.g., T is red, A is blue, and G is green, such that A, T, and C in the identifier sequence are associated with red, blue, and green signals, respectively (all ON signals), while the dark base G in the identifier sequence is associated with an OFF signal. Two exemplary identifier (e.g., barcode) sequences are shown in Table 2B below, and the signals detected using the three-color chemistry are indicated (dark is indicated by "-").

[0198] Table 2B

[0199]

[0200]

[0201] In some cases, the nucleotide mixture comprises unlabeled C nucleotides and A, T, and G nucleotides labeled with two different colored fluorophores, e.g., T is red, A is red and green (e.g., each nucleotide comprising A is labeled with a red fluorophore and a green fluorophore), and G is green, such that A, T, and C in the identifier sequence are associated with red, red and green, and green signals, respectively (all ON signals), while the dark base G in the identifier sequence is associated with an OFF signal. Table 2A below shows the signals detected for sequencing two exemplary barcode sequences using two-color chemistry.

[0202] Table 2A

[0203]

[0204]

[0205] In some cases, the nucleotide mixture includes unlabeled C nucleotides and A, T and G nucleotides labeled with a color of fluorophore. For example, each sequencing cycle can include two chemical steps and two imaging steps. The first chemical step exposes the sample to a nucleotide mixture with fluorescently labeled A and T nucleotides, wherein the C and G nucleotides in the mixture are not fluorescently labeled, but the C nucleotide includes a functional group or part for attaching a fluorescent label thereto. During the first imaging step, the signal or its absence at multiple positions in the sample is detected (image 1). The second chemical step removes the fluorescent label from the incorporated A nucleotides and adds a fluorescent label to the incorporated C nucleotides. In both chemical steps, the G nucleotides are not labeled. The signal or its absence at multiple positions in the sample is detected again (image 2). The combination of image 1 and image 2 is processed to identify which bases are incorporated at each position. For example, the signal code (image 1 / image 2) in the sequencing cycle can be ON / OFF for T in the identifier sequence (e.g., barcode sequence), OFF / ON for G, ON / ON for A, and OFF / OFF for C. In some embodiments, nucleotides in a first barcode sequence detected at a particular cycle correspond to ON signals (e.g., T is ON / OFF, G is OFF / ON, A is ON / ON), and corresponding nucleotides in a second barcode sequence detected at a particular cycle correspond only to OFF signals (e.g., C is OFF / OFF).

[0206] In some embodiments, different nucleotide mixtures can be used in multiple sequencing cycles. For example, a first nucleotide mixture can include nucleotides comprising a first base that is not detectably labeled, and the nucleotides in the first nucleotide mixture that include bases other than the first base are each labeled with one or more detectable labels (e.g., one, two or three different colors), and a subsequent nucleotide mixture can include nucleotides comprising subsequent bases that are not detectably labeled, and the nucleotides in the second nucleotide mixture that include bases other than the subsequent base are each labeled with one or more detectable labels (e.g., one, two or three different colors), and the subsequent base is different from the first base.

[0207] In some embodiments, provided herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a probe that directly or indirectly binds to an analyte at a position in the biological sample, wherein the probe or a product thereof comprises a priming site and a barcode sequence, and a sequencing primer hybridizes to the priming site; b) contacting the biological sample with a first nucleotide mixture comprising nucleotides comprising different bases, wherein: in the first nucleotide mixture, the nucleotide comprising the first base is not detectably labeled, and the nucleotide comprising one or more other bases is detectably labeled and forms a complex comprising i) a sequencing primer that hybridizes to the probe or a product thereof, ii) a polymerase, and iii) a homologous nucleotide that base pairs with the first nucleotide in the barcode sequence, and detecting a signal (ON signal) and / or a lack of signal (OFF signal) associated with the homologous nucleotide at the position, wherein the ON signal, the OFF signal, or a combination thereof corresponds to a difference between the homologous nucleotide and the first nucleotide in the barcode sequence. base; c) contacting the biological sample with a subsequent nucleotide mixture including nucleotides comprising different bases, wherein: in the second nucleotide mixture, the nucleotides comprising the subsequent base are not detectably labeled, while the nucleotides comprising one or more other bases are detectably labeled, the second base is different from the first base, forming a complex comprising i) an extension product of a sequencing primer hybridized to the probe or its product, ii) a polymerase, and iii) a homologous nucleotide that base pairs with the subsequent nucleotide in the barcode sequence, and detecting a signal (ON signal) and / or a lack of signal (OFF signal) associated with the homologous nucleotide at the position, wherein the ON signal, the OFF signal, or a combination thereof corresponds to the homologous nucleotide and the base in the subsequent nucleotide in the barcode sequence; and d) generating a signal code sequence, the signal code sequence comprising a signal code corresponding to the ON signal, the OFF signal, or a combination thereof in step b) and step c) at the position, thereby detecting the barcode sequence at the position in the biological sample.

[0208] In some embodiments, a biological sample can be contacted with two or more of the following nucleotide mixtures, in any order, in consecutive cycles: nucleotide mixture 1, in which nucleotides comprising G are not detectably labeled, while nucleotides comprising A, C, or T are detectably labeled (e.g., using single-color, two-color, or three-color chemistry); nucleotide mixture 2, in which nucleotides comprising T are not detectably labeled, while nucleotides comprising A, C, or G are detectably labeled (e.g., using single-color, two-color, or three-color chemistry); nucleotide mixture 3, in which nucleotides comprising C are not detectably labeled, while nucleotides comprising A, G, or T are detectably labeled (e.g., using single-color, two-color, or three-color chemistry); and nucleotide mixture 4, in which nucleotides comprising A are not detectably labeled, while nucleotides comprising G, C, or T are detectably labeled (e.g., using single-color, two-color, or three-color chemistry).

[0209] In some embodiments, any nucleotide mixture (e.g., nucleotide mixture 1 to nucleotide mixture 4) can be used in two or more consecutive base-by-base sequencing cycles, preceded and / or followed by cycles using different nucleotide mixtures. An exemplary order can be nucleotide mixture 4-nucleotide mixture 2-nucleotide mixture 2-nucleotide mixture 1-nucleotide mixture 1-nucleotide mixture 1-nucleotide mixture 3.

[0210] B. Multiple sequencing primers targeting different identifier sequences

[0211] In some embodiments, a method is provided herein, which includes determining the sequence of a plurality of different identifier sequences (e.g., barcode sequences) in situ in a cell or tissue sample, wherein a first subset of the different identifier sequences is sequenced using a first sequencing primer and a second subset of the different identifier sequences is sequenced using a second sequencing primer. In some aspects, the identifier sequences sequenced using different sequencing primers may contain bases that do not produce detectable signals in corresponding base-by-base sequencing cycles, for example, these bases are "dark", as described in Section III.A. In some embodiments, the first sequencing primer and the second sequencing primer are different, for example, they bind to different priming sites, and when the first subset of the different identifier sequences is sequenced using the first sequencing primer, no signal associated with the second subset of the different identifier sequences is detected. Similarly, when the second subset of the different identifier sequences is sequenced using the second sequencing primer, no signal associated with the first subset of the different identifier sequences is detected. Therefore, optical crowding of signals can be improved by using a plurality of sequencing primers across different identifier sequences and detecting signals separately using each sequencing primer.

[0212] In some embodiments, provided herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a plurality of probes each of which directly or indirectly binds to an analyte at a position in the biological sample, wherein the first probe or its product comprises a first priming site and a first barcode sequence, the second probe or its product comprises a second priming site and a second barcode sequence, and the first priming site and the second priming site are different; b) hybridizing a first sequencing primer to the first priming site; c) contacting the biological sample with nucleotides in successive cycles, wherein in each cycle: a complex is formed at a first position, the complex comprising i) the first sequencing primer or its extension product hybridized to the first probe or its product, ii) a polymerase, and iii) a homologous nucleotide that base pairs with a nucleotide in the first barcode sequence, and a signal (ON signal) and / or a lack of signal (OFF signal) associated with the homologous nucleotide is detected at the first position, wherein the ON signal, the OFF signal, or a combination thereof corresponds to a base in the homologous nucleotide and a corresponding nucleotide in the first barcode sequence; d) removing or blocking the first sequencing primer or its extension product; e) hybridizing the second sequencing primer to the second priming site; f) contacting the biological sample with nucleotides in continuous cycles, wherein in each cycle: forming a complex at a second position, the complex comprising i) the second sequencing primer or its extension product hybridized to the second probe or its product, ii) a polymerase, and iii) a homologous nucleotide that base pairs with the nucleotide in the second barcode sequence, and detecting a signal (ON signal) and / or a lack of signal (OFF signal) associated with the homologous nucleotide at the second position, wherein the ON signal, the OFF signal, or a combination thereof corresponds to a base in the homologous nucleotide and the corresponding nucleotide in the second barcode sequence; g) generating a first signal code sequence and a second signal code sequence, the first signal code sequence comprising a signal code corresponding to the signal at the first position in the continuous cycles, the second signal code sequence comprising a signal code corresponding to the signal at the second position in the continuous cycles, thereby detecting the first barcode sequence at the first position and the second barcode sequence at the second position in the biological sample.

[0213] In some embodiments, the first probe and the second probe are directly or indirectly bound to the same analyte. In some embodiments, the first probe and the second probe are directly or indirectly bound to different analytes. In some embodiments, the first barcode sequence and the second barcode sequence have the same sequence, but the 3' initiation site is different. Therefore, different sequencing primers can be used to sequence the same barcode sequence. In some embodiments, the first barcode sequence and the second barcode sequence are different and are sequenced using different sequencing primers. The first barcode sequence and the second barcode sequence can be associated with the same analyte or different analytes, correspond to the same analyte or different analytes, and / or identify the same analyte or different analytes. In some cases, a plurality of different probes include the same first initiation site (e.g., primer binding sequence), but each of the plurality of different probes includes a different barcode sequence. In some cases, a first plurality of different probes comprises the same first priming site (e.g., primer binding sequence), but each of the first plurality of different probes comprises a different barcode sequence, and a second plurality of different probes comprises the same second priming site (e.g., primer binding sequence), but each of the second plurality of different probes comprises a different barcode sequence.

[0214] In some embodiments, the method may include hybridizing a first sequencing primer to a first priming site and performing base-by-base sequencing (e.g., SBS or SBB) to generate an extension product of the first sequencing primer. Figure 3 As shown in , a first sequencing primer can hybridize to a first priming site in a molecule or complex (e.g., a probe or its product) comprising a first set ("block") of identifier sequences, for example, barcode sequences of genes 1 to 3 (gene block 1 in the figure) and be used to sequence the first set of identifier sequences without detecting signals associated with bases in a second set ("block") of identifier sequences, for example, barcode sequences of genes 4 to 6 (gene block 2 in the figure). In some embodiments, the extension product of the first sequencing primer is prevented from generating signals for base-by-base sequencing such as SBS and SBB. In some embodiments, the method includes removing, cutting, or blocking the extension product of the first sequencing primer. In some embodiments, the method also includes hybridizing a second sequencing primer to the second priming site and performing base-by-base sequencing (e.g., SBS or SBB) to generate an extension product of the second sequencing primer. As Figure 3As shown in , the second sequencing primer can hybridize to a second priming site in a molecule or complex (e.g., a probe or a product thereof) comprising a second set of identifier sequences (e.g., barcode sequences in gene block 2) and be used to sequence the second set of identifier sequences without detecting signals associated with bases in the first set of identifier sequences (e.g., barcode sequences in gene block 1). In some embodiments, the extension product of the second sequencing primer is prevented from generating signals for base-by-base sequencing such as SBS and SBB. In some embodiments, the method includes removing, cutting, or blocking the extension product of the second sequencing primer.

[0215] In some embodiments, the method further comprises hybridizing a third sequencing primer to the third priming site and performing base-by-base sequencing (e.g., SBS or SBB) to generate an extension product of the third sequencing primer. In some embodiments, the third sequencing primer is different in sequence from the first sequencing primer and the second sequencing primer. The third sequencing primer can be hybridized to the third priming site in a molecule or complex (e.g., a probe or a product thereof) comprising a third set of identifier sequences and used to sequence the third set of identifier sequences without detecting signals associated with bases in the first set or the second set of identifier sequences. For example, the third sequencing primer can be used to sequence a third set of identifier sequences that is different from the first set or the second set of identifier sequences. Figure 3 The barcode sequences of genes 1 to 6 shown in the sequence were sequenced.

[0216] In some embodiments, probes for a first plurality of analytes or their products can share a common first priming site, and probes for a second plurality of analytes or their products can share a common second priming site. In some embodiments, the second plurality of analytes can include two or more different analytes that are different from the two or more different analytes in the first plurality of analytes. For example, Figure 3 As shown in the figure, probes or their products against gene block 1 (comprising genes 1 to 3) share a common first priming site ("sequencing primer 1" binding site), probes or their products against gene block 2 (comprising genes 4 to 6) share a common second priming site ("sequencing primer 2" binding site), and at least one, two or all genes in gene block 1 are different from those in gene block 2.

[0217] In some embodiments, a first sequencing primer (e.g., Figure 3 ) and a second sequencing primer (e.g., Figure 3The "sequencing primer 2" in the sequencer can be pre-mixed or contacted with the biological sample separately but simultaneously, followed by multiple decoding cycles. In some embodiments, one of the sequencing primers can be selectively blocked from generating a signal in base-by-base sequencing, while sequencing is performed from another sequencing primer. Once sequencing from one of the sequencing primers is completed, the blocked sequencing primer can be unblocked and used to sequence the identifier sequence.

[0218] In some embodiments, the plurality of identifier sequences comprises multiple subsets, and each subset can be sequenced in a separate series of sequencing cycles using a different sequencing primer / priming site, thereby reducing optical crowding compared to methods in which all identifier sequences in the plurality of identifier sequences are sequenced in the same series of sequencing cycles.

[0219] In some cases, two different sequencing primers can be used to sequence the barcode sequences corresponding to two analytes (e.g., genes), and the barcode sequences can be shared between the two genes. In some cases, the barcode sequences can be repeated for different analytes.

[0220] C. Sequencing primer combinations targeting the same identifier sequence

[0221] In some embodiments, two or more different sequencing primers can be used to sequence the same identifier sequence. For example, a molecule or complex can include two different priming sites, e.g. Figure 4 As shown in , each of the molecules or complexes of genes 1 to 5 can contain two different priming sites for sequencing the identifier sequence corresponding to the analyte (e.g., the transcript of the corresponding gene). The molecule or complex can be any of those disclosed herein, for example, Figure 2 In the RCA product or probe complex shown in . In each molecule or complex, two different priming sites can be 3' (e.g., barcode sequence) of the identifier sequence, and one of the priming sites can be 3' or 5' of the other priming site. In some embodiments, different priming sites can partially overlap. In some embodiments, in each molecule or complex, one of the priming sites can be 3' of a copy of the identifier sequence, and the other priming site can be 3' of another copy of the identifier sequence. In some aspects, the identifier sequence sequence sequenced using different priming sites can be included in the bases that do not produce detectable signals in the corresponding base-by-base sequencing cycle, for example, these bases are "dark", as described in Section III.A.

[0222] In some embodiments, a biological sample can be contacted with a plurality of probes each configured to bind directly or indirectly to a different analyte, and each probe or its product can comprise a combination of different priming sites. In some embodiments, the combination comprises two, three, four, five or more different priming sites. For example, Figure 4 It is shown that the probe or its product of each gene contains two different priming sites, such as the "sequencing primer 1" binding site and the "sequencing primer 2" binding site of gene 1, and the "sequencing primer 1" binding site and the "sequencing primer 3" binding site of gene 2, etc.

[0223] In some embodiments, the first probe or its product may include a first combination of different priming sites including a first priming site. Figure 4 The "sequencing primer 1" and "sequencing primer 2" shown in Figure 1 sequence the barcode sequence of gene 1. In some embodiments, the second probe or its product may include a second combination of different priming sites including a second priming site. For example, the Figure 4 The "sequencing primer 1" and "sequencing primer 3" shown in the sequence sequence of the barcode of gene 2. In some embodiments, the biological sample can be contacted with a third probe that directly or indirectly binds to a third analyte. In some embodiments, the third probe or its product can include a third combination of different priming sites including a first priming site, a second priming site, and / or a third priming site. In some embodiments, any two or more of the first combination, the second combination, and the third combination can share one or more common priming sites. For example, the barcode sequence of gene 3 can be sequenced using "sequencing primer 1" (shared by gene 1 and gene 2) and "sequencing primer 4" (not shared by gene 1 and gene 2).

[0224] In some embodiments, the method may include contacting the biological sample with a first sequencing primer for base-by-base sequencing, thereby hybridizing the first sequencing primer to a first priming site in a first probe or its product and in one or more other probes or their products, and generating extension products of the first sequencing primer. Figure 4As shown in , "sequencing primer 1" can hybridize with the priming site in the probes or their products of genes 1 to 3, and sequence the identifier sequence (e.g., barcode sequence) of genes 1 to 3. In some embodiments, the method can also include removing, cutting, or blocking the extension products of the first sequencing primer, thereby preventing them from generating signals for base-by-base sequencing (e.g., using SBS and SBB). In some embodiments, the method can also include contacting the biological sample with a second sequencing primer for base-by-base sequencing, thereby hybridizing the second sequencing primer with the second priming site in the second probe or its product and in one or more other probes or their products, and generating extension products of the second sequencing primer. For example, as Figure 4 As shown in , "sequencing primer 2" can hybridize with the priming sites in the probes or products thereof of genes 1, 4, and 5, and sequence the identifier sequences (e.g., barcode sequences) of these genes. Similarly, "sequencing primer 3" can hybridize with the priming sites in the probes or products thereof of genes 2, 4, and 6, and sequence the identifier sequences (e.g., barcode sequences) of these genes.

[0225] In some embodiments, the method may include contacting the biological sample with a first sequencing primer and a second sequencing primer, so that both sequencing primers hybridize to corresponding priming sites in the first probe or its product, in the second probe or its product, and optionally in one or more other probes or their products, and generate an extension product of the first sequencing primer and an extension product of the second sequencing primer. In some embodiments, the first sequencing primer and the second sequencing primer can be pre-mixed or contacted with the biological sample separately but simultaneously, followed by multiple decoding cycles. In some embodiments, the extension products of the first sequencing primer and the second sequencing primer can be removed (e.g., stripped), cut or blocked, or otherwise prevented from generating signals for base-by-base sequencing, followed by hybridization of a different mixture of sequencing primers (e.g., a third sequencing primer and a fourth sequencing primer). For example, as Figure 4 As shown in , "sequencing primer 1" and "sequencing primer 2" can hybridize to the priming site in the probe or its product, "sequencing primer 1" for genes 1 to 3, and "sequencing primer 2" for genes 1, 4 and 5. For the identifier sequence (e.g., barcode sequence) of gene 1 where sequencing can be performed from two sequencing primers, the signals can be distinguished, for example, "ACG" from "sequencing primer 1" and "CGA" from "sequencing primer 2". This can be achieved, for example, in the case where the identifier sequence comprises multiple subsequences separated by the priming site, for example, 3'-"sequencing primer 1" binding site-"ACG"-"sequencing primer 2" binding site-"CGA"-5'.

[0226] In some embodiments, multiple cycles of nucleotide incorporation or combination can be performed. In some embodiments, the method includes contacting the biological sample with nucleotides in continuous cycles, detecting the signal associated with the nucleotide incorporation or combination of each continuous cycle, and generating a signal code sequence of multiple analytes (e.g., the first multiple analytes and the second multiple analytes). In some embodiments, the first multiple analytes and the second multiple analytes include one or more common analytes. In some embodiments, the first multiple analytes and the second multiple analytes do not include a common analyte.

[0227] In some embodiments, the use of a combination of sequencing primers for the same identifier sequence allows different portions of the identifier sequence to be sequenced in a separate series of sequencing cycles using different sequencing primers or different combinations of sequencing primers, and helps reduce optical crowding compared to methods in which all identifier sequences in a plurality of identifier sequences are sequenced in the same series of sequencing cycles. In some embodiments, the method is not constrained by a block diagonal code. For example, the use of a combination of sequencing primers can allow for increased complexity of the identifier sequence.

[0228] In some embodiments, any identifier sequence disclosed herein, or portion thereof, can be sequenced multiple times using the same sequencing primer or one or more different sequencing primers, using the same mixture of nucleotides or one or more different mixtures of nucleotides, and / or using the same polymerase or one or more different polymerases (e.g., including the same polymerase with different labels or modifications).

[0229] In some embodiments, after sequencing the identifier sequence or a portion thereof, for example, in round N, the extension product (or a portion thereof) of the round 1 sequencing primer can be removed from the template strand to allow hybridization of the sequencing primer of round (N+1). In some embodiments, the round (N+1) sequencing primer and the round N sequencing primer are identical in sequence, so that the identifier sequence or a portion thereof can be sequenced again. In some embodiments, the identifier sequence or a portion thereof is sequenced in round (N+1) using the same nucleotide mixture as in round N. In some embodiments, the identifier sequence or a portion thereof is sequenced in round (N+1) using different nucleotide mixtures than in round N, for example, as described in Section III-A. In some embodiments, the round (N+1) sequencing primer and the round N sequencing primer are different in sequence, and the nucleotide mixtures in round (N+1) and round N can be the same or different. In some embodiments, the round (N+1) priming site and the round N priming site are both 3' of the identifier sequence, and the round (N+1) priming site can be 3' or 5' of the round N priming site. In some embodiments, the (N+1)th round priming site and the Nth round priming site may partially overlap.

[0230] IV. Testing and Analysis

[0231] Typically in a sequencing by synthesis method, a first population of detectably labeled nucleotides (e.g., dNTPs) is introduced to contact the template nucleotides hybridized with the sequencing primer, and the first detectably labeled nucleotides (e.g., A, T, C, or G nucleotides) are incorporated by a polymerase to extend the sequencing primer in the 5' to 3' direction using the complementary nucleotides (first nucleotide residues) in the template nucleotides as a template. The signal from the first detectably labeled nucleotide can then be detected. The first population of nucleotides can be continuously introduced, but in order to allow the second detectably labeled nucleotides to be incorporated into the extended sequencing primer, the nucleotides that are not incorporated into the sequencing primer in the first population of nucleotides are usually removed (e.g., by washing), and the second population of detectably labeled nucleotides is introduced into the reaction. Then, the second detectably labeled nucleotides (e.g., A, T, C, or G nucleotides) are incorporated by the same or different polymerases to extend the already extended sequencing primer in the 5' to 3' direction using the complementary nucleotides (second nucleotide residues) in the template nucleotides as a template. Therefore, in some embodiments, a cycle of introducing and removing detectably labeled nucleotides is performed.

[0232] In some cases, the cycle of introducing and removing detectably labeled nucleotides in a time-sequential manner is carried out to realize the in-situ analysis of the analyte (for example, the target nucleic acid in the cell in the intact tissue) in the biological sample. In some respects, a method for detecting detectably labeled nucleotides is provided herein to generate a signal signature associated with the oligonucleotides of the mark. In some cases, the signal signature corresponds to an analyte in a variety of analytes. In some cases, the method described herein is based in part on the development of multiple biological assays and reading, wherein first the sample is contacted with a variety of nucleic acid probes, so as to allow the probe to directly or indirectly bind to the target analyte, and then the target analyte can be optically detected (for example, by sequencing) in a time-sequential manner. In some respects, because nucleic acid probes are exogenously added and their sequence can be controlled and designed, this allows the use of identifier sequences optimized for each analyte (for example, compared with direct sequencing of endogenous molecules). In some respects, a method for multiple biological assays and sequencing readings is provided herein, and the method includes optically detecting labeled oligonucleotides in a time-sequential manner. In some cases, since the position of the analyte, probe and / or its product can be maintained in the sample during multiple sequencing cycles, the fluorescent spots corresponding to the analyte, probe or its product remain in place during multiple rounds of cycles and can be compared to read the signal string associated with each target analyte. The signal string observed at a certain position (e.g., an ON or OFF signal in each round) can be compared with a codebook containing identifier sequences assigned to multiple analytes.

[0233] In some embodiments, the methods disclosed herein include the use of one or more nucleotides or their analogs, including natural nucleotides or nucleotide analogs or modified nucleotides (e.g., labeled with one or more detectable labels). In some embodiments, the nucleotide analogs include nitrogenous bases, pentoses, and phosphate groups, wherein any component of the nucleotides can be modified and / or replaced. In some embodiments, the methods disclosed herein may include the use of one or more non-incorporable nucleotides. Non-incorporable nucleotides can be modified to be incorporable at any time point during the sequencing method.

[0234] Nucleotide analogs include, but are not limited to, α-phosphate modified nucleotides, α-β nucleotide analogs, β-phosphate modified nucleotides, β-γ nucleotide analogs, γ-phosphate modified nucleotides, caged nucleotides, or ddNTPs. Examples of nucleotide analogs are described in U.S. Pat. No. 8,071,755, which is incorporated herein by reference in its entirety.

[0235] In some embodiments, the methods disclosed herein may include the use of a terminator that will reversibly prevent the incorporation of a nucleotide at the 3′-end of a primer. One type of reversible terminator is a 3′-O-blocked reversible terminator. Here, the terminator portion is connected to the oxygen atom at the 3′-OH end of the 5-carbon sugar of the nucleotide. For example, U.S. Patent Nos. 7,544,794 and 8,034,923 (the disclosures of these patents are incorporated by reference) describe a reversible terminator dNTP in which the 3′-OH group is replaced with a 3′-ONH2 group. Another type of reversible terminator is a 3′-unblocked reversible terminator, in which the terminator portion is connected to the nitrogenous base of the nucleotide. For example, U.S. Patent No. 8,808,989 (the disclosure of which is incorporated by reference) discloses a specific example of a base-modified reversible terminator nucleotide that can be used in combination with the methods described herein. Other reversible terminators that can similarly be used in conjunction with the methods described herein include those described in U.S. Pat. Nos. 7,956,171, 8,071,755, and 9,399,798, which are incorporated herein by reference.

[0236] In some embodiments, the methods disclosed herein may include the use of nucleotide analogs having a terminator portion that will irreversibly prevent the incorporation of a nucleotide at the 3'-end of the primer. Irreversible nucleotide analogs include 2', 3'-dideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs that is essential for polymerase-mediated synthesis.

[0237] In some embodiments, the methods disclosed herein may include the use of a non-incorporable nucleotide comprising a blocking moiety that inhibits or prevents the nucleotide from forming a covalent bond with a second nucleotide (the 3′-OH of the primer) during the incorporation step of the nucleic acid polymerization reaction. The blocking moiety can be removed from the nucleotide, thereby allowing the nucleotide to be incorporated.

[0238] In some embodiments, the methods disclosed herein may include using 1, 2, 3, 4 or more nucleotide analogs present in the SBS reaction. In some embodiments, the nucleotide analogs are replaced, diluted or isolated during the incorporation step. In some embodiments, the nucleotide analogs are replaced with natural nucleotides. In some embodiments, the nucleotide analogs are modified during the incorporation step. The modified nucleotide analogs may be similar or identical to natural nucleotides.

[0239] In some embodiments, the methods disclosed herein may include the use of nucleotide analogs that have different binding affinity for polymerases compared to natural nucleotides. In some embodiments, the interaction of the nucleotide analogs with the next base is different from that of natural nucleotides. Nucleotide analogs and / or non-incorporable nucleotides can be base paired with complementary bases of the template nucleic acid.

[0240] In some embodiments, one or more nucleotides can be marked with a distinguishing and / or detectable label or mark. The label can be distinguished by means of its difference in fluorescence, Raman spectroscopy, charge, quality, refractive index, luminescence, length or any other measurable properties. The label can be attached to one or more different positions on the nucleotide, as long as the fidelity of the combination with the polymerase-nucleic acid complex is fully maintained so that the complementary base on the template nucleic acid can be correctly identified. In some embodiments, the label is attached to the core base of the nucleotide. Or, the label is attached to the gamma phosphate position of the nucleotide.

[0241] Detectable label can be applicable to small-scale detection and / or to high-throughput screening.Therefore, suitable detectable label includes but is not limited to radioisotope, fluorophore, chemiluminescent compound, bioluminescent compound and dye.Detectable label can be qualitatively detected (for example, optically or spectrally), or can be quantitative.Qualitative detection generally includes the detection method of wherein confirming the existence of detectable label or occurrence, and quantifiable detection generally includes the detection method with quantifiable (for example, numerical value can report) value such as intensity, duration, polarization and / or other properties.In some embodiments, detectable label is combined with another part (for example, nucleotide or nucleotide analog), and can include fluorescence, colorimetric or chemiluminescent label.

[0242] In some embodiments, the detectable label can be attached to another moiety, for example, a nucleotide or a nucleotide analog. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be from the group consisting of: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), 350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, 750, allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, aniline blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Feulgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBO TM -1 / BO-PRO TM -1.BOBO TM -3 / BO-PRO TM -3. FL, TMR, TR-X, 530 / 550, 558 / 568, 564 / 570, 581 / 591, 630 / 650-X, 650-665-X, BTC, Calcium Crimson, Calcium Crimson Blue TM 、Calcium Green-1 TM Calcium Orange TM , White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow TM 、CCF2(GeneBLAzer TM ), CFP (cyan fluorescent protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC alpha Cychrome (PE-Cy5), Dansylamide, Dansylcadaverine, Dansyl chloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18 (5)), DIDS, Dil (DilC18 (3)), DiO (DiOC18 (3)), DiR (DilC18 (7)), Di-4ANEPPS, Di-8ANEPPS, DM-NERF (4.5-6.5pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, alcohol, eosin, erythrosine, ethidium bromide, ethidium homodimer-1 (EthD-1), europium (III) chloride, 5-FAM (5-carboxyfluorescein), fast blue, fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo-4, Fluoro-Gold TM (High pH), Fluoro-Gold TM (Low pH), Fluoro-Jade, 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red TM (High calcium), Fura Red TM / Fluo-3、GeneBLAzer TM (CCF2), GFP Red Shifted (rsGFP), GFP wild type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst33342&33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751(+DNA), LDS751(+RNA), LOLO TM -1 / LO-PRO TM -1. Fluorescent yellow, LysoSensor TM Blue (pH 5), LysoSensor TMGreen (pH 5), LysoSensor TM Yellow / blue (pH 4.2), green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM Marina 4-Methylumbelliferone, mithramycin, green, orange, Red, NBD (amine), Nile Red, 488, 500, 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinyl Chlorophyll Protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1. POPO TM -3 / PO-PRO TM -3, propidium iodide (PI), PyMPO, pyrene, pyronine Y, Quantam Red (PE-Cy5), quinacridone mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), red fluorescent protein (DsRed), resorufin, RH 414, Rhod-2, rhodamine B, Rhodamine Green TM 、RhodamineRed TM , rhodamine phalloidin, rhodamine 110, rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, (High pH), (High pH), (Low pH), Sodium Green TM , #1, #2, 11. 13. 17. 45. blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), Thiadicarbocyanine, Thiazole Orange, Tri-color (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO542, ATTO 550, ATTO 565, ATTO Rhol01, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE665, TYE 705, 700, 800, 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, 640 (NHS ester) and Dy 750 (NHS ester).

[0243] Detectable label can be itself directly detectable (for example, radioisotope labeling or fluorescent labeling), or in the case of enzymatic labeling, can be indirectly detectable, for example, by catalyzing the chemical change of substrate compound or composition, the substrate compound or composition is directly detectable. Label can send a signal or change the signal delivered to the label, so that the presence or absence of the label can be detected. In some cases, coupling can be carried out via a joint, and the joint can be cleavable, such as light cleavable (for example, cleavable under ultraviolet light), chemical cleavable (for example, via a reducing agent, such as dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP)) or enzymatic cleavable (for example, via esterase, lipase, peptidase or protease).

[0244] In addition to the sequencing by synthesis method described herein, in some cases, alternative sequencing biochemistry can be used for nucleic acid sequencing. Examples include, but are not limited to, the "sequencing by binding" (SBB) method described in WO 2017 / 117235, U.S. Patent Nos. 9,951,385 and 10,655,176, and the "affinity sequencing" (SBA) method described in U.S. Patent Nos. 10,768,173 and 10,982,280, all of which are incorporated herein by reference. The sequencing chemistry described in US 2020 / 0370113, entitled "Polymerase-nucleotide conjugates for sequencing bytrapping" and incorporated herein by reference, can also be used.

[0245] In some embodiments, the SBB method herein is based on performing the following repeated cycles: detecting a stable complex formed at each position along the template (e.g., a ternary complex comprising a triggered template (anchored to a sample support structure), a polymerase, and a homologous nucleotide at the position) under conditions that prevent the covalent incorporation of homologous nucleotides into the primer, and then extending the primer to allow detection of the next position along the template (see, e.g., U.S. Pat. Nos. 9,951,385 and 10,655,176). In the sequencing-by-binding method, detection of the nucleotide at each position of the template is performed before the primer is extended to the next position. Typically, the method is used to distinguish four different nucleotide types that may be present at a position along the nucleic acid template by uniquely labeling each type of ternary complex (i.e., different types of ternary complexes differ in the types of nucleotides they contain) or by separately delivering the reagents required to form each type of ternary complex. In some cases, the label may include, for example, a fluorescent label of a homologous nucleotide or polymerase that participates in the ternary complex.

[0246] In some cases, for example, a method for sequencing a nucleic acid molecule using a sequencing-by-binding method may include the steps of: (a) forming a mixture under ternary complex stabilizing conditions, wherein the mixture comprises a primed template nucleic acid, a polymerase, and nucleotide homologs of the first, second, and third base types in the template; (b) examining the mixture to determine whether a ternary complex has been formed; (c) identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first, second, or third base type, and wherein based on the absence of formation of a ternary complex in step (b), the next correct nucleotide is inferred to be a nucleotide homolog of the fourth base type; (d) adding the next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby generating an extension primer; and (e) repeating steps (a) to (d) on the primed template nucleic acid comprising the extension primer. In some cases, the mixture formed in step (a) may comprise a nucleotide homolog of the first base type in the template. In some cases, the mixture formed in step (a) can include nucleotide homologs of the first base type and the second base type in the template.

[0247] "Affinity sequencing" (or SBA) methods rely on the increased affinity (or "functional affinity") resulting from the formation of a complex comprising multiple individual non-covalent binding interactions (see, e.g., U.S. Pat. Nos. 10,768,173 and 10,982,280). Affinity sequencing methods are based on the detection of multivalent binding complexes formed between fluorescently labeled polymer-nucleotide conjugates, polymerases, and multiple primed target nucleic acid molecules anchored to a sample support structure, which allows the detection / base recognition step to be separated from the nucleotide incorporation step. Fluorescence imaging is used to detect the bound complex and thereby determine the identity of the N+1 nucleotides in the target nucleic acid sequence (where the primer extension chain is N nucleotides in length).

[0248] In some cases, for example, nucleic acid sequencing using an affinity sequencing method may include the steps of: (a) providing a composition comprising: (i) two or more copies of a target nucleic acid sequence; (ii) two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and (iii) two or more polymerase molecules; (b) contacting the composition with a polymer-nucleotide conjugate under conditions sufficient to allow formation of a multivalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition of (a), wherein the polymer-nucleotide conjugate comprises two or more nucleotide portions; and (c) detecting the multivalent binding complex (e.g., by fluorescence imaging), thereby determining the identity of the nucleotides in the target nucleic acid sequence. After the imaging step, the multivalent binding complex is destroyed and washed away, the correct nucleotide (e.g., blocked nucleotide) is incorporated into the primer extension chain (e.g., after unblocking of the previously incorporated nucleotide), and the cycle is repeated.

[0249] Any suitable enzyme with polymerase activity can be used in the sequencing reactions described herein, and exemplary polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and bacteriophage DNA polymerases. Bacterial DNA polymerases include E. coli DNA polymerases I, II and III, IV and V, Klenow fragments of E. coli DNA polymerases, Clostridium dunghill (Cst) DNA polymerases, Clostridium thermocellum (Cth) DNA polymerases, and Sulfolobus solfataricus (Sso) DNA polymerases. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, ε, η, ζ, λ, σ, μ, and K, as well as Rev1 polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerase, PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase and T4 polymerase. Other DNA polymerases include thermostable and / or thermophilic DNA polymerases, such as DNA polymerases isolated from Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermmus thermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, and TurboPfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus GB-D polymerase, Thermotoga marine (Tli) DNA polymerase, maritima)(Tmma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus go N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcusthermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D.Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase; Thermococcusfumicolans DNA polymerase; Aeropyrum pernix) DNA polymerase; and heterodimeric DNA polymerase DP 1 / DP2. Engineered and modified polymerases can also be used in conjunction with the disclosed technology. For example, a modified form of the extremely thermophilic marine archaeon Thermococcus species 9°N (e.g., Therminator™ DNA polymerase from New England BioLabs Inc. (Ipswich, Massachusetts)) can be used. Still other useful DNA polymerases, including 3PDX polymerase, are disclosed in U.S. Pat. No. 8,703,461, the disclosure of which is incorporated by reference in its entirety. Additional examples include viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and K11 polymerase; eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V, archaeal RNA polymerases, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastosis virus, and telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes.;

[0250] Fluorescence detection in tissue samples is often hindered by the presence of strong background fluorescence. "Autofluorescence" is a general term for distinguishing background fluorescence (which can be caused by a variety of sources (including aldehyde fixation, extracellular matrix components, erythrocytes, lipofuscin, etc.) from the desired immunofluorescence of fluorescently labeled antibodies or probes. Tissue autofluorescence may cause difficulty in distinguishing signals caused by fluorescent antibodies or probes from general background. In some embodiments, the methods disclosed herein utilize one or more reagents to reduce tissue autofluorescence, such as autofluorescence eliminators (Sigma / EMDMillipore), TrueBlack lipofuscin autofluorescence quenchers (Biotium), MaxBlock autofluorescence reduction kits (MaxVision Biosciences) and / or very strong black dyes (e.g., Sudan Black or comparable dark chromophores).

[0251] Examples of fluorescent labels and nucleotides and / or polynucleotides conjugated to such fluorescent labels include those described in, for example, Hoagland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, Second Edition (Stockton Press, New York, 1993); Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259 (1991). In some embodiments, exemplary techniques and methods suitable for use with the provided embodiments include, for example, those described in US 4,757,141, US 5,151,507, and US 5,091,519. In some embodiments, one or more fluorescent dyes are used as labels for labeled target sequences, for example, as described in: US 5,188,934 (4,7-dichlorofluorescein dye); US 5,366,860 (spectrally resolvable rhodamine dye); US 5,847,162 (4,7-dichlororhodamine dye); US 4,318,846 (ether-substituted fluorescein dye); US 5,800,996 (energy transfer dye); US 5,066,580 (xanthine dye); and US 5,688,648 (energy transfer dye). Quantum dots can also be used for labeling, as described in the following documents: US 6,322,901, US 6,576,291, US 6,423,551, US 6,251,303, US 6,319,426, US 6,426,513, US 6,444,143, US 5,990,479, US 6,207,392, US 2002 / 0045045 and US 2003 / 0017264. As used herein, the term "fluorescent label" includes a signal transduction portion that transmits information through the fluorescence absorption and / or emission characteristics of one or more molecules. Exemplary fluorescence characteristics include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer.

[0252] In some aspects, detection (including imaging) is performed using any of a number of different types of microscopy, such as confocal microscopy, two-photon microscopy, light-field microscopy, intact tissue expansion microscopy, and / or CLARITY TM -Optimized light sheet microscopy (COLM).

[0253] In some embodiments, fluorescence microscopy is used to detect and image samples. In some aspects, a fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of or in addition to reflection and absorption to study the properties of organic or inorganic substances. In fluorescence microscopy, the sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescence is then imaged by a microscope objective, and the fluorescence wavelength is usually longer than the illumination wavelength. Two filters can be used in this technology; an illumination (or excitation) filter that ensures that the illumination is close to monochromatic and at the correct wavelength, and a second emission (or barrier) filter that ensures that no excitation light source reaches the detector. Alternatively, these functions can all be achieved by a single dichroic filter. "Fluorescence microscope" includes any microscope that uses fluorescence to generate images, whether it is a simpler device like an epifluorescence microscope or a more complex design like a confocal microscope, which uses optical sectioning to obtain better resolution of fluorescent images.

[0254] In some embodiments, confocal microscopy is used to perform detection and imaging of samples. Confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signals. Since only light generated by fluorescence very close to the focal plane can be detected, the optical resolution of the image, especially in the direction of sample depth, is much better than that of wide-field microscopy. However, since much of the light from sample fluorescence is blocked at the pinhole, this increase in resolution comes at the expense of reduced signal intensity - so long exposure times are usually required. Since only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning in the specimen with a regular raster (i.e., a rectangular pattern of parallel scan lines). The achievable thickness of the focal plane is primarily limited by the wavelength of the light used divided by the numerical aperture of the objective, but is also limited by the optical properties of the specimen. Thin optical sections make these types of microscopes particularly good at 3D imaging and surface profile analysis of samples. CLARITY TM - Optimized Light Sheet Microscopy (COLM) provides an alternative microscopy technique for rapid 3D imaging of large clarified samples. COLM interrogates large immunostained tissues, allowing for increased acquisition speeds and producing higher quality generated data.

[0255] Other types of microscopy that may be employed include bright field microscopy, oblique illumination microscopy, dark field microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, reflection interference microscopy (also known as reflected interference contrast or RIC), single plane illumination microscopy (SPIM), ultra-high resolution microscopy, laser microscopy, electron microscopy (EM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM), scanning transmission electron microscopy (STEM), and low voltage electron microscopy. (LVEM), scanning probe microscopy (SPM), atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), feature-oriented scanning probe microscopy (FOSPM), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM ), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscopy, SNOM, piezoelectric response force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microspectroscopy / microscopy (PTMS), SCM, scanning capacitance microscopy (SCM), SECM, scanning electrochemical microscopy (SECM), SGM, scanning gate microscopy (SGM), SHPM, scanning Hall probe microscopy ( The invention relates to the present invention to the invention to the invention of the present invention. The invention relates to the present invention to the ...

[0256] In some embodiments, the method herein includes performing extended microscopy methods and techniques on the sample. Expansion allows single targets (e.g., mRNA or RNA transcripts) densely packed in the cell to be spatially resolved in a high-throughput manner. Extended microscopy techniques are known in the art and can be performed as described in US 2016 / 0116384 and Chen et al., Science, 347, 543 (2015), each of which is incorporated herein by reference in its entirety. In some embodiments, the method does not include performing extended microscopy on the sample. In some embodiments, the method does not include dissociating cells from samples such as tissues or cell microenvironments. In some embodiments, the method does not include cracking samples or cells therein. In some embodiments, the method does not include embedding samples or molecules from samples in exogenous matrix.

[0257] V. Samples and Sample Handling

[0258] The methods and compositions disclosed herein can be used to analyze biological samples, which can be obtained from a subject using any of a variety of techniques (including but not limited to biopsy, surgery, and laser capture microscopy (LCM)), and typically include cells and / or other biological materials from the subject. Biological samples can also be obtained from eukaryotic organisms, such as tissue samples, patient-derived organoids (PDOs), or patient-derived xenografts (PDXs). Biological samples from organisms can include one or more other organisms or components thereof. For example, in addition to mammalian cells and non-cellular tissue components, mammalian tissue sections can include prions, viroids, viruses, bacteria, fungi, or components from other organisms.

[0259] The subject from whom a biological sample can be obtained can be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., a patient having a disease such as cancer) or susceptible to a disease, and / or an individual in need of therapy or suspected of being in need of therapy.

[0260] In some embodiments, biological sample corresponds to cells (e.g., derived from cell culture, tissue sample or cells deposited on the surface). In a cell sample with multiple cells, individual cells may not aggregate naturally. For example, cells may be derived from a suspension of cells (e.g., body fluids, such as blood) and / or may be cells dissociated or disaggregated from tissue or tissue sections. The number of cells in a biological sample may vary. Some biological samples include a large number of cells, such as blood samples, while other biological samples include less or only a small amount of cells or may only be suspected of containing cells, such as plasma, serum, urine, saliva, synovial fluid, amniotic fluid, tears, lymph, liquid (liquor), cerebrospinal fluid, etc.

[0261] In some embodiments, the biological sample containing cells can include body fluids or cell samples derived from body fluids, for example, whole blood, samples derived from blood such as plasma or serum, buffy coat, urine, sputum, tears, lymph, sweat, liquid, cerebrospinal fluid, ascites, milk, feces, bronchial lavage fluid, saliva, amniotic fluid, nasal secretions, vaginal secretions, semen, wound secretions, cell culture and swab samples, or any cell-containing sample derived from the aforementioned samples. In some embodiments, the biological sample containing cells can be body fluids, body secretions or body excretions, for example, lymph, blood, buffy coat, blood plasma or serum. In some embodiments, the biological sample containing cells can be a circulating body fluid, such as blood or lymph, for example, peripheral blood obtained from a mammal such as a human.

[0262] Biological samples can include any number of macromolecules, for example, cellular macromolecules and organelles (for example, mitochondria and nuclei). Biological samples can be obtained as tissue samples (such as tissue sections, biopsy samples, core needle biopsy samples, needle aspirates or fine needle aspirates). The sample can be a fluid sample, such as a blood sample, a urine sample or a saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histological sample, a histopathological sample, a plasma or serum sample, a tumor sample, a living cell, a cultured cell, a clinical sample (for example, whole blood or blood-derived products, hemocytes or cultured tissues or cells, including cell suspensions). In some embodiments, biological samples can include cells deposited on the surface. In some embodiments, biological samples can include transcripts of antigen receptor molecules.

[0263] The biological sample may be derived from a homogenous culture or population of the subjects or organisms mentioned herein, or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0264] Biological samples can include one or more diseased cells. Diseased cells may have altered metabolic properties, gene expression, protein expression and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

[0265] The biological sample may include an analyte (e.g., protein, RNA, and / or DNA) embedded in a 3D matrix. In some embodiments, an amplicon (e.g., rolling circle amplification product) derived from an analyte (e.g., protein, RNA, and / or DNA) or associated with an analyte may be embedded in a 3D matrix. In some embodiments, the 3D matrix may include natural molecules and / or synthetic molecular networks chemically and / or enzymatically connected (e.g., by crosslinking). In some embodiments, the 3D matrix may include a synthetic polymer. In some embodiments, the 3D matrix includes a hydrogel.

[0266] In some embodiments, the substrate herein can be any support that is insoluble in aqueous liquids, and allows positioning biological samples, analytes, features and / or reagents (e.g., probes) on the support. In some embodiments, biological samples can be attached to substrates. The attachment of biological samples can be irreversible or reversible, depending on the subsequent steps in the properties of the sample and the analytical method. In certain embodiments, by applying a suitable polymer coating to substrates and contacting the sample with the polymer coating, the sample can be reversibly attached to the substrate. Then, for example, using an organic solvent that at least partially dissolves the polymer coating, the sample can be separated from the substrate. Hydrogels are examples of polymers suitable for this purpose.

[0267] In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0268] Various steps may be performed to prepare or process biological samples for and / or during an assay. Unless otherwise indicated, the preparation or processing steps described below may generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.

[0269] (i) Tissue sections

[0270] Biological samples can be obtained from subjects (e.g., by surgical biopsy, whole subject sectioning) or grown in vitro as cell populations on growth substrates or culture dishes and prepared for analysis as tissue slices or tissue sections. The grown samples may be thin enough to be used for analysis without further processing steps. Alternatively, grown samples and samples obtained via biopsy or sectioning can be prepared into thin tissue sections using mechanical cutting devices such as vibrating blade microtomes. As another alternative, in some embodiments, thin tissue sections can be prepared by applying a touch impression of the biological sample to a suitable substrate material.

[0271] The thickness of the tissue section can be a fraction of the maximum cross-sectional dimension of the cells (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections having a thickness greater than the maximum cross-sectional cell dimension can also be used. For example, cryostat sections can be used, which can be, for example, 10-20 μm thick.

[0272] More generally, the thickness of the tissue section usually depends on the method for preparing the section and the physical properties of the tissue, so sections with various thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40 or 50 μm. If necessary or convenient, thicker sections can also be used, such as at least 70, 80, 90 or 100 μm or thicker. Typically, the thickness of the tissue section is between 1-100 μm, 1-50 μm, 1-30 μm, 1-25 μm, 1-20 μm, 1-15 μm, 1-10 μm, 2-8 μm, 3-7 μm or 4-6 μm, but as mentioned above, sections with thickness greater than or less than these ranges can also be analyzed.

[0273] Multiple slices can also be obtained from a single biological sample. For example, multiple tissue slices can be obtained from a surgical biopsy sample by serially slicing the biopsy sample using a microtome blade. Spatial information between serial slices can be preserved in this way, and the slices can be analyzed serially to obtain three-dimensional information about the biological sample.

[0274] (ii) Freezing

[0275] In some embodiments, biological samples (e.g., tissue sections as described above) can be prepared by deep freezing at a temperature suitable for maintaining or preserving the integrity (e.g., physical properties) of the tissue structure. Any number of suitable methods can be used to slice (e.g., thin slices) frozen tissue samples onto the substrate surface. For example, a freezing microtome (e.g., a cryostat) can be used to prepare tissue samples at a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of the nucleic acid in the sample. Such a temperature can be, for example, below -15 ° C, below -20 ° C, or below -25 ° C.

[0276] (iii) Fixation and post-fixation

[0277] In some embodiments, established method formalin fixation and paraffin embedding (FFPE) can be used to prepare biological samples. In some embodiments, formalin fixation and paraffin embedding can be used to prepare cell suspensions and other non-tissue samples. After fixing the sample and being embedded in paraffin or resin blocks, the sample can be sliced ​​as described above. Before analysis, it is possible to remove paraffin embedded materials (e.g., dewaxing) from tissue sections by incubating tissue sections in appropriate solvents (e.g., xylene), then rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

[0278] As an alternative to formalin fixation described above, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, the sample can be fixed by immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0279] In some embodiments, acetone fixation is used for fresh frozen samples, which may include but are not limited to cortical tissue, mouse olfactory bulb, human brain tumor, human postmortem brain and breast cancer samples. When acetone fixation is performed, a pre-permeabilization step (as described below) may not be performed. Alternatively, acetone fixation may be performed in combination with a permeabilization step.

[0280] In some embodiments, the method provided herein includes one or more post-fixing (post-fixing) (also referred to as post-fixation) steps. In some embodiments, one or more post-fixing steps are performed after contacting the sample with a polynucleotide disclosed herein (e.g., one or more probes, such as circular or padlock probes). In some embodiments, one or more post-fixing steps are performed after a hybridization complex comprising a probe and a target is formed in the sample. In some embodiments, one or more post-fixing steps are performed before a ligation reaction disclosed herein (such as a connection with a padlock probe circularization).

[0281] In some embodiments, one or more post-fixation steps are performed after contacting the sample with a binding agent or a labeling agent (e.g., an antibody or its antigen binding fragment) for a non-nucleic acid analyte such as a protein analyte. The labeling agent may include a nucleic acid molecule (e.g., a reporter oligonucleotide) comprising a sequence corresponding to the labeling agent and therefore corresponding to the analyte (e.g., uniquely identified). In some embodiments, the labeling agent may include a reporter oligonucleotide containing one or more barcode sequences.

[0282] The post-fixation step can be performed using any suitable fixation reagent disclosed herein (eg, 3% (w / v) paraformaldehyde in DEPC-PBS).

[0283] (iv) Embedding

[0284] As an alternative to paraffin embedding described above, biological samples can be embedded in any of a variety of other embedding materials to provide a structural substrate for the sample before sectioning and other processing steps. In some cases, the embedding material can be removed, for example, before analyzing the tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, wax, resin (e.g., methacrylic resin), epoxy resin, and agar.

[0285] In some embodiments, the biological sample can be embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this way generally involves contacting the biological sample with a hydrogel so that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material and activating the polymer material to form a hydrogel. In some embodiments, forming a hydrogel allows the hydrogel to be internalized in the biological sample.

[0286] In some embodiments, the biological sample is immobilized in the hydrogel by cross-linking of the hydrogel-forming polymeric material. The cross-linking may be performed chemically and / or photochemically, or alternatively by any other hydrogel-forming method.

[0287] The composition of the hydrogel-matrix and the application to the biological sample generally depend on the nature and preparation (e.g., sliced, non-sliced, fixed type) of the biological sample. As an example, when the biological sample is a tissue slice, the hydrogel-matrix may include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, when the biological sample is composed of cells (e.g., cultured cells or cells dissociated from a tissue sample), the cells may be incubated with a monomer solution and an APS / TEMED solution. For cells, the hydrogel-matrix gel is formed in a compartment, which includes, but is not limited to, a device for culturing, maintaining or transporting cells. For example, a hydrogel-matrix may be formed to a depth range of about 0.1 μm to about 2 mm with a monomer solution added to the compartment plus APS / TEMED.

[0288] Additional methods and aspects of hydrogel embedding of biological samples are described, for example, in Chen et al., Science 347(6221): 543-548, 2015, the entire contents of which are incorporated herein by reference.

[0289] (v) Staining and immunohistochemistry (IHC)

[0290] For the convenience of visualization, various staining agents and staining techniques can be used to stain biological samples. For example, in some embodiments, any number of staining agents and / or immunohistochemical reagents can be used to stain samples. One or more staining steps can be performed to prepare or process biological samples for determinations described herein, or can be performed during and / or after determination. In some embodiments, samples can be contacted with one or more nucleic acid stains, membrane stains (for example, cell membrane or nuclear membrane), cytological stains, or a combination thereof. In some instances, staining can be specific to the compartment of proteins, phospholipids, DNA (for example, dsDNA, ssDNA), RNA, organelles, or cells. Samples can be contacted with one or more labeled antibodies (for example, a first antibody specific to an analyte of interest and a second antibody specific to a label of the first antibody). In some embodiments, one or more images taken of the stained sample can be used to segment the cells in the sample.

[0291] In some embodiments, lipophilic dye is used for dyeing. In some instances, lipophilic carbocyanine or aminostyrene dye or their analog (for example, DiI, DiO, DiR, DiD) are used for dyeing. Other cell membrane stains can include FM and RH dye or immunohistochemical reagents specific to cell membrane proteins. In some instances, staining agent can include but is not limited to acridine orange, acid fuchsin, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hoechst stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (for example, rhodamine B) or safranin or their derivatives. In some embodiments, sample can be dyed with hematoxylin and eosin (H&E).

[0292] Samples can be stained using hematoxylin and eosin (H&E) staining techniques, using Papanicolaou staining techniques, Masson's trichrome staining techniques, silver staining techniques, Sudan staining techniques, and / or using periodic acid Schiff (PAS) staining techniques. PAS staining is usually performed after formalin or acetone fixation. In some embodiments, Romanovsky stains (including Wright's stain, Jenner's stain, Can-Grunwaldstain, Leishman stain, and Giemsa stain) can be used to stain samples.

[0293] In some embodiments, the biological sample can be decolorized. The method of decolorizing or fading the biological sample generally depends on the nature of the stain applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample by antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide bonds by washing with a reducing agent and a detergent, treatment with a chaotropic salt, treatment with an antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiple staining and destaining are described in, for example, Bolognesi et al., J. Histochem. Cytochem. 2017; 65 (8): 431-444; Lin et al., Nat Commun. 2015; 6: 8390; Pirici et al., J. Histochem. Cytochem. 2009; 57: 567-75; and Glass et al., J. Histochem. Cytochem. 2009; 57: 899-905, the entire contents of each document are incorporated herein by reference.

[0294] (vi) Isometric Expansion

[0295] In some embodiments, the biological sample embedded in the matrix (e.g., hydrogel) can be isovolumetrically expanded. The isovolumetric expansion method that can be used includes hydration, which is a preparation step in expansion microscopy, as described in Chen et al., Science 347 (6221): 543-548, 2015.

[0296] Isovolumetric expansion can be performed by anchoring one or more components of the biological sample to a gel, followed by gel formation, proteolysis, and swelling. In some embodiments, the analyte in the sample, the product of the analyte, and / or the probe associated with the analyte in the sample can be anchored to a matrix (e.g., a hydrogel). Isovolumetric expansion of the biological sample can occur before the biological sample is fixed to the substrate or after the biological sample is fixed to the substrate. In some embodiments, the isochoric expanded biological sample can be removed from the substrate before contacting the substrate with the probe disclosed herein.

[0297] In general, the steps used to perform isochoric expansion of a biological sample may depend on the characteristics of the sample (e.g., thickness of tissue sections, fixation, cross-linking) and / or the analytes of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to a gel).

[0298] In some embodiments, proteins in biological samples are anchored to swellable gels (such as polyelectrolyte gels). Antibodies can be directed to proteins before, after, or in conjunction with anchoring to swellable gels. DNA and / or RNA in biological samples can also be anchored to swellable gels via suitable linkers. Examples of such linkers include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X SE) (available from ThermoFisher, Waltham, MA), Label-IT Amine (available from MirusBio, Madison, WI), and Label X (e.g., Chen et al., Nat. Methods 13: 679-684, 2016, the entire contents of which are incorporated herein by reference).

[0299] Isovolumetric expansion of a sample can increase the spatial resolution of subsequent analysis of the sample. The increase in resolution in the spatial distribution can be determined by comparing an isochoric expanded sample with a sample that has not been isochoric expanded.

[0300] In some embodiments, the biological sample is isovolumetrically expanded to a size of at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x of its unexpanded size. In some embodiments, the sample is isovolumetrically expanded to at least 2x and less than 20x of its unexpanded size.

[0301] (vii) Crosslinking and decrosslinking

[0302] In some embodiments, before or during the original position determination, biological sample is reversibly cross-linked. In some respects, the amplified product (for example, amplicon) of analyte, polynucleotide and / or analyte or the probe combined therewith can be anchored on the polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more of polynucleotide probe and / or its amplified product (for example, amplicon) can be modified to contain the functional group that can be used as anchoring point to attach polynucleotide probe and / or amplified product to the polymer matrix. In some embodiments, the modified probe comprising oligodT can be used for combining interested mRNA molecules, followed by the reversible cross-linking of mRNA molecules.

[0303] Hydrogels can include macromolecular polymer gels containing networks. In the network system, some polymer chains can be optionally cross-linked, but cross-linking does not always occur.

[0304] In some embodiments, the hydrogel can include hydrogel subunits such as, but not limited to, acrylamide, bisacrylamide, polyacrylamide and its derivatives, poly(ethylene glycol) and its derivatives (e.g., PEG-acrylate (PEG-DA), PEG-RGD), methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof.

[0305] In some embodiments, the hydrogel comprises a hybrid material, for example, the hydrogel material comprises elements of a synthetic polymer and a natural polymer. Examples of suitable hydrogels are described in, for example, U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422 and U.S. Patent Application Publication Nos. 2017 / 0253918, 2018 / 0052081, and 2010 / 0055733, the entire contents of each document being incorporated herein by reference.

[0306] In some embodiments, the hydrogel can form a substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on the top of the one or more second materials. For example, the hydrogel can be preformed and then placed on the top, bottom or any other configuration with the one or more second materials. In some embodiments, the hydrogel forms after contacting the one or more second materials during the formation of the substrate. The hydrogel formation can also occur in the structure (e.g., hole, ridge, protrusion and / or texture) located on the substrate.

[0307] In some embodiments, hydrogel formation on the substrate occurs before, simultaneously with, or after providing the probe to the sample. For example, hydrogel formation can be performed on a substrate that already contains the probe.

[0308] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue section) is embedded in a hydrogel. In some embodiments, a hydrogel subunit is injected into a biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

[0309] In the embodiment of forming hydrogel in biological sample, functionalization chemistry can be used.In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC).Any hydrogel-tissue skeleton (for example, synthetic or natural) suitable for HTC can be used for anchoring biomacromolecules and regulating functionalization.Non-limiting examples of methods using HTC skeleton variants include CLARITY, PACT, ExM, SWITCH and ePACT.In some embodiments, the hydrogel formation in biological sample is permanent.For example, biomacromolecules can be permanently adhered to hydrogel, allowing multiple rounds of inquiry.In some embodiments, the hydrogel formation in biological sample is reversible.

[0310] In some embodiments, before, simultaneously and / or after polymerization, additional reagents are added to the hydrogel subunits. For example, additional reagents may include, but are not limited to, oligonucleotides (e.g., probes), endonucleases for fragmenting DNA, fragmentation buffers for DNA, DNA polymerases, dNTPs for amplifying nucleic acids and connecting barcodes to amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, ligases, proteinase K, and DNA enzymes. Additional reagents may also include reverse transcriptases (including enzymes with terminal transferase activity), primers, and switch oligonucleotides. In some embodiments, before, simultaneously and / or after polymerization, optical labels are added to the hydrogel subunits.

[0311] In some embodiments, the HTC agent is added to the hydrogel before, simultaneously with, and / or after polymerization. In some embodiments, the cell labeling agent is added to the hydrogel before, simultaneously with, and / or after polymerization. In some embodiments, the cell permeation agent is added to the hydrogel before, simultaneously with, and / or after polymerization.

[0312] Any suitable method can be used to make the hydrogel embedded in the biological sample transparent. For example, the electrophoretic tissue clearing method can be used to remove biomacromolecules from the hydrogel-embedded sample. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixing medium, methylcellulose or other semi-solid medium) before or after the hydrogel is cleared.

[0313] In some embodiments, the methods disclosed herein include decrosslinking a reversibly crosslinked biological sample. Decrosslinking does not need to be complete. In some embodiments, only a portion of the crosslinked molecules in the reversibly crosslinked biological sample are decrosslinked and allowed to migrate.

[0314] (viii) Tissue permeabilization and processing

[0315] In some embodiments, biological samples can be permeabilized to facilitate the transfer of substances (such as probes) into the sample. If the sample is not fully permeabilized, the amount of substances (such as probes) in the sample may be too low to be fully analyzed. On the contrary, if the tissue sample permeability is too strong, the relative spatial relationship of the analyte in the tissue sample may be lost. Therefore, it is ideal to fully permeabilize the tissue sample to obtain a good signal intensity while still maintaining a balance between the spatial resolution of the analyte distribution in the sample.

[0316] Typically, biological samples can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100, TM or Tween-20 TM ) and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample can be incubated with a cell permeabilizing agent to facilitate permeabilization of the sample. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588: 63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable sample permeabilization method can generally be used in conjunction with the samples described herein.

[0317] In some embodiments, the biological sample can be permeabilized by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive agents such as lysing enzymes for cracking different cell types (e.g., Gram-positive or negative bacteria, plants, yeasts, mammals, etc.), such as lysozyme, colorless peptidase, lysostaphin, labiase, kitalase, lysoplasmin, and a variety of other commercially available lysing enzymes.

[0318] Other lysing agents can be added to the biological sample additionally or alternatively to promote permeabilization. For example, a lysing solution based on a surfactant can be used to lyse the sample cells. The lysing solution can include an ionic surfactant, such as sodium lauroyl sarcosinate and sodium dodecyl sulfate (SDS). More generally, chemical lysing agents can include but are not limited to organic solvents, chelating agents, detergents, surfactants and chaotropic agents.

[0319] In some embodiments, the biological sample can be permeabilized by non-chemical permeabilization methods. The non-chemical permeabilization methods that can be used include, but are not limited to, physical lysis techniques (such as electroporation), mechanical permeabilization methods (e.g., bead milling using a homogenizer and grinding balls to mechanically destroy the sample tissue structure), acoustic permeabilization (e.g., ultrasonic treatment), and thermal lysis techniques (such as heating to induce thermal permeabilization of the sample).

[0320] Before analyzing the sample, additional reagents can be added to the biological sample to perform various functions. In some embodiments, DNA enzyme and RNA enzyme inactivators or inhibitors (such as proteinase K) and / or chelating agents (such as EDTA) can be added to the sample. For example, the method disclosed herein can include a step of increasing the accessibility of the nucleic acid for binding, for example, opening the DNA in the cell for the denaturation step of probe hybridization. For example, proteinase K treatment can be used to release DNA bound to proteins.

[0321] (ix) Selective enrichment of RNA or cDNA species

[0322] In some embodiments, when RNA or cDNA is analyte, one or more RNA or cDNA analyte species of interest can be selectively enriched. For example, one or more RNA or cDNA species of interest can be selected by adding one or more oligonucleotides to the sample. In some embodiments, other oligonucleotides are sequences for initiating reactions by enzyme (e.g., polymerase). For example, one or more RNA or cDNA of interest can be amplified using one or more primer sequences with sequence complementarity to one or more RNA or cDNA of interest, thereby selectively enriching these RNA or cDNA.

[0323] In some aspects, when analyzing two or more analytes, a first probe and a second probe specific to each RNA or cDNA analyte (e.g., specifically hybridized with each RNA or cDNA analyte) are used. For example, in some embodiments of the method provided herein, templated connection is used to detect gene expression in a biological sample. An analyte of interest (such as a protein) that can be targeted and combined by a labeling agent or a binding agent (e.g., an antibody or its epitope binding fragment) is used for analysis, wherein the binding agent is conjugated or otherwise associated with a reporter oligonucleotide containing a reporter sequence for identifying the binding agent. The probe can be hybridized with the reporter oligonucleotide and connected in a templated connection reaction to produce a product for analysis. In some embodiments, before connection, the gap between the probe oligonucleotides can be filled using, for example, Mu polymerase, DNA polymerase, RNA polymerase, reverse transcriptase, VENT polymerase, Taq polymerase and / or any combination thereof, derivatives and variants (e.g., engineered mutants). In some embodiments, the assay can also include the amplification (e.g., by multiplex PCR) of the templated connection product.

[0324] In some embodiments, the analyte can be further enriched by immobilization at a location in the biological sample for in situ readout. In one non-limiting example, the analyte can include one or more fragments that are unique to a location in the biological sample.

[0325] Alternatively, one or more RNA species can be selected (e.g., removed) using any of a variety of methods. For example, a probe can be applied to the sample that selectively hybridizes to ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. Additionally and alternatively, double-stranded specific nuclease (DSN) treatment can remove rRNA (see, e.g., Archer et al., Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage, BMC Genomics, 15 401, (2014), the entire contents of which are incorporated herein by reference). In addition, hydroxyapatite chromatography can remove abundant species (e.g., rRNA) (see, e.g., Vandemoot, V..A., cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53 (6) 373-80, (2012), the entire contents of which are incorporated herein by reference).

[0326] The biological sample may contain one or more analytes of interest. Methods are provided for performing multiplex assays to analyze two or more different analytes in a single biological sample.

[0327] VI. Compositions, Kits, and Systems

[0328] Provided herein is a kit, for example, comprising one or more oligonucleotides (for example, any oligonucleotide described in Section IV) and instructions for performing the method provided herein. In some embodiments, the kit also includes one or more reagents (for example, nucleotide mixtures, probes, etc.) for performing the method provided herein. In some embodiments, the kit also includes one or more reagents required for one or more steps, and the one or more steps include hybridization, connection, extension, amplification, detection and / or sample preparation as described herein. In some embodiments, the kit also includes an enzyme, such as a ligase and / or a polymerase described herein. In some embodiments, the kit includes a polymerase, such as a polymerase for extending a primer and incorporating nucleotides. In some embodiments, the kit may contain reagents for forming a functionalized matrix (for example, a hydrogel), such as any suitable functional part. In some instances, a buffer and reagent for anchoring a probe and a product (for example, an RCA product) to a functionalized matrix are also provided. The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined in a single container. In some embodiments, the kit also contains instructions for implementing the provided method using the kit components.

[0329] In some embodiments, the test kit may contain reagents and / or consumables required for one or more steps of the method provided. In some embodiments, the test kit contains reagents for fixing, embedding and / or permeabilizing biological samples. In some embodiments, the test kit contains reagents, such as enzymes and buffers for connection and / or amplification, such as ligases and / or polymerases. In some respects, the test kit may also include any reagent as described herein, such as wash buffer and connection buffer. In some embodiments, the test kit contains reagents for detection and / or sequencing, such as detectably labeled nucleotides, polymerases or conjugates. In some embodiments, the test kit optionally contains other components, such as nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, reagents for other determinations.

[0330] VII. Optofluidic Instruments for Analyzing Biological Samples

[0331] Provided herein is an instrument with an integrated optical and fluidics module ("optical fluidic instrument" or "optical fluidic system") for detecting target molecules (e.g., nucleic acids, proteins, antibodies, etc.) in biological samples (e.g., one or more cell or tissue samples) as described herein. In the optical fluidic instrument, the fluidics module is configured to deliver one or more reagents (e.g., detectably labeled nucleotides, polymerases, or conjugates) to the biological sample and / or remove used reagents from the biological sample. In addition, the optical module is configured to irradiate the biological sample with light having one or more spectral emission curves (within a certain wavelength range), and then capture one or more images of the emitted light signal from the biological sample during one or more sequencing cycles (e.g., as described in Section 1II). In some embodiments, the in situ assays disclosed herein (e.g., sequencing by synthesis) can be performed using an automated instrument or system, for example, an optical fluidic instrument or system disclosed herein.

[0332] In various embodiments, the captured images can be processed in real time and / or later to determine the presence of one or more target molecules in the biological sample, as well as three-dimensional position information associated with each detected target molecule. In addition, the optofluidic instrument includes a sample module configured to receive (and optionally fix) one or more biological samples. In some cases, the sample module includes an XY stage configured to move the biological sample along an XY plane (e.g., perpendicular to the objective lens of the optical module).

[0333] In various embodiments, the optofluidic instrument is configured to analyze one or more target molecules (e.g., any of the analytes described in Section III) within a biological sample where they naturally occur (i.e., in situ). For example, the optofluidic instrument can be an in situ analytical system for analyzing a biological sample and detecting target molecules (e.g., analytes), including but not limited to DNA, RNA, proteins, antibodies, and / or the like.

[0334] Optofluidic instruments can be used for in situ target molecule detection via base-by-base sequencing (e.g., sequencing of identifier sequences such as barcode sequences) and / or other imaging or target molecule detection techniques. That is, for example, an optofluidic instrument may include a fluidics module that includes fluids required to establish experimental conditions required to detect target molecules in a sample. In addition, such an optofluidic instrument may also include a sample module configured to receive a sample, and an optical module that includes an imaging system for irradiating the sample (e.g., exciting one or more fluorescently labeled nucleotides within the sample) and / or imaging light signals received from the sample. The in situ analysis system may also include other auxiliary modules configured to facilitate the operation of the optofluidic instrument, such as but not limited to a cooling system, a motion calibration system, and the like.

[0335] Figure 5An example workflow for analyzing a biological sample 510 (e.g., a cell or tissue sample) using an optofluidic instrument or system 500 according to various embodiments is shown. In various embodiments, the sample 510 can be a biological sample (e.g., a tissue) that includes molecules such as DNA, RNA, proteins, antibodies, etc. For example, the sample 510 can be a sliced ​​tissue that is processed to obtain its RNA for probe hybridization and sequencing as described herein (e.g., in Section III).

[0336] In various embodiments, the sample 510 can be placed in an optofluidic instrument or system 500 to analyze and detect molecules in the sample 510. In various embodiments, the optofluidic instrument or system 500 can be a system configured to facilitate experimental conditions that are conducive to target molecule detection. For example, the optofluidic instrument or system 500 can include a fluidics module 540, an optical module 550, a sample module 560, and an auxiliary module 570, and these modules can be operated by a system controller 530 to create experimental conditions for base-by-base sequencing of nucleic acid molecules in the sample 510 and facilitate imaging of the sample (e.g., by an imaging system of the optical module 550). In various embodiments, the various modules of the optofluidic instrument or system 500 can be separate components that communicate with each other, or at least some of them can be integrated together.

[0337] In various embodiments, the sample module 550 can be configured to receive the sample 510 into the optofluidic instrument or system 500. For example, the sample module 560 may include a sample interface module (SIM) configured to receive a sample device (e.g., a box) onto which the sample 510 can be deposited. That is, the sample 510 can be placed in the optofluidic instrument or system 500 by depositing the sample 510 (e.g., a sliced ​​tissue) on the sample device and then inserting the sample device into the SIM of the sample module 560. In some cases, the sample module 560 may also include an XY stage onto which the SIM is mounted. The XY stage may be configured to move the SIM mounted thereon (e.g., and the sample device containing the sample 510 inserted therein) in a vertical direction along a two-dimensional (2D) plane of the optofluidic instrument or system 500.

[0338] Experimental conditions that are conducive to detecting molecules in the sample 510 may depend on the target molecule detection technology employed by the optofluidic instrument or system 500. For example, in various embodiments, the optofluidic instrument or system 500 may be a system configured to detect molecules in the sample 510 (e.g., by detecting nucleotides incorporated into an extended sequencing primer using the identifier sequence as a template).

[0339] In various embodiments, the fluidics module 540 may include one or more components that can be used to store reagents and to transport the reagents to and from a sample device containing a sample 510. For example, the fluidics module 540 may include a reservoir configured to store reagents and a waste container configured to collect reagents (e.g., and other wastes) after the optical fluidic instrument or system 500 is used to analyze and detect molecules of the sample 510. In addition, the fluidics module 540 may also include pumps, tubes, pipettes, etc. configured to facilitate the transport of reagents to the sample device (e.g., and thus the sample 510). For example, the fluidics module 540 may include a pump ("reagent pump") configured to pump washing / stripping reagents to the sample device for washing / stripping the sample 510 (e.g., and other washing functions, such as washing the objective lens of the imaging system of the optical module 550).

[0340] In various embodiments, the auxiliary module 570 may be a cooling system of the optofluidic instrument or system 500, and the cooling system may include a network of coolant-carrying pipes configured to deliver coolant to the various modules of the optofluidic instrument or system 500 to regulate their temperatures. In such cases, the fluidics module 540 may include a coolant reservoir for storing coolant and a pump (e.g., a "coolant pump") for generating a pressure differential, thereby forcing the coolant to flow from the reservoir to the various modules of the optofluidic instrument or system 500 via the coolant-carrying pipes. In some cases, the fluidics module 540 may include a return coolant reservoir, which may be configured to receive and store return coolant, i.e., heated coolant that flows back into the return coolant reservoir after absorbing heat rejected by the various modules of the optofluidic instrument or system 500. In such cases, the fluidics module 540 may also include a cooling fan configured to force air (e.g., cooling and / or ambient air) into the return coolant reservoir to cool the heated coolant stored therein. In some cases, the fluidics module 540 may also include a cooling fan configured to force air directly into components of the optofluidic instrument or system 500 to cool the components. For example, the fluidics module 540 may include a cooling fan configured to direct cooling or ambient air into the system controller 530 to cool the system controller.

[0341] As discussed above, the optofluidic instrument or system 500 may include an optical module 550 that includes various optical components of the optofluidic instrument or system 500, such as, but not limited to, a camera, an illumination module (e.g., an LED), an objective lens, and / or the like. The optical module 550 may include a fluorescence imaging system configured to image fluorescence emitted by detectably labeled nucleotides incorporated into an extended sequencing primer in the sample 510 after the detectable labels are excited by light from an illumination module of the optical module 550.

[0342] In some cases, optical module 550 may also include an optical frame upon which the camera, illumination module, and / or XY stage of sample module 560 may be mounted.

[0343] In various embodiments, the system controller 530 can be configured to control the operation of the optofluidic instrument or system 500 (e.g., and the operation of one or more modules thereof). In some cases, the system controller 530 can take various forms, including a processor, a single computer (or computer system), or multiple computers that communicate with each other. In various embodiments, the system controller 530 can be communicatively coupled to a data storage device, an input device group, a display system, or a combination thereof. In some cases, some or all of these components can be considered part of the system controller 530 or otherwise integrated with the system controller, can be separate components that communicate with each other, or can be integrated together. In other examples, the system controller 530 can be a cloud computing platform or can communicate with a cloud computing platform.

[0344] In various embodiments, the optofluidic instrument or system 500 can analyze the sample 510 and can generate an output 590 that includes an indication of the presence of target molecules in the sample 510. For example, with respect to the example embodiments discussed above in which the optofluidic instrument or system 500 employs hybridization techniques to detect molecules, the optofluidic instrument or system 500 can subject the sample 510 to consecutive sequencing cycles, wherein during the same sequencing cycle, the sample is imaged to detect at least some locations (e.g., Figure 3 1) where one or more genes in gene block 1 shown in FIG. 1 are present, and signals associated with nucleotide binding and / or incorporation events at other locations in the sample (e.g., Figure 3 2 (e.g., the absence of signal may be due to the use of unmodified nucleotides as dark bases or the absence of reagents capable of detecting incorporated nucleotides). In such a case, output 590 may include an optical signature (e.g., codeword) unique to each identifier sequence (e.g., barcode sequence), thereby allowing identification of the target molecule.

[0345] VIII. Terminology

[0346] Unless otherwise defined, all specialized terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference from what is generally understood in the art.

[0347] The terms "polynucleotide", "polynucleotides" and "nucleic acid molecules" used interchangeably herein refer to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, eDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups.

[0348] As used herein, "hybridization" may refer to a process in which two single-stranded polynucleotides are non-covalently bound to form a stable double-stranded polynucleotide. In one aspect, the resulting double-stranded polynucleotide may be a "hybrid" or "duplex." "Hybridization conditions" typically include a salt concentration that is approximately less than 1M, typically less than about 500mM, and may be less than about 200mM. "Hybridization buffer" includes a buffered saline solution, such as 5% SSPE, or other such buffers. The hybridization temperature may be as low as 5°C, but is typically greater than 22°C, more typically greater than about 30°C, and typically greater than 37°C. Hybridization is typically performed under stringent conditions (i.e., under which the sequence will hybridize with its target sequence, but will not hybridize with other non-complementary sequences). Stringent conditions are sequence-dependent and are different in different situations. For example, for specific hybridization, longer fragments may require higher hybridization temperatures than shorter fragments. Since other factors may affect the stringency of hybridization, including the base composition and length of the complementary chain, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of any one parameter alone. Generally, stringent conditions are selected to be greater than the T for the specific sequence at a defined ionic strength and pH. m About 5°C lower. Melting temperature T m It can be the temperature at which half of the population of double-stranded nucleic acid molecules dissociate into single strands. As indicated in standard references, when nucleic acids are in an aqueous solution of 1 M NaCl, T m A simple estimate of the value can be obtained by using the equation T m = 81.5 + 0.41 (% G + C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawi and SantaLucia, Jr., Biochemistry, 36: 10581-94 (1997)) include alternative methods of calculation that take into account structural and environmental as well as sequence characteristics. m 's calculation.

[0349] Typically, the stability of the hybrid is a function of ion concentration and temperature. Typically, the hybridization reaction is carried out under conditions of lower stringency, followed by washing with different but higher stringency. Exemplary stringent conditions include a salt concentration of at least 0.01M to no more than 1M sodium ion concentration (or other salts) at a pH of about 7.0 to about 8.3 and a temperature of at least 25°C. For example, 5×SSPE (750mM NaCl, 50mM sodium phosphate, 5mM EDTA, pH 7.4) and a temperature condition of about 30°C are suitable for allele-specific hybridization, although the appropriate temperature depends on the length and / or GC content of the hybridized region. In one aspect, the "hybridization stringency" for determining the percentage mismatch can be as follows: 1) high stringency: 0.1×SSPE, 0.1%SDS, 65°C; 2) medium stringency: 0.2×SSPE, 0.1%SDS, 50°C (also referred to as medium stringency); and 3) low stringency: 1.0×SSPE, 0.1%SDS, 50°C. It should be understood that equivalent stringency can be achieved using alternative buffers, salts and temperatures. For example, medium stringency hybridization can refer to conditions that allow nucleic acid molecules such as probes to bind complementary nucleic acid molecules. The hybridized nucleic acid molecules generally have at least 60% identity, including, for example, at least 70%, 75%, 80%, 85%, 90% or 95% identity. Medium stringency conditions can be conditions equivalent to hybridization at 42°C in 50% formamide, 5× Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 42°C in 0.2× SSPE, 0.2% SDS. For example, high stringency conditions can be provided by hybridization at 42°C in 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS, followed by washing at 65°C in 0.1× SSPE and 0.1% SDS. Low stringency hybridization can refer to conditions equivalent to hybridization in 10% formamide, 5× Denhardt's solution, 6× SSPE, 0.2% SDS at 22° C., followed by washing in 1× sSPE, 0.2% SDS at 37° C. Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20× SSPE (sodium chloride, sodium phosphate, ethylenediaminetetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA.Other suitable moderate and high stringency hybridization buffers and conditions are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, NY (1989); and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0350] Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize to its complementary sequence under selective hybridization conditions. Typically, selective hybridization will occur when there is at least about 65% complementarity, preferably at least about 75%, and more preferably at least about 90% complementarity over a stretch of at least 14 to 25 nucleotides. See M. Kanehisa, Nucleic Acids Res. 12: 203 (1984).

[0351] As used herein, a "primer" may be a natural or synthetic oligonucleotide that is capable of serving as a starting point for nucleic acid synthesis when forming a duplex with a polynucleotide template and extending from its 3' end along the template to form an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers are typically extended by a DNA polymerase.

[0352] "Ligation" can refer to the formation of a covalent bond or connection between the termini of two or more nucleic acids (e.g., oligonucleotides and / or polynucleotides) in a template-driven reaction. The nature of the bond or connection can vary widely, and the ligation can be performed enzymatically or chemically. As used herein, ligati...

Claims

1. A method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: The biological sample is a cell or tissue sample, The biological sample comprises a first analyte and a second analyte at a first location and a second location in the biological sample, respectively, The first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively. The first probe or its product comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and The second probe or a product thereof comprises i) a second priming site for a second sequencing primer and ii) a second identifier sequence associated with the second analyte; b) performing base-by-base sequencing of the first identifier sequence and the second identifier sequence using the first sequencing primer and the second sequencing primer, thereby generating a first signal code sequence and a second signal code sequence, each signal code sequence comprising a signal code corresponding to a signal (ON signal), a signal absence (OFF signal), or a combination thereof, detected at the first position and the second position, respectively, in consecutive cycles, wherein in one or more of said consecutive cycles, an ON signal is detected at said first position and an OFF signal is detected at said second position; and c) detecting the first identifier sequence and the second identifier sequence in the biological sample based at least on the first signal code sequence and the second signal code sequence.

2. The method of claim 1, wherein the first analyte and the second analyte are the same or different.

3. The method of claim 1 or 2, wherein the first identifier sequence and the second identifier sequence are different.

4. The method of any one of claims 1-3, wherein the first identifier sequence and the second identifier sequence comprise an analyte sequence or a complementary sequence thereof.

5. The method of any one of claims 1-4, wherein the first identifier sequence and the second identifier sequence comprise barcode sequences assigned to the first analyte and the second analyte, respectively, or complementary sequences thereof. 6 . The method of claim 5 , comprising assigning a first barcode sequence to the first analyte and assigning a second barcode sequence to the second analyte.

7. The method of claim 6, wherein assigning the barcode sequence is based on a decision rule designed to minimize the maximum predicted density of ON signals detected in each of one or more of the consecutive cycles.

8. The method of claim 6, wherein assigning the first barcode sequence to the first analyte and assigning the second barcode sequence to the second analyte comprises assignments based on expression data of the first analyte and the second analyte.

9. The method of claim 8, wherein assigning the first barcode sequence to the first analyte and assigning the second barcode sequence to the second analyte comprises assignment based on expression data of the first analyte and the second analyte in clustered cell types.

10. The method of claim 9, wherein the clustered cell types represent the distribution of cell types present in the biological sample.

11. The method of any one of claims 8-10, wherein the expression data of the first analyte and the second analyte at least partially overlap.

12. The method according to any one of claims 8-11, wherein the expression data of the first analyte and the second analyte comprises bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single cell gene expression data, single cell protein expression data or any combination thereof.

13. The method of any one of claims 6-12, wherein the nucleotides in the first barcode sequence detected in a specific cycle correspond to a signal code comprising an ON signal, and the corresponding nucleotides in the second barcode sequence detected in the specific cycle correspond to a signal code comprising an OFF signal.

14. The method of claim 13, wherein nucleotides in the first barcode sequence detected in the specific cycle correspond only to ON signals, and corresponding nucleotides in the second barcode sequence detected in the specific cycle correspond only to OFF signals.

15. The method of any one of claims 6-14, wherein one or more pairs of corresponding nucleotides in the first barcode sequence and the second barcode sequence to be detected in the same cycle are selected to reduce optical crowding of signals detected in the cycle.

16. The method according to any one of claims 1 to 15, wherein base-by-base sequencing is performed by contacting the biological sample with nucleotides in continuous cycles, wherein in each cycle a complex is formed, the complex comprising i) the first sequencing primer or the second sequencing primer hybridized to the first priming site or the second priming site, respectively, or an extension product thereof, ii) a polymerase, and iii) a homologous nucleotide that base pairs with a nucleotide in the first identifier sequence or the second identifier sequence, and A signal (ON signal) and / or a lack of signal (OFF signal) associated with the homologous nucleotide and / or the polymerase in the complex is detected at a specific position in the biological sample, wherein the ON signal, the OFF signal, or a combination thereof corresponds to a base in the homologous nucleotide and the corresponding nucleotide in the first identifier sequence or the second identifier sequence.

17. The method according to any one of claims 1 to 16, wherein 25% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence correspond to an OFF signal.

18. The method of claim 17, wherein 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the nucleotides in the first identifier sequence and / or the second identifier sequence are assigned to correspond to an OFF signal.

19. The method of any one of claims 1-18, wherein a plurality of different identifier sequences are detected in the biological sample, and each different identifier sequence is detected at one or more locations in the biological sample.

20. The method of claim 19, wherein 50% or more of the different identifier sequences each contain 50% or more of the nucleotides corresponding to an OFF signal in the identifier sequence.

21. The method of claim 19 or 20, wherein 80% or more of the different identifier sequences each comprise 80% or more of the nucleotides corresponding to an OFF signal in the identifier sequence.

22. The method of any one of claims 1-21, wherein the signal codes each correspond to a signal of a first color, a signal of a second color, a signal of a third color, or an absence of a signal, and wherein the first color, the second color, and the third color are different.

23. The method of any one of claims 1-21, wherein the signal codes each correspond to a signal of a first color, a signal of a second color, a combination of signals of the first color and the second color, or an absence of a signal, wherein the first color and the second color are different.

24. A method according to any one of claims 1-23, wherein the signal codes each correspond to a combination of a signal (ON signal) and / or a signal absence (OFF signal), wherein the combination of ON and / or OFF signals is detected in two or more imaging steps.

25. The method of any one of claims 1-24, further comprising detecting the first analyte and the second analyte in the biological sample based on detecting the first identifier sequence and the second identifier sequence.

26. The method of any one of claims 1-25, wherein the first identifier sequence is a first barcode sequence or a complement thereof, and the second identifier sequence is a second barcode sequence or a complement thereof.

27. The method of claim 26, wherein: The first barcode sequence identifies the first analyte, and / or The second barcode sequence identifies the second analyte.

28. The method according to claim 26 or 27, wherein: The first probe is provided as a first plurality of probes that bind directly or indirectly to the first analyte, the first plurality of probes together comprise a first combination of barcode sequences, and the first combination of barcode sequences identifies the first analyte, and / or The second probe is provided as a second plurality of probes that bind directly or indirectly to the second analyte, the second plurality of probes collectively comprise a second combination of barcode sequences, and the second combination of barcode sequences identifies the second analyte.

29. The method of any one of claims 1-28, wherein the first analyte and the second analyte comprise nucleic acid sequences, the first identifier sequence is a sequence of the first analyte or its complement, and the second identifier sequence is a sequence of the second analyte or its complement.

30. The method of any one of claims 1-29, wherein the base-by-base sequencing comprises: using a fluorescently labeled polymerase and one or more non-fluorescently labeled nucleotides; using a polymerase-nucleotide conjugate comprising a fluorescently labeled polymerase linked to a non-fluorescently labeled nucleotide moiety; or Multivalent polymer-nucleotide conjugates are used, comprising a polymer core, a plurality of nucleotide moieties, and one or more fluorescent labels.

31. The method of claim 30, wherein the polymerase does not incorporate a cognate nucleotide into the first sequencing primer or the second sequencing primer or an extension product thereof.

32. The method of claim 30, wherein incorporation of a cognate nucleotide by the polymerase into the first sequencing primer or the second sequencing primer or an extension product thereof is attenuated or inhibited.

33. The method of any one of claims 1-32, wherein the base-by-base sequencing comprises contacting the biological sample with a mixture of nucleotides comprising fluorescently labeled nucleotides and non-fluorescently labeled nucleotides.

34. The method of claim 33, wherein the polymerase incorporates homologous nucleotides into the first sequencing primer or the second sequencing primer or an extension product thereof, and the homologous nucleotides are fluorescently labeled or non-fluorescently labeled.

35. The method of claim 33 or 34, wherein the base-by-base sequencing comprises: contacting the biological sample with a first nucleotide mixture, wherein nucleotides comprising a first base are not detectably labeled, and nucleotides comprising bases other than the first base are each labeled with one or more detectable labels, and contacting the biological sample with a subsequent nucleotide mixture, wherein nucleotides comprising the subsequent base are not detectably labeled, and nucleotides comprising bases other than the subsequent base are each labeled with one or more detectable labels, wherein the subsequent base is the same as the first base, optionally wherein the first base and the subsequent base are A, T, C or G.

36. The method of claim 33 or 34, wherein the base-by-base sequencing comprises: contacting the biological sample with a first nucleotide mixture, wherein the nucleotide comprising the first base is not detectably labeled and the other nucleotides in the first nucleotide mixture are each labeled with one or more detectable labels, and contacting the biological sample with a subsequent nucleotide mixture, wherein the nucleotide comprising the subsequent base is not detectably labeled and the other nucleotides in the subsequent nucleotide mixture are each labeled with one or more detectable labels, wherein the subsequent base is different from the first base.

37. The method of claim 36, wherein the biological sample is contacted with two or more of the following nucleotide mixtures in any order in consecutive cycles: Nucleotide mixture 1, wherein nucleotides comprising G are not detectably labeled and nucleotides comprising A, C or T are detectably labeled; nucleotide mixture 2, wherein nucleotides comprising T are not detectably labeled and nucleotides comprising A, C, or G are detectably labeled; Nucleotide mixture 3, wherein nucleotides comprising C are not detectably labeled, and nucleotides comprising A, G, or T are detectably labeled; and Nucleotide mixture 4, wherein nucleotides comprising A are not detectably labeled, and nucleotides comprising G, C, or T are detectably labeled.

38. The method of claim 37, wherein independently of each other, each nucleotide mixture is contacted with the biological sample in one or more cycles, wherein the cycles are continuous or non-continuous.

39. The method of claim 37 or 38, wherein independently of each other, in each nucleotide mixture, the detectably labeled nucleotides comprise: i) three different colored fluorescent labels, one for each of the three bases; ii) two different colors of fluorescent labeling, one color for each of two of the three bases, with the nucleotide containing the remaining base labeled with two colors; or iii) a fluorescent label of the same color, wherein the fluorescent label on the nucleotide comprising one of the three bases is configured to be cleaved, and the nucleotide comprising another of the three bases is configured to be labeled with the fluorescent label.

40. The method of claim 36, wherein the biological sample is contacted with two or more of the following nucleotide mixtures in any order in consecutive cycles: Nucleotide mixture 1, wherein nucleotides comprising G or A are not detectably labeled, and nucleotides comprising C or T are detectably labeled; nucleotide mixture 2, wherein nucleotides comprising G or T are not detectably labeled and nucleotides comprising C or A are detectably labeled; nucleotide mixture 3, wherein nucleotides comprising G or C are not detectably labeled and nucleotides comprising A or T are detectably labeled; nucleotide mixture 4, wherein nucleotides comprising C or A are not detectably labeled and nucleotides comprising G or T are detectably labeled; Nucleotide mixture 5, wherein nucleotides comprising C or T are not detectably labeled, and nucleotides comprising G or A are detectably labeled; and Nucleotide mixture 6, wherein nucleotides comprising A or T are not detectably labeled, and nucleotides comprising G or C are detectably labeled.

41. The method of any one of claims 1-40, wherein the first priming site and the second priming site are different, and the method comprises: b1) hybridizing the first sequencing primer to the first priming site and performing base-by-base sequencing to generate an extension product of the first sequencing primer and the first signal code sequence; b2) removing, cutting or blocking the extension product of the first sequencing primer in b1); as well as b3) hybridizing the second sequencing primer to the second priming site and performing base-by-base sequencing to generate an extension product of the second sequencing primer and the second signal code sequence.

42. The method of claim 41, wherein probes for a first plurality of analytes, or products thereof, share a common first priming site, and probes for a second plurality of analytes, or products thereof, share a common second priming site.

43. The method of claim 42, wherein the second plurality of analytes comprises two or more different analytes that are different from the two or more different analytes in the first plurality of analytes.

44. The method according to any one of claims 1 to 43, wherein: In a), the biological sample is contacted with a plurality of probes each configured to bind directly or indirectly to a different analyte, and Each probe or its product contains a different combination of priming sites.

45. The method of claim 44, wherein: The first probe or its product comprises a first combination of different priming sites including the first priming site, and / or The second probe or its product comprises a second combination of different priming sites including the second priming site.

46. ​​The method of claim 45, wherein: In a), the biological sample is contacted with a third probe that directly or indirectly binds to a third analyte, The third probe or its product comprises a third combination of different priming sites including the first priming site, the second priming site and / or a third priming site.

47. The method of claim 46, wherein any two or more of the first combination, the second combination, and the third combination share one or more common priming sites.

48. The method according to any one of claims 44 to 47, comprising: b') contacting the biological sample with the first sequencing primer for base-by-base sequencing, so that the first sequencing primer hybridizes with the first priming site in the first probe or its product and in one or more other probes or their products, and generates an extension product of the first sequencing primer; b”) removing, cutting or blocking the extension product of the first sequencing primer in b’); as well as b'') contacting the biological sample with the second sequencing primer for base-by-base sequencing, thereby hybridizing the second sequencing primer to the second priming site in the second probe or its product and in one or more other probes or their products, and generating an extension product of the second sequencing primer.

49. The method according to claim 48, wherein the base-by-base sequencing in b') is performed by: contacting the biological sample with nucleotides in continuous cycles, detecting a signal associated with each successive cycle of nucleotide incorporation or incorporation, and Signal code sequences are generated for a first plurality of analytes.

50. The method according to claim 48 or 49, wherein the base-by-base sequencing in b″′) is performed by: contacting the biological sample with nucleotides in continuous cycles, detecting a signal associated with each successive cycle of nucleotide incorporation or incorporation, and Signal code sequences are generated for the second plurality of analytes.

51. The method of claim 50, wherein the first plurality of analytes and the second plurality of analytes comprise one or more common analytes.

52. The method of claim 50, wherein the first plurality of analytes and the second plurality of analytes do not comprise common analytes.

53. The method of any one of claims 1-52, wherein each analyte is independently a nucleic acid analyte or a non-nucleic acid analyte.

54. The method of any one of claims 1-53, wherein each probe is independently i) a primary probe that binds directly to its corresponding analyte, or ii) a probe that binds directly or indirectly to the primary probe.

55. A method according to claim 54, wherein the primary probe and the probe that directly or indirectly binds to the primary probe are independently selected from: a probe comprising a 3' or 5' protrusion, optionally wherein the 3' or 5' protrusion comprises one or more barcode sequences; a probe comprising a 3' protrusion and a 5' protrusion, optionally wherein the 3' protrusion and the 5' protrusion each independently comprise one or more barcode sequences; a circular probe; a circularizable probe or probe set; a probe or probe set comprising a split hybridization zone configured to hybridize to a splint, optionally wherein the split hybridization zone comprises one or more barcode sequences; and combinations thereof.

56. The method of any one of claims 1-55, wherein the product of each probe is a rolling circle amplification (RCA) product generated in situ in the biological sample or a component of a branched structure formed in situ in the biological sample.

57. The method according to any one of claims 1-56, wherein in b), the base-by-base sequencing is performed in situ in the biological sample.

58. A method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: The biological sample is a cell or tissue sample, The biological sample comprises a first analyte and a second analyte at a first location and a second location in the biological sample, respectively, The first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively. The first probe or its product comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and The second probe or a product thereof comprises i) a second priming site for a second sequencing primer and ii) a second identifier sequence associated with the second analyte; b) performing base-by-base sequencing of the first identifier sequence using the first sequencing primer to generate a first signal code sequence, the first signal code sequence comprising a signal code detected at the first position in consecutive cycles, wherein the signal code corresponds to a signal (ON signal), an absence of a signal (OFF signal), or a combination thereof; c) subsequently performing base-by-base sequencing of the second identifier sequence using the second sequencing primer to generate a second signal code sequence, the second signal code sequence comprising the signal code detected at the second position in a further consecutive cycle; wherein in at least one or more of said continuous cycles in b) and said further continuous cycles in c), an OFF signal is detected; and d) detecting the first identifier sequence and the second identifier sequence at the first location and the second location in the biological sample based at least in part on the first signal code sequence and the second signal code sequence, respectively.

59. The method of claim 58, wherein the first identifier sequence and the second identifier sequence are identical.

60. The method of claim 58, wherein the first identifier sequence and the second identifier sequence are different.

61. The method of any one of claims 58-60, wherein the first identifier sequence and the second identifier sequence comprise a barcode sequence or a complement thereof assigned to the first analyte and the second analyte, respectively.

62. A method according to claim 61, wherein the first priming site and / or the first barcode sequence is assigned to the first analyte, and the second priming site and / or the second barcode sequence is assigned to the second analyte based on a decision rule designed to minimize the maximum predicted density of ON signals detected in each of one or more of the consecutive cycles.

63. The method of claim 61, wherein the assigning comprises an assignment based on expression data for the first analyte and the second analyte.

64. The method of claim 63, wherein the assigning comprises assigning based on expression data of the first analyte and the second analyte in clustered cell types.

65. The method of claim 64, wherein the clustered cell types represent the distribution of cell types present in the biological sample.

66. The method of any one of claims 63-65, wherein the expression data for the first analyte and the second analyte at least partially overlap.

67. The method of any one of claims 63-66, wherein the expression data of the first analyte and the second analyte comprises bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single cell gene expression data, single cell protein expression data, or any combination thereof.

68. The method of any one of claims 58-67, wherein the nucleotides in the first barcode sequence or the second barcode sequence detected in a particular cycle correspond to a signal code comprising an ON signal.

69. The method of any one of claims 58-68, wherein the nucleotides in the first barcode sequence or the second barcode sequence detected in a particular cycle correspond to a signal code comprising an OFF signal.

70. The method of claim 68 or claim 69, wherein the nucleotides in the first barcode sequence or the second barcode sequence detected in the particular cycle correspond only to ON signals.

71. The method of claim 69 or claim 70, wherein the nucleotides in the first barcode sequence or the second barcode sequence detected in the particular cycle correspond only to OFF signals.

72. A method for decoding an identifier sequence in a biological sample while minimizing optical crowding, the method comprising: a) contacting the biological sample with a first probe and a second probe, wherein: The biological sample is a cell or tissue sample, The biological sample comprises a first analyte and a second analyte at a first location and a second location in the biological sample, respectively, The first probe and the second probe bind directly or indirectly to the first analyte and the second analyte, respectively. The first probe or its product comprises i) a first priming site of a first sequencing primer and ii) a first identifier sequence associated with the first analyte, and The second probe or a product thereof comprises i) a second priming site for a second sequencing primer and ii) a second identifier sequence associated with the second analyte; b) performing base-by-base sequencing of the first identifier sequence and the second identifier sequence using the first sequencing primer and the second sequencing primer, thereby generating a first signal code sequence and a second signal code sequence, each signal code sequence comprising a signal code corresponding to a signal (ON signal), a signal absence (OFF signal), or a combination thereof detected at the first position and the second position, respectively, in consecutive sequencing cycles, wherein the base-by-base sequencing comprises contacting the biological sample with a polymerase and a mixture of nucleotides in each successive cycle, wherein: i) at least one nucleotide (e.g., A, T / U, CC, or G) is not detectably labeled; ii) at least one nucleotide (e.g., A, T / U, C, or G) is omitted from the mixture; or iii) at least one nucleotide (e.g., A, T / U, C, or G) is not detected in c); and c) detecting the first identifier sequence and the second identifier sequence in the biological sample based at least on the first signal code sequence and the second signal code sequence.

73. The method of claim 72, wherein the first analyte and the second analyte are the same or different.

74. A method according to claim 72 or 73, wherein the first identifier sequence and the second identifier sequence are different.

75. The method of any one of claims 72-74, wherein the first identifier sequence and the second identifier sequence comprise an analyte sequence or a complement thereof.

76. The method of any one of claims 72-75, wherein the first identifier sequence and the second identifier sequence comprise a barcode sequence or a complement thereof assigned to the first analyte and the second analyte, respectively.

77. The method of claim 76, comprising assigning a first barcode sequence to the first analyte and assigning a second barcode sequence to the second analyte.

78. The method of claim 77, wherein assigning the barcode sequence is based on a decision rule designed to minimize the maximum predicted density of ON signals detected in each of one or more of the consecutive cycles.

79. The method of claim 77, wherein the decision rule to assign the first barcode sequence to the first analyte and the second barcode sequence to the second analyte comprises an assignment based on expression data for the first analyte and the second analyte.

80. The method of claim 79, wherein assigning the first barcode sequence to the first analyte and assigning the second barcode sequence to the second analyte comprises assignments based on expression data of the first analyte and the second analyte in clustered cell types.

81. The method of claim 80, wherein the clustered cell types represent the distribution of cell types present in the biological sample.

82. The method of any one of claims 79-81, wherein the expression data for the first analyte and the second analyte at least partially overlap.

83. The method of any one of claims 79-82, wherein the expression data of the first analyte and the second analyte comprises bulk gene expression data, bulk protein expression data, spatial gene expression data, spatial protein expression data, single cell gene expression data, single cell protein expression data, or any combination thereof.

84. The method of any one of claims 72-83, wherein the nucleotide mixture comprises at least two nucleotides that are not detectably labeled.

85. The method of any one of claims 72-83, wherein the biological sample is contacted with two or more of the following nucleotide mixtures in any order in consecutive sequencing cycles: Nucleotide mixture 1, wherein nucleotides comprising G are not detectably labeled and nucleotides comprising A, C or T are detectably labeled; nucleotide mixture 2, wherein nucleotides comprising T are not detectably labeled and nucleotides comprising A, C, or G are detectably labeled; Nucleotide mixture 3, wherein nucleotides comprising C are not detectably labeled, and nucleotides comprising A, G, or T are detectably labeled; and Nucleotide mixture 4, wherein nucleotides comprising A are not detectably labeled, and nucleotides comprising G, C, or T are detectably labeled.

86. The method of claim 85, wherein independently of each other, each nucleotide mixture is contacted with the biological sample in one or more cycles, wherein the cycles are continuous or non-continuous.

87. The method of claim 85 or 86, wherein independently of each other, in each nucleotide mixture, the detectably labeled nucleotides comprise: i) three different colored fluorescent labels, one for each of the three bases; ii) two different colors of fluorescent labeling, one color for each of two of the three bases, with the nucleotide containing the remaining base labeled with two colors; or iii) a fluorescent label of the same color, wherein the fluorescent label on the nucleotide comprising one of the three bases is configured to be cleaved, and the nucleotide comprising another of the three bases is configured to be labeled with the fluorescent label.

88. The method of any one of claims 72 to 83, wherein the biological sample is contacted with two or more of the following nucleotide mixtures in any order in consecutive cycles: Nucleotide mixture 1, wherein nucleotides comprising G or A are not detectably labeled, and nucleotides comprising C or T are detectably labeled; nucleotide mixture 2, wherein nucleotides comprising G or T are not detectably labeled and nucleotides comprising C or A are detectably labeled; nucleotide mixture 3, wherein nucleotides comprising G or C are not detectably labeled and nucleotides comprising A or T are detectably labeled; as well as nucleotide mixture 4, wherein nucleotides comprising C or A are not detectably labeled and nucleotides comprising G or T are detectably labeled; nucleotide mixture 5, wherein nucleotides comprising C or T are not detectably labeled, and nucleotides comprising G or A are detectably labeled; as well as Nucleotide mixture 6, wherein nucleotides comprising A or T are not detectably labeled, and nucleotides comprising G or C are detectably labeled.

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