Compositions for spatial omics

Abstract

The present invention relates to compositions comprising a population of capture barcode units and a population of localization barcode units, and their use for determining the spatial location of any analyte from a biological sample.

Description

Technical Field

[0001] This invention relates to the spatial positioning of barcode units and spatial omics. Background Technology

[0002] Omics is a powerful tool that can deeply characterize cells or cell populations by dynamically identifying and quantifying their biological components, such as RNA and proteins.

[0003] There is growing interest in further developing these methods when applied to tissues or other complex biological samples, in order to map changes in the transcriptome, proteome, metabolome, and other sequences based on the location of cells within the sample. In other words, spatial omics aims to add spatial information to omics analysis.

[0004] Among existing technologies, there are several methods that can be used to implement space omics.

[0005] For example, the Visium spatial gene expression technology developed by 10x Genomics (see, for example, 10xGenomics, 2019, “Visium Spatial Gene Expression Solution”; or WO2021 / 102039) relies on oligonucleotide microarrays. Typically, microarrays are formed by “spotting” droplets containing oligonucleotides onto a glass slide; the position of each oligonucleotide is known, thus enabling the reconstruction of the spatial information of the RNA captured by the oligonucleotides.

[0006] However, this method is limited by the resolution of the microarray itself due to the local diffusion of droplets during the spotting process.

[0007] Other methods aim to overcome this limitation. For example, Rodriques et al. (Science vol.363,6434(2019):1463-1467) described a slide-seq method in which barcode beads are randomly deposited on a slide and then indexed, i.e., the barcodes are sequenced, to associate oligonucleotides with a given location on the slide. The slide is then contacted with a sample (tissue section); the captured analytes are then sequenced. Another example is Liao et al. 2023 (bioRxiv preprint doi:https: / / doi.org / 10.1101 / 2023.04.28.538364), which described a stereo-seq method based on a similar approach, except that the barcode beads are replaced by DNA nanospheres.

[0008] While these methods improve resolution, they inevitably involve indexing the position of each oligonucleotide on the slide with a first sequencing step, followed by a second sequencing once the analyte is captured, thus complicating the process.

[0009] Patent US, 11,624,088 provides an alternative that uses beads containing two types of barcoded oligonucleotides on the same bead: one type is responsible for capturing the analyte, and the other type is responsible for linking the beads together, thereby associating barcode information on two or more different beads and reconstructing the relative spatial positions of the barcoded bead array. Since the spatial information is inferred from the connections between the beads, rather than through pre-indexing, this method avoids the need for two sequencing steps.

[0010] However, in this method, nothing prevents the self-hybridization of the linked oligonucleotides. This is important because a large portion of the library generated for sequencing these barcode beads will not provide spatial information. Furthermore, having two oligonucleotides per bead increases manufacturing cost and complexity.

[0011] Also worth mentioning is another method called GPS-seq, disclosed by Greenstreet et al. (bioRxiv preprint doi:https: / / doi.org / 10.1101 / 2022.03.22.485380). This method is based on a monolayer of barcode-laden beads that are contacted with a hydrogel sheet containing dots (or satellites) of spatially indexed oligonucleotides, and then with a biological sample. The spatial index diffuses from its initial spatial location in the hydrogel onto the beads. Therefore, proximity relationships are reconstructed based on the shared spatial index.

[0012] However, this approach appears to be technically challenging and difficult to implement. In particular, the quality of the resolution depends heavily on the uniformity of spatial indexed oligonucleotide diffusion, a parameter notoriously difficult to control.

[0013] Therefore, there is an unmet need for space omics, particularly space transcriptomics, for improved solutions that are easy to manufacture and implement while maintaining high resolution. This invention provides such a solution. Summary of the Invention

[0014] Therefore, the present invention relates to a composition comprising:

[0015] - A group of capture barcode units, wherein each capture barcode unit contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0016] - A group of positioning barcode units, wherein each positioning barcode unit contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a first linker sequence capable of hybridizing on the capturing oligonucleotide;

[0017] Each barcode unit contains a different barcode sequence, and all oligonucleotides on the same barcode unit contain the same barcode sequence.

[0018] In some embodiments, the capture sequence on the first capturing oligonucleotide cannot hybridize with the capture sequence of the second capturing oligonucleotide, and the first linker sequence on the first localizing oligonucleotide cannot hybridize with the first linker sequence of the second localizing oligonucleotide.

[0019] In some implementations, the capture sequence cannot self-hybridize. In some implementations, the first ligation sequence cannot self-hybridize.

[0020] In some implementations, the first linker sequence of the localized oligonucleotide is capable of hybridizing to the capture sequence of the capture oligonucleotide.

[0021] In some embodiments, the capturing oligonucleotide includes a second linker sequence, wherein the second linker sequence is different from the barcode sequence and the capturing sequence, and wherein the first linker sequence that locates the oligonucleotide is capable of hybridizing to the second linker sequence of the capturing oligonucleotide.

[0022] In some implementations, the capture sequence is the same for all captured barcode units, and the first connection sequence is the same for all positioned barcode units.

[0023] In some implementations, the capture sequence comprises a poly(dT) sequence or a poly(dU) sequence, and wherein the first connection sequence comprises a poly(dA) sequence.

[0024] In some implementations, the capture oligonucleotide and / or localization oligonucleotide also contain at least one PCR primer sequence.

[0025] In some implementations, each oligonucleotide from the group of captured oligonucleotides and / or the group of localized oligonucleotides also contains at least one unique molecular identifier (UMI).

[0026] In some implementations, the capturing oligonucleotide and / or positioning oligonucleotide also include at least one adapter.

[0027] In some embodiments, the capturing oligonucleotide comprises, in order from 5'-P to 3'-OH: optionally at least one adapter, optionally at least one PCR primer sequence, barcode sequence, optionally at least one UMI, and a capturing sequence; and wherein the positioning oligonucleotide comprises, in order from 5'-P to 3'-OH: optionally at least one adapter, optionally at least one PCR primer sequence, barcode sequence, and a first ligation sequence.

[0028] In some implementations, the barcode unit is a composite barcode unit.

[0029] In some implementations, the barcode unit is a bead or a DNA nanosphere.

[0030] In some implementations, the capture barcode unit and / or positioning barcode unit have different sizes.

[0031] In some implementations, groups of capture barcode units and groups of positioning barcode units are assembled on a substrate.

[0032] In some implementations, the captured barcode unit comes into contact with at least one, preferably at least two, more preferably at least three positioning barcode units.

[0033] The present invention also relates to a method for determining the spatial location of a group of captured barcode units, comprising the following steps:

[0034] (i) to bring a group of capturing barcode units from the composition according to the invention into contact with a group of positioning barcode units;

[0035] (ii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide;

[0036] (iii) At the multiple contact points formed in step (ii), at least one linker sequence of the localized oligonucleotide is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0037] (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acid sequences comprising a barcode sequence of a capture barcode unit and a barcode sequence of a positioning barcode unit;

[0038] (v) Optionally, the multiple nucleic acid sequences obtained in step (iv) are amplified to obtain multiple amplicones;

[0039] (vi) Sequencing the multiple nucleic acid sequences obtained in step (iv) or the multiple amplicon obtained in step (v);

[0040] (vii) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0041] (viii) Inferring the relative position of each captured barcode cell and / or each positioned barcode cell; and

[0042] (ix) Optionally, infer the absolute position of each captured barcode unit and / or each positioned barcode unit.

[0043] The present invention also relates to a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes, the method comprising the steps of:

[0044] (i) To bring the group of capturing barcode units from the composition according to the invention into contact with the group of positioning barcode units;

[0045] (ii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide;

[0046] (iii) At the multiple contact points formed in step (ii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0047] (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acid sequences comprising a barcode sequence of a capture barcode unit and a barcode sequence of a positioning barcode unit;

[0048] (v) Optionally, the multiple nucleic acid sequences obtained in step (iv) are amplified to obtain multiple amplicones;

[0049] (vi) Sequencing the multiple nucleic acid sequences obtained in step (iv) or the multiple amplicon obtained in step (v);

[0050] (vii) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0051] (viii) Inferring the relative position of each captured barcode cell and / or each positioned barcode cell; and

[0052] (ix) Optionally, infer the absolute position of each captured barcode unit and / or each positioned barcode unit.

[0053] And it also includes the following steps:

[0054] a) In any step prior to step (v), bring the group of captured barcode units and the group of positioned barcode units into contact with the biological sample;

[0055] b) In any step after step (a) and before step (v), multiple analytes are captured from a biological sample by binding multiple analytes to a capture sequence of multiple capture barcode units;

[0056] c) Optionally, after step (b), the capture sequence is extended onto the sequences of multiple analytes to obtain multiple nucleic acids containing a barcode sequence of the capture barcode unit and an analyte sequence;

[0057] d) Optionally, after step (c), multiple nucleic acids are amplified to obtain multiple amplicones;

[0058] e) Sequencing the multiple nucleic acid sequences obtained in step (c) or the multiple amplicon obtained in step (d);

[0059] f) In any step after step (viii), infer the relative position of each analyte; and optionally, infer the absolute position of each analyte in the biological sample.

[0060] In some implementations, the biological sample is a tissue, tissue slice, monolayer of cells, multilayer of cells including organoids, or a single cell, including syncytiosomes.

[0061] definition

[0062] In this disclosure, unless the context clearly indicates otherwise, elements without a quantifier include both singular and plural forms. Thus, for example, references to "reagent" include both a single reagent and multiple such reagents.

[0063] In this invention, the following terms have the following meanings:

[0064] The term "about" or "approximately" can refer to an acceptable range of error for a particular value as determined by a person skilled in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can refer to within one or more standard deviations. Alternatively, "about" preceding a number means ±10% of that numerical value. Or, particularly in biological systems or processes, the term can refer to within orders of magnitude, within 5 times, and more preferably within 2 times. When a particular value is described in an application and claims, unless otherwise stated, the term "about" should be assumed to mean an acceptable range of error for that particular value.

[0065] "Amplification" refers to the process of generating multiple copies of the desired template sequence, i.e., at least two copies. Techniques for amplifying nucleic acids are well-known to those skilled in the art, including specific amplification methods and random amplification methods.

[0066] A "barcode" is a sequence of biomolecules, such as nucleic acids or amino acid sequences, preferably nucleic acid sequences. Barcodes can be attached to analytes in a reversible or irreversible manner. They can be added to fragments of molecules such as DNA or RNA before and / or during sequencing to convey information about the origin or source of the DNA or RNA molecule.

[0067] A "barcode unit" refers to an entity (body) containing at least one copy of a barcode sequence. In one embodiment, the barcode unit comprises a core, a support, or a substrate, and at least one oligonucleotide containing at least one copy of the barcode sequence, wherein the oligonucleotide is attached to or bound to the core, support, or substrate. The barcode unit is typically a barcode-bearing bead. In another embodiment, the barcode unit consists of multiple repeats of a nucleic acid sequence containing a barcode sequence, preferably consecutive repeats, and typically the barcode unit is a DNA nanosphere. In a preferred embodiment, the barcode unit has an overall spherical shape. In a preferred embodiment, the barcode unit is a synthetic barcode unit.

[0068] "Barcoding" refers to attaching a barcode, such as a nucleic acid barcode, to an analyte or nucleic acid sequence through primer template annealing, primer-dependent DNA synthesis, and / or ligation. In some embodiments, the analyte is mRNA. In some embodiments, the nucleic acid sequence is an oligonucleotide, preferably a capture oligonucleotide or a localizing oligonucleotide according to the present invention.

[0069] "Beads" refer to particles that can be spherical (e.g., microspheres) or have irregular shapes. The diameter of beads can be as small as about 10 nm or as large as about a few millimeters.

[0070] A "library" refers to a collection, group, plurality, or cluster of nucleic acid fragments used for sequencing. In some embodiments, the nucleic acid fragments contain analyte information, such as complementary DNA reverse transcribed from mRNA to cDNA. In some embodiments, the nucleic acid fragments contain location barcode information from all or part of the contact points between capture barcode units and location barcode units. In some embodiments, the nucleic acid fragments contain both analyte information and location barcode information.

[0071] "Clonal copy" refers to a group of identical copies of the oligonucleotides of each barcode unit. As those skilled in the art will appreciate, clonal copies can also be nearly identical, possessing sufficient identity to hybridize with the same sequence and / or perform the same function (e.g., initiation, recognition, etc.). In a preferred embodiment, the barcode sequences of the two clonal copies share at least 70% sequence identity, preferably at least 80% sequence identity, preferably at least 90% sequence identity, preferably at least 93% sequence identity, and even more preferably at least 95% sequence identity. In a preferred embodiment, at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, and even more preferably at least 95% of the clonal copies are identical.

[0072] "Complementary" or "complementary" refers to a polynucleotide sequence capable of forming a base pair with another polynucleotide sequence via hydrogen bonds, preferably a Watson-Crick base pair, unless otherwise explicitly specified. For example, guanine (G) is the Watson-Crick complementary base of cytosine (C), and adenine (A) is the Watson-Crick complementary base of thymine (T) and uracil (U). Other types of base pairing include, but are not limited to, wobble base pairing. For example, inosine (I) is a nucleoside capable of wobble base pairing with any native base (C, T, U, A, or G). Depending on the context, and as will be clearly understood by those skilled in the art, "complementary" can mean "reverse complementary," meaning the sequence is complementary when read in the opposite direction.

[0073] "Reagent kit" or "kit" refers to any article (e.g., packaging or at least one container) containing the different reagents required to perform the methods described herein, packaged in a manner that allows for its transport and storage. The terms "reagent kit" and "kit" should cover physically separate components intended for individual use but functionally related to each other. Reagent kits may be promoted, distributed, or sold as units for performing the methods described herein. Furthermore, any or all reagent kit reagents may be provided in containers that protect them from external environmental influences, such as sealed and sterile containers. Reagent kits may also include a package insert describing the kit and its usage.

[0074] "Lysis" refers to the disruption of a biological sample, particularly the cells contained within it, to obtain materials that would otherwise be unavailable. Typically, lysis involves the rupture of the cell membrane and may include the nuclear membrane. Lysis methods are well known to those skilled in the art and include, but are not limited to, protein hydrolysis, chemical lysis, thermal lysis, mechanical lysis, and osmotic lysis. In some embodiments, lysis constitutes the permeation of the biological membrane. In a preferred embodiment, lysis does not alter the spatial position of the analyte.

[0075] "Nucleic acid sequence primers" or simply "primers" are oligonucleotides that, under appropriate conditions, can hybridize or anneal with nucleic acids and serve as the starting site for nucleotide polymerization. These appropriate conditions include, for example, the presence of nucleoside triphosphates and an enzyme for polymerization, such as DNA polymerase, RNA polymerase, or reverse transcriptase, in a suitable buffer and at a suitable temperature.

[0076] "Oligonucleotide" refers to a polymer of nucleotides, typically a single-chain nucleotide polymer. In some embodiments, the oligonucleotide comprises 2 to 500 nucleotides, preferably 10 to 150 nucleotides, and more preferably 20 to 100 nucleotides. The oligonucleotide can be synthetic or enzymatically prepared. In some embodiments, the oligonucleotide may comprise ribonucleotide monomers, deoxyribonucleotide monomers, or a mixture of both. As those skilled in the art will appreciate, nucleotide analogs (e.g., peptide nucleic acids, locked nucleic acids, glycol nucleic acids, etc.) can replace some or all of the nucleotides in the oligonucleotide.

[0077] The terms "PCR primer sequence," "primer sequence," or "universal tag sequence" are used interchangeably to refer to nucleic acid sequences that facilitate amplification, preferably PCR amplification and further sequencing of nucleic acid sequences extracted from or derived from biological units. In one implementation, the PCR primers lack homology with the template sequence.

[0078] Polymerase chain reaction, abbreviated as "PCR", refers to methods including but not limited to: allele-specific PCR, asymmetric PCR, hot-start PCR, sequence-specific PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, quantitative PCR, reverse transcription PCR, and / or landing PCR. DNA polymerases suitable for amplifying nucleic acids include, but are not limited to: Taq polymerase Stoffel fragment, Taq polymerase, Advantage DNA polymerase, AmpliTaq, AmpliTaq Gold, Titanium Taq polymerase, KlenTaq DNA polymerase, Platinum Taq polymerase, Accuprime Taq polymerase, Pfu polymerase, Pfu polymerase turbo, Vent polymerase, Vent exo polymerase, Pwo polymerase, 9 Nm DNA polymerase, Therminator, Pfx DNA polymerase, Expand DNA polymerase, rTth DNA polymerase, DyNAzyme-EXT polymerase, Klenow fragment, DNA polymerase I, T7 polymerase, Sequenase™, Tfi polymerase, T4 DNA polymerase, Bst polymerase, Bca polymerase, BSU polymerase, phi-29 DNA polymerase, and DNA polymerase β, or modified forms thereof. In one embodiment, the DNA polymerase has 3' to 5' proofreading, i.e., exonuclease activity. In one embodiment, the DNA polymerase has 5' to 3' proofreading, i.e., exonuclease activity. In another embodiment, the DNA polymerase has strand displacement activity, i.e., when the DNA polymerase is bound to and in proximity to template-dependent nucleic acid synthesis, it causes the pairing nucleic acid to dissociate from its complementary strand in the 5' to 3' direction. For example, E. coli DNA polymerase I, the Klenow fragment of DNA polymerase I, T7 or T5 phage DNA polymerases, and the DNA polymerase of HIV reverse transcriptase are enzymes that possess both polymerase and strand displacement activities. For example, helicase reagents can be used in combination with inducers that do not possess strand displacement activity to produce a strand displacement effect, i.e., the substitution of nucleic acid is coupled to the synthesis of a nucleic acid with the same sequence. Similarly, proteins such as RecA or single-stranded DNA-binding proteins (SSBs) from E. coli or another organism can be used to bind with other inducers to produce or promote strand displacement.

[0079] A “group” or “plurality” refers to a group of barcode units that contain at least two of the same species, type, or model. In one implementation, all units in the group are identical. In another implementation, units in the group may contain minor structural differences (e.g., labels, markers, unique unit identifiers, or any suitable means to distinguish one unit from another within the group) but share the same functionality and / or characteristics.

[0080] "Primer-guided extension" refers to any method known in the art in which primers are used to initiate the replication of a nucleic acid sequence in the amplification of linear or logarithmic nucleic acid molecules. Primer-guided extension can be accomplished by any of several protocols known in the art, including but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), and strand displacement amplification (SDA). "Primer-guided extension" can be performed using the DNA polymerases described herein.

[0081] "Random amplification techniques" include, but are not limited to, multiple displacement amplification (MDA), random PCR, random amplification of polymorphic DNA (RAPD), or multiple annealing circular amplification (MALBAC). In contrast, "specific amplification techniques" include, but are not limited to, methods requiring temperature cycling (e.g., polymerase chain reaction (PCR), ligase chain reaction, transcription-based amplification) and / or isothermal amplification systems (e.g., self-sustaining sequence replication, replicase systems, helicase systems, strand displacement amplification, rolling circle amplification, and NASBA).

[0082] "Reverse transcription" refers to the replication of RNA to produce a complementary DNA strand (cDNA), particularly using an RNA-directed DNA polymerase (e.g., reverse transcriptase, RT). Reverse transcription of RNA can be performed using techniques well known to those skilled in the art, using a reverse transcriptase and a mixture of four deoxyribonucleoside triphosphates (dNTPs): deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and (deoxy)thymidine triphosphate (dTTP). In some embodiments, reverse transcription of RNA includes a first step of first-strand cDNA synthesis. Methods for first-strand cDNA synthesis are well known to those skilled in the art. First-strand cDNA synthesis reactions can use combination of sequence-specific primers, oligomeric (dT) primers, or random primers. Examples of reverse transcriptases include, but are not limited to, MMLV reverse transcriptase, SuperScript II (Invitrogen), SuperScript III (Invitrogen), SuperScript IV (Invitrogen), Maxima (ThermoFisher Scientific), ProtoScript II (New England Biolabs), and PrimeScript (ClonTech). In some embodiments, reverse transcription can be a primer-guided extension. In these embodiments, reverse transcription requires a single-stranded DNA molecule containing at least one probe sequence capable of hybridizing with a portion of the RNA and serving as an amplification primer site. The single-stranded DNA molecule may also contain other functional sequences. In some embodiments, the single-stranded DNA molecule is an oligonucleotide that binds to or otherwise attaches to a particle as described herein, particularly containing a unique identifier, such as a nucleic acid barcode. cDNA polymerization begins at the amplification primer site, and the resulting cDNA molecule (the so-called "first-strand cDNA molecule") contains (i) the original single-stranded DNA molecule, which, when present, includes a functional sequence, and (ii) a complementary DNA sequence to the hybridized RNA.

[0083] "Second-strand synthesis" refers to the synthesis of a sequence complementary to a first-strand cDNA molecule to produce a DNA molecule that is at least partially double-stranded. Second-strand synthesis can be performed to obtain cDNA replicas in solution without intermediate steps, such as extraction steps (e.g., in the case of chemical contamination) or molecular cleavage (e.g., in the case of particle-grafted cDNA). In some embodiments, second-strand synthesis can be a primer-guided extension. In these embodiments, second-strand synthesis requires a single-stranded DNA molecule that contains a probe sequence at least at its 3' end, which is capable of hybridizing with a portion of the first-strand cDNA molecule and serving as an amplification primer site. In some embodiments, these primers are random oligonucleotides. In some embodiments, the primers are specific to some target sequence. The single-stranded DNA molecule may also contain other functional sequences in the 5' direction of the probe sequence, such as unique molecular identifiers (UMIs), PCR primers, and / or nucleic acid barcodes. In these implementations, second-strand synthesis begins at the amplification primer site, and the resulting DNA molecule (the so-called "second-strand cDNA molecule") contains (i) the original single-stranded DNA molecule, which includes a functional sequence when present, and (ii) the complementary DNA sequence of the first-strand cDNA molecule. Methods for second-strand cDNA synthesis are well known to those skilled in the art and can be implemented using DNA-dependent DNA polymerases, such as the Taq polymerase Stoffel fragment, Taq polymerase, Advantage DNA polymerase, AmpliTaq, AmpliTaqGold, Titanium Taq polymerase, KlenTaq DNA polymerase, Platinum Taq polymerase, Accuprime Taq polymerase, Pfu polymerase, Pfu polymerase turbo, Vent polymerase, Vent exo polymerase, Pwo polymerase, 9 Nm DNA polymerase, Therminator, Pfx DNA polymerase, Expand DNA polymerase, rTth DNA polymerase, DyNAzyme-EXT polymerase, Klenow fragment, DNA polymerase I, T7 polymerase, Sequenase™, Tfi polymerase, T4 DNA polymerase, Bst polymerase, Bca polymerase, BSU polymerase, phi-29 DNA polymerase, and DNA polymerase β, or modified forms thereof. In some embodiments, the DNA-dependent DNA polymerase has 3' to 5' proofreading activity. In some embodiments, the DNA-dependent DNA polymerase has 5' to 3' proofreading activity. In some embodiments, the DNA-dependent DNA polymerase has 5' to 3' exonuclease activity.In some implementations, DNA-dependent DNA polymerases possess strand displacement activity, meaning that when bound to and near a template-dependent nucleic acid synthesizer, the DNA-dependent DNA polymerase causes the pairing nucleic acid to dissociate from its complementary strand in the 5' to 3' direction. Examples of DNA-dependent DNA polymerases such as E. coli DNA polymerase I, the Klenow fragment of DNA polymerase I, T7 or T5 phage DNA polymerases, and HIV reverse transcriptase are enzymes that possess both polymerase and strand displacement activities.

[0084] "Spatial location" or "location" refers to the position or coordinates of an object (such as an analyte or barcode unit) in two-dimensional or planar space (2D) or three-dimensional space (3D). Without limitation, three-dimensional space can be a volume or a single plane that includes some degree of irregularity (e.g., protrusions, holes, etc.) along the Z-axis. Alternatively, a location in three-dimensional space can refer to a 3D projection of a 2D location, typically a volume reconstructed or extrapolated from a set of continuous 2D information.

[0085] A "template" or "template sequence" refers to the nucleic acid sequence that needs to be amplified. The template can contain DNA or RNA. The template sequence can be known or unknown. Detailed Implementation

[0086] This invention relates to a composition comprising:

[0087] - A group of capture barcode units, wherein each capture barcode unit contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0088] - A group of positioning barcode units, wherein each positioning barcode unit contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of hybridizing on the capturing oligonucleotide;

[0089] In a preferred embodiment, each barcode unit contains a different barcode sequence, and all oligonucleotides on the same barcode unit contain the same barcode sequence.

[0090] It should be understood that the present invention provides a useful scheme for decoding the relative positions of barcode units. By combining this method with the capture and detection of analytes (e.g., mRNA) at high resolution, the present invention is able to decode the location and distribution of analytes in complex biological samples, such as tissues or sections of these tissues. Therefore, this application provides a useful method for spatial multi-omics, such as spatial transcriptomics, spatial genomics, etc.

[0091] This invention enables the capture of analytes from biological samples while preserving the spatial information of the original biological sample and individually barcoding each analyte. This capture involves using capture sequences, including but not limited to nucleic acid sequences complementary to the sequences contained on the analyte.

[0092] It will be apparent to those skilled in the art that the present invention is able to decode the position of each barcode unit by reproducing the proximity relationships between each barcode unit, and thus decode the position of each analyte.

[0093] The solution of this invention relies on using two distinct groups of barcode units: a first group dedicated to the capture and labeling of analytes (capture barcode units), and a second group dedicated to providing spatial information (location barcode units). Therefore, each captured analyte is associated with: (i) information about its associated capture barcode unit and optionally a unique molecular identifier, and (ii) information about the proximity relationship between its associated capture barcode unit and surrounding location barcode units.

[0094] It will be apparent to those skilled in the art that capture barcode units (containing capture oligonucleotides) and positioning barcode units (containing positioning oligonucleotides) are structurally and functionally distinct. A capture oligonucleotide, more specifically, a capture sequence contained on a capture oligonucleotide, is designed to bind an analyte from a biological sample; illustratively and non-limitingly, the capture sequence may contain or be configured as a poly(dT) sequence capable of binding and capturing a poly(A) tail of mRNA. Furthermore, it should be understood that capture oligonucleotides (and their corresponding capture barcode units) are not designed to interact or interfere with each other; that is, a capture oligonucleotide will not hybridize with another capture oligonucleotide, whether present on the same capture barcode unit or different capture barcode units. A positioning oligonucleotide, more specifically a first linker sequence, is designed to bind a capture sequence (or optionally a second linker sequence) on a capture oligonucleotide; illustratively and non-limitingly, the first linker sequence may contain or be configured as a poly(dA) sequence capable of binding a poly(dT) sequence on a capture oligonucleotide. Furthermore, it should be understood that positioning oligonucleotides (and their corresponding positioning barcode units) are not designed to interact or interfere with each other; that is, a positioning oligonucleotide will not hybridize with another positioning oligonucleotide, whether it is present on the same positioning barcode unit or a different positioning barcode unit. In addition, positioning oligonucleotides do not imply the capture of analytes from biological samples.

[0095] Therefore, it should be understood that in the compositions of the present invention, the links between capture barcode units and positioning barcode units (i.e., links formed by hybridization of a first linker sequence contained on a positioning oligonucleotide with a capture sequence or optional second linker sequence contained on a capture oligonucleotide) form a linking network. Thus, a given capture barcode unit can be linked or associated with, for example, two, three, four, five, or more than five positioning barcode units; consequently, the capture oligonucleotides hybridized with various positioning oligonucleotides will be extended and optionally amplified to obtain nucleic acids containing both the barcode sequence of the capture barcode unit and the barcode sequence of the positioning barcode unit hybridized therewith. For example, capturing barcode unit "A" may be associated with locating barcode unit "1", locating barcode unit "2", and locating barcode unit "3", which can be determined by obtaining: first, at least one nucleic acid containing the barcode from "A" and the barcode from "1"; second, at least one nucleic acid containing the barcode from "A" and the barcode from "2"; and third, at least one nucleic acid containing the barcode from "A" and the barcode from "3".

[0096] Furthermore, it should be understood that determining the exact interaction between the capturing barcode unit and the positioning barcode unit can determine the proximity relationship between the barcode units, thereby determining the relative positions of the capturing barcode units to each other. For example, the first capturing barcode unit "A" is associated with positioning barcode units "1", "2", and "3" (see above), and the second barcode unit "B" is associated with positioning barcode units "3", "4", and "5"; in this example, since both "A" and "B" share a connection with at least one positioning barcode unit ("3"), it can be determined that "A" and "B" are adjacent.

[0097] Capture oligonucleotides and localization oligonucleotides exist on different barcode units, so the risk of hybridization between capture oligonucleotides and localization oligonucleotides from the same barcode unit is zero.

[0098] Once the relative positions of the capture barcode units and the location barcode units are known, it becomes possible to infer the relative positions of analytes (such as mRNA molecules) from biological samples. In fact, nucleic acid molecules formed by the hybridization of, for example, capture oligonucleotides with analytes such as mRNA contain both the sequence of the analyte and a unique barcode from the barcode units. Therefore, the adjacency and relative positions of the capture barcode units are identical to the adjacency and relative positions of the analytes from the biological sample.

[0099] Therefore, in some embodiments, the compositions according to the invention enable spatial transcriptomics, i.e., detecting changes in gene expression based on changes in the location of cells in a biological sample (typically a tissue).

[0100] In some implementations, each capture barcode unit in a group of capture barcode units contains a group of capture oligonucleotides. As used herein, a "group of capture oligonucleotides" means at least 100, at least 1000, at least 10000, at least 100000, at least 1000000, at least 1000000, at least 10000000, at least 100000000, at least 1000000000, at least 10000000000, at least 100000000000, or more than 10 ...

[0101] In some embodiments, each capturing oligonucleotide contains at least one capturing sequence. In a preferred embodiment, each capturing oligonucleotide contains one capturing sequence.

[0102] Within the scope of this invention, the capture sequence serves as an analyte capture probe. In a preferred embodiment, the capture sequence is a nucleic acid sequence. In some embodiments, the capture sequence is a DNA sequence or an RNA sequence. In a more preferred embodiment, the capture sequence is a DNA sequence.

[0103] In some implementations, the capture sequence is single-stranded or double-stranded, preferably single-stranded.

[0104] The capture sequence can be specifically designed to capture mRNA.

[0105] In a preferred embodiment, the capture sequence is configured for non-specific capture of nucleic acids, preferably mRNA. In some embodiments, the capture sequence is capable of binding to the poly(A) tail of mRNA. In some embodiments, the capture sequence comprises or constitutes a poly(T) sequence.

[0106] In another, less preferred embodiment, the capture sequence is configured to capture a specific target nucleic acid, preferably a specific target mRNA molecule. In some embodiments, the capture sequence comprises or is composed of a sequence complementary to a target sequence on the target nucleic acid. In some embodiments, the capture sequence is degenerate (i.e., has a random sequence) or specific to the nucleic acid sequence of interest.

[0107] In a preferred embodiment, the capture sequence can hybridize at the first linker sequence. In a preferred embodiment, the capture sequence of the capture oligonucleotide can hybridize at the first linker sequence of the localizing oligonucleotide.

[0108] In a preferred embodiment, the capture sequence cannot hybridize with the capture sequence. In some embodiments, the capture sequence on the first capture oligonucleotide on the capture barcode unit cannot hybridize with the capture sequence on the second capture oligonucleotide on the same capture barcode unit. In some embodiments, the capture sequence on the capture oligonucleotide on the first capture barcode unit cannot hybridize with the capture sequence on the capture oligonucleotide on the second capture barcode unit.

[0109] In some implementations, the captured sequence cannot hybridize with itself. In some implementations, the captured sequence cannot form secondary structures with itself, such as loops.

[0110] In a preferred embodiment, the capture sequence is the same for all barcode units in the group of captured barcode units.

[0111] In some implementations, the barcode sequence and the capture sequence are arranged in a 5' to 3' order on the capture oligonucleotide.

[0112] In some embodiments, the capture sequence comprises or consists of a poly(dT) sequence or a poly(U) sequence. Therefore, in some embodiments, the capture sequence is specific to the poly(dA) sequence. The poly(dA) sequence may be present, for example, within a poly-A tail at the 3' end of the mRNA.

[0113] In some embodiments, the capture sequence comprises or is composed of the sequence (dT)nVN or (U)nVN (from 5' to 3'), where n is 5 to 50, V represents any nucleotide other than dT / U (i.e., dA, dC, or dG), and N represents any nucleotide (i.e., dA, dT, U, dC, or dG). Thus, in some embodiments, the capture sequence is specific to the NB(A)n sequence (from 5' to 3'), where n is 5 to 50, B represents any nucleotide other than dA (i.e., dT, U, dC, or dG), and N represents any nucleotide (i.e., dA, dT, U, dC, or dG). The NB(A)n sequence can be found, for example, at the 3' end of the mRNA, overlapping between the poly-A tail and the 3' UTR or CDS.

[0114] In some embodiments, the capture sequence comprises or constitutes a poly(I) sequence. Inosine is a nucleoside capable of swing pairing with any natural base. In some embodiments, the capture sequence is nonspecific and can trigger any nucleic acid sequence.

[0115] In some embodiments, the capture sequence of each capture oligonucleotide of a given capture barcode unit is one of a set of capture sequences containing at least one different capture sequence. In a preferred embodiment, the set of capture sequences contains at least two, five, ten, twenty, fifty, or one hundred different capture sequences. Such a set of capture sequences allows, in particular, each given barcode unit to capture different sets of nucleic acid sequences from a given biological unit.

[0116] In some embodiments, the capture sequence is the same for all capture oligonucleotides. In some embodiments, the capture sequence is the same for all capture barcode units. In some embodiments, the capture sequence is the same for all capture oligonucleotides.

[0117] In some implementations, the capture sequence comprises 5 to 50 or more nucleotides. In some cases, the length of the capture sequence can be 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some cases, the length of the captured sequence can be 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 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 nucleotides or longer than 50 nucleotides. In some cases, the length of the capture sequence can be up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 characters. Up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 41, up to 42, up to 43, up to 44, up to 45, up to 46, up to 47, up to 48, up to 49, up to 50 nucleotides or less.

[0118] In a preferred embodiment, the length of the captured sequence can be from 5 to 100 nucleotides, preferably from 10 to 50 nucleotides, and more preferably from 20 to 40 nucleotides.

[0119] In some implementations, each capture barcode unit can capture approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10%, approximately 5%, approximately 2%, or approximately 1% of the total amount of analyte in the cell.

[0120] In some implementations, the capturing oligonucleotide also includes a second linker sequence.

[0121] In some embodiments, the first linker sequence can hybridize onto the second linker sequence. In some embodiments, the first linker sequence that locates the oligonucleotide can hybridize onto the second linker sequence that captures the oligonucleotide.

[0122] In some embodiments, the second linker sequence from the first capturing oligonucleotide cannot hybridize with the second linker sequence of the second capturing oligonucleotide. In some embodiments, the second linker sequence cannot hybridize with itself. In some embodiments, the second linker sequence cannot hybridize with the capturing sequence.

[0123] In some embodiments, the second linker sequence comprises 5 to 50 or more nucleotides. In some embodiments, the second linker sequence differs from the barcode sequence and the capture sequence.

[0124] In some implementations, each positioning barcode unit in a group of positioning barcode units contains a group of positioning oligonucleotides. As used herein, a "group of positioning oligonucleotides" means at least 100, 1000, 10000, 100000, 1000000, 10000000, 10000000, 100000000, 1000000000, 10000000000, 100000000000, 100000000000, or more than 10000000000000 positioning oligonucleotides.

[0125] In some embodiments, each positioning oligonucleotide contains at least one first linker sequence. In a preferred embodiment, each positioning oligonucleotide contains one first linker sequence.

[0126] In some implementations, the first linker sequence is single-stranded or double-stranded, preferably single-stranded.

[0127] In a preferred embodiment, the first linker sequence can hybridize onto the capture sequence. In a preferred embodiment, the first linker sequence of the localizing oligonucleotide can hybridize onto the capture sequence of the capture oligonucleotide.

[0128] In some embodiments, the first linker sequence cannot hybridize with the first linker sequence. In some embodiments, the first linker sequence on the first positioning oligonucleotide on the positioning barcode unit cannot hybridize with the first linker sequence on the second positioning oligonucleotide on the same positioning barcode unit. In some embodiments, the first linker sequence on the positioning oligonucleotide on the first positioning barcode unit cannot hybridize with the first linker sequence on the positioning oligonucleotide on the second positioning barcode unit.

[0129] In some implementations, the first linker sequence cannot hybridize with itself.

[0130] In some implementations, the barcode sequence and the first linker sequence are arranged in a 5' to 3' order on the positioning oligonucleotide.

[0131] In some implementations, the first connection sequence is the same for all barcode cells in a group of locating barcode cells.

[0132] In some implementations, the first ligation sequence cannot bind to at least one analyte from a biological sample.

[0133] In some implementations, the capture sequence contains a poly(dT) sequence or a poly(U) sequence, and the first connection sequence contains a poly(dA) sequence.

[0134] In a preferred embodiment, each barcode unit contains a different barcode sequence.

[0135] In a preferred embodiment, all oligonucleotides on the same barcode unit contain the same barcode sequence. In some embodiments, all capture oligonucleotides on the same capture barcode unit contain the same barcode sequence. In some embodiments, all positioning oligonucleotides on the same positioning barcode unit contain the same barcode sequence.

[0136] In some implementations, the number of clone copies of the barcode sequence is approximately 2 to approximately 10. 12 One clone copy.

[0137] In some embodiments, the barcode sequence is preferably a non-optical identifier, particularly a nucleic acid barcode. In some embodiments, the nucleic acid barcode is single-stranded or double-stranded, preferably single-stranded. In some embodiments, the nucleic acid barcode is a DNA barcode, an RNA barcode, or a mixture of both.

[0138] In some implementations, the first linker sequence is the same for all locating barcode units. In some implementations, the first linker sequence is the same for all locating oligonucleotides.

[0139] In some embodiments, the capture sequence is the same for all capture barcode units, and the first linker sequence is the same for all positioning barcode units. In some embodiments, the capture sequence is the same for all capture oligonucleotides, and the first linker sequence is the same for all positioning oligonucleotides.

[0140] In some implementations, the nucleic acid barcode may contain 5 to 20 or more nucleotides. In some cases, the length of the nucleic acid barcode may be 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, or 30 nucleotides. In some cases, the length of a nucleic acid barcode can be 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 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 nucleotides or longer than 30 nucleotides. In some cases, the length of the nucleic acid barcode can be up to 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, or up to 30 nucleotides, or less than 30 nucleotides. In a preferred embodiment, the length of the nucleic acid barcode can be 5 to 20 nucleotides, preferably 8 to 16 nucleotides, 9 to 16 nucleotides, and more preferably 10 to 14 nucleotides. These nucleotides can be continuous, i.e., in a single fragment of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by one or more nucleotides.

[0141] In some implementations, all nucleic acid barcodes attached to a particular barcode unit contain the same nucleic acid barcode sequence (i.e., a clone of the nucleic acid barcode), but each barcode unit in the group of barcode units, or most barcode units in the group of barcode units, contains different nucleic acid barcode sequences, such that a large number of different nucleic acid barcode sequences are embodied in the group of barcode units, for example, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 1,000,000, at least about 5,000,000, at least about 1,000,000, at least about 5,000,000, at least about 1,000,000, at least about 5,000,000, at least about 1,000,000,000, or more than 100,000,000, different nucleic acid barcode sequences.

[0142] In one implementation, the barcode units have a consistent size (same or nearly the same) during use.

[0143] In another embodiment, the barcode units have different sizes. In some embodiments, the barcode sequence for capturing oligonucleotides and the barcode sequence for locating oligonucleotides have different sizes.

[0144] In some implementations, the location barcode unit cannot be combined with the analyte.

[0145] In some implementations, the localized oligonucleotide does not contain an analyte capture sequence.

[0146] In some embodiments, the capture oligonucleotide and / or positioning oligonucleotide further comprise at least one PCR primer sequence. In other words, each capture oligonucleotide and each positioning oligonucleotide comprises at least one PCR primer sequence.

[0147] "PCR primer sequences" refer to nucleic acid sequences that facilitate the amplification of nucleic acid sequences extracted or derived from biological units, and are preferred for PCR amplification and further sequencing.

[0148] In some implementations, at least one PCR primer sequence contains 10 to 30 or more nucleotides. In some cases, the length of the PCR primer sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some cases, the length of PCR primer sequences can be at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 nucleotides or longer than 50 nucleotides. In some cases, the length of at least one PCR primer sequence can be up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. Up to 30, up to 31, up to 32, up to 33, up to 34, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 41, up to 42, up to 43, up to 44, up to 45, up to 46, up to 47, up to 48, up to 49, up to 50 nucleotides or less.

[0149] In a preferred embodiment, at least one PCR primer sequence is 15 to 35 nucleotides in length, preferably 20 to 30 nucleotides, and more preferably 25 nucleotides in length.

[0150] In a preferred embodiment, at least one PCR primer sequence is identical in each oligonucleotide of a given capture barcode unit and / or location barcode unit; preferably, at least one PCR primer sequence is identical in each oligonucleotide of a group of capture barcode units and / or in each oligonucleotide of a group of location barcode units.

[0151] In one embodiment, at least one PCR primer sequence on the capturing oligonucleotide is identical to at least one PCR primer sequence on the localizing oligonucleotide. In another embodiment, at least one PCR primer sequence on the capturing oligonucleotide is different from at least one PCR primer sequence on the localizing oligonucleotide.

[0152] In some implementations, the PCR primer sequence is a primer sequence.

[0153] In some implementations, each oligonucleotide from the group of captured oligonucleotides and / or the group of localized oligonucleotides also contains at least one unique molecular barcode (UMI). In other words, each captured oligonucleotide and / or each localized oligonucleotide contains at least one UMI.

[0154] A "unique molecular identifier" or "UMI" is a nucleic acid sequence used as an index, which helps reduce errors and quantitative biases introduced by the amplification step.

[0155] In a preferred embodiment, at least one UMI is different in each oligonucleotide of each barcode unit. In some embodiments, at least one UMI is different in each capture oligonucleotide of each capture barcode unit. In some embodiments, at least one UMI is different in each positioning oligonucleotide of each positioning barcode unit.

[0156] In some implementations, the oligonucleotides that capture the barcode unit and / or locate the barcode unit contain a variety of different UMIs, particularly more than 100, more than 1,000, more than 2,000, more than 5,000, or more than 10,000 different UMIs.

[0157] In some implementations, the unique molecular identifier contains 3 to 30 or more nucleotides. In some cases, the length of the unique molecular identifier can be 3, 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, or 30 nucleotides. In some cases, the length of a unique molecular identifier can be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 nucleotides or longer than 30 nucleotides. In some cases, the length of a unique molecular identifier can be up to 3, 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, or up to 30 nucleotides or less.

[0158] In a preferred embodiment, the length of the unique molecular identifier is 5 to 25 nucleotides, preferably 7 to 15 nucleotides, and more preferably 8 to 13 nucleotides.

[0159] In one implementation, each oligonucleotide from the group of captured oligonucleotides and / or the group of localized oligonucleotides contains a UMI.

[0160] In another implementation, each oligonucleotide from the group of captured oligonucleotides and / or the group of localized oligonucleotides contains more than one UMI. The presence of several UMIs on the oligonucleotide can facilitate the sequencing of the oligonucleotide.

[0161] In some embodiments, each oligonucleotide from the group of capture oligonucleotides and / or the group of localization oligonucleotides further comprises at least one linker. In other words, each capture oligonucleotide and / or each localization oligonucleotide comprises at least one linker.

[0162] In some embodiments, the capture oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, a barcode sequence, optionally at least one UMI, and a capture sequence. In some embodiments, the capture oligonucleotide comprises, in a 5' to 3' order: at least one adapter, at least one PCR primer sequence, a barcode sequence, at least one UMI, and a capture sequence. In some embodiments, the capture oligonucleotide comprises, in a 5' to 3' order: at least one adapter, at least one PCR primer sequence, a barcode sequence, at least one UMI, a second ligation sequence, and a capture sequence.

[0163] In some embodiments, the targeting oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, a barcode sequence, optionally at least one UMI, and a first linker sequence.

[0164] In some embodiments, the capture oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, barcode sequence, optionally at least one UMI, and capture sequence; and the localization oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, barcode sequence, optionally at least one UMI, and first ligation sequence.

[0165] In some embodiments, the capturing oligonucleotide comprises, in a 5' to 3' order, a barcode sequence and a capturing sequence; and the positioning oligonucleotide comprises, in a 5' to 3' order, a barcode sequence and a first linker sequence.

[0166] In some embodiments, the capture oligonucleotide comprises, in a 5' to 3' order: at least one PCR primer sequence, a barcode sequence, at least one UMI, and a capture sequence; and the localization oligonucleotide comprises, in a 5' to 3' order: at least one PCR primer sequence, a barcode sequence, at least one UMI, and a first ligation sequence.

[0167] In some implementations, the density and spacing of oligonucleotide coupling to the barcode unit itself or the core of the barcode unit can be tuned by adapter molecules to ensure a sufficient number of target capture sites. Adapters also serve to prevent the formation of unwanted structures (e.g., secondary or tertiary structures) on the surface of the entity or to cause molecular folding. Therefore, adapters can be designed in various ways, either as linear fragments or as branched fragments (e.g., dendritic polymers or other branched fragments). Furthermore, adapter molecules can be configured for selective attachment, using specific functional groups tailored to specific chemicals. They can also be designed for activated cleavage (similar to molecular scissors) to enable the controlled release of captured target material from the core of the barcode unit. This controlled cleavage can be triggered by various mechanisms, including thermal cleavage, pH changes, photocleaving, enzymatic cleavage, or other suitable cleavage mechanisms.

[0168] As used herein, the term "cleavage" refers to the separation of the covalent backbone of a DNA molecule. Cleavage can be initiated in various ways, such as enzymatic or chemical hydrolysis of phosphodiester bonds. Single-stranded and double-stranded cleavages can be achieved, the latter being the result of two independent single-stranded cleavage events. DNA cleavage can produce blunt ends or sticky ends. In certain embodiments, fusion peptides are used to achieve targeted double-stranded DNA cleavage.

[0169] In one embodiment, the captured oligonucleotide and / or targeted oligonucleotide binds to the core of the barcode unit, such as a bead. In another embodiment, the captured oligonucleotide and / or targeted oligonucleotide bind to each other in a linear sequence, such as DNA nanospheres.

[0170] Capture oligonucleotides and / or localize oligonucleotides can contain 5 to 300 or more nucleotides. In some cases, the length of the capturing oligonucleotide and / or positioning oligonucleotide can be 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 5... 9, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300 nucleotides. In some cases, the length of the capturing oligonucleotide and / or positioning oligonucleotide can be 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 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, or at least 46. At least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92.At least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118, at least 119, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129 1, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141, at least 142, at least 143, at least 144, at least 145, at least 146, at least 147, at least 148, at least 149, at least 150, at least 151, at least 152, at least 153, at least 154, at least 155, at least 156, at least 157, at least 158, at least 159, at least 160, at least 161, at least 162, at least 163, at least 164, at least 1 65, at least 166, at least 167, at least 168, at least 169, at least 170, at least 171, at least 172, at least 173, at least 174, at least 175, at least 176, at least 177, at least 178, at least 179, at least 180, at least 181, at least 182, at least 183, at least 184, at least 185, at least 186, at least 187, at least 188, at least 189, at least 190, at least 191, at least 192, at least 193, at least 194, at least 195, at least 196, at least 197, at least 198, at least 199, at least 200, up to At least 201, at least 202, at least 203, at least 204, at least 205, at least 206, at least 207, at least 208, at least 209, at least 210, at least 211, at least 212, at least 213, at least 214, at least 215, at least 216, at least 217, at least 218, at least 219, at least 220, at least 221, at least 222, at least 223, at least 224, at least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236.At least 237, at least 238, at least 239, at least 240, at least 241, at least 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least 249, at least 250, at least 251, at least 252, at least 253, at least 254, at least 255, at least 256, at least 257, at least 258, at least 259, at least 260, at least 261, at least 262, at least 263, at least 264, at least 265, at least 266, at least 267, at least 268, at least 269 At least 270, at least 271, at least 272, at least 273, at least 274, at least 275, at least 276, at least 277, at least 278, at least 279, at least 280, at least 281, at least 282, at least 283, at least 284, at least 285, at least 286, at least 287, at least 288, at least 289, at least 290, at least 291, at least 292, at least 293, at least 294, at least 295, at least 296, at least 297, at least 298, at least 299, at least 300 nucleotides or longer than 300 nucleotides. In some cases, the length of the capturing oligonucleotide and / or positioning oligonucleotide can be up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or more. 3, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 41, up to 42, up to 43, up to 44, up to 45 At most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 6 8, up to 69, up to 70, up to 71, up to 72, up to 73, up to 74, up to 75, up to 76, up to 77, up to 78, up to 79, up to 80, up to 81, up to 82, up to 83, up to 84, up to 85, up to 86, up to 87, up to 88, up to 89, up to 90.At most 91, at most 92, at most 93, at most 94, at most 95, at most 96, at most 97, at most 98, at most 99, at most 100, at most 101, at most 102, at most 103, at most 104, at most 105, at most 106, at most 107, at most 108, at most 109. At most 110, at most 111, at most 112, at most 113, at most 114, at most 115, at most 116, at most 117, at most 118, at most 119, at most 120, at most 121, at most 122, at most 123, at most 124, at most 125, at most 126, at most 127 At most 128, at most 129, at most 130, at most 131, at most 132, at most 133, at most 134, at most 135, at most 136, at most 137, at most 138, at most 139, at most 140, at most 141, at most 142, at most 143, at most 144, at most 145 1, at most 146, at most 147, at most 148, at most 149, at most 150, at most 151, at most 152, at most 153, at most 154, at most 155, at most 156, at most 157, at most 158, at most 159, at most 160, at most 161, at most 162, at most 16 3, up to 164, up to 165, up to 166, up to 167, up to 168, up to 169, up to 170, up to 171, up to 172, up to 173, up to 174, up to 175, up to 176, up to 177, up to 178, up to 179, up to 180, up to 1 81, at most 182, at most 183, at most 184, at most 185, at most 186, at most 187, at most 188, at most 189, at most 190, at most 191, at most 192, at most 193, at most 194, at most 195, at most 196, at most 197, at most 198, at most 199, at most 200, at most 201, at most 202, at most 203, at most 204, at most 205, at most 206, at most 207, at most 208, at most 209, at most 210, at most 211, at most 212, at most 213, at most 214, at most 215, at most 216, and so on. More than 217, at most 218, at most 219, at most 220, at most 221, at most 222, at most 223, at most 224, at most 225, at most 226, at most 227, at most 228, at most 229, at most 230, at most 231, at most 232, at most 233, at most 234.At most 235, at most 236, at most 237, at most 238, at most 239, at most 240, at most 241, at most 242, at most 243, at most 244, at most 245, at most 246, at most 247, at most 248, at most 249, at most 250, at most 251, at most 252, at most 253, at most 254, at most 255, at most 256, at most 257, at most 258, at most 259, at most 260, at most 261, at most 262, at most 263, at most 264, at most 265, at most 266, at most 267, at most 268 Up to 269, up to 270, up to 271, up to 272, up to 273, up to 274, up to 275, up to 276, up to 277, up to 278, up to 279, up to 280, up to 281, up to 282, up to 283, up to 284, up to 285, up to 286, up to 287, up to 288, up to 289, up to 290, up to 291, up to 292, up to 293, up to 294, up to 295, up to 296, up to 297, up to 298, up to 299, up to 300 nucleotides or less.

[0171] It should be understood that the composition according to the invention comprises two different groups of barcode units: capture barcode units and positioning barcode units.

[0172] In a preferred embodiment, the capture barcode unit contains only one type of oligonucleotide. In some embodiments, the capture barcode unit contains only capture oligonucleotides. In some embodiments, the capture barcode unit does not contain positioning oligonucleotides.

[0173] In a preferred embodiment, the positioning barcode unit contains only one type of oligonucleotide. In some embodiments, the positioning barcode unit contains only positioning oligonucleotides. In some embodiments, the positioning barcode unit does not contain capture oligonucleotides.

[0174] These two types of barcode units can share many features described below, but they are functionally different. Capture barcode units and location barcode units can be combined with each other. However, while capture barcode units can be combined with analytes from biological samples, location barcode sequences cannot.

[0175] In some implementations, each capture barcode unit contains 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000, 10000000000, 10000000000, 100000000000, 10000000000000, or more than 10 ... In some implementations, each positioning barcode unit contains 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000, 10000000000, 10000000000, 100000000000, 10000000000000, or more than 100000000000000 positioning oligonucleotides.

[0176] In some implementations, the barcode unit can have any suitable shape or form.

[0177] In some implementations, the barcode unit is a 3D barcode unit.

[0178] In some embodiments, the barcode unit has a spherical or non-spherical shape (e.g., elliptical, prism, polyhedral, amorphous, etc.). In some embodiments, the barcode unit has a generally spherical shape. In some embodiments, the barcode unit may have a pelota or ball shape. In some embodiments, the barcode unit may have a regular or irregular form or shape.

[0179] In some implementations, the barcode unit is either synthetic or natural.

[0180] In a preferred embodiment, the barcode unit is a synthetic barcode unit. As used herein, "synthetic" means that the barcode is synthesized in vitro. In some embodiments, the barcode unit is not generated naturally (e.g., by cells).

[0181] In another, less preferred embodiment, the barcode unit is a natural barcode unit, meaning the barcode unit is generated naturally. In some embodiments, the barcode unit is synthesized by cells, such as bacteria.

[0182] In some implementations, a barcode unit is an entity containing at least one copy of a barcode sequence.

[0183] In one embodiment, the barcode unit includes a core, a support or substrate, and a group of oligonucleotides, each group of oligonucleotides containing at least one copy of the barcode sequence, wherein the oligonucleotides are attached to or bound to the core, support or substrate.

[0184] In another embodiment, the barcode unit consists of multiple repeats of a nucleic acid sequence containing at least one barcode sequence, preferably consecutive repeats.

[0185] In some embodiments, the capture barcode unit and / or positioning barcode unit are beads or DNA nanospheres. As used herein, a "DNA nanosphere" refers to a nucleic acid sequence containing more than one oligonucleotide repeat as described herein. As used herein, the term "bead" may be used interchangeably with "bead with barcode."

[0186] In some implementations, the capture barcode units are dots on a DNA microarray or a glass slide. As used herein, a "dot" refers to a discrete location on a DNA microarray or glass slide, typically designed to retain DNA molecules.

[0187] In some implementations, the positioning barcode unit is a bead, a DNA nanosphere, or a dot on a DNA microarray.

[0188] In some implementations, when the positioning barcode unit is a bead, the oligonucleotides defined herein are attached to the bead (i.e., the bead's nucleus) by any suitable means known in the art.

[0189] In some implementations, when the positioning barcode unit is a DNA nanosphere, the nucleic acid sequence of the DNA nanosphere contains oligonucleotides and therefore contains the barcode sequence itself.

[0190] In some embodiments, the bead (i.e., the bead core) is made of a material selected from the group consisting of: acrylic resin, carbon, cellulose, ceramics, controlled-pore glass, cross-linked polysaccharides, gels, glass, gold, graphite, inorganic glass, inorganic polymers, latex, metal oxides, semi-metals, metals, mica, molybdenum sulfide, nanomaterials, nitrocellulose, fiber bundles, organic polymers, paper, plastics, polyacryloylmorpholide, poly(4-methylbutene), polyethylene terephthalate, poly(vinyl butyrate), polybutene, polydimethylsiloxane, polyethylene, polyoxymethylene, polymethyl methacrylate, polypropylene, polysaccharides, polystyrene, polyurethane, polyvinylidene fluoride, quartz, rayon, resins, rubber, semiconductor materials, silicon dioxide, silicon, sulfides, and combinations thereof.

[0191] In some embodiments, all or part of the barcode unit is magnetic. In some embodiments, the barcode unit is made of a magnetic material. In some embodiments, the capturing barcode unit is a magnetic bead. In some embodiments, the positioning barcode unit is a magnetic bead.

[0192] In one embodiment, the capture barcode unit and the positioning barcode unit are made of different materials. In another embodiment, the capture barcode unit and the positioning barcode unit are made of the same material.

[0193] In some implementations, the barcode unit can be a solid (or rigid, hard) or semi-solid (or flexible, soft) barcode unit.

[0194] In some implementations, both the capture barcode unit and the positioning barcode unit are solid barcode units. In other implementations, both the capture barcode unit and the positioning barcode unit are semi-solid barcode units.

[0195] In a preferred embodiment, one type of barcode unit is rigid, while the other type is semi-rigid. In one embodiment, the capture barcode unit is rigid, while the positioning barcode unit is semi-rigid. In another embodiment, the positioning barcode unit is rigid, while the capture barcode unit is semi-rigid.

[0196] In some implementations, the barcode cells from the group of captured barcode cells and / or the group of positioned barcode cells have different sizes (i.e., multi-dispersion sizes).

[0197] In some implementations, the barcode cells from the group of capturing barcode cells and / or the group of positioning barcode cells have the same size (i.e., monodisperse size).

[0198] In some implementations, the average size of the barcode unit is from about 1 nm to about 500 µm.

[0199] In some implementations, the average size of the barcode unit is from about 10 nm to about 100 µm.

[0200] In some implementations, the average diameter of the barcode unit is approximately 1µm, approximately 2µm, approximately 3µm, approximately 4µm, approximately 5µm, approximately 6µm, approximately 7µm, approximately 8µm, approximately 9µm, approximately 10µm, approximately 11µm, approximately 12µm, approximately 13µm, approximately 14µm, approximately 15µm, approximately 16µm, approximately 17µm, approximately 18µm, approximately 19µm, approximately 20µm, approximately 21µm, approximately 22µm, approximately 23µm, approximately 24µm. Approximately 25µm, approximately 26µm, approximately 27µm, approximately 28µm, approximately 29µm, approximately 30µm, approximately 31µm, approximately 32µm, approximately 33µm, approximately 34µm, approximately 35µm, approximately 36µm, approximately 37µm, approximately 38µm, approximately 39µm, approximately 40µm, approximately 41µm, approximately 42µm, approximately 43µm, approximately 44µm, approximately 45µm, approximately 46µm, approximately 47µm, approximately 48µm, approximately 49µm, approximately 50µm.

[0201] In some implementations, the average size of the barcode unit is from about 10 nm to about 50 nm, preferably from 20 nm to 40 nm.

[0202] In some implementations, the average diameter of the barcode unit is approximately 10 nm, approximately 11 nm, approximately 12 nm, approximately 13 nm, approximately 14 nm, approximately 15 nm, approximately 16 nm, approximately 17 nm, approximately 18 nm, approximately 19 nm, approximately 20 nm, approximately 21 nm, approximately 22 nm, approximately 23 nm, approximately 24 nm, approximately 25 nm, approximately 26 nm, approximately 27 nm, approximately 28 nm, approximately 29 nm, approximately 30 nm, approximately 31 nm, approximately 32 nm, approximately 33 nm, approximately 34 nm, approximately 35 nm, approximately 36 nm, approximately 37 nm, approximately 38 nm, approximately 39 nm, approximately 40 nm, approximately 41 nm, approximately 42 nm, approximately 43 nm, approximately 44 nm, approximately 45 nm, approximately 46 nm, approximately 47 nm, approximately 48 nm, approximately 49 nm, or approximately 50 nm. In some implementations, the average diameter of the barcode unit is approximately 20 nm, approximately 21 nm, approximately 22 nm, approximately 23 nm, approximately 24 nm, approximately 25 nm, approximately 26 nm, approximately 27 nm, approximately 28 nm, approximately 29 nm, approximately 30 nm, approximately 31 nm, approximately 32 nm, approximately 33 nm, approximately 34 nm, approximately 35 nm, approximately 36 nm, approximately 37 nm, approximately 38 nm, approximately 39 nm, or approximately 40 nm.

[0203] It will be apparent to those skilled in the art that the size of the barcode unit can be adjusted according to the application and / or the type of analyte.

[0204] In some embodiments, barcode units smaller than 1 µm are preferred, especially for intracellular and / or subcellular applications. In some embodiments, barcode units larger than 1 µm are preferred, especially for cellular and / or tissue-scale applications.

[0205] In some implementations, barcode units smaller than 10µm are preferred, as capturing barcode units enables high resolution of spatial information for each analyte. In some implementations, barcode units smaller than 1µm are preferred, as capturing barcode units enables even higher resolution of spatial information for each analyte.

[0206] In some implementations, the group of positioning barcode units is combined, attached, or otherwise connected to a substrate, preferably a support.

[0207] In some embodiments, the substrate or support is a solid support. In some embodiments, the solid support may be made of any suitable material, such as glass, polymer, plastic, metal, silica, or a combination thereof.

[0208] In some embodiments, the substrate or support is a planar support or a two-dimensional support. In some embodiments, the planar support or two-dimensional support is selected from glass slides, coverslips, arrays, culture flasks, culture dishes, etc., preferably glass slides or coverslips.

[0209] In some embodiments, the substrate or support is covered or coated with at least one material that enables bonding or connection between the substrate or support and the barcode unit. The at least one material enabling bonding or connection can be of any suitable nature, such as polymers, peptides, sugars, etc., or any combination thereof.

[0210] In some embodiments, groups of capture barcode units and groups of positioning barcode units are assembled. As used herein, “assembly” is intended to refer to the formation of a heterogeneous structure comprising at least one capture barcode unit and at least one positioning barcode unit. Within the scope of this invention, the term “assembly” may be used interchangeably with the terms “packaging,” “arrangement,” “distribution,” etc.

[0211] In some embodiments, the assembly is formed by the connection or contact between a plurality of capture barcode units and a plurality of positioning barcode units. In some embodiments, a contact point is formed between the capture barcode units and the positioning barcode units. In one embodiment, the contact point involves a capture sequence of at least one capture oligonucleotide of the capture barcode unit and a first linker sequence of at least one positioning oligonucleotide of the positioning barcode unit. In another embodiment, the contact point involves a second linker sequence of at least one capture oligonucleotide of the capture barcode unit and a first linker sequence of at least one positioning oligonucleotide of the positioning barcode unit. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the capture oligonucleotides on the capture barcode unit participate in forming a contact point with the positioning barcode unit. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the capture oligonucleotides participate in forming a contact point with the positioning barcode unit. In some embodiments, at least 100, at least 1000, at least 10000, at least 100000, or more than 100000 capture oligonucleotides on the capture barcode unit participate in forming a contact point with the positioning barcode unit. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the capture oligonucleotides participate in forming a contact point with the positioning barcode unit. In some embodiments, approximately 100, 1000, 10000, 100000, or more than 100000 capture oligonucleotides on the capture barcode unit participate in forming contact points with the positioning barcode unit. In some embodiments, at least 100, 1000, 10000, 100000, or more than 100000 positioning oligonucleotides on the positioning barcode unit participate in forming contact points with the capture barcode unit. In some embodiments, approximately 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the capture oligonucleotides on the positioning barcode unit participate in forming contact points with the capture barcode unit. In some implementations, approximately 100, 1,000, 10,000, 100,000, or more than 100,000 positioning oligonucleotides on the positioning barcode unit participate in forming contact points with the capture barcode unit.

[0212] In some embodiments, capture oligonucleotides and positioning oligonucleotides hybridize. In a preferred embodiment, hybridization occurs at the contact point. It should be understood that hybridization binds or tethers a capture barcode unit to a positioning barcode unit, thereby ensuring physical connectivity between barcode units contained in the assembly. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the contact points are hybridized. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the contact points are hybridized.

[0213] It should be understood that the assembly methods listed below are not restrictive, but illustrative.

[0214] In some implementations, barcode units are assembled in one or more layers. As used herein, “one or more layers” includes 1 layer (i.e., single layer), 2 layers (i.e., double layer), 3 layers, 4 layers, 5 layers, 10 layers, or more than 10 layers.

[0215] In one embodiment, each layer is homogeneous, meaning that a layer contains more than 90%, more than 95%, more than 99%, more than 99.5%, or 100%, preferably 100%, of one type of barcode unit. In another embodiment, each layer is heterogeneous, meaning that a layer contains both positioning barcode units and capturing barcode units. In some embodiments, one or more layers simultaneously contain homogeneous and heterogeneous layers.

[0216] In some implementations, one or more layers can be configured in any suitable way. It will be apparent to those skilled in the art that any configuration may be used depending on the application required.

[0217] In some embodiments, one or more layers can be configured as one homogeneous positioning barcode unit and another homogeneous capture barcode unit. In some embodiments, one or more layers can be configured as two, three, four, or more than four heterogeneous barcode units. In some embodiments, one or more layers can be configured as a first homogeneous layer of positioning barcode units, one or two layers of capture barcode units, and another layer of positioning barcode units.

[0218] In one implementation, barcode units are assembled in a random, ordered, or crystalline manner. The type of assembly may depend on the number of layers.

[0219] In some implementations, the barcode units are assembled in a random manner, that is, the distribution of the positioning barcode units and the capture barcode units on the substrate is uncontrolled or random, and may result in uneven distribution of the positioning barcode units and the capture barcode units on the substrate.

[0220] In some embodiments, the barcode units are assembled in an ordered manner, i.e., the distribution of positioning and capture barcode units on the substrate is controlled, resulting in a uniform distribution of positioning and capture barcode units on the substrate. In some embodiments, when barcode units are formed in one or more layers, the layers can be heterogeneous (i.e., one layer simultaneously contains positioning and capture barcode units) or homogeneous (i.e., one layer contains more than 90%, more than 95%, more than 99%, more than 99.5%, or 100%, preferably 100%, of one type of barcode unit). Therefore, in some embodiments, the assembly of barcode units is designed to maximize the contact opportunities between capture and positioning barcode units.

[0221] In some implementations, the barcode units are assembled in a crystalline manner, that is, the positioning barcode units and the capturing barcode units are held together in an ordered three-dimensional arrangement.

[0222] It should be understood that the purpose of assembling the two types of barcode units (positioning and capture) together is to enable them to be positioned relative to each other. Therefore, this step requires different types of barcode units to be interconnected. Thus, it should be understood that a large number of connections helps to reconstruct the proximity relationships between barcode units.

[0223] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, 80%, at least 90%, or 100% of the barcode units from the group of captured barcode units are in contact with at least one, preferably at least two, more preferably at least three positioning barcode units. In some embodiments, the barcode units from the group of captured barcode units are in contact with at least one, preferably at least two, more preferably at least three positioning barcode units. In a preferred embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the barcode units from the group of captured barcode units have at least one hybridized oligonucleotide, which has at least one, preferably at least two, more preferably at least three positioning barcode units. In a preferred embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the hybridized oligonucleotide is sequenced.

[0224] Therefore, in some embodiments, contact and / or physical proximity between the capture barcode unit and the positioning barcode unit is facilitated by at least one external force. Non-limiting examples of such forces include mechanical forces, pressure, gravity, centrifugal forces, clamping forces, magnetic forces, etc. In some embodiments, at least one external force increases or ensures the probability of contact between the capture barcode unit and the positioning barcode unit, which are spatially close.

[0225] In some implementations, the external force is gravity, meaning the barcode units are settled. In some implementations, the external force is centrifugal force, meaning the barcode units are centrifuged at an appropriate speed. In some implementations, the external force is clamping force. In some implementations, the external force is magnetic force, meaning the barcode units themselves are magnetic or are held together by a magnetic field.

[0226] In some implementations, groups of capture barcode units and groups of positioning barcode units are assembled on a substrate.

[0227] In one implementation, the substrate is a support that provides solid mechanical support for the barcode unit. In some implementations, the support can be planar (i.e., two-dimensional) or three-dimensional.

[0228] In some embodiments, the substrate is a support, preferably a planar support. In some embodiments, the support has a smooth or rough surface. In some embodiments, the support has a uniform or non-uniform surface. In some embodiments, the support includes irregularities or structures on its surface, such as micropores, microarrays, supports, etc. It should be understood that these structures are intended, for example, to position, retain, or expose barcode units of the composition according to the invention. In some embodiments, the support includes at least one means on its surface for incorporating and / or positioning barcode units according to the invention.

[0229] In some embodiments, the support is a glass slide, array, chip, etc. In some embodiments, the support is made of a material selected from glass, carbon, ceramics, metals, nitrocellulose, polymers, plastics, silicates, and combinations thereof.

[0230] In one implementation, the support is rigid.

[0231] In another embodiment, the support is flexible, meaning it can be folded. This embodiment is suitable for applications involving thick biological samples, where the support can be folded to cover or wrap the biological sample.

[0232] In some embodiments, the dimensions of the support are from about 1 mm to about 10 cm. In some embodiments, the dimensions of the support are about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 2 cm, about 3 cm, about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, or greater than 10 cm. In some embodiments, the surface area of ​​the support is about 1 mm². 2 Approximately 2mm 2 Approximately 3mm 2 Approximately 4mm 2 Approximately 5mm 2 Approximately 6mm 2 Approximately 7mm 2 Approximately 8mm 2 Approximately 9mm 2 Approximately 1cm 2 Approximately 2cm 2 Approximately 3cm 2 Approximately 4cm 2 Approximately 5cm 2 or greater than 5cm 2 .

[0233] In some embodiments, the substrate is a three-dimensional substrate or support. In one embodiment, the substrate is a matrix. Non-limiting examples of matrices include hydrogels, collagen, etc.

[0234] In some embodiments, the assembly of the barcode unit occupies about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or more than 80% of the total surface area or volume of the substrate or support. In some embodiments, the assembly of the barcode unit occupies about 1 mm² of the total surface area of ​​the substrate or support. 2 Approximately 2mm 2 Approximately 3mm 2 Approximately 4mm 2 Approximately 5mm 2 Approximately 6mm 2 Approximately 7mm 2 Approximately 8mm 2 Approximately 9mm 2 Approximately 1cm 2 Approximately 2cm 2 Approximately 3cm 2 Approximately 4cm 2 Approximately 5cm 2 or greater than 5cm 2 .

[0235] In some embodiments, when assembled as described herein, the composition according to the invention comprises a defined ratio of positioning barcode units and capturing barcode units. It should be understood that this ratio reflects the relative quantity of one type of barcode unit relative to another type of barcode unit. This ratio may also be referred to as the level or degree of heterogeneity.

[0236] In one embodiment, the ratio of the positioned barcode unit to the captured barcode unit is approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:7, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:20, approximately 1:30, approximately 1:40, approximately 1:50, approximately 1:100, or greater than 1:100. In another embodiment, the ratio of the captured barcode unit to the positioned barcode unit is approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:7, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:20, approximately 1:30, approximately 1:40, approximately 1:50, approximately 1:100, or greater than 1:100.

[0237] In some implementations, the ratio of the located barcode unit to the captured barcode unit is approximately 1:1.

[0238] In one embodiment, the composition or assembly comprises approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 30 times, approximately 40 times, approximately 50 times, approximately 100 times, approximately 200 times, approximately 500 times, approximately 1000 times, or approximately 10000 times more positioning barcode units than the positioning barcode units. In another embodiment, the composition or assembly comprises approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 30 times, approximately 40 times, approximately 50 times, approximately 100 times, approximately 200 times, approximately 500 times, approximately 1000 times, or approximately 10000 times more positioning barcode units than the positioning barcode units.

[0239] In some implementations, the degree of heterogeneity is adjusted according to the application and / or assembly type. Such an adjustment can be readily conceived by those skilled in the art. It will be apparent to those skilled in the art that the degree of heterogeneity can be adjusted based on the relative dimensions of the barcode units.

[0240] Therefore, in some embodiments, when barcode units are assembled randomly, the ratio of locating barcode units to capturing barcode units is preferably about 1:1, meaning that the number of each type of barcode unit is equal or nearly equal. In some embodiments, when barcode units are assembled in an ordered or crystalline manner, the ratio of locating barcode units to capturing barcode units is different from 1:1, meaning that one type of barcode unit is more representative than another type of barcode unit.

[0241] In some embodiments, the assembly of barcode units is performed using any suitable technique selected from: pick-and-place processes, self-assembly, sedimentation, emulsion layering, entropy-driven colloidal assembly, solvent evaporation-assisted deposition, spin coating, dip coating, electrostatic deposition, electrophoretic deposition, capillary assembly, topographical-templated assembly, wettability-templated assembly, chemical template assembly, magnetic assembly, or any combination thereof. It will be apparent to those skilled in the art which techniques are suitable based on the desired results.

[0242] It should be understood that the total number of barcode units can be adjusted based on the application, the size of the barcode units, the density of the analyte, and / or the biological sample. In some embodiments, both the number of captured barcode units and the number of positioned barcode units are adjusted. In some embodiments, the number of captured barcode units is adjusted. In some embodiments, the number of positioned barcode units is adjusted.

[0243] In some implementations, the total number of positioned barcode units is maintained at a minimum required level, specifically the minimum required to capture positioned barcode units. In other implementations, the total number of captured barcode units is maintained at a higher level in order to capture as many analytes as possible.

[0244] In some implementations, the number of captured barcode units is inversely proportional to the size of the captured barcode units (i.e., the number of captured barcode units increases when the size of the captured barcode units decreases, and the number of captured barcode units decreases when the size of the captured barcode units increases).

[0245] Therefore, in some implementations, when the captured barcode units have a submicron size, the number of captured barcode units increases by 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times compared to when the captured barcode units have a size greater than 1µm.

[0246] It will be apparent to those skilled in the art that the number of captured oligonucleotides in a group of barcode units can be adapted to the quantity and / or density of analytes present in a biological sample by adjusting the number of captured oligonucleotides contained in each captured barcode unit to effectively capture the analytes. In some embodiments, the number of captured oligonucleotides contained in each captured barcode unit is increased when the analyte density is high; in some embodiments, the number of captured oligonucleotides contained in each captured barcode unit is decreased when the analyte density is low. As used herein, “high” means an increase of 2, 4, 10, or greater than 10 times in the concentration of the analyte compared to the average concentration in the biological sample. As used herein, “low” means a decrease of 2, 4, 10, or greater than 10 times in the concentration of the analyte compared to the average concentration in the biological sample.

[0247] In some implementations, when the sample contains low-density analytes, the number of captured barcode units is increased by 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times.

[0248] In some implementations, the group of barcode units comprises at least about 1,000, about 5,000, about 10,000, about 50,000, about 100,000, about 1,000,000, about 5,000,000, about 10,000,000, or more than 10,000,000 barcode units.

[0249] In one embodiment, the ratio of each captured barcode oligonucleotide to the analyte is about 1:1, about 2:1, about 5:1, about 10:1, about 100:1, or greater than 100:1, preferably about 10:1. In some embodiments, the number of barcode units is at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, or at least 1000 times less than the number of analytes.

[0250] In another embodiment, the capture barcode oligonucleotide ratio for each analyte is about 1:1, about 2:1, about 5:1, about 10:1, about 100:1, or greater than 100:1, preferably about 10:1. In some embodiments, the number of barcode units is at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, or at least 1000 times more than the number of analytes.

[0251] It will be apparent to those skilled in the art that increasing the concentration of the capture barcode oligonucleotide and / or increasing the number of capture oligonucleotides per capture barcode unit increases the probability of capturing oligonucleotides binding to analytes.

[0252] In some embodiments, the group of barcode units contains at least 1,000 barcode units. In some embodiments, the group of barcode units contains at least 10,000 barcode units. In some embodiments, the group of barcode units contains at least 100,000 barcode units. In some embodiments, the group of barcode units contains at least 1,000,000 barcode units.

[0253] In some implementations, the assembly of the capture barcode unit and the positioning barcode unit described herein is stable. As used herein, "stable" means that the assembly is not altered or significantly changed by external and / or internal constraints, damage and / or stress.

[0254] In some implementations, the assembly of the capture barcode unit and the positioning barcode unit described herein is stable over time, i.e., it does not change or remains substantially unchanged after 1 hour, 1 day, 1 week, 1 month, 1 year or more.

[0255] In some embodiments, the assembly of the capture barcode unit and the positioning barcode unit described herein is stable when exposed to external damage such as mechanical stress or temperature. In some embodiments, the assembly of the capture barcode unit and the positioning barcode unit described herein is stable when exposed to mechanical damage such as mechanical shock, vibration, or friction. In some embodiments, the assembly of the capture barcode unit and the positioning barcode unit described herein is thermally stable, i.e., it remains stable at temperatures up to 10°C, 20°C, 30°C, 35°C, 37°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or above 90°C.

[0256] In a preferred embodiment, the assembly of the capture barcode unit and the positioning barcode unit described herein is stable during storage. In some embodiments, the assembly of the capture barcode unit and the positioning barcode unit described herein is stored in a frozen state. "Frozen state" means maintained at a temperature of -200°C to 0°C, -200°C to -20°C, -200°C to -80°C, or at a temperature of about -200°C, about -196°C, about -150°C, about -80°C, or about -20°C.

[0257] In a preferred embodiment, the assembly of the capture barcode unit and the positioning barcode unit described herein is stable when transported and / or shipped by conventional means, i.e., in suitable packaging, which may or may not be refrigerated by conventional means (e.g., dry ice) and transported by land, air or sea.

[0258] In some implementations, the assembly of the capture barcode unit and the positioning barcode unit described herein further includes one or more components to increase stability.

[0259] In some embodiments, one or more components may be any suitable molecule or mixture of molecules that increase the robustness of the assembly. In some embodiments, one or more components are selected from any kind of hydrogel, preferably a depolymerizable hydrogel, a thickener or any agent that increases viscosity, such as polyethylene glycol (PEG), pectin, gelatin, agar, cellulose, gum, sugars, peptides or other polymers, or a freezing liquid, optionally containing a cryoprotectant.

[0260] In one implementation, the groups of captured barcode units and the groups of positioned barcode units are stored separately, i.e., in separate containers, tubes, vials, etc. It should be understood that for applications requiring a heterogeneous ratio of captured and positioned barcode units, having separate containers to allow for adjustment of the ratio as needed is more convenient.

[0261] In another embodiment, the group of capture barcode units and the group of positioning barcode units are stored in the same container, tube, or vial, wherein the groups of both barcode units are maintained under conditions preventing hybridization. In some embodiments, the conditions preventing hybridization can be achieved by any suitable means, such as chemical, freezing, or retention on another support. In a preferred embodiment, the conditions preventing hybridization are reversible; in particular, the conditions preventing hybridization can be reversed before using the composition according to the invention. It should be understood that for applications requiring a 1:1 ratio of capture barcode units to positioning barcode units, it is more convenient to have a ready-made 1:1 ratio composition in one container.

[0262] In some embodiments, the contact area between the capturing barcode unit and the positioning barcode unit has a surface area equivalent to 1 / 4, 1 / 8, 1 / 16, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 110, 1 / 120, 1 / 130, 1 / 140, 1 / 150, 1 / 160, 1 / 170, 1 / 180, 1 / 190, 1 / 200, or less than 1 / 200 of the total surface area of ​​the barcode unit. In some embodiments, the contact area between the capturing barcode unit and the positioning barcode unit has a surface area equivalent to at least 1 / 200, at least 1 / 100, or at least 1 / 50 of the total surface area of ​​the barcode unit.

[0263] In some implementations, the contact area between the capturing barcode unit and the positioning barcode unit has a minimum of 1 nm. 2 At least 5nm 2 At least 10nm 2 At least 50nm 2 At least 100nm 2 At least 500nm 2At least 1µm 2 At least 5µm 2 At least 10µm 2 At least 50µm 2 At least 100µm 2 or greater than 100µm 2 The surface area. In some implementations, the contact area between the capturing barcode unit and the positioning barcode unit has a maximum of 500 µm. 2 Up to 100µm 2 or at most 50µm 2 Surface area.

[0264] In some implementations, the contact area between the capture barcode unit and the positioning barcode unit is 1 nm. 2 Up to 100µm 2 Preferably 10nm 2 up to 10µm 2 .

[0265] In some embodiments, the composition of the present invention comprises:

[0266] - A group of capture barcode units, wherein each capture barcode unit contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0267] - A group of positioning barcode units, wherein each positioning barcode unit contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of binding the capture sequence;

[0268] Each barcode unit contains a different barcode sequence, and all oligonucleotides on the same barcode unit contain the same barcode sequence.

[0269] In some embodiments, the composition of the present invention comprises:

[0270] - A group of capture barcode units, wherein each capture barcode unit contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0271] - A group of positioning barcode units, wherein each positioning barcode unit contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of binding the capture sequence;

[0272] Each barcode unit contains a different barcode sequence, and all oligonucleotides on the same barcode unit contain the same barcode sequence.

[0273] The captured sequence cannot hybridize with other captured sequences, and

[0274] The linker sequence cannot hybridize with other linker sequences.

[0275] In some embodiments, the composition of the present invention comprises:

[0276] - A group of capture barcode units, wherein each capture barcode unit contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0277] - A group of positioning barcode units, wherein each positioning barcode unit contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of binding the capture sequence;

[0278] Each barcode unit contains a different barcode sequence, and all oligonucleotides on the same barcode unit contain the same barcode sequence.

[0279] The captured sequence cannot hybridize with other captured sequences.

[0280] The linker sequence cannot hybridize with other linker sequences.

[0281] The captured oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, a barcode sequence, optionally at least one UMI, and a capture sequence, and

[0282] The localization oligonucleotides described herein comprise, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, barcode sequence, and linker sequence.

[0283] In some embodiments, the composition of the present invention comprises:

[0284] - A group of capture barcode beads, wherein each capture barcode bead contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0285] - A group of positioning barcode beads, wherein each positioning barcode bead contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of binding the capture sequence;

[0286] Each barcode bead contains a different barcode sequence, and all oligonucleotides on the same barcode bead contain the same barcode sequence.

[0287] In some embodiments, the composition of the present invention comprises:

[0288] - A group of capture barcode beads, wherein each capture barcode bead contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0289] - A group of positioning barcode beads, wherein each positioning barcode bead contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of binding the capture sequence;

[0290] Each barcode bead contains a different barcode sequence, and all oligonucleotides on the same barcode bead contain the same barcode sequence.

[0291] The captured sequence cannot hybridize with other captured sequences, and

[0292] The linker sequence cannot hybridize with other linker sequences.

[0293] In some embodiments, the composition of the present invention comprises:

[0294] - A group of capture barcode beads, wherein each capture barcode bead contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample;

[0295] - A group of positioning barcode beads, wherein each positioning barcode bead contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a linker sequence capable of binding the capture sequence;

[0296] Each barcode bead contains a different barcode sequence, and all oligonucleotides on the same barcode bead contain the same barcode sequence.

[0297] The captured sequence cannot hybridize with other captured sequences.

[0298] The linker sequence cannot hybridize with other linker sequences.

[0299] The captured oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, a barcode sequence, optionally at least one UMI, and a capture sequence, and

[0300] The localization oligonucleotides described herein comprise, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, barcode sequence, and linker sequence.

[0301] The present invention also relates to a substrate comprising a composition according to the invention, wherein a group of capturing barcode units and a group of positioning barcode units are assembled on the substrate as described herein.

[0302] In some implementations, the biological sample is selected from tissues, tissue sections, monolayers of cells, multilayers of cells, organoids, syncytia, or single cells.

[0303] In some embodiments, the analyte is released from the biological sample. It should be understood that the analyte must be readily accessible to the composition according to the invention, particularly to the capture barcode unit.

[0304] In some implementations, the release of analytes from biological units includes the step of lysing a biological sample or the cells that make up it.

[0305] In some embodiments, lysing a biological sample or otherwise releasing an analyte from a biological sample includes disrupting, permeating, or dissolving the lipid membrane (e.g., lipid bilayer) of the biological sample. In some embodiments, lysis constitutes permeation of the biological membrane. In a preferred embodiment, lysis does not alter the spatial position of the analyte.

[0306] Means and methods for lysing biological samples or otherwise releasing analytes from biological samples are well known in the art. In some embodiments, the method is chemical or mechanical. In some embodiments, the method is selected from: chemical lysis (e.g., using one or more of a detergent, a dissociating agent, an alkaline reagent, a solvent, etc.), physical lysis (e.g., by heating, one or more freeze / thaw cycles, osmotic shock, cavitation, sonication, etc.), and enzymatic lysis (e.g., using an enzyme).

[0307] In some embodiments, the analyte is selected from nucleic acids, amino acids, and small molecules. In other embodiments, the analyte is selected from DNA, RNA, miRNA, peptides or proteins, and small molecules.

[0308] Non-restricted examples of DNA include genomic DNA and organelle DNA, namely mitochondrial DNA or chloroplast DNA. Non-restricted examples of RNA include messenger RNA, transfer RNA, ribosomal RNA, microRNA, small interfering RNA, Piwi-interacting RNA, antisense RNA, small nucleolar RNA, small Cahalson RNA, and enhancer RNA.

[0309] In some implementations, the analyte is a nucleic acid molecule, preferably selected from DNA, RNA, and microRNA.

[0310] In some embodiments, the analyte is RNA. In some embodiments, the analyte is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA). In a preferred embodiment, the analyte is mRNA.

[0311] In some implementations, the analyte is DNA.

[0312] In one embodiment, the analyte is a single analyte. In another embodiment, the analyte is a multi-analyte, meaning that the analyte contains more than one type of molecule (e.g., DNA molecules and peptides), optionally, the molecules are linked together.

[0313] In some embodiments, the analyte is a nucleic acid bound to another molecule selected from peptides, polypeptides, proteins, antibodies, small molecules, carbohydrates, lipids, and combinations thereof. In some embodiments, the nucleic acid is a tag or label for other molecules, such as peptides, polypeptides, proteins, antibodies, small molecules, carbohydrates, and lipids. A “tag” or “label” refers to the covalent or non-covalent attachment or linkage of a nucleic acid to the analyte. In some embodiments, the tag may include, for example, annealing, hybridizing, or ligating the nucleic acid to the analyte.

[0314] In some implementations, the steps of releasing the analyte from the biological sample, and optionally lysing the biological sample, are performed on the substrate or support described herein.

[0315] In some embodiments, the step of releasing the analyte from the biological sample, and optionally lysing the biological sample, includes or is followed by a sub-step of freezing the biological sample and / or releasing the analyte from the biological sample. Freezing is defined as placing the sample at temperatures from -200°C to 0°C, -200°C to -20°C, -200°C to -80°C, or approximately -200°C, approximately -196°C, approximately -80°C, or approximately -20°C. In some embodiments, the biological sample and / or the analyte released from the biological sample is rapidly frozen (e.g., with liquid nitrogen, dry ice, etc.).

[0316] In some implementations, the mRNA analyte bound to the capture sequence is reverse transcribed to form complementary DNA (cDNA).

[0317] The present invention also relates to a method for determining the spatial location of a group of captured barcode units, comprising the following steps:

[0318] (i) To bring the group of capturing barcode units from the composition according to the invention into contact with the group of positioning barcode units;

[0319] (ii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide;

[0320] (iii) At the multiple contact points formed in step (ii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0321] (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acid sequences comprising a barcode sequence of a capture barcode unit and a barcode sequence of a positioning barcode unit;

[0322] (v) Optionally, the multiple nucleic acid sequences obtained in step (iv) are amplified to obtain multiple amplicones;

[0323] (vi) Sequencing the multiple nucleic acid sequences obtained in step (iv) or the multiple amplicon obtained in step (v);

[0324] (vii) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0325] (viii) Inferring the relative position of each captured barcode cell and / or each positioned barcode cell; and

[0326] (ix) Optionally, infer the absolute position of each captured barcode unit and / or each positioned barcode unit.

[0327] In one implementation, the complementary sequence is a capture sequence or a fragment thereof.

[0328] In another embodiment, the complementary sequence is a second linker sequence or a fragment thereof. In some embodiments, the second linker sequence is located on the capturing oligonucleotide. In some embodiments, the positioning oligonucleotide does not contain a second linker sequence.

[0329] As described above, after hybridization of the captured oligonucleotide with the localizing oligonucleotide (step (iii)) and extension of the hybridized oligonucleotide (step (iv)), multiple nucleic acid molecules are obtained comprising a barcode sequence from the captured barcode unit and a barcode sequence from the localizing barcode unit. These multiple obtained nucleic acid molecules form a library or network that summarizes the proximity relationships of all barcode units, thereby summarizing their relative positions. Therefore, the step (vii) of inferring the proximity relationship between the captured barcode unit and the localizing barcode unit, and the step (viii) of inferring the relative position of each captured barcode unit and / or localizing barcode unit, are based on determining which localizing barcode units a given captured barcode unit interacts with or does not interact with, and then determining which common localizing barcode units two or more captured barcode units interact with or do not interact with.

[0330] In some implementations, step (vi) of sequencing the plurality of nucleic acid sequences obtained in step (iv) or the plurality of amplicones obtained in step (v) further includes detecting or identifying barcode sequences from capture barcode units and barcode sequences from positioning barcode units on each of the plurality of nucleic acid sequences obtained in step (iv) or on each of the plurality of amplicones obtained in step (v).

[0331] In some implementations, in step (vii) of inferring the proximity relationship between the captured barcode unit and the positioned barcode unit, the captured barcode unit and the positioned barcode unit are inferred, determined, or established as:

[0332] - If at least one nucleic acid sequence containing the barcode sequence of the captured barcode unit and the barcode sequence of the positioned barcode unit is detected, it is either adjacent or neighboring; or

[0333] - If no nucleic acid sequence containing the barcode sequence of the captured barcode unit and the barcode sequence of the located barcode unit is detected, then they are neither adjacent nor neighboring.

[0334] In some implementations, in step (viii) of inferring the relative positions of each captured barcode unit and / or each positioned barcode unit, the two captured barcode units are inferred, determined, or established as:

[0335] - If they share a connection with at least one, preferably at least two, more preferably at least three positioning barcode units (i.e., if a first nucleic acid molecule containing a barcode sequence of a first capture barcode unit and a positioning barcode unit, and a second nucleic acid molecule containing a barcode sequence of a second capture barcode unit and the same positioning barcode unit can be detected in step (vi), they are adjacent, neighboring, or closely positioned; or

[0336] - They are not adjacent if they share a connection with zero positioning barcode units.

[0337] In some implementations, steps (i) and (ii) are performed simultaneously.

[0338] In some implementations, step (i) is performed before step (ii).

[0339] In some implementations, in step (i), the group of captured barcode units and the group of located barcode units are added simultaneously.

[0340] In one implementation, in step (i), the group of captured barcode units is added before the group of positioned barcode units. In another implementation, in step (i), the group of positioned barcode units is added before the group of captured barcode units.

[0341] In some implementations, in step (i), at least one layer is formed by a group of captured barcode units, and at least one layer is formed by a group of positioned barcode units.

[0342] In some implementations, the biological sample is a tissue, tissue slice, single-layer cell or multi-layer cell.

[0343] In some implementations, multiple contact points are formed between groups of captured and positioned barcode units, wherein each contact point involves a captured oligonucleotide and a positioned oligonucleotide.

[0344] In one embodiment, the contact point relates to a capture sequence of at least one capture oligonucleotide of the capture barcode unit and a first linker sequence of at least one positioning oligonucleotide of the positioning barcode unit.

[0345] In another embodiment, the contact point relates to a second linker sequence of at least one capturing oligonucleotide of the capturing barcode unit and a first linker sequence of at least one positioning oligonucleotide of the positioning barcode unit.

[0346] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the capture oligonucleotides on the capture barcode unit participate in forming a contact point with the positioning barcode unit. In some embodiments, at least 100, at least 1000, at least 10000, at least 100000, or more than 100000 capture oligonucleotides on the capture barcode unit participate in forming a contact point with the positioning barcode unit. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the capture oligonucleotides on the capture barcode unit participate in forming a contact point with the positioning barcode unit. In some implementations, approximately 100, 1,000, 10,000, 100,000, or more than 100,000 capture oligonucleotides on the capture barcode unit participate in forming contact points with the positioning barcode unit.

[0347] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the capture oligonucleotides on the positioning barcode unit participate in forming a contact point with the capture barcode unit. In some embodiments, at least 100, at least 1000, at least 10000, at least 100000, or more than 100000 positioning oligonucleotides on the positioning barcode unit participate in forming a contact point with the capture barcode unit. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the capture oligonucleotides on the positioning barcode unit participate in forming a contact point with the capture barcode unit. In some implementations, approximately 100, 1,000, 10,000, 100,000, or more than 100,000 positioning oligonucleotides on the positioning barcode unit participate in forming contact points with the capture barcode unit.

[0348] In some embodiments, each capture barcode unit forms at least one, preferably at least two, more preferably at least three contact points with different positioning barcode units. In some embodiments, each capture barcode unit is connected to at least two, more preferably at least three different positioning barcode units. In some embodiments, each capture barcode unit forms at least one contact point with at least one, preferably at least two, more preferably at least three positioning barcode units. In some embodiments, each capture barcode unit forms one contact point with at least one, preferably at least two, more preferably at least three positioning barcode units. In some embodiments, each capture barcode unit forms at least one contact point with one, two, three, four, five, six, seven, eight, nine, or more than nine positioning barcode units. In some embodiments, each capture barcode unit forms one contact point with one, two, three, four, five, six, seven, eight, nine, or more than nine positioning barcode units. As used in this article, "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0349] It should be understood that the number of contact points mentioned above represents the average value of the entire group of barcode units.

[0350] In some implementations, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 100% of the captured barcode units have formed at least one contact point with at least one positioning barcode unit.

[0351] In some implementations, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 100% of the positioning barcode units have formed at least one contact point with at least one capturing barcode unit.

[0352] It should be understood that as long as the majority of the barcode units are connected as described above, the group of capturing barcode units and the group of positioning barcode units are considered connected or assembled; strictly speaking, not 100% of the barcode units need to be connected. In some implementations, up to 30%, up to 20%, up to 10%, up to 5%, or less than 5% of the capturing barcode units are not connected to any positioning barcode units.

[0353] In some implementations, the contact points are evenly distributed within a group of barcode cells. In other implementations, the average number of contact points within a group of barcode cells is similar or equal.

[0354] In some implementations, the assembly of a group of captured barcode units with a group of positioned barcode units increases the chance of forming contact points.

[0355] In some implementations, capture oligonucleotides and localization oligonucleotides hybridize.

[0356] In a preferred embodiment, hybridization occurs at the contact points as defined herein. In some embodiments, hybridization occurs at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the contact points. In some embodiments, hybridization occurs at about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the contact points.

[0357] Therefore, in a preferred embodiment, each capture barcode unit forms at least one, preferably at least two, and more preferably at least three hybrid contact points with different positioning barcode units.

[0358] In one embodiment, the capture sequence or a fragment thereof of the capture oligonucleotide hybridizes with the first linker sequence or a fragment thereof of the localization oligonucleotide.

[0359] In another embodiment, the second linker sequence or a fragment thereof of the captured oligonucleotide hybridizes with the first linker sequence or a fragment thereof of the localized oligonucleotide.

[0360] In some embodiments, the capture oligonucleotide cannot hybridize with localization oligonucleotides other than the capture sequence and the first linker sequence. In some embodiments, the capture oligonucleotide cannot hybridize with localization oligonucleotides other than the first linker sequence and the second linker sequence. In some embodiments, the capture oligonucleotide cannot hybridize with localization oligonucleotides other than the capture sequence and the first and second linker sequences.

[0361] In some implementations, hybridization is stable. In some implementations, the capturing oligonucleotide that hybridizes with the localizing oligonucleotide does not de-hybridize. In some implementations, the capturing oligonucleotide that hybridizes with the localizing oligonucleotide does not spontaneously de-hybridize.

[0362] In some implementations, the hybridization of the group of capture barcode units and the group of location barcode units stabilizes the assembly of the group of capture barcode units and the group of location barcode units.

[0363] This method includes the steps of extending the capture oligonucleotide and / or positioning the oligonucleotide. Methods for extending oligonucleotides will be apparent to those skilled in the art.

[0364] In some implementations, an extension step may be performed on one or two oligonucleotides.

[0365] In one embodiment, the capturing oligonucleotide extends onto the positioning oligonucleotide. In some embodiments, the positioning oligonucleotide serves as a template for the extension of the capturing oligonucleotide.

[0366] In some embodiments, the nucleic acid derived from the extension of the capture oligonucleotide comprises a barcode sequence of the capture oligonucleotide and a barcode sequence of the localization oligonucleotide in the same strand. In some embodiments, the nucleic acid derived from the extension of the capture oligonucleotide further comprises one or more elements selected from the capture sequence, one or more UMIs from the capture oligonucleotide, one or more UMIs from the localization oligonucleotide, one or more PCR primer sequences from the capture oligonucleotide, and one or more PCR primer sequences from the localization oligonucleotide. In some embodiments, the nucleic acid derived from the extension of the capture oligonucleotide further comprises one or more elements selected from a second linker sequence, one or more UMIs from the capture oligonucleotide, one or more UMIs from the localization oligonucleotide, one or more PCR primer sequences from the capture oligonucleotide, and one or more PCR primer sequences from the localization oligonucleotide.

[0367] In some implementations, the nucleic acids resulting from the elongation of the captured oligonucleotides are comprised in a 5' to 3' sequence:

[0368] -Optionally, from one or more PCR primer sequences that capture oligonucleotides;

[0369] - Capture the barcode sequence of oligonucleotides;

[0370] -Optionally, from one or more UMIs that capture oligonucleotides;

[0371] -Capture sequence;

[0372] -Optionally, from one or more UMIs of a localized oligonucleotide;

[0373] - The barcode sequence for locating oligonucleotides; and

[0374] -Optionally, from one or more PCR primer sequences derived from the localization oligonucleotide.

[0375] In some implementations, the nucleic acids resulting from the elongation of the captured oligonucleotides are comprised in a 5' to 3' sequence:

[0376] -Optionally, from one or more PCR primer sequences that capture oligonucleotides;

[0377] - Capture the barcode sequence of oligonucleotides;

[0378] -Optionally, from one or more UMIs that capture oligonucleotides;

[0379] -Second connection sequence;

[0380] -Optionally, from one or more UMIs of a localized oligonucleotide;

[0381] - The barcode sequence for locating oligonucleotides; and

[0382] -Optionally, from one or more PCR primer sequences derived from the localization oligonucleotide.

[0383] In another embodiment, the localizing oligonucleotide extends onto the capturing oligonucleotide. In some embodiments, the capturing oligonucleotide serves as a template for the extension of the localizing oligonucleotide.

[0384] In some embodiments, the nucleic acid derived from the extension of the targeting oligonucleotide comprises a barcode sequence of the targeting oligonucleotide and a barcode sequence of the capturing oligonucleotide. In some embodiments, the nucleic acid derived from the extension of the targeting oligonucleotide further comprises one or more elements selected from a first linker sequence, one or more UMIs from the targeting oligonucleotide, one or more UMIs from the capturing oligonucleotide, one or more PCR primer sequences from the targeting oligonucleotide, and one or more PCR primer sequences from the capturing oligonucleotide. In some embodiments, the nucleic acid derived from the extension of the targeting oligonucleotide further comprises one or more elements selected from a second linker sequence, one or more UMIs from the targeting oligonucleotide, one or more UMIs from the capturing oligonucleotide, one or more PCR primer sequences from the targeting oligonucleotide, and one or more PCR primer sequences from the capturing oligonucleotide.

[0385] In some implementations, the nucleic acids resulting from the extension of the localizing oligonucleotides comprise, in a 5' to 3' sequence:

[0386] -Optionally, from one or more PCR primer sequences derived from the localization oligonucleotide;

[0387] -Locating the barcode sequence of oligonucleotides;

[0388] -Optionally, from one or more UMIs of a localized oligonucleotide;

[0389] - First connection sequence;

[0390] -Optionally, from one or more UMIs that capture oligonucleotides;

[0391] - Capture the barcode sequence of oligonucleotides; and

[0392] -Optionally, from one or more PCR primer sequences that capture oligonucleotides.

[0393] In another embodiment, both the targeting oligonucleotide and the capturing oligonucleotide are extended. In some embodiments, both the targeting oligonucleotide and the capturing oligonucleotide serve as templates for each other's extension. In some embodiments, the nucleic acid resulting from the extension of the targeting oligonucleotide and the capturing oligonucleotide may contain any of the configurations described herein.

[0394] It will be apparent to those skilled in the art that, after extension, the sequence corresponding to the sequence contained on the template sequence is a complementary sequence on the nucleic acid formed after extension.

[0395] It should be understood that forming a nucleic acid that contains both a barcode sequence of a capture oligonucleotide and a barcode sequence of a localizing oligonucleotide is important for retrieving information about which capture oligonucleotide interacts with which localizing oligonucleotide, thereby reconstructing the proximity relationships between barcode units.

[0396] The present invention also relates to a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes.

[0397] It should be understood that the determination of the spatial location of the analyte depends on (i) the method according to the invention for determining the spatial location of a group of captured barcode units, and (ii) the ability of the captured barcode units to combine with the analyte.

[0398] It should be understood that obtaining the relative or absolute position of a particular captured barcode unit can infer the relative or absolute position of the analyte associated with or otherwise linked to that barcode.

[0399] Therefore, in some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the following steps:

[0400] (i) To bring the group of capturing barcode units and the group of positioning barcode units of the composition according to the invention into contact with a biological sample;

[0401] (ii) Determine the spatial location of the captured barcode unit using the method according to the invention;

[0402] (iii) Detecting the capture barcode unit to which each analyte is bound; and

[0403] (iv) Inferring the spatial location of the object being analyzed.

[0404] In an alternative implementation, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the following steps:

[0405] (i) bringing the group of capture barcode units and the group of positioning barcode units of the composition according to the invention into contact with the biological sample; and

[0406] (ii) Determine the spatial location of the captured barcode unit using the method according to the invention;

[0407] (iii) Detecting the capture barcode unit to which each analyte is bound; and

[0408] (iv) Inferring the spatial location of the object being analyzed.

[0409] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the step of contacting a group of captured and localized barcode units with the biological sample, wherein this step is performed prior to the step of amplifying multiple nucleic acid sequences generated by the extension of the captured and localized oligonucleotides through hybridization.

[0410] In a preferred embodiment, the biological sample is lysed as described herein to release the analyte.

[0411] In some embodiments, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes a step of capturing multiple analytes from the biological sample by incorporating the multiple analytes into a capture sequence of multiple capture barcode units, wherein this step is performed prior to a step of amplifying multiple nucleic acid sequences resulting from the extension of hybridized capture and localization oligonucleotides, wherein this step is performed after a step of hybridizing at least one linker sequence on the localization oligonucleotide with at least one complementary sequence on the capture oligonucleotide.

[0412] In some embodiments, the analyte binds to the capture sequence. In some embodiments, the analyte hybridizes with the capture sequence. Illustratively but not limitingly, the poly(dT) capture sequence may hybridize with the poly(A) tail of the mRNA. In a preferred embodiment, no more than one analyte binds to one capture oligonucleotide.

[0413] In some embodiments, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the step of extending a capture sequence onto the sequences of the multiple analytes to obtain multiple nucleic acids comprising a barcode sequence of a capture barcode unit and the sequence of the analytes, wherein this step is performed prior to the step of amplifying multiple nucleic acid sequences resulting from the extension of capture and localization oligonucleotides by hybridization, wherein this step is performed after the step of hybridizing at least one linker sequence on the localization oligonucleotide with at least one complementary sequence on the capture oligonucleotide.

[0414] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes a step of amplifying multiple nucleic acids comprising a barcode sequence containing a capture barcode unit and a sequence of the analyte, wherein this step is performed after a step of capturing and locating oligonucleotides by extension hybridization.

[0415] In some implementations, methods for determining the spatial location of multiple analytes in a biological sample containing multiple analytes include sequencing multiple nucleic acid sequences or multiple amplicones containing a barcode sequence of a capture barcode unit and a sequence of the analyte.

[0416] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the steps of inferring the relative location of each analyte and optionally inferring the absolute location of each analyte, wherein this step is performed after the step of inferring the relative location of each capture barcode unit and / or positioning barcode unit.

[0417] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the following steps:

[0418] (i) To bring the group of capturing barcode units from the composition according to the invention into contact with the group of positioning barcode units;

[0419] (ii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide;

[0420] (iii) At the multiple contact points formed in step (ii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0421] (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acid sequences comprising a barcode sequence of a capture barcode unit and a barcode sequence of a positioning barcode unit;

[0422] (v) Optionally, the multiple nucleic acid sequences obtained in step (iv) are amplified to obtain multiple amplicones;

[0423] (vi) Sequencing the multiple nucleic acid sequences obtained in step (iv) or the multiple amplicon obtained in step (v);

[0424] (vii) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0425] (viii) Inferring the relative position of each captured barcode cell and / or each positioned barcode cell; and

[0426] (ix) Optionally, infer the absolute position of each captured barcode unit and / or each positioned barcode unit;

[0427] And it also includes the following steps:

[0428] a) In any step prior to step (v), bring the group of captured barcode units and the group of positioned barcode units into contact with the biological sample;

[0429] b) In any step after step (a) and before step (v), multiple analytes from a biological sample are captured by combining multiple analytes into a capture sequence of multiple capture barcode units;

[0430] c) Optionally, after step (b), the capture sequence is extended onto the sequences of multiple analytes to obtain multiple nucleic acids containing a barcode sequence of the capture barcode unit and the sequence of the analyte;

[0431] d) Optionally, after step (c), multiple nucleic acids are amplified to obtain multiple amplicones;

[0432] e) Sequencing the multiple nucleic acid sequences obtained in step (c) or the multiple amplicon obtained in step (d);

[0433] f) In any step after step (viii), infer the relative position of each analyte; and optionally, infer the absolute position of each analyte.

[0434] In some embodiments, the biological sample is mapped before step (a) or before step (b). As used herein, “mapped” means any suitable means known in the art that provides topological or any other locational information of a biological sample. In some embodiments, the biological sample is mapped using any technique selected from immunohistochemistry, immunofluorescence, in situ hybridization, affinity labeling, etc.

[0435] It will be apparent to those skilled in the art that mapping of biological samples or any spatial information is useful for further optimizing the determination of the spatial locations of multiple analytes. In some embodiments, the mapping information associated with the biological sample intersects with the relative or absolute locations of barcode units and / or analytes. In some embodiments, the mapping information associated with the biological sample defines or helps define the boundaries of the analytes.

[0436] In some embodiments, at least one specific target is detected in a biological sample. In some embodiments, the at least one specific target is a protein, nucleic acid, lipid, organelle, or a combination thereof. Methods for detecting specific targets in cells or tissues are well known in the art. In some embodiments, the at least one specific target is used to improve the relative or absolute positioning of barcode units and / or analytes (e.g., the specific target acts as a waypoint). In some embodiments, the at least one specific target defines the center of the biological sample.

[0437] In some embodiments, the biological sample is lysed prior to step (b) as described herein. In other words, in some embodiments, the method further includes a step (a') of lysing the biological sample.

[0438] In one implementation, the barcoding of the analyte is achieved by annealing the barcode to the primer template of the analyte, wherein the analyte is preferably a nucleic acid. In another implementation, the barcoding of the analyte is achieved by guiding the barcode to extend to the analyte using primers, wherein the analyte is preferably a nucleic acid. In yet another implementation, the barcoding of the analyte is achieved by concatenating the barcode with the analyte, wherein the analyte is preferably a nucleic acid.

[0439] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the following steps:

[0440] (i) to bring the group of the first barcode unit and the group of the second barcode unit from the composition according to the invention into contact;

[0441] (ii) Contact the group of the first barcode unit and the group of the second barcode unit from step (i) with the biological sample;

[0442] (iii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide;

[0443] (iv) At the multiple contact points formed in step (iii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0444] (v) Capturing the plurality of analytes in the biological sample by incorporating the plurality of analytes into a capture sequence of a plurality of capture barcode units;

[0445] (vi) Optionally, the capture sequence is extended on the analyte sequence to obtain multiple nucleic acids comprising a barcode sequence of the capture barcode unit and the sequence of the analyte;

[0446] (vii) Extend the multiple hybrid oligonucleotides obtained in step (iv) to obtain multiple nucleic acids containing a barcode sequence of capturing barcode units and a barcode sequence of locating barcode units;

[0447] (viii) Optionally, the multiple nucleic acids obtained in step (vii) are amplified, and the multiple nucleic acids obtained in step (vi) are amplified to obtain multiple amplicones;

[0448] (ix) Sequencing the multiple nucleic acid sequences obtained in step (vii), and optionally the multiple nucleic acid sequences obtained in step (vi) or the multiple amplicon obtained in step (viii);

[0449] (x) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0450] (xi) Infer the relative position of each analyte; and

[0451] (xii) Optionally, infer the absolute position of each analyte.

[0452] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the following steps:

[0453] (i) to bring the group of the first barcode unit and the group of the second barcode unit of the composition according to the invention into contact;

[0454] (ii) Multiple contact points are formed between groups of capture and positioning barcode units, wherein each contact point involves a capture oligonucleotide and a positioning oligonucleotide;

[0455] (iii) At the multiple contact points formed in step (iii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0456] (iv) Contact the group of the first barcode unit and the group of the second barcode unit from step (i) with the biological sample;

[0457] (v) The plurality of analytes are captured from the biological sample by combining the plurality of analytes into a capture sequence of a plurality of capture barcode units;

[0458] (vi) Optionally, the capture sequence is extended on the analyte sequence to obtain a plurality of nucleic acids comprising a barcode sequence and an analyte sequence containing capture barcode units;

[0459] (vii) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acids containing a barcode sequence of capturing barcode units and a barcode sequence of locating barcode units;

[0460] (viii) Optionally, the multiple nucleic acids obtained in step (vii) and the multiple nucleic acids obtained in step (vi) are amplified to obtain multiple amplicones;

[0461] (ix) Sequencing the multiple nucleic acid sequences obtained in step (vii), and optionally the multiple nucleic acid sequences obtained in step (vi) or the multiple amplicon obtained in step (viii);

[0462] (x) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0463] (xi) Infer the relative position of each analyte; and

[0464] (xii) Optionally, infer the absolute position of each analyte.

[0465] In some implementations, a method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes includes the following steps:

[0466] (i) to bring the group of the first barcode unit and the group of the second barcode unit of the composition according to the invention into contact;

[0467] (ii) Multiple contact points are formed between groups of capture and positioning barcode units, wherein each contact point involves a capture oligonucleotide and a positioning oligonucleotide;

[0468] (iii) At the multiple contact points formed in step (iii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides;

[0469] (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acids comprising a barcode sequence for capturing barcode units and a barcode sequence for locating barcode units;

[0470] (v) Contact the group of the first barcode unit and the group of the second barcode unit from step (i) with the biological sample;

[0471] (vi) The plurality of analytes are captured from the biological sample by combining the plurality of analytes into a capture sequence of a plurality of capture barcode units;

[0472] (vii) Optionally, the capture sequence is extended on the analyte sequence to obtain multiple nucleic acids comprising a barcode sequence of the capture barcode unit and the sequence of the analyte;

[0473] (viii) Optionally, the multiple nucleic acids obtained in step (iv) and the multiple nucleic acids obtained in step (vii) are amplified to obtain multiple amplicones;

[0474] (ix) Sequencing the multiple nucleic acid sequences obtained in step (iv), and optionally the multiple nucleic acid sequences obtained in step (vii) or the multiple amplicon obtained in step (viii);

[0475] (x) Infer the proximity relationship between the captured barcode unit and the located barcode unit;

[0476] (xi) Infer the relative position of each analyte; and

[0477] (xii) Optionally, infer the absolute position of each analyte.

[0478] In some embodiments, the capture oligonucleotide bound to the analyte is extended. In some embodiments, the extension of the capture oligonucleotide bound to the analyte yields one or two nucleic acid molecules.

[0479] In some embodiments, the nucleic acid derived from the extension of the capture oligonucleotide bound to the analyte comprises a barcode sequence of the capture oligonucleotide, a capture sequence, and the sequence of the analyte. In some embodiments, the nucleic acid derived from the extension of the capture oligonucleotide bound to the analyte further comprises one or more UMIs and one or more primer sequences.

[0480] In some implementations, the nucleic acid resulting from the extension of the capture oligonucleotide bound to the analyte comprises, in a 5' to 3' sequence:

[0481] -Optionally, from one or more PCR primer sequences that capture oligonucleotides;

[0482] - Capture the barcode sequence of oligonucleotides;

[0483] -Optionally, from one or more UMIs that capture oligonucleotides;

[0484] -Capture sequence; and

[0485] - The sequence of the analyte.

[0486] It will be apparent to those skilled in the art that, after extension, the sequence of the analyte is a form of complementary sequence on the nucleic acid formed after extension.

[0487] In some embodiments, the method further includes the step of forming at least one, preferably two, complementary DNAs from the extension of the captured and localized oligonucleotides of hybridization, and / or from the extension of the captured oligonucleotides bound to the analyte.

[0488] In a preferred embodiment, nucleic acids formed by the extension of captured and localized oligonucleotides from hybridization, and / or nucleic acids formed by the extension of captured oligonucleotides bound to the analyte, are amplified.

[0489] In some implementations, the amplification of nucleic acids formed by the capture and extension of hybridization oligonucleotides creates a "localization amplicon".

[0490] In some implementations, an "analyte amplicon" is formed by the extension of a capture oligonucleotide bound to the analyte or by the amplification of the capture oligonucleotide linked to the analyte sequence.

[0491] Methods for amplifying nucleic acids are well known to those skilled in the art, including, for example, PCR. As used herein, "amplifier" refers to the nucleic acid produced by amplification.

[0492] In some implementations, amplification is primer-dependent. Therefore, in some implementations, amplification begins with PCR primer sequences that capture and / or locate oligonucleotides.

[0493] In some embodiments, the amplicon has a fixed or variable length. In some embodiments, the amplicon has a monodisperse or polydisperse length. Within the scope of this invention, "monodisperse length" and "fixed length" are used interchangeably. Within the scope of this invention, "polydisperse length" and "variable length" are used interchangeably. It should be understood that the length of the capture-analyte conjugate is variable because the length of mRNA from different genes is naturally variable.

[0494] In some embodiments, the positioning amplicons have a fixed or variable size. In a preferred embodiment, the positioning amplicons have a fixed size. In fact, it should be understood that since the sequences of the capturing oligonucleotide and the positioning oligonucleotide are predetermined, extended oligonucleotides or amplicons of the desired length can be designed.

[0495] In some embodiments, the analyte amplicon has a variable or fixed size. In a preferred embodiment, the analyte amplicon has a variable size. It will be apparent to those skilled in the art that the source of the length variability is that the length of the analyte sequence may vary from analyte to analyte.

[0496] In one implementation, an amplification is performed when the PCR primer sequence on the capturing oligonucleotide is the same as the PCR primer sequence on the localizing oligonucleotide.

[0497] In some embodiments, the localization amplicon and the capture amplicon are separated or isolated based on their size. In some embodiments, the localization amplicon and the capture amplicon are separated by size exclusion methods known in the art (e.g., electrophoresis).

[0498] In another implementation, when the PCR primer sequence on the capturing oligonucleotide and the PCR primer sequence on the localizing oligonucleotide are different, two amplifications are performed, typically using two different sets of primers.

[0499] In some implementations, the primer set used includes:

[0500] - Universal forward primers (e.g., primers for capturing oligonucleotides);

[0501] - First reverse primer (e.g., primer for locating oligonucleotides); and

[0502] - Second reverse primer (e.g., primer for the analyte).

[0503] Because the sequences of analytes are naturally variable, in some embodiments, universal PCR primer sequences are added to all barcoded analytes. For example, this can be accomplished by second-strand synthesis using synthetic oligonucleotides with a degenerate sequence at the 3' end and a universal common sequence at the 5' end. In another, less preferred embodiment, a partial cDNA sequence of all or part of the gene of interest is used as a set of selective PCR primers for the barcoded analyte.

[0504] In some implementations, amplicones from two different amplifications are separated or isolated. In some implementations, the localization amplicon and the capture amplicon are formed separately.

[0505] In some embodiments, the method according to the invention may include a pre-amplification step of the analyte.

[0506] It will be apparent to those skilled in the art that, for analytes captured by the captured oligonucleotide, elongation occurs primarily via reverse transcription from the 3' end of the captured oligonucleotide, followed by the addition of a cDNA sequence to the 3' end of the captured oligonucleotide. This addition can be performed in any manner, such as ligation.

[0507] In some implementations, the method further includes the step of forming a library of at least one, preferably two, nucleic acids or amplicones as defined herein, preferably amplicones.

[0508] In a preferred embodiment, the method further includes the step of forming two libraries of nucleic acids or amplicones as defined herein, preferably amplicon libraries. In some embodiments, these two libraries include:

[0509] - A first library containing nucleic acids, or localization amplicones, formed by the extension of hybridized capture and localization oligonucleotides; and

[0510] - A second library, which contains nucleic acids formed by the extension of capture oligonucleotides bound to the analyte, or analyte amplicon.

[0511] Within the scope of this invention, the first library is interchangeably referred to as the "location library" and the second library is interchangeably referred to as the "analyte library".

[0512] In some implementations, the first library includes location information of the captured barcode units, and the second library includes analyte sequence information associated with each captured barcode unit.

[0513] In some implementations, the method further includes the step of sequencing a nucleic acid or amplicon, preferably an amplicon library, as defined herein. As used herein, for the sequencing step, nucleic acid or localization amplicon formed by the extension of capture and localization oligonucleotides of hybridization, and nucleic acid or analyte amplicon formed by the extension of capture oligonucleotides bound to an analyte, are interchangeably referred to as a sequencing “fragment” or “read”.

[0514] In a preferred embodiment, each capture barcode unit forms at least one, preferably at least two, and more preferably at least three sequencing contact points with different positioning barcode units.

[0515] Methods for nucleic acid sequencing are well known to those skilled in the art.

[0516] In one implementation, the nucleic acid formed by the extension of the capture and localization oligonucleotide of hybridization, or the localization amplicon, and / or the nucleic acid formed by the extension of the capture oligonucleotide bound to the analyte, or the analyte amplicon, is sequenced in its entire length, i.e., sequenced along its entire length of nucleic acid sequence.

[0517] In one embodiment, the nucleic acid formed by the extension of the capture and localizing oligonucleotides of hybridization, or the localizing amplicon, and / or the nucleic acid formed by the extension of the capture oligonucleotides bound to the analyte, or the analyte amplicon, is partially sequenced. In a preferred embodiment, the unsequential portion corresponds to the contact point and / or the sequence that hybridizes between the capture sequence and the analyte.

[0518] It should be understood that, for the purpose of sequencing, the libraries, preferably the first and second libraries as defined above, are separated. As mentioned above, separation can be achieved in the following ways:

[0519] - Amplicons with different sizes are separated by a size exclusion method, wherein the localized amplicon has a fixed size;

[0520] - Perform separate amplification reactions (and, for example, using different primer sets); or

[0521] - Separate the capture barcode unit and the location barcode unit before amplification.

[0522] To further optimize this method, constructing a library containing the localization information of the captured barcode units requires minimal sequencing to reconstruct the localization information; however, constructing a library containing the analyte sequence information associated with each captured barcode unit requires extensive and complete sequencing to maximize the amount of information obtained from the biological sample. Therefore, it is important to control the ratio between the localization library and the analyte library to sequence both libraries simultaneously, or to sequence each library at the optimal depth in a separate sequencing run.

[0523] In one embodiment, nucleic acids or amplicones as defined herein are separated based on their length. In one embodiment, nucleic acids or amplicones as defined herein are separated based on their length when the PCR primer sequences on the capturing oligonucleotide and the PCR primer sequences on the positioning oligonucleotide are identical and a single amplification is performed. Separation of nucleic acids based on length or size is a conventional technique well known to those skilled in the art.

[0524] In another embodiment, when the PCR primer sequences on the capturing oligonucleotide and the PCR primer sequences on the localizing oligonucleotide are different and two different amplifications are performed, the localizing amplicons and analyte amplicons are formed separately. Therefore, in some embodiments, the method further includes the following sub-steps:

[0525] As described in this article, localization amplicon is generated;

[0526] Collect and / or isolate localization amplicon;

[0527] As described in this article, generate analyte amplicon; and

[0528] Collect and / or separate analyte amplicon.

[0529] or

[0530] As described in this article, generate analyte amplicon;

[0531] Collect and / or separate analyte amplicon;

[0532] As described in this article, localization amplicon is generated; and

[0533] Collect and / or isolate localization amplicon.

[0534] In another embodiment, the nucleic acid formed by the extension of the capture and localization oligonucleotides of hybridization, and the nucleic acid formed by the extension of the capture oligonucleotides bound to the analyte, are separated prior to amplification. In some embodiments, the nucleic acid formed by the extension of the capture and localization oligonucleotides of hybridization, and the nucleic acid formed by the extension of the capture oligonucleotides bound to the analyte, are separated prior to sequencing.

[0535] For separation prior to library construction, methods such as magnetic separation, centrifugation, sedimentation, and size exclusion may be practical. In some implementations, groups of two types of barcode units are separated magnetically, where one type of barcode unit is magnetic and the other is non-magnetic. In some implementations, groups of two types of barcode units are separated by size exclusion, where the capturing barcode units and the positioning barcode units have different sizes. In some implementations, groups of two types of barcode units are separated by centrifugation or sedimentation, where the capturing barcode units and the positioning barcode units have different densities.

[0536] Therefore, one PCR from the isolated capture barcode unit generates an analyte library, while another PCR from the isolated positioning barcode unit generates a positioning library.

[0537] In some embodiments, capturing and / or locating barcode units includes methods for their separation and / or isolation. In some embodiments, one type of barcode unit is magnetic and the other is non-magnetic, and the method further includes at least one method for separating magnetic barcode units.

[0538] It will be apparent to those skilled in the art that pre-construction separation is useful for maximizing the information gathered from the biological sample while collecting the minimum required localization information. In other words, pre-construction separation allows for greater freedom or flexibility in independently adjusting the sequencing depth of each library.

[0539] It should be understood that the present invention aims to obtain information regarding the spatial location of barcode units in an assembly of two types of barcode units, and / or the spatial location of analytes in biological samples. The spatial location can be relative (e.g., barcode units relative to each barcode unit and / or analytes relative to each other captured barcode unit) or absolute (e.g., barcode units relative to a known reference point and / or analytes relative to a known reference point).

[0540] In some implementations, the relative positions of capturing and / or locating barcode units are defined relative to each barcode unit.

[0541] In some implementations, the absolute position of capturing and / or locating the barcode unit is defined relative to the absolute position of a known reference point. In one implementation, the known reference point is the center point of the assembly of barcode units, preferably the geometric center point of the assembly of barcode units. In another implementation, the known reference point belongs to the boundary of the assembly of barcode units.

[0542] In some implementations, the relative position of the analyte is defined relative to each captured barcode unit.

[0543] In some embodiments, the absolute position of the analyte is defined relative to the absolute position of a known reference point. In one embodiment, the known reference point is the center point of an assembly of barcode units, preferably the geometric center point of the assembly of barcode units. In another embodiment, the known reference point is located at the boundary of the assembly of barcode units. In another less preferred embodiment, the absolute position of the analyte is defined relative to the absolute position of a biological reference point on a biological sample, wherein the biological reference point is a protein, protein complex, nucleic acid, organelle, etc., preferably marked or labeled by any method known in the art. In some embodiments, the absolute position of the analyte reflects its position within the biological sample, for example, its position within a defined region of the biological sample, or its position within a defined cell of the biological sample.

[0544] It should be understood that determining the position of barcode units relative to each other and / or relative to a known reference point requires obtaining and summarizing the proximity relationships between barcode units, i.e., representing the network or array of connections between capture and positioning barcode units. As used herein, "connection" means a hybridization contact point, preferably a sequencing contact point. In some embodiments, the connections between capture and positioning barcode units are represented in the form of at least one graph, preferably a single graph. In a preferred embodiment, all barcode units that have formed at least one connection are included in at least one graph, preferably a single graph.

[0545] Therefore, in some implementations, the method for determining the relative or absolute position of a barcode unit, preferably capturing the relative or absolute position of the barcode unit or analyte, includes using at least one algorithm and at least one input for the algorithm.

[0546] As used in this article, the variables used in the algorithm include the following:

[0547] : The number of barcode units captured;

[0548] : The number of location barcode units;

[0549] : The quantity of analytes;

[0550] The radius of each captured barcode unit;

[0551] The radius of each positioning barcode unit;

[0552] The spatial position of the i-th captured barcode relative to the center of the barcode unit group, preferably wherein the center is the geometric center;

[0553] The spatial position of the m-th analyzer relative to the center of the barcode unit group, preferably wherein the center is the geometric center;

[0554] : The number of sequencing fragments (i.e., reads) containing the barcode sequence of the i-th capture barcode unit and the barcode sequence of the j-th positioning barcode unit;

[0555] : The number of sequencing fragments (i.e., reads) containing the barcode sequence of the i-th captured barcode unit and the sequence information of the m-th analyte.

[0556] In some implementation schemes, and / or Denominated in µm. In some implementations, and / or The coordinate values ​​are in µm.

[0557] In some implementations, the spatial resolution of the analyte's spatial location is at the level of capturing barcode units. In some implementations, if Then it is approximately considered that .

[0558] In some embodiments, at least one algorithm includes or comprises at least one graph for capturing interactions between barcode units. In some embodiments, this graph is used as input to at least one algorithm.

[0559] In one implementation, graph G is used as input. In some implementations, graph G reflects nodes as capturing barcode units and positioning barcode units, and edges connect the i-th capturing barcode unit and the j-th positioning barcode unit if and only if .

[0560] It should be understood that by applying state-of-the-art manifold learning or graph embedding methods to graph G, the relative position of each captured barcode i can be estimated. Then, the relative position of each captured analyte m is estimated. .

[0561] In some implementations, diagrams are used. The weighted version of graph G is used as input to account for some connection uncertainties. In some implementations, the weighted graph... This reflects that the node is a capture barcode unit and a positioning barcode unit, and the edge connects the i-th capture barcode unit and the j-th positioning barcode unit if and only if And the weights of these edges are .

[0562] In some implementations, diagrams are used. The weighted version of Figure G is used as input, preferably when the captured oligonucleotide and the localizing oligonucleotide contain UMI, so that PCR clones are ignored. In some implementations, It is the number of distinct sequencing fragments (i.e., reads) containing the barcode sequence of the i-th capture barcode unit and the barcode sequence of the j-th location barcode unit, after removing PCR clones based on the UMI associated with the i-th capture barcode unit and the UMI associated with the j-th location barcode unit.

[0563] In another implementation, a diagram is used. As input. In some implementations, the figure This reflects that nodes are limited to capturing barcode units, and an edge connects the i-th capturing barcode unit and the k-th capturing barcode unit if and only if there exists a positioning barcode unit j, such that... and .

[0564] In some implementations, diagrams are used. As input, the graph It is a picture The weighted version, where the edge weights are .

[0565] In some implementations, diagrams are used. As input, it is preferable when the captured oligonucleotide and the localized oligonucleotide contain UMI, so as to ignore PCR clones, wherein the figure It is a picture The weighted version, of which .

[0566] In some implementations, diagrams are used. As input, the graph It is a picture The weighted version, where the edge weights are ,in It is the Kronecker delta function, if but It equals 1, otherwise It equals 0.

[0567] In some embodiments, determining the relative or absolute position of barcode cells involves using at least one multidimensional scaling technique for graph embedding and manifold learning. In some embodiments, the at least one multidimensional scaling technique is selected from local methods (e.g., Laplacian feature maps, LLE, and diffusion maps), global methods (e.g., isometric feature maps and landmark isometric feature maps), or neural network-based methods (e.g., DIMAL: Deep Isometric Manifold Learning). Such methods are known in the art (see, for example, Pai et al., 2022, “Deep Isometric Maps”). In some embodiments, at least one algorithm is any suitable algorithm known in the art. In some embodiments, at least one algorithm is a manifold learning algorithm. In some embodiments, at least one algorithm is ISOMAP.

[0568] In some implementations, the use of the algorithm includes the step of estimating the geodesic distance between nodes of the graph in order to infer the relative spatial position of the barcode units relative to each other, wherein the geodesic distance between two nodes is the distance of the shortest path connecting the two nodes via the edges of the graph.

[0569] Therefore, in some implementations, these distances can be described using the following distance function:

[0570] It is the geodesic distance between the center of the i-th captured barcode unit and the center of the j-th positioned barcode unit; and

[0571] It is the geodesic distance between the center of the i-th captured barcode cell and the center of the k-th captured barcode cell.

[0572] In some implementation schemes, based on the triangle inequality, and .

[0573] In some implementations, for rigid and spherical barcode units:

[0574] -if ,but ;

[0575] -if and ,but ;

[0576] - If the captured barcode cells form a dense hexagonal grid, where each captured barcode cell is in physical contact with 6 other barcode cells, then: if and ,but .

[0577] As used in this paper, the following variables are defined to describe the relationship between groups of the two types of barcode units:

[0578] This indicates the number of captured barcode units that have at least one sequencing contact point with the positioning barcode unit j;

[0579] This indicates the number of positioning barcode units that have at least one sequencing contact point with the capture barcode unit i.

[0580] In one implementation, if each positioning barcode unit is connected to at most N capturing barcode units, then: if The sequence barcode of the location barcode unit j may have barcode conflicts at the level of the location barcode unit (i.e., two oligonucleotides of two different location barcode units have two identical barcode sequences), and it can be decided to exclude the location barcode unit j from the graph of interest.

[0581] In another implementation, if each capture barcode unit is connected to at most N positioning barcode units, then: if The sequence barcode of captured barcode unit i may have barcode conflicts at the level of the capture barcode unit (i.e., two different captured barcode units have two identical barcode sequences on their oligonucleotides), and it can be decided to exclude captured barcode unit i from the graph of interest.

[0582] In some implementation schemes, The predefined similarity score represents the biological characteristics of an analyte captured by the i-th capture barcode unit and the biological characteristics of an analyte captured by the k-th capture barcode unit, wherein the analyte is said to be captured by the i-th capture barcode unit if there exists at least one sequencing fragment (i.e., a read) containing the barcode sequence of the i-th capture barcode and the sequence information of the analyte.

[0583] In some implementations, determining the relative or absolute position of the barcode unit includes the following steps:

[0584] i. Use at least one predefined graph as defined in this paper, and optionally use a similarity matrix as defined in this paper. To identify the connected components of the input graph, where a connected component in the graph is defined as a group of barcode cells that are linked to each other by paths in the graph;

[0585] ii. For each connected component, estimate the geodesic distance between the centers of any two captured barcode cells belonging to that connected component (i.e., the shortest path in the graph of interest);

[0586] iii. For each of the connected components, infer the spatial location of the center of each captured barcode unit belonging to that connected component, wherein the spatial location is defined as the absolute location relative to the center of the connected component;

[0587] iv. For each of the components, infer the spatial location of the center of each analyte captured by the capture barcode unit belonging to that connected component, wherein the spatial location is defined as the absolute location relative to the center of the connected component;

[0588] v. Optionally, use a similarity matrix And / or the boundary shape of each of the connected components, estimate the geodesic distance between the centers of any two captured barcode cells belonging to the boundaries of two different connected components, and then estimate the spatial location of the center of each analyte captured by the captured barcode cells, wherein the spatial location is defined relative to the absolute location of the group of barcode cells; and

[0589] vi. Optionally, knowledge of the absolute positions of at least three barcode units may be used as spatial landmarks and / or supplementary mapping information, such as microscopic images of biological samples (e.g., fluorescence images), may be used to apply local adjustments (e.g., translation, rotation, shearing) to the estimated spatial position.

[0590] It should be understood that determining the relative or absolute position of a barcode cell may require several conditions, or may be facilitated by several conditions, such as the non-restrictive conditions detailed below.

[0591] In a preferred embodiment, for the majority of capture barcode units, each capture barcode unit is connected to at least one positioning barcode unit, which in turn is connected to at least one other capture barcode unit, and each capture barcode unit is connected to at least one analyte of the biological sample. As used herein, "majority" means at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or greater than 99% of the total number of capture barcode units. In some embodiments, each positioning barcode unit is in physical contact with three capture barcode units. In some embodiments, each capture barcode unit is in physical contact with six capture barcode units, except those at the boundaries of the assembly. In some embodiments, each capture barcode unit is in physical contact with five positioning barcode units, except those at the boundaries of the assembly.

[0592] Certain configurations of the capture barcode units can help determine their relative or absolute positions. In some embodiments, all capture barcode units have the same radius, and all positioning barcode units have the same radius. In some embodiments, the capture barcode units are rigid spherical units, while the positioning barcode units are semi-solid units. In some embodiments, the positioning barcode units are smaller than the capture barcode units, for example, twice the diameter.

[0593] In some implementations, the biological sample is located on a flat surface. In some implementations, the assembly of the captured barcode unit is a dense hexagonal mesh.

[0594] In some implementations, the graph G representing the physical contact between the captured and positioned barcode units consists of only one connected component. As used herein, a "connected component" refers to an ensemble of interconnected barcode units. In some implementations, the graph G representing the physical contact between the captured and positioned barcode units contains as many loops as possible, where a loop is defined as a closed path in the graph (i.e., starting and ending at the same barcode unit) and contains at least three distinct barcode units.

[0595] In some implementations, the graph G representing the physical contact between the captured barcode unit and the positioned barcode unit is homogeneous. In some implementations, the graph G representing the physical contact between the captured barcode unit and the positioned barcode unit is biconnected. In some implementations, the graph G representing the physical contact between the captured barcode unit and the positioned barcode unit is multi-ringed.

[0596] In some implementations, one, two, three, or four distinct connected components are formed in the graph of interest. In a preferred implementation, only one connected component is formed in the graph of interest.

[0597] In some implementations, when more than one connected component is formed in the graph of interest, the topology is reconstructed in other ways.

[0598] Some configurations for capturing barcode units may also help determine the relative or absolute position of the barcode units, but this is optional and not strictly required by the method of the present invention.

[0599] In some implementations, the absolute spatial location of the center of the group of barcode units relative to the center of the biological sample or any anchor point is known.

[0600] In some implementations, the absolute spatial positions of at least two barcode units (capture or position barcode units) are known, wherein the absolute spatial position is defined relative to the absolute spatial position of the assembly of barcode units.

[0601] In some implementations, the absolute spatial position of at least one barcode cell belonging to each connected component of graph G is known, wherein the absolute spatial position is defined relative to the absolute spatial position of the assembly of barcode cells.

[0602] In some implementations, similarity scores are known between biological characteristics of analytes captured by different capture barcode units and / or between different connected components of the graph of interest.

[0603] The present invention also relates to a kit comprising a composition according to the invention and instructions for use.

[0604] The present invention also relates to a kit comprising a substrate according to the invention and instructions for use. Attached Figure Description

[0605] Figures 1A to 1B These are a set of schematic diagrams illustrating an example of an assembly of a captured barcode unit and a positioned barcode unit. Figure 1A A single-layer positioning barcode bead (BC) containing a poly(dA) sequence as a connecting sequence is shown; Figure 1B A single-layer capture barcode unit containing a poly(dT) sequence as the capture sequence is shown.

[0606] Figures 2A to 2B This illustrates the hybridization of a localizing oligonucleotide (BC1) and a capturing oligonucleotide (BCA). Figure 2A ) and extensions ( Figure 2B A set of schematic diagrams of examples of ).

[0607] Figures 3A to 3B This is a set of schematic diagrams showing examples of the packing of positioning barcode units (BC1, BC2, BC3, etc.) and capturing barcode units (BCA, BCB, BCC, etc.), where the contact surface area is indicated by an asterisk (*). Figure 3A ), and relative position reconstruction ( Figure 3B The numbers on the edges represent the identifier of a given positioned barcode cell that is incorporated into the captured barcode cell.

[0608] Figures 4A to 4D These are a set of schematic diagrams showing an example of the arrangement of barcode cells. Figure 4A and Figure 4B This illustrates a first-type barcode unit combined with three second-type barcode units. Figure 4C and Figure 4D This illustrates when two types of barcode units have the same size ( Figure 4C ) or different sizes ( Figure 4DWhen the two types of barcode units are arranged closely together, the white beads are the capture barcode units; the black beads are the positioning barcode units.

[0609] Example

[0610] The present invention is further illustrated by the following embodiments.

[0611] Example 1:

[0612] Figures 1A to 1B An example of a positioning barcode unit is shown, which contains a poly(A) sequence as a first connection sequence and is deposited on a support to form a layer. Figure 1A This layer is completed by the second layer of capture barcode units, containing a poly(T) sequence as the capture sequence. Figure 1B ).

[0613] This two-layer support can be used for spatial omics applications of biological samples. In fact, it facilitates hybridization between locating barcode units and capturing barcode units (…). Figure 2A ) and subsequent oligonucleotide elongation ( Figure 2B This allows for the reconstruction of connections between barcode units of two groups by forming and sequencing nucleic acid sequences that contain both the location (A, B, C, D, etc.) and the capture barcode unit (1, 2, 3, 4, etc.).

[0614] For example, various types of assembly can be performed based on the density and size of the barcode units (see Figure 4).

[0615] Example 2:

[0616] Figure 3 illustrates an example of determining the spatial location of captured barcode cells. This occurs in a complex mixture of groups containing two types of barcode cells. Figure 3A The proximity relationships between captured barcode units (1, 2, 3, 4, etc.) are defined by their connection with the positioning barcode units (A, B, C, D, etc.). Figure 3A ).

Claims

1. A composition comprising: - A group of capture barcode units, wherein each capture barcode unit contains a group of capture oligonucleotides, wherein each capture oligonucleotide contains a barcode sequence and a capture sequence capable of binding an analyte from a biological sample; - A group of positioning barcode units, wherein each positioning barcode unit contains a group of positioning oligonucleotides, wherein each positioning oligonucleotide contains a barcode sequence and a first linker sequence capable of hybridizing on the capturing oligonucleotide; Each barcode unit contains a different barcode sequence, and all oligonucleotides on the same barcode unit contain the same barcode sequence.

2. The composition of claim 1, wherein the capture sequence cannot hybridize with the capture sequence, and wherein the first ligation sequence cannot hybridize with the first ligation sequence.

3. The composition according to claim 1 or 2, wherein the first linker sequence of the localized oligonucleotide is capable of hybridizing on the capture sequence of the capture oligonucleotide.

4. The composition according to claims 1 to 3, wherein the capturing oligonucleotide comprises a second linker sequence, wherein the second linker sequence is different from the barcode sequence and the capturing sequence, and wherein the first linker sequence that locates the oligonucleotide is capable of hybridizing on the second linker sequence of the capturing oligonucleotide.

5. The composition according to any one of claims 1 to 4, wherein, The capture sequence is the same for all captured barcode units, and the first connection sequence is the same for all positioned barcode units.

6. The composition according to any one of claims 1 to 5, wherein the capturing oligonucleotide and / or the positioning oligonucleotide further comprises at least one PCR primer sequence.

7. The composition according to any one of claims 1 to 6, wherein each oligonucleotide from the group of said capturing oligonucleotides and / or from the group of said positioning oligonucleotides further comprises at least one unique molecular identifier (UMI).

8. The composition according to any one of claims 1 to 7, wherein the capturing oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, the barcode sequence, optionally at least one UMI, and the capturing sequence; and wherein the positioning oligonucleotide comprises, in a 5' to 3' order: optionally at least one adapter, optionally at least one PCR primer sequence, the barcode sequence, and the first linker sequence.

9. The composition according to any one of claims 1 to 8, wherein the barcode unit is a synthetic barcode unit.

10. The composition according to any one of claims 1 to 9, wherein the barcode unit is a bead or a DNA nanosphere.

11. The composition according to any one of claims 1 to 10, wherein the group of capturing barcode units and the group of positioning barcode units are assembled on a substrate.

12. The composition according to any one of claims 1 to 11, wherein the capturing barcode unit is in contact with at least one, preferably at least two, more preferably at least three of the positioning barcode units.

13. A method for determining the spatial location of a group of captured barcode cells, comprising the following steps: (i) bringing the group of capturing barcode units from the composition according to any one of claims 1 to 12 into contact with the group of positioning barcode units; (ii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide; (iii) At the multiple contact points formed in step (ii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides; (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acid sequences comprising a barcode sequence of a capture barcode unit and a barcode sequence of a positioning barcode unit; (v) Optionally, the multiple nucleic acid sequences obtained in step (iv) are amplified to obtain multiple amplicones; (vi) Sequencing the multiple nucleic acid sequences obtained in step (iv) or the multiple amplicon obtained in step (v); (vii) Infer the proximity relationship between the captured barcode unit and the located barcode unit; (viii) Inferring the relative position of each captured barcode cell and / or each positioned barcode cell; and (ix) Optionally, infer the absolute position of each captured barcode unit and / or each positioned barcode unit.

14. A method for determining the spatial location of multiple analytes in a biological sample containing multiple analytes, comprising the steps of: (i) bringing the group of capturing barcode units from the composition according to any one of claims 1 to 12 into contact with the group of positioning barcode units; (ii) Multiple contact points are formed between the group of capturing barcode units and the group of positioning barcode units, wherein each contact point involves a capturing oligonucleotide and a positioning oligonucleotide; (iii) At the multiple contact points formed in step (ii), at least one linker sequence is hybridized with at least one complementary sequence on the captured oligonucleotide to obtain multiple hybrid oligonucleotides; (iv) Extend the multiple hybrid oligonucleotides obtained in step (iii) to obtain multiple nucleic acid sequences comprising a barcode sequence of a capture barcode unit and a barcode sequence of a positioning barcode unit; (v) Optionally, the multiple nucleic acid sequences obtained in step (iv) are amplified to obtain multiple amplicones; (vi) Sequencing the multiple nucleic acid sequences obtained in step (iv) or the multiple amplicon obtained in step (v); (vii) Infer the proximity relationship between the captured barcode unit and the located barcode unit; (viii) Inferring the relative position of each captured barcode cell and / or each positioned barcode cell; and (ix) Optionally, infer the absolute position of each captured barcode unit and / or each positioned barcode unit; And it also includes the following steps: a) In any step prior to step (v), bring the group of capturing barcode units and the group of positioning barcode units into contact with the biological sample; b) In any step after step (a) and before step (v), the plurality of analytes are captured from the biological sample by combining the plurality of analytes into a capture sequence of a plurality of capture barcode units; c) Optionally, after step (b), the capture sequence is extended on the sequences of the plurality of analytes to obtain a plurality of nucleic acids comprising a barcode sequence of a capture barcode unit and the sequence of the analyte; d) Optionally, after step (c), multiple nucleic acids are amplified to obtain multiple amplicones; e) Sequencing the multiple nucleic acid sequences obtained in step (c) or the multiple amplicon obtained in step (d); f) In any step after step (viii), infer the relative position of each analyte; and optionally, infer the absolute position of each analyte in the biological sample.

15. The method of claim 14, wherein the biological sample is selected from tissues, tissue sections, monolayers of cells, multilayers of cells, organoids, syncytia, or single cells.

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