Orthogonal Section Ligation Sequencing (OCLS)

Abstract

This disclosure relates to a method and system for spatial coding / decoding features. The provided orthogonal section-ligation sequencing (OCLS) system encodes two or more orthogonal recognition sites for restriction enzymes, such as IIS-type enzymes, for decoding visual barcodes. Decoding employs specific orthogonal ligation of differentially labeled probes, which enables visual distinction of barcodes at each feature. This process can be repeated over multiple cycles and in parallel. TIFF2026514381000021.tif86134

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to and the benefit of its earlier filing of U.S. Provisional Application No. 63 / 492,777, filed on March 28, 2023, the entire content of which is incorporated herein by reference.

[0002] Incorporation by Reference of Sequence Listing A computer - readable text file named "O046 - 0082PCT.xml", created around March 26, 2024, with a file size of 28,237 bytes, contains the sequence listing of this application, and the entire content thereof is incorporated herein by reference.

[0003] Field of the Disclosure This disclosure generally relates to molecular barcodes such as visual barcodes, and related methods and systems. More specifically, this relates to labeling compositions, methods, and workflows that overcome the limitations of previously described visual barcoding.

Background Art

[0004] Background of the Disclosure Determining the identity and / or location of target molecules (such as proteins or nucleic acids) in a sample can be essential for clinical applications, diagnostics, and biomedical research. In - situ hybridization (ISH), immunohistochemistry, laser capture microdissection, and such techniques enable visualization of the location of target molecules within a sample such as a biological sample.

[0005] The identity of target molecules can also be determined by labeling them and tracking them through amplification and / or sequencing processes (such as probabilistic barcoding). However, research is still ongoing to develop new tools for improved spatial tracking for its applications toward single-cell biology and spatial biology. Thus, there is a need for methods and systems that reliably correlate the identity of target molecules with their positions within a sample, such as a substantially two-dimensional (2D) biological sample.

[0006] Next-generation sequencing (NGS) technology can be used to determine the sequences of nucleic acid-coated barcoded beads. However, conventional NGS sequencing requires expensive reagents for accurate determination of DNA sequences, including nucleotides with and / or labeled reversible terminators; high temperatures (typically 65°C) are required during decoding; multiple different reagents are introduced during each decoding cycle, and these reagents do not function properly when mixed (e.g., the cleavage solution cannot be mixed with the incorporation solution), which requires the use of advanced fluid dynamics; and the NGS cycle time is significantly longer than 5 minutes for each cycle. To read a large surface area of ​​1 μm beads via NGS, for example, a 1.75 cm × 1.75 cm surface composed of densely packed 1 μm beads with approximately 10 billion (10B) individual 1 μm beads present, the length of the NGS position barcode must be greater than 24 bp to reach the diversity of at least 200 trillion different beads in the library (possible combinations of position barcodes) to reduce the possibility of barcode redundancy within the large surface area. This necessitates a large number of decoding cycles, more than six.

[0007] For example, there is still a strong need in this field for developing further methods for visually barcoding targets in the analysis of biological molecules. [Overview of the project]

[0008] Summary of Disclosure Disclosed herein are methods and systems for visual barcoding with orthogonal cleavage-ligation sequencing (OCLS), as well as various components used in such methods and systems, including double-stranded DNA oligonucleotides and hairpin-loop single-stranded oligonucleotides. Typical barcodes used for the OCLS embodiments described herein are made from dsDNA. Visually decoded barcodes used in the OCLS workflow are constructed across multiple rounds of splitting and pooling (similar to those described in WO2022 / 187719), but the segments constituting the OCLS barcode are not labeled. However, similar to the OCS method in WO2022 / 187719, each segment of the OCLS library can be bound to a bead (or other solid support) or ligated to a previous segment to construct a strand, and these segments can be co-coded together with NGS-captured oligo (CO) barcodes.

[0009] This disclosure further provides a method for coding and decoding (sequencing) visual barcodes.

[0010] Methods including orthogonal cleavage-ligation sequencing (OCLS) of visual barcodes can be practiced to overcome problems associated with other sequencing methods, such as orthogonal cleavage sequencing (OCS) for decrypting identimerm strands. This OCLS method is disclosed herein, including in Figures 1–6. The OCS identimerm strand may consist of a non-nucleic acid polymer (such as a peptide linker using an orthogonal protease or a chemical linker using an orthogonal chemical cleavage agent) for alternative decryption, but the barcodes used for OCLS described herein must consist of double-stranded or partially double-stranded DNA (dsDNA). Briefly, visually decrypted barcodes used in the OCLS workflow are also constructed across multiple rounds of splitting and pooling, but the segments that make up the OCLS barcodes are not labeled (because they use OCS identimerms). Each segment in the OCLS library is ligated with the previous segment to construct a strand, and these segments can be co-coded together with NGS-captured barcodes.

[0011] Similar to the split-and-pool method outlined in WO2022 / 187719, over several rounds of splitting and pooling, more than 100 different wells can be used in each round to construct the OCLS bead library. For decoding, this method requires the use of at least one, but preferably two or more, orthogonal REs in a single reaction mixture, such as IIS-type enzymes (examples of which are considered and shown herein). This step is followed by specific orthogonal ligation of differentially labeled probes, which allows for visual distinction of barcodes at each feature. This process is repeated over multiple cycles as described herein, including in the accompanying drawings.

[0012] A visual barcode configuration is provided herein, including: at least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to one another, each containing a recognition site (RS) for specific restriction endonuclease (RE), a designed cleavage site (CS), or both RS and CS within the sequence of the dsDNA segment; and an unfixed end at one end of the visual barcode. Optionally, the visual barcode is immobilized on a solid substrate by a flexible linker attached to or near the dsDNA end of the visual barcode that is not an unfixed end. The visual barcode configuration provided does not include a visually detectable label, for visually detectable labels are provided by probes used in conjunction with the barcode, as described herein.

[0013] In various visual barcode embodiments, the RE is an IIS-type restriction endonuclease, and the CS does not overlap with the corresponding RS. Optionally, in a given visual barcode embodiment, at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to the IIS-type RE, which is appropriately positioned so that a homologous IIS-type RE can cut the designed CS based on its position relative to the RS.

[0014] A set of visual barcodes is also provided, the set comprising multiple visual barcodes, each comprising a different set of dsDNA segments having different recognition sites (RS), designed cleavage sites (CS), or both for specific restriction endonucleases (REs). In an example of a set of visual barcodes, at least two different visual barcodes are immobilized on the same solid substrate.

[0015] Another embodiment is an orthogonal cleavage-ligation sequencing (OCLS) barcode comprising two or more dsDNA segments (cassettes), each dsDNA segment comprising a recognition site (RS) for a specific restriction endonuclease (RE) and one or more overlapping regions configured to allow ligation to adjacent dsDNA segments to form a segmental strand, the segmental strands constituting the OCLS barcode.

[0016] Another embodiment is a visually detectable orthogonal ligation probe comprising: a complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein the sequence comprises a recognition site (RS) for specific restriction endonucleases (RE), a designed cleavage site (CS), or both RS and CS; and a visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide (which may optionally be attached to the oligonucleotide via a flexible linker). In an example of the visually detectable orthogonal ligation probe embodiment, the complete or partial double-stranded DNA oligonucleotide comprises: a linear double-stranded DNA oligonucleotide having a 5' overhang; a linear double-stranded DNA oligonucleotide having a 3' overhang; a single-stranded DNA oligonucleotide with a hairpin stem-loop configuration having a 5' overhang; or a single-stranded DNA oligonucleotide with a hairpin stem-loop configuration having a 3' overhang.

[0017] In further examples of visually detectable orthogonal ligation probes provided, the visually detectable label includes one or more fluorescent labels, bioluminescent labels, chemiluminescent labels, chromophores, quantum dots, Raman labels, biotin moieties, or radioisotopes.

[0018] Optionally, in a visually detectable orthogonal ligation probe, RE is an IIS-type limiting endonuclease, and RS is specific to the IIS-type RE. As a further example, a visually detectable orthogonal ligation probe may include an RS specific to an IIS-type RE, which is appropriately positioned so that the homologous IIS-type RE can be cut based on its position relative to the RS.

[0019] Another embodiment is a set of visually detectable orthogonal ligation probes, such as those provided in any one of the probe embodiments, the set comprising a plurality of visually detectable orthogonal ligation probes, each comprising different recognition sites (RSs) for specific restriction endonucleases (REs), or both different RSs and different cleavage sites (CSs). Optionally, at least two different detectable orthogonal ligation probes in such a set comprising visually distinguishable detectable labels.

[0020] A set of visually detectable orthogonal ligation probes is also intended, the set comprising several different probes, each configured such that cutting the probe by RE generates overhangs having a different sequence from at least 5, at least 7, at least 10, at least 12, at least 15, or more than 15 other probes in the set. These sets of unique probes (within the set) can be used to “read” results based on the detection of different visual signals, depending on the specific complementarity of the sequences between the probes and the visual barcodes to which the probes ligate through their respective overhangs.

[0021] Another aspect is an orthogonal cleavage-ligation sequencing (OCLS) oligonucleotide pair, which is: A visual barcode comprising at least two or more functionally linearly linked double-stranded DNA (dsDNA) oligonucleotide segments (cassettes), each containing a recognition site (RS) for specific restriction endonucleases (RE), a designed cleavage site (CS), or both RS and CS within the sequence of the dsDNA segment; and a visual barcode comprising an unfixed end at one end of the visual barcode. A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein the sequence comprises a recognition site (RS) for a specific restriction endonuclease (RE), or both an RS and a cleavage site (CS); and a visually detectable orthogonal ligation probe comprising a visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide. The visual barcode includes a segment, and the cutting of the RS or CS in the visual barcode generates a single-stranded "sticky end" overhang having an arrangement that is perfectly complementary to the sticky end (overhang) generated by the cutting of the RS in the visually detectable orthogonal ligation probe.

[0022] A set of OCLS oligonucleotide pairs is also provided, in which each pair of visual barcodes and visually detectable orthogonal ligation probes has a different, fully complementary sequence overlap, and each visually detectable orthogonal ligation probe contains a different, visually distinguishable, detectable label.

[0023] Another aspect is a method for coding a visual barcode, which includes: A double-stranded DNA (dsDNA) oligonucleotide, which is fixed to a solid support at its first end and has a single-stranded overhang at its unfixed second end, A first overhanging end that is compatible with the binding of the immobilized dsDNA oligonucleotide to the single-stranded overhang, and a second overhanging end, and A recognition site (RS), a designed cleavage site (CS), or both RS and CS for a specific restriction endonuclease (RE) The first dsDNA segment, which contains within the sequence of the first dsDNA segment A step of contacting, wherein the contacting occurs under conditions sufficient to allow specific hybridization of the single-stranded overhang of the first dsDNA segment to the single-stranded overhang of the immobilized dsDNA oligonucleotide; A step of ligating the first dsDNA segment to the immobilized dsDNA oligonucleotide to form a first captured barcode segment that includes the second overhanging end of the first dsDNA segment; A step of contacting the first captured barcode segment with a second dsDNA segment having a first overhanging end that is compatible with the binding of the first captured barcode segment oligonucleotide to the single-stranded overhang, and a second overhanging end, and including within the sequence of the second dsDNA segment a RS for a specific RE that is different from the RS / RE in the first dsDNA segment, a designed CS that is different from the designed CS in the first dsDNA segment, or both; and A step of ligating the second dsDNA segment to the first captured barcode segment to form a second captured barcode segment that includes the second overhanging end of the second dsDNA segment, wherein the first captured barcode segment and the second captured barcode segment constitute the visual barcode.

[0024] In an example of such a method for encoding a visual barcode, the method further includes the step of repeating the contacting and ligating steps one or more additional times to form the visual barcode, each time by attaching an additional dsDNA segment to the captured barcode segment. Optionally, in any method for encoding a visual barcode, at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to an IIS type RE, which is appropriately positioned, so that the cognate RE can cut the designed CS based on its position relative to the RS.

[0025] An example of a method for encoding visual barcodes is also intended, in which visual barcodes are constructed using multiple rounds of splitting and pooling with unlabeled DNA segments. For example, one or more rounds of splitting and pooling include: ligating one barcode cassette onto beads at once; splitting the resulting beads into individual compartments, optionally into wells of a plate; ligating different first compartment-specific barcode cassettes onto the beads in each individual compartment to result in a collection of beads containing two different pairs of barcode cassettes; washing the beads containing the two barcode cassettes; pooling the beads containing the two barcode cassettes; splitting the pooled beads containing the two barcode cassettes into individual compartments, optionally into wells of a plate; and repeating the ligating, washing, pooling, and splitting steps to increase the diversity of the barcode set.

[0026] In any method for encoding a visual barcode, optionally, the visual barcode includes a continuous strand of dsDNA segments (cassettes), or the visual barcode includes at least two separate cassettes attached directly and separately to the solid support.

[0027] In any method of encoding a visual barcode pattern, the segment (cassette) is optionally co-coded with a next-generation sequence (NGS) captured barcode.

[0028] Visual barcodes produced by any of the methods provided are also included within the scope of this disclosure.

[0029] Another aspect is a method for decrypting a visual barcode, which includes the following: A step of bringing at least one double-stranded DNA (dsDNA) orthogonal cleavage-ligation sequencing (OCLS) barcode containing at least one restriction site (RS) into contact with a restriction endonuclease (RE) that recognizes the RS, under conditions sufficient to cause the RE to cleave the dsDNA OCLS barcode, wherein the RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode; The step of bringing a partial ssDNA-partial dsDNA OCLS barcode into contact with at least one orthogonal ligation probe comprising a first terminal overhang and a dsDNA oligonucleotide having a visually detectable label, under conditions sufficient to allow the overhang of the orthogonal ligation probe to bond to the overhang on the partial ssDNA-partial dsDNA OCLS barcode by base-pair-mediated hydrogen bonding when the sequence of the overhang of the orthogonal ligation probe is the reverse complement of the sequence of the overhang of the partial ssDNA-partial dsDNA OCLS barcode; If base pair-mediated binding occurs, the orthogonal ligation probe is ligated to the partially single-stranded (ss)DNA-partial dsDNA OCLS barcode to generate a captured probe; and, A step of detecting the presence, absence, and / or quantity of a captured probe by imaging the visually detectable label.

[0030] Optionally, in such a method embodiment, the method may further include one or more repetitions of the cycle of steps: contacting / cutting, contacting / base-pair-mediated binding, ligating, and detecting, each additional cycle involving cutting of the dsDNA OCLS barcode at different cleavage sites (CS), base-pair-mediated binding of different orthogonal ligation probes, and / or detection of the presence, absence, and / or quantity of different labeled and captured probes.

[0031] Further examples of methods for decoding a visual barcode morphology include one or more of the following: a dsDNA OCLS barcode is attached to a bead or another solid surface; multiple different dsDNA OCLS barcodes are attached to a single bead or to a single address on another solid surface; a dsDNA OCLS barcode contains one or more non-overlapping RSs; at least one RS in a dsDNA OCLS barcode is recognized by an IIS-type RE and a break occurs outside the RS at a predetermined location; at least one RS in a dsDNA OCLS barcode is recognized by an RE that breaks within the RS; or a visually detectable label contains at least one of a fluorescent label, a bioluminescent label, a chemiluminescent label, a chromophore, a quantum dot, a Raman label, or a radioisotope.

[0032] In an example of a method for decoding a visual barcode, the orthogonal ligation probe comprises a single-stranded DNA oligonucleotide having a stem-loop hairpin structure, with the overhang located at the end of the stem of the hairpin. For example, optionally, after the ligation step, the method may further include contacting the environment containing the captured probe with a 5'-exonuclease.

[0033] Examples of methods for decoding visual barcodes, including one or more of the following, are also provided: a step of bringing at least one dsDNA OCLS barcode into contact with two or more REs, each recognizing a different non-overlapping RS within the dsDNA OCLS, under conditions sufficient to cause each RE to cleave the dsDNA OCLS barcode, such that each RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode; the ligating step includes chemical ligation; or the ligating step includes enzyme-mediated ligation.

[0034] An example of a method for decoding a visual barcode is also provided, including: a step of bringing two or more dsDNA OCLS barcodes, each containing at least one RS, into contact with an RE that recognizes the RS, under conditions sufficient to cause the RE to cleave the dsDNA OCLS barcode, wherein the RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode, and the RS / RE differs for each dsDNA OCLS barcode. For example, such a method may include a step of bringing two or more dsDNA OCLS barcodes into contact with two or more orthogonal REs. Optionally, the step of bringing two or more orthogonal REs into contact may be simultaneous or sequential.

[0035] An example in which a differentially labeled probe is ligated using a specific orthogonal reaction following a step of contacting one or more orthogonal REs is also intended in an example of a method for decoding visual barcodes. For example, and furthermore, the specific orthogonal ligation of a differentially labeled probe enables visual distinction of barcodes in each feature in an array of visual barcodes.

[0036] A method is also provided in which the process of ligating an orthogonal ligation probe to a partially ssDNA-partially dsDNA OCLS barcode is used to add a new RS to the resulting captured probe.

[0037] A method for decoding a visual barcode using orthogonal section-ligation sequencing (OCLS), essentially as described herein, is also provided.

[0038] Another embodiment is an improved system for molecular barcoding, comprising repeated cycles of labeling, orthogonal cleavage, ligation, and imaging for identifying individual features, wherein the orthogonal cleavage comprises cleaving a double-stranded DNA barcode having an IIS-type restriction endonuclease. In an example of this system embodiment, two or more labeling and orthogonal probe-ligation specific cycles occur sequentially or simultaneously.

[0039] Also provided are kits for performing one or more of the methods described herein, for example, a kit comprising one or more of the visual barcodes described herein (optionally attached to beads or other solid supports), one or more of the visually detectable orthogonal ligation probes described herein, and optionally other components useful for performing a method of encoding or decoding an OCLS barcode. [Brief explanation of the drawing]

[0040] [Figure 1A]A method for creating (encoding) OCLS barcodes is described, where each dsDNA segment used to construct the barcode comprises a recognition site (RS) for specific and distinct restriction endonucleases (REs) and one or more overlapping regions for ligation to adjacent dsDNA segments to form a chain. Three rounds of barcode encoding via attachment of different segments for forming a barcode chain are shown. The described embodiments are performed using a first dsDNA segment attached to a bead or other solid support via a flexible linker. The described method is described more fully herein. [Figure 1B] A method for constructing OCLS barcodes is described, where each dsDNA segment used to construct the barcode comprises a cassette containing RS (a designed cassette that positions the RS at an appropriate distance from the CS to a congeneral IIS-type RE) designed so that the RS is at a specific distance (number of bases) from the cleavage site (CS) when recognized by an IIS-type RE, and one or more overlapping regions for ligation to adjacent dsDNA segments to form a strand. Three rounds of barcode encoding via attachment of different segments to form a barcode strand are shown. When producing a combination of OCLS barcodes that comprises a designed cassette with an manipulated CS, the RS is optionally included in the barcode strand. The shown embodiment reflects that the first dsDNA segment is attached to a bead or other solid support via a flexible linker. The shown method is described more fully herein. [Figure 2]Two different OCLS barcode types are shown. The barcode type shown on the left includes an RS for a RE that cuts the OCLS barcode at a corresponding RS that is designed (designed to exist). The barcode type shown on the right includes an RS for an IIS type RE that cuts at a specially designed CS located at a predetermined set distance (a distance selected based on the RE used) from that RS. The methods for encoding and decoding these OCLS barcodes are described more fully herein. [Figure 3A] Figures 3A–3B show orthogonal ligation probes that can be used in the OCLS decoding workflow. The dsDNA probe shown on the left of Figure 3A is formed and labeled by hybridization of two complementary strands of ssDNA or by folding a single ssDNA to form a hairpin with a double-stranded stem. The label is shown to be attached to the probe via a flexible linker. Probe embodiments include labeled dsDNA or ssDNA in a hairpin structure, as shown below the dsDNA probe in Figures 3A and 3B. An alternative embodiment shown in Figure 3B provides a probe containing an encoded RS. The methods shown are described more fully herein. [Figure 3B] Please refer to the explanation in Figure 3A. [Figure 4A] The images show beads (or other solid supports) to which unlabeled barcodes have been attached, each having a different RS (RS1, RS2, RS3); the barcodes are orthogonally cleaved with one or more restriction enzymes (REs), and the resulting products are imaged. [Figure 4B] This demonstrates the stepwise identification of orthogonally cut barcodes using a first labeled orthogonal ligation probe (Identification (ID) Cycle 1). [Figure 4C] This shows the second cycle of sequentially identifying orthogonally cut barcodes using a second labeled orthogonal ligation probe (ID cycle 2). [Figure 4D]This shows the third cycle of sequentially identifying orthogonally cut barcodes using a third labeled orthogonal ligation probe (ID cycle 3). [Figure 4E] This demonstrates an option to identify orthogonally cut barcodes using more than one labeled orthogonal ligation probe in a single ID cycle. [Figure 5A]Figures 5A–5E show embodiments of orthogonal cleavage-ligation sequencing (OCLS) reflecting two different barcodes (of the type shown in Figure 1B and the right side of Figure 2), each containing a different RS designed for recognition by different IIS-type REs, as well as a designed cleavage site (CS) located a precise number of bases away from the RS, determined by the enzyme used. In Figure 5A, two different IIS-type REs are added to a single reaction mixture to produce cleavage products 1 and 2 (also known as orthogonally cleaved products 1 and 2), which are removed in a subsequent washing step. Images are taken after cleavage by the enzyme to establish a baseline signal prior to decoding by orthogonal ligation with a labeled probe. Figure 5B shows both OCLS barcodes (top: stem 1 oligo, bottom: stem 2 oligo), with arrows pointing to both the RS and the designed CS of each barcode oligo. In the first barcode oligo containing RS1 (top), the RS is located 10 bp upstream of the encoded CS. After recognition at the RS element, cleavage by this enzyme (BsmFI) occurs at the designed CS element exactly 10 bp downstream of RS, resulting in a designed 4-base-length 5' overhang. Similarly, in the second barcode oligo (below) containing RS2, RS is located 14 bp upstream of the encoded CS. After recognition at the RS element, cleavage by this enzyme (BpuEI) occurs at the designed CS element exactly 14 bp downstream of RS, resulting in a designed 2-base-length 3' overhang. Figure 5C shows the ligation of an orthogonally labeled probe of the type shown in Figure 3B, where the probe contains an additional RS encoded within its dsDNA region. Here, the encoded RS is specific to the same enzyme that cleaved the barcode in the previous cycle (although this is not essential to the system). Each probe shown in Figure 5C also includes a unique, detectable label (in the reactant) that corresponds to a specific overhanging base, designed to be recognized (hybridized) by the probe attached to the cut OCLS barcode (i.e., the label indicates a specific overhanging base).Therefore, the overhangs on the probe and the overhangs on the barcode are matched by correct base complementarity, and based on this association, the barcoded feature can be identified by imaging, as described elsewhere. After ligation of the first series of probes, a washing step is performed, followed by an imaging step to determine which labels or combinations of labels are present on each bead. As shown here (an example of one bead in the theoretical library of barcoded beads), a single label is attached to the sticky end that appeared on the first OCLS barcode (stem 1) via an encoded matching sticky end in probe 1. Following the first imaging step, ligation of the second series of probes is performed in the same manner as the first, followed by an imaging step to determine which sticky ends appeared after digestion of the second OCLS barcode (stem 2). As shown in Figure 5D, both the first series ligated probe (top) and the second series ligated probe (bottom) are orthogonally labeled probes of the type shown in Figure 3B, where the probes contain further RS ​​encoded within their dsDNA region. As shown in Figure 5D, the probe ligation creates new CSs of 10 bp or 14 bp for OCLS barcode 1 (top) and OCLS barcode 2 (bottom), respectively. Here, in both cases, the encoded RS contained within the labeled ligated probe is specific to the same enzyme that cleaved the barcode in the previous cycle (although this is illustrative and not mandatory). As shown in Figure 5E, in the next cycle of enzymatic digestion by these two IIS-type REs, the next designed sticky end (a 4-base 5' overhanging stretch) appears on OCLS barcode 1, and the next designed sticky end (a 2-base 3' overhanging stretch) appears on OCLS barcode 2.Following a washing step to remove cleavage products 1 and 2, these two barcodes are then subjected to a subsequent round of orthogonal ligation, beginning with a series of sticky-end-specific labeled probes revealed by digestion with a first IIS-type enzyme (BsmFI), followed by an imaging step, followed by ligation with a series of sticky-end-specific labeled probes revealed by digestion with a second IIS-type enzyme (BpuEI). In Figure 5E, the probes shown (probes 3 and 4) are orthogonal labeled probes of the type shown in Figure 3B, where these probes contain further RS ​​encoded within their dsDNA region. The decoding cycle may be continued until all the designed decoding sites of the OCLS barcode are revealed and specifically detected, as described herein. In summary, Figures 5A and 5B: Unlabeled barcodes orthogonally cleaved with one or more IIS-type REs: Step 1. Figures 5C and 5D: Barcodes orthogonally cleaved with one or more IIS-type REs identified by orthogonal ligation: Step 2. This allows the stem oligo to be "ratcheted" down towards the bead throughout the entire cycle, with each cycle including the following steps: cutting, ligation, and imaging for decoding. Figure 5E: Cutting, (ligation, imaging) × 2: All subsequent cycles. Although shown with only two stem / RS pairs, this system can be operated with several different stem / RS pairs (e.g., simultaneously). The two pairs shown form different overhangs after cutting (one leaving a 5' "sticky end" for specific ligation, and the other leaving a 3' "sticky end" for ligation). The overhang lengths are also different. Thus, this method ensures that there are no incorrect ligation events (i.e., no crossovers between the two stem and ligation oligos) by this combination. This figure illustrates an aspect of the OCLS method with multiplexing of (two) different "orthogonal" stem / RS pairs. [Figure 5B] Please refer to the explanation in Figure 5A. [Figure 5C] Please refer to the explanation in Figure 5A. [Figure 5D] Please refer to the explanation in Figure 5A. [Figure 5E] Please refer to the explanation in Figure 5A. [Figure 6] The OCLS method and workflow, as well as the resulting graphs described in Example 1, are shown. An ssDNA hairpin probe of the type shown in Figure 3B (bottom), labeled with AF-488, is demonstrated. The labeled ssDNA hairpin probe contains an encoded RS and is used for orthogonal ligation after cutting an OCLS barcode of the type shown in Figure 1B (Figure 2, right), which contains multiple encoded CSs. For the experiment shown in Figure 6, an imaging step was performed in the 488 nm (luminescence) channel to determine the bead signal intensity after the first OCLS barcode cutting with BsmFI, an IIS-type RE. After probe ligation and the subsequent washing step, the beads were imaged in the 488 nm channel to determine the signal obtained from the label and therefore the overhang that appeared on the cut OCLS barcode. Since the same RS for the IIS-type RE (BsmFI) used in the first cutting reaction was encoded in the ligated probe, this enzyme was added to the beads after the next washing to remove components from the previous reactants to digest the OCLS barcode for the second time. After the second digestion and washing process, an imaging process was performed in the 488 nm channel, and the signal obtained from the beads was measured. The signals obtained from all three imaging processes in the 488 nm channel are plotted in the graph in Figure 6, with relative fluorescence units (RFU) on the y-axis. [Modes for carrying out the invention]

[0041] References about arrays Nucleic acid and / or amino acid sequences described herein are indicated using standard letter abbreviations as defined in 37 CFR §1.822. Although only one strand of each nucleic acid sequence is shown, it should be understood that the complementary strand is included in any applicable form. Representative oligonucleotides that may be used with the OCLS workflow are listed below. Legend: 5AmMC6 = 5' flexible 6-carbon linker supporting a reactive primary amine; 5Phos = 5' phosphorylation added so that the oligo can be a substrate for T4 DNA ligase; iAmMC6T = internal 6-carbon amine-modified thymine.

[0042] SEQ ID NO: 1 is TIIS stem 1 (BsmFI): TIFF2026514381000002.tif9144

[0043] SEQ ID NO: 2 is TIIS stem 1 comp: TIFF2026514381000003.tif3130

[0044] SEQ ID NO: 3 is TIIS s1hp1 (BsmFI): TIFF2026514381000004.tif3145

[0045] SEQ ID NO: 4 is TIIS s1hp2 (BsmFI): TIFF2026514381000005.tif3145

[0046] SEQ ID NO: 5 is TIIS s1hp3 (BsmFI): TIFF2026514381000006.tif3145

[0047] SEQ ID NO: 6 is TIIS s1p1 (BsmFI): TIFF2026514381000007.tif3128

[0048] SEQ ID NO: 7 is TIIS s1p1 comp: TIFF2026514381000008.tif3128

[0049] SEQ ID NO: 8 is TIIS s1p2 (BsmFI): TIFF2026514381000009.tif3128

[0050] SEQ ID NO: 9 is TIIS s1p2 comp: TIFF2026514381000010.tif3128

[0051] SEQ ID NO: 10 is TIIS s1p3 (BsmFI): TIFF2026514381000011.tif3128

[0052] SEQ ID NO: 11 is TIIS s1p3 comp c: TIFF2026514381000012.tif3128

[0053] SEQ ID NO: 12 is TIIS stem 2 (BpuEI): TIFF2026514381000013.tif9145

[0054] SEQ ID NO: 13 is TIIS stem 2 comp: TIFF2026514381000014.tif3128

[0055] SEQ ID NO: 14 is TIIS s2p1 (BpuEI): TIFF2026514381000015.tif3128

[0056] SEQ ID NO: 15 is TIIS s2p1 comp: TIFF2026514381000016.tif3128

[0057] SEQ ID NO: 16 is TIIS s2p2 (BpuEI): TIFF2026514381000017.tif3128

[0058] SEQ ID NO: 17 is TIIS s2p2 comp: TIFF2026514381000018.tif3128

[0059] SEQ ID NO: 18 is TIIS s2p3 (BpuEI): TIFF2026514381000019.tif3128

[0060] SEQ ID NO: 19 is TIIS s2p3 comp: TIFF2026514381000020.tif3128

[0061] Detailed explanation Efforts are underway to provide a visual barcode system that demonstrates improvements over conventional NGS, such as reduced processing time, lower material and reagent costs, the absence of temperature cycling (i.e., isothermal reaction) and lower overall operating temperature, as well as designing the system so that all decoding cycles occur in the same buffer (which allows for the simple addition of each "next cleavage reagent" without the removal of the previous one). These efforts have led to significant improvements over NGS, including the advancement described in WO2022 / 187719 as orthogonal cleavage sequencing (OCS). After decoding the OCS identimerm strands on a bead to determine the bead's position on the surface, an NGS capture oligo (CO) containing information (color code) about which identimerm strand was on that bead remains for subsequent capture of the nucleic acid (the CO escapes the decoding workflow described, for example, in WO2022 / 187719). The previously disclosed method for creating, coding, and decoding visual barcodes relies on identimers and orthogonal cleavage sequencing (OCS), as described in WO2022 / 187719. In the previously disclosed method, beads containing combined or linear identimers are combinatorially coded with a visually detectable phosphor / label before introduction of the cleavage agent for use in OCS. OCS involves introduction of one cleavage agent at a time, followed by an imaging step to decode the sequence of the identimer chains. In the OCS workflow, the order of detectable labels within the identimer chains is determined by the disappearance of the signal after each cycle (because a specific label is removed from the identimer chain during each cycle of orthogonal cleavage); orthogonal cleavage is present, but a ligation step is not required. The identimer chain (used in OCS) may be composed of a non-nucleic acid polymer for alternative decoding (such as a peptide linker using an orthogonal protease or a chemical linker using an orthogonal chemical linker cleavage agent).

[0062] Disclosed herein are methods involving orthogonal cleavage-ligation sequencing (OCLS) of visual barcodes that can be practiced to overcome various limitations associated with other sequencing methods, such as identifier strand decoding by orthogonal cleavage sequencing (OCS); see WO2022 / 187719. The barcodes used in the OCLS described herein are, in some embodiments, dsDNA. The double-stranded nature may arise from two paired single-stranded DNA molecules, or from single-stranded DNA that itself folds back to form a hairpin (the stem of the hairpin is double-stranded). In short, the visually decoded barcodes used in the OCLS workflow are constructed through multiple rounds of splitting and pooling (similar to those described in WO2022 / 187719; see also Rosenberg et al., Science 360(6385): 176-182, 2018; Kuchina et al., Science 371(6531): doi:10.1126 / science.aba5257, 2020; USPN 11,692,214; US Patent Application Publication US20220403452A1), however, the segments that make up the OCLS barcode are not labeled (they use the identifiers of WO2022 / 187719). Each segment in the OCLS library is ligated with the previous segment to build a chain, and these segments can be co-coded together with the NGS-captured barcode.

[0063] Similar to the split-and-pool method outlined in WO2022 / 187719, over several rounds of splitting and pooling, more than 100 different wells can be used in each round to construct the bead library. For decoding, this method involves using at least one, but optionally two or more, orthogonal REs, such as IIS-type enzymes (e.g., those used in the drawings and legend described herein) in a single reaction mixture. This step is followed by specific orthogonal ligation of differentially labeled probes, which allows for visual distinction of barcodes in each feature based on their encoded sticky ends (overhanging bases). This process is repeated over multiple cycles as described in the drawings and legend.

[0064] Different barcodes suitable for use in orthogonal cleavage ligation sequencing (OCLS) workflows can be created by attaching dsDNA segments containing coded restriction endonuclease (RE) recognition sites (RS) and / or cleavage sites (CS) to beads or other solid support features in different combinations. The dsDNA OCLS barcode stem oligos shown in Figures 1A and 1B can be used to construct such barcodes. Here, the dsDNA stem region is flexible (e.g., polyethylene glycol (PEG)). 12The dsDNA oligo is attached to a bead at one end of its 5' via a linker; in this diagram, the stem dsDNA oligo is common to all beads contained within the library. At opposite ends, the dsDNA stem oligo contains a 5' overhang, or "sticky end" (region of single-stranded DNA bases), designed for ligation to the next barcode segment. Optionally, different stems (distinguishable by sequence contents and / or "sticky ends") or different cleavable segments of the OCLS barcode can be attached to different positions on the beads to increase coding diversity. In this way, the split-and-pool approach can be used to construct many different individual segments or multiple strands with different contents or types in various combinations.

[0065] Although enzymatic ligation is illustrated in this specification, the use of chemical ligation to capture probes on OCLS barcodes is also intended.

[0066] Figure 1A illustrates a method for creating OCLS barcodes, where each dsDNA segment contains a recognition site (RS) for a specific restriction endonuclease (RE) and one or more overlapping regions for ligation to adjacent dsDNA segments to form a chain. This figure shows three rounds of barcode coding via attachment of different segments to form a barcode chain. When creating combinations of OCLS barcodes that contain RS for recognition and cleavage by a specific RE (the enzyme cleaves at the recognition site), it is necessary to include many different RS in each round of combinatorial coding. For example, since two OCLS barcodes are decoded by ligation to differentially labeled probes, the variety of barcodes shown in Figure 1A is limited by the number of different options for probe ligation (probes containing sticky ends that fit the ligation to the cleaved barcodes), and therefore each round of information coding is directly proportional to the number of different RS used. During the decoding of OCLS barcodes by orthogonal ligation, a single uniquely distinguishable marker (or a unique combination of distinguishable markers) attached to the probe, which contains an overlapping region or "sticky end" that fits the ligation of each decoding probe (contained within the probe pool) with one or more detached barcode oligos, is used to determine which sticky ends are available / exposed on each bead. For each detachable barcode segment (added during each round of coding) in this OCLS barcode type, each unique RS corresponds to only a single marker or combination of markers during decoding: a single orthogonal RS is determined by only one distinguishable marker (or combination of distinguishable markers) during decoding.

[0067] Figure 1B illustrates how to create an OCLS barcode, where each dsDNA segment contains a cleavage site (CS) that can be designed to be present in the sequence, and one or more overlapping regions for ligation with adjacent dsDNA segments to form a strand. This figure shows three rounds of barcode encoding via attachment of different segments to form a barcode strand. When creating a combination of OCLS barcodes that contain a designed CS, it is optional but not required to include an RS inside the barcode strand itself, because the CS can be designed for cleavage by IIS-type REs, and these enzymes have a recognition site (RS) that is located away from the site where the enzyme cleaves the dsDNA. These enzymes cleave any site a certain number of bases away from their RS (specific to each enzyme), leaving a certain number of 5'- or 3'-overhanging bases, depending on the specific enzyme / RS used. Thus, the orthogonality of barcode / probe pairs can be designed for this OCLS barcode type, which greatly increases the number of different ligation probes that can be used for each RS. When using an IIS-type RE, the RS can be included in the outermost segment, which allows for recognition by the enzyme at this included RS and dsDNA cleavage by the enzyme at the designed cleavage site (the location of which is selected based on the IIS-type RE and RS used). Furthermore, the IIS-type enzyme-specific RS can be included (encoded within it) on the incoming labeled orthogonal ligation probe to allow for subsequent cleavage reactions by the same or a different IIS-type RE. This makes it possible to encode only the designed CS (along with the adjacent sequences necessary to construct the barcode) within this OCLS dsDNA barcode type.

[0068] Figure 2 shows two different OCLS barcode types. These barcode types can be attached to beads at different sites around the beads via a split-and-pool approach to create different combinations, or segments of these barcodes can be attached to form barcode strands as shown in Figures 1A and 1B. After barcode construction, the barcode-attached beads can be immobilized, for example, on a surface in a flow cell, for decoding. The barcode type shown on the left contains recognition sites (RSs) for restriction enzymes (REs) that cleave the OCLS barcode with the corresponding designed RSs. After cleavage, all dsDNA (or ssDNA hairpins) downstream of the RSs (from the bead attachment site) are removed during the washing step, revealing sticky ends for ligation to a specifically labeled probe during decoding. The OCLS barcode type shown on the left requires multiple different RSs (and therefore multiple different orthogonal REs) to reveal multiple different sticky ends for downstream decoding by orthogonal ligation with differentially labeled probes. The barcode type shown on the right encompasses RSs for IIS-type REs that cleave at a specifically designed site away from the RS. After cleavage by the IIS-type RE, all dsDNA (or ssDNA hairpins) downstream of the encoded cleavage site are removed during the washing process, potentially revealing a series of differentially designed sticky ends that can be recognized by a single probe in a designed set of ligation probes.

[0069] Figure 3A shows orthogonal ligation probes that can be used in the OCLS decoding workflow. As outlined above, decoding of individual OCLS barcodes involves recognition by one designed probe in a set of probes, each probe in the set containing one or more distinguishable labels, and each probe's distinguishable label (or distinguishable combination of related labels) corresponds to one of the differentially designed sticky ends that appear on the cleaved OCLS barcode (containing complementary bases). The dsDNA probes shown in Figure 3A are formed by the hybridization of two complementary ssDNA strands and are labeled with an NHS-modified fluoropolymer reagent for covalent attachment to the dsDNA via a flexible 6-carbon linker at one end (carrying a 5'-amino modification). At the opposite ends of the double helix, these probes contain overhanging bases in a specific region to create a designed 5' or 3' overhang. The overhanging bases of the probe correspond to a known label or a known combination of labels (attached to the probe), and as a result, during decoding (following the ligation and imaging steps), the detectable label is used to determine the overhanging bases that appear on different OCLS barcodes. The probe may consist of labeled dsDNA DNA, or it may consist of ssDNA in a hairpin structure as shown below the dsDNA probe in Figure 3A and Figure 3B. Alternatively, the probe may contain an encoded RS, as shown in Figure 3B. The encoded RS in the orthogonal ligation probe used for decoding the OCLS barcode may correspond to an RS recognized by an IIS-type RE. The RS may be one used in a previous decoding cycle or may be used in a future decoding cycle. In this way, the same IIS-type RE site (RS) can be used throughout the decoding cycle, thereby providing a ratchet effect as the OCLS barcode is decoded.Alternatively, different IIS-type RSs can be encoded in the probes used in each decoding cycle to reduce abnormal cleavage throughout the cycle (in this case, any OCLS barcodes that remained uncleaved in the previous cycle will not be recognized in the next cycle). Similarly, using labeled hairpin probes during decoding produces labeled products (after ligation) that do not have open 5'-phosphorylated ends. In this case, all oligos that did not receive the hairpin ligation probe can be removed by using a 5'-exonuclease, such as a lambda exonuclease that recognizes only the 5'-phosphorylated ends of the oligos, for digestion. By varying the IIS-type RS encoded in the probe during each decoding cycle, and by including a cleanup step with a 5'-exonuclease after ligation, any phasing during the OCLS decoding process (observation of signals corresponding to incorrect cycles) can be reduced.

[0070] Figure 4A shows beads containing three different segments of the OCLS barcode type shown in Figure 1A (and Figure 2, left), attached to three different positions on the bead. The beads can be immobilized on the surface before decoding, for example, in a flow cell. Each segment contains a unique RS (RS1-RS3), which is recognized and cleaved by one of three different specific orthogonal REs. These three enzymes can be used sequentially or included in a single reaction mixture containing a commonly compatible buffer supplied by the manufacturer (e.g., NEB 1X CutSmart Buffer). After digestion with all three enzymes, three different cleavage events result in overhanging bases (orthogonal overhangs) of different compositions, thereby allowing each type of stem to be detected by orthogonal ligation to differentially labeled detection probes of the type shown in Figures 3A and 3B. Figure 4B shows the ligation of a first labeled probe to a cleaved OCLS barcode segment, which contains overhangs compatible with ligation with the first probe. The label on the first probe is used to determine which overhanging bases on the OCLS barcode appeared after digestion and were subsequently recognized by the probe for ligation. In Figure 4B, the image of the beads taken after ligation will show a single label or combination of labels corresponding to the detectable label contained on probe 1. Figure 4C shows the ligation of the second labeled probe to a cut OCLS barcode segment, which contains the overhangs that are compatible with ligation with the second probe. Similar to the first probe, the label on the second probe is used to determine which overhanging bases on the OCLS barcode appeared after enzymatic digestion. In Figure 4C, the image of the beads taken after ligation of the second probe will show a single label or combination of labels corresponding to the detectable labels contained on both probe 1 and probe 2.Figure 4D shows the ligation of a third labeled probe onto a cleaved OCLS barcode segment, which includes an overhang that fits the ligation with the third probe. Similar to the first and second probes, the label on the third probe is used to determine which overhanging bases on the OCLS barcode appeared after enzymatic digestion. In Figure 4D, the image of the bead taken after ligation of the third probe would show a single label or combination of labels corresponding to the detectable labels included on probes 1, 2, and 3. In this example, a single cleavage event may include multiple enzymes in a single reaction mixture, but the number of decoding cycles (ligation followed by imaging) is repeated as many times as necessary to decode the number of stems [overhangs that appear on top] that are intended to be detected on the bead.

[0071] Alternatively, as described above, one or more REs may be used in a single reaction mixture, but following this step, multiple different labeled probes can be used, contained within a pool (or series of probes) designed to produce combinations of labels used for decoding OCLS barcodes. Figure 4E shows the use of three different segments of the OCLS barcode type shown in Figure 1A (and Figure 2, left) attached to three different positions on a bead. These three OCLS barcodes can then be digested (cut) by one or more REs to create combinations of sticky ends for detection by the corresponding probes during one or more cycles of OCLS for decoding.

[0072] Figures 5A–5E illustrate the orthogonal cleavage-ligation sequencing (OCLS) configuration. The stem 1 oligo (paired with RS1), once hybridized, encapsulates the formed dsDNA BsmFI recognition site (RS1); this IIS-type enzyme cleaves 10 bp downstream of the recognition site, leaving a 4-base 5' overhang. The stem 2 oligo (paired with RS2), once hybridized, encapsulates the formed dsDNA BpuEI recognition site (RS2); this IIS-type enzyme cleaves 14 bp downstream of the recognition site, leaving a 2-base 3' overhang (Figures 5A and 5B; unlabeled barcode orthogonally cleaved with one or more IIS-type REs: Step 1). Each time these two enzymes cleave the DNA, they cleave precisely this distance downstream of the recognition site, although the cleavage site is not sequence-specific. This enables collaborative design between orthogonal cleavage sites (overhangs, or "sticky ends") by labeled orthogonal probes that encompass the matching overhangs, so that each distinctly labeled probe recognizes a specifically designed cleavage site. dsDNA barcodes (stem) that encompass 5' or 3' overhangs defined in different orders (e.g., from 5' to 3') can be coded by a split-and-pool ligation strategy for constructing a library of dsDNA barcodes. Labeled ligation probes covalently attach to the cleaved oligos by orthogonal ligation, and this newly formed ligation product contains detectable labels that are imaged to identify specific overhang sequences. Ligable probes may also encompass dsDNA regions encoding IIS-type recognition sites, allowing for subsequent cleavage at different locations on the stem oligo after ligation (Figures 5C and 5D; barcodes orthogonally cleaved at one or more IIS-type REs and identified by orthogonal ligation: Step 2).This allows the stem oligo to be "ratcheted" down toward the beads throughout the entire cycle, with each cycle encompassing the following steps: cleavage, ligation, and imaging for decoding (Figure 5E; OCLS is preferred: cleavage, (ligation, imaging) × 2: all subsequent cycles). If more than one stem type is used in the same OCLS decoding workflow, cleavage is performed by more than one enzyme in a single reaction mixture, and the ligation and imaging cycles are performed for each stem type included. Labeled, hybridized dsDNA ligation probes are used for both BsmFI and BpuEI-specific stems (stem 1 and stem 2, respectively), and labeled DNA hairpin ligation probes are also designed for BsmFI stems. Although shown with only two stem / RS pairs, the system can be operated with several different stem / RS pairs (e.g., simultaneously). The two pairs shown form different overhangs after cleavage (one leaving a 5' "sticky end" for specific ligation, and the other leaving a 3' "sticky end" for ligation). The overhang lengths are also different. Thus, in this combination, incorrect ligation events are effectively eliminated (i.e., there can be no crossover between the two stems and the ligation oligo). Therefore, this figure demonstrates a method involving multiplexing of (two) different "orthogonal" stem / RS pairs.

[0073] Figure 6 shows a preferred embodiment of the OCLS method and workflow, as well as the results of Example 1. Here, the use of an AF-488-labeled ssDNA hairpin probe of the type shown in Figure 3B (bottom) is demonstrated. The labeled ssDNA hairpin probe contains an encoded RS and is used for orthogonal ligation after cutting (Figure 2, right) of an OCLS barcode of the type shown in Figure 1B, which contains multiple encoded CSs. In the experiment outlined in Figure 6, an imaging step was performed in the 488 nm (luminescence) channel to measure the bead signal intensity after the first OCLS barcode cutting with BsmFI, an IIS-type RE. After probe ligation and the subsequent washing step, the beads were imaged in the 488 nm channel to determine the signal obtained from the label and thus the overhangs that appeared on the cut OCLS barcode. Since the same RS for the IIS-type RE (BsmFI) used in the first cleavage reaction was also encoded in the ligated probe, this enzyme was added to the beads after subsequent washing to remove components from the previous reactant, and the OCLS barcode was digested for a second time. After the second digestion and washing steps, an imaging step was performed in the 488 nm channel, and the signal obtained from the beads was measured. The signals obtained over all three imaging steps in the 488 nm channel are plotted in the graph in Figure 6, with relative fluorescence units (RFU) on the y-axis. These experiments clearly demonstrated successful probe ligation and subsequent probe cleavage. After the first cleavage with BsmFI (TIIS RE cut 1), the beads contained almost the same average intensity value (<1,000 RFU) as after the second cleavage with BsmFI (TIIS RE cut 2), but after ligation with the AF-488-labeled ssDNA hairpin probe, the average intensity value of the beads was approximately 33,000 RFU. In the ligation reaction involving a labeled incorrect ssDNA hairpin probe (hairpin probe 3, which includes an incorrect overhang relative to the OCLS barcode), no ligation product was produced (as also determined by gel analysis).This experiment demonstrates the possibility of decoding many different beads in a library encoded with different combinations of OCLS barcodes containing different CSs in a highly parallel manner using the OCLS workflow outlined here. This method involves ratcheting down the OCLS barcode by adding labeled orthogonal ligation probes containing encoded RSs for use with IIS-type REs. In a preferred embodiment, RSs corresponding to IIS-type enzymes are used as described above, and the individual probes used in each decoding cycle consist of labeled ssDNA hairpin oligos containing specific overhangs for recognition of overhangs appearing on the cleaved OCLS barcode, as shown in Figure 6. Preferably, different IIS-type RSs are included in each subsequent probe used in each subsequent cycle to reduce abnormal cleavage throughout the cycle (not shown here). As mentioned elsewhere, this approach reduces the tendency for enzymes used in the previous cycle to cleave intact OCLS barcodes that may have escaped digestion (remained uncleaved) after the previous cleavage cycle. Furthermore, the use of labeled ssDNA hairpin oligos as ligation probes allows for a cleanup step after the ligation step, which can be performed before or after the imaging step (as previously mentioned, along with the use of 5'-exonucleases such as lambda exonucleases, which recognize 5'-phosphorylated ends for digestion and removal of the strand, thereby preventing these barcodes from interacting with all subsequent enzymes and probes used in future cycles). Since the ligated product (containing labeled ssDNA hairpin oligos) can be designed not to contain open 5'-phosphorylated ends, the cleanup step removes any remaining dsDNA after the ligation step, which was not 100% efficient.In summary, a preferred embodiment outlining the use of a ligation probe consisting of labeled ssDNA hairpins containing different RSs in each cycle to reduce abnormal cleavage in subsequent cycles, combined with the described cleanup step to remove 5'-phosphorylated DNA performed after the ligation step, should enable a workflow capable of achieving multiple cycles of accurate OCLS barcode decoding.

[0074] The method provided herein was developed for multiplexing a large number of differentially barcoded beads in a single experiment. In such applications, it is a critical requirement to determine which bead / position on a solid support is which of many beads / positions; therefore, each bead / position in the library must have a unique code (there may be many copies of each unique bead / position, but each individual addressable position in the library must have a unique code). Described herein are methods, components, and systems useful for determining which bead / position has which barcode using visual labels ligated to specific sticky ends that become visible on the barcode after restriction enzyme digestion. Thus, what is read in the described approach is a specific sticky end on the barcode using one unique label (or combination of unique labels) for each different sticky end that appears. This relies on the following:

[0075] (1) Cuttable barcode a) Since barcodes are read using labeled probes that must find their homologous matches, the simplest way to do this is to use dsDNA barcodes that have been enzymatically cut, against which probes specifically labeled probes can be designed to find their matches through base pairing and subsequent ligation. The reason for this is that this can be done in cycles; over many cycles, it is possible to read barcodes with greater diversity. If only one cycle is desired, there are other ways to do so: i) This can also be achieved using specific orthogonal recognition by antibodies that find the congeneral analytes, thereby labeling the antibodies and, by binding to the analytes in different combinations, creating combinations of labels (color codes) on the feature. ii) This can be achieved using differentially labeled DNA-binding proteins or RNA-binding proteins, preferably proteins that recognize specific DNA or RNA elements from among a number of different elements. b) Orthogonality is employed to multiplex multiple barcodes. This means that the system can include one or more different sticky ends (single-strand overhangs) to ensure at least one difference. Three are shown in the diagram, because one of the following can be done to create many differences: i) Encode many different sticky ends using many different restriction sites (and corresponding restriction enzymes) (as shown in the figure). ii) Alternatively, a restriction enzyme (RE) that cuts the dsDNA at a site distant from its recognition site (RS), as exemplified by the IIS-type enzymes herein, is used. In this case, the cleavage site (CS) can be designed to encompass any sequence as long as it is in the appropriate position (relative to the RS) (since TIIS-type REs cut at a specified distance away from the RS rather than at a set sequence), which is not possible with conventional restriction enzymes. By designing the sequence of each CS, a single IIS-type RE can produce many different sticky ends (overhang sequences) depending on the placement of the RS relative to the designed / operated / encoded CS.

[0076] (2) A labeled probe that can ligate the cut barcode a) With typical restriction enzymes (which cut at their recognition site), this is straightforward. When a dsDNA molecule is cut with XhoI, it will have an overhang, which will ligate with a probe that has the same overhang. The overhangs of XhoI are well known and consistent, and this has been a method used in cloning for decades. The problem is that typical REs simply cut the dsDNA at each RS, so only one probe is needed for each enzyme used (to distinguish a specific overhang on the OCLS barcode). b) A predetermined number of probes are used in each decoding cycle. For example, if there are 10 different barcode overhang possibilities in a given cycle, 10 different probe possibilities are added - all 10 must match (i.e., all 10 have overhangs that can bind to and ligate to a particular exposed overhang on the barcode), but only on the correct barcode (only if the correct base complementarity occurs between the overhang on the barcode and the overhang on the probe). This system is artificial, as is the case with next-generation DNA sequencing, and the result read out is which beads or features have which barcodes on them. c) In IIS-type ligation probes, the ligation sites are designed as barcode sequences, which correspond to customized (designed) overhangs that appear after digestion with IIS-type restriction enzymes. This is truly scalable multiplexing because it eliminates the need to find 10 differentially labeled restriction enzymes to create 10 different overhangs for detection by 10 different ligation probes. Here, a single enzyme can produce all 10 ligable ends that can be recognized by 10 differentially labeled probes. i) It is also possible to multiplex a large number (e.g., 10) different probes using only four different fluorophore colors. When using only four different fluorophores, it is necessary to combine and mix them to create 10 different distinguishable probes that can be read out in a single image. This can be done as follows: If two differentially labeled probes are mixed in different combinations for each individual probe type, then 10 different combinations can be distinguished using four distinguishable labels (labels 1-4): 1+1, 1+2, 1+3, 1+4, 2+2, 2+3, 2+4, 3+3, 3+4, 4+4. Using five different labels, 15 combinations can be distinguished. However, for example, 1+2 and 2+1 cannot be distinguished, so they will appear the same to the observer. (1) Figures 1A-1B show multiplexing, which can reach a number of different restriction enzymes (REs) used throughout all cycles in the experiment. Recognition sites (RSs) can be in different combinations, but this aspect can only be extended to a limited level - there is no scalability beyond using a known number of RS / RE pairs, and it is only possible to increase the number of combinations by directly increasing the number of RS / RE pairs used, in combination with further multiplexing of distinguishable visual markers (such as fluorophores). (2) On the other hand, Figure 2 shows the use of IIS-type barcodes and enzymes to scale up possible coded different sticky ends for ligation, thereby potentially allowing billions of different beads to be coded in a combinatorial manner, for example, as shown in Figure 1B, and decoded over multiple cycles of the method. This is because one enzyme can actually be used to code many different overhangs (the "cut" site can be designed to have a different sequence each time, and the IIS-type RE simply recognizes its RS and cuts it at a fixed set distance, whatever the distant sequence may be). The same enzyme can be used repeatedly if it is necessary to code billions of different combinations. This can also be done by reintroducing another IIS-type enzyme recognition site into the labeled probe, so that when ligated to the barcode, another IIS-type recognition site can be created at the correct distance from the corresponding coded "next cut site" on the barcode.

[0077] (3) An imaging step to determine the probe ligation. The imaging step can be performed using any appropriate means, taking into account the label used and the sample to be analyzed.

[0078] (4) A method and system for decoding a barcode when it is attached to a bead or other positionally identifiable solid surface. a) The described method is designed to be extensible for coding when performed within a cycle. The cycle involves: cutting the barcode; ligating the cut barcode with a labeled probe; and imaging the barcode with the labeled probe ligated. b) If there are multiple (n) distinct overhangs in one bead / location, the ligation and imaging process is repeated n times depending on the maximum number of distinct overhangs present on a single bead in the library.

[0079] Embodiments of the methods for visual molecular barcoding provided are shown in Figures 4A–4E and 5A–5E. These methods involve orthogonal cleavage and ligation, and are therefore referred to herein as orthogonal cleavage ligation sequencing (OCLS). The OCLS method uses detectable labels attached to an incoming ligation probe, and these labels are used to determine specific overhang sequences on the beads after restriction enzyme cleavage. Figures 1A and 1B show one method to achieve this, involving the use of multiple different orthogonal restriction enzyme sites (RS1–RS3 shown here). Briefly, a series of OCLS tags present on the beads are cleaved through the use of orthogonal restriction enzymes (RE1–RS3 here) that specifically recognize one site on each tag. This makes it possible to reveal specific “sticky ends” (overhangs) for ligating a particular labeled probe (Figure 4B). The decoding cycle (ID cycle) involves ligation and imaging following the cleavage. Three of these decoding cycles are shown in Figures 4A–4E. The cleavage process occurs once per cycle, followed by the introduction of a ligation probe and ligase, and then imaging (imaging cycle), which occurs depending on the number of different enzymes used during cleavage. By employing various combinations, this method allows for the visual encoding and decoding of a large number of beads. Furthermore, as shown in Figure 4E, it is also possible to decode smaller bead libraries using a single ligation and imaging step after cleavage.

[0080] Figures 5A-5E illustrate the configuration of OCLS. Once hybridized, the stem 1 oligo encompasses the formed dsDNA BsmFI recognition site (RS1); this IIS-type enzyme cleaves 10 bp downstream of the recognition site, leaving a 4-base 5' overhang. Once hybridized, the stem 2 oligo encompasses the formed dsDNA BpuEI recognition site (RS2); this IIS-type enzyme cleaves 14 bp downstream of the recognition site, leaving a 2-base 3' overhang (Figures 5A and 5B; unlabeled barcodes orthogonally cleaved by one or more IIS-type REs: Step 1). Each time these two enzymes cleave the DNA, they precisely cleave this distance downstream of the recognition site, although the cleavage site is not sequence-specific. This allows for the collaborative design of orthogonal cleavage sites (overhangs, or "sticky ends") with labeled orthogonal probes that encompass the matching overhang, so that each distinctly labeled probe recognizes a specifically designed cleavage site on the OCLS barcode. dsDNA barcodes (OCLS barcode stems) that encompass 5' or 3' overhangs defined in different orders (e.g., from 5' to 3') can be encoded by a split-and-pool ligation strategy for constructing a library of dsDNA barcodes for OCLS. Labeled OCLS ligation probes covalently attach to the cleaved OCLS barcode oligos via orthogonal ligation, and this newly formed ligation product contains a detectable label that is imaged to identify a specific overhang sequence. Ligable probes may also contain dsDNA regions encoding other IIS-type recognition sites, allowing for subsequent cleavage at different locations on the stem oligo after ligation (Figures 5C and 5D; barcodes identified by orthogonal ligation after orthogonal cleavage at one or more IIS-type REs: Step 2).This allows the stem oligo to be "ratcheted" down towards the beads throughout the entire cycle, with each cycle encompassing the following steps: cleavage, ligation, and imaging for decoding (Figure 5E; OCLS is preferred: cleavage, (ligation, imaging) × (number of stems used) - explained by two stems: all subsequent cycles). Although shown with two stems (thus X=2 in Figures 5A-5E), it is understood that only one stem may be used, or multiple different stems may be employed to increase method multiplexing. Since two different stems are shown in Figures 5A-5E, it is intended that both be seen for each cycle. Thus, here this is X2. When more than one stem type is used in the same OCLS decoding workflow, cleavage is performed by more than one enzyme in one reaction mixture, and the ligation and imaging cycles are performed for each stem type involved. Labeled, hybridized dsDNA ligation probes are used for both BsmFI and BpuEI-specific stems (stem 1 and stem 2, respectively), and labeled DNA hairpin ligation probes are also designed for BsmFI stems.

[0081] In a preferred embodiment, the orthogonal ligation probe comprises an ssDNA hairpin oligo containing a detectable label, as shown in Figure 6. The detectable label can attach to the oligo through internal amino modification within the loop region of the hairpin oligo, as shown here. Figure 6 shows one cycle of decoding using the OCLS hairpin probe. In the graph of Figure 6, data obtained from imaging beads after ligation of the correct OCLS hairpin probe labeled with Alexa Fluor 488 to the cleaved OCLS stem barcode after the first cleavage event, and after the second cleavage event are graphed side by side. As disclosed here and throughout this specification, the incoming OCLS probe not only distinguishes the correct overhanging ligation site but also codes for another restriction site. In this example, the enzyme recognition site (RS) is recognized by an IIS-type restriction enzyme (RE). Thus, as shown here, the second digestion removes the detectable label from the OCLS stem barcode, subsequently exposing the coded sticky end. This prepares the OCLS stem oligo for the subsequent decoding round by orthogonal ligation. The use of hairpin oligos is beneficial in many ways. Primarily, these advantages include: 1) the hybridized strand of the ssDNA hairpin oligo is less prone to dehybridization than a pair of antiparallel oligos hybridized to form a dsDNA double-stranded species; and 2) the hairpin structure ensures that the entire ligation product does not contain an open 5' end. This is important when decoding beads from a large library using the OCLS method described above, because incomplete digestion by restriction enzymes and incomplete ligation by ligase enzymes will lead to errors during subsequent cycles. After the ligation step, to remove non-ligated stem species, a 5'-exonuclease, such as a lambda exonuclease that recognizes the phosphorylated 5' end of the oligonucleotide to remove DNA by digestion, is used.During the final imaging step of each cycle, a lambda exonuclease can be introduced, which "cleans up" all remaining non-ligated stems (stems that did not receive the hairpin probe). In this case, the ligated species does not contain an open 5' end and is protected from the 5'-exonuclease activity of the lambda exonuclease. This operational "cleanup" step can also be achieved by using a labeled dsDNA probe that contains a label at its 5' end (blocking any 5'-exonuclease activity), or does not contain a 5'-phosphate group, or is otherwise blocked at its 5' end.

[0082] In other embodiments, the same enzyme can be used for decoding all stems by OCLS in a given reaction environment. For example, digestion with RNAse H as a cleavage agent can expose encoded ligation sites on the stem or probe, which, by design, allows for the encoding of sticky ends (overhanging bases). In this case, the inclusion of a labeled probe for orthogonal ligation would involve including complementary sticky ends for the purpose of decoding the bases that appear on the stem after digestion with RNAse H. This approach can be used in single-cycle OCLS substitution, for example, as described here.

[0083] As used herein, "orthogonal" means that, although all components of a multicomponent system are present in the same environment, some components of the multicomponent system exhibit chemical reactivity with a particular reagent under a specific set of reaction conditions, while at least one other component of the multicomponent system exhibits limited or no reactivity with the reagent.

[0084] As used herein, "orthogonal reactivity" refers to a situation where, even though all components of a multicomponent system are present in the same environment, some components in the system exhibit chemical reactivity with a particular reagent under a specific set of reaction conditions, while at least one or more components in the system do not. Similarly, "orthogonal reactivity" refers to a substance that exhibits orthogonal reactivity.

[0085] The term "detectable label" refers to a substance that, when associated with another substance, can indicate the presence of that substance. A detectable label may be a substance linked to or incorporated into the substance to be detected. In some embodiments, detectable labels are suitable for enabling detection and also quantification, for example, detectable labels that emit a detectable and measurable signal. Detectable labels include bioluminescent labels, biotin / avidin labels, chemiluminescent labels, chromophores, coenzymes, dyes, electroactive groups, electrochemiluminescent labels, enzyme labels, fluorescent labels, latex particles, magnetic particles, quantum dots, Raman labels, metals, metal chelates, phosphorescent dyes, protein labels, radioisotopes, elements or parts, and stable radicals. Visually detectable labels are those that label the detection of electromagnetic energy (such as light) wavelengths / photons, for example, through imaging. In certain embodiments, fluorescent labels are preferred.

[0086] "Fluorescence" refers to the emission of visible light by a substance that absorbs light of a different wavelength. In some embodiments, fluorescence provides a non-destructive means of tracking and / or analyzing biological molecules based on fluorescence emission at specific wavelengths. Proteins (including antibodies), peptides, nucleic acids, oligonucleotides (including single-stranded and double-stranded oligonucleotides), etc., can be "labeled" with any of the diverse foreign fluorescent molecules called fluorophores. Isothiocyanate derivatives of fluorescein, such as carboxyfluorescein, are examples of fluorophores that can be conjugated to proteins (such as antibodies for immunohistochemistry) or nucleic acids. In some embodiments, fluorescein can be conjugated to nucleoside triphosphates and incorporated into nucleic acid probes (such as "fluorescent conjugated primers") for in-situ hybridization.

[0087] Direct and indirect attachment (for example, of a detectable label to an oligonucleotide or other substance) may include covalent or non-covalent interactions. Covalent bonding includes the sharing of electrons in chemical bonding. Non-covalent interactions include dispersed electromagnetic interactions such as hydrogen bonding (such as those occurring between paired nucleic acid chains), ionic bonding, van der Waals interactions, and hydrophobic bonding.

[0088] The term "linker" refers to one or more nucleotides, nucleotide analogs, amino acids, peptides, polypeptides, polymers, or non-nucleotide chemical moieties used to link two molecules together. Linkers can be used to link nucleic acids (such as barcodes) to a support, or to link a detection agent to a nucleic acid (such as a probe), and so on. In certain embodiments, linkers link two molecules via enzymatic or chemical reactions (e.g., click chemistry).

[0089] As used herein, "next-generation sequencing" (NGS) refers to high-throughput sequencing methods capable of sequencing millions to billions of molecules in parallel. Examples of next-generation sequencing methods include synthesis sequencing, ligation sequencing, hybridization sequencing, polony sequencing, ion semiconductor sequencing, and pyrosequencing. By attaching primers to a solid substrate and complementary sequences to nucleic acid molecules, nucleic acid molecules can be hybridized to the solid substrate via the primers. Then, by amplification using polymerase, multiple copies can be created within a discontinuous region on the solid substrate (these groups are sometimes called polymerase colonies or polonys). As a result, nucleotides at a specific position can be sequenced multiple times (e.g., hundreds or thousands of times) during the sequencing process—this depth of coverage is called "deep sequencing." Examples of high-throughput nucleic acid sequencing technologies include platforms offered by Illumina, BGI, Qiagen, Thermo-Fisher, and Roche, which include forms such as parallel bead arrays, synthesis sequencing, ligation sequencing, capillary electrophoresis, electronic microchips, "biochips," microarrays, parallel microchips, and single-molecule arrays. (See, for example, Service, Science 311:1544-1546, 2006).

[0090] As used herein, “single-molecule sequencing” or “third-generation sequencing” refers to next-generation sequencing methods in which reads from single-molecule sequencing instruments are produced by sequencing a single molecule, typically DNA. Unlike next-generation sequencing methods that rely on amplification to clone a large number of DNA molecules in parallel for sequencing in a stepwise approach, single-molecule sequencing examines a single molecule (e.g., DNA) and does not require amplification or synchronization. Single-molecule sequencing includes methods that require pausing the sequencing reaction after each base introduction (“wash-and-scan” cycle) and methods that do not require pausing between reading steps. Examples of single-molecule sequencing methods include single-molecule real-time sequencing (Pacific Biosciences), nanopore-based sequencing (Oxford Nanopore), double-strand interrupted nanopore sequencing, and direct imaging of DNA using high-performance microscopy.

[0091] For example, an NGS capture barcode is an oligonucleotide containing a capture region, which is a sequence of nucleotides that is complementary to the oligonucleotide used in a biological, chemical, or biochemical assay, or found within a biological sample. Generally, a capture oligo comprises a 3' capture region (from 3' to 5'), a UMI, and then a region corresponding to the visual barcode information (so that it is operably linked on the same bead), and then a 5' PCR handle. Other elements within a capture oligonucleotide are described in the shared international application PCT / US24 / 17772 entitled "COMPOSITIONS AND METHODS FOR MOLECULAR BARCODING".

[0092] As used herein, the terms “solid support,” “solid surface,” “solid substrate,” or “sequencing substrate” refer to any solid material, including porous and non-porous materials, to which polypeptides can directly or indirectly associate by any means known in the Art, including covalent and non-covalent interactions or any combination thereof. A solid support may be two-dimensional (e.g., planar) or three-dimensional (e.g., gel matrix or beads). A solid support may be any support surface, including beads, microbeads, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, PTFE membranes, PTFE films, nitrocellulose membranes, nitrocellulose polymer surfaces, nylon, silicon wafer chips, flow-through chips, flow cells, biochips including signal transduction electronics, channels, microtiter wells, ELISA plates, rotational interferometer disks, nitrocellulose membranes, nitrocellulose polymer surfaces, polymer matrices, nanoparticles, or microspheres. Materials for solid supports include acrylamide, agarose, cellulose, dextran, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polyester, polymethacrylate, polyacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, polyvinyl alcohol (PVA), Teflon, fluorocarbon, nylon, silicone rubber, polyanhydride, polyglycolic acid, polyvinyl chloride, polylactic acid, polyorthoester, functionalized silane, polypropyl fumaric acid, collagen, glycosaminoglycan, polyamino acid, dextran, or any combination thereof. Solid supports also include thin films, membranes, bottles, dishes, fibers, fabrics, molded polymers, such as tubes, particles, beads, microspheres, fine particles, or any combination thereof.

[0093] For example, if the solid surface is beads, the beads may include ceramic beads, polystyrene beads, polymer beads, polyacrylate beads, methylstyrene beads, agarose beads, cellulose beads, dextran beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, controlled pore beads, silica-based beads, or combinations thereof. The beads may be spherical or amorphous. The beads or support may be porous. The size of the beads may range from nanometers, e.g., 100 nm, to millimeters, e.g., 1 mm. In some embodiments, the size of the beads ranges from 0.2 microns to 200 microns, or from 0.5 microns to 5 microns. In some embodiments, the diameter of the beads may be 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 15, or 20 μm. In certain embodiments, the “bead” solid support may refer to individual beads or a group of beads. In some embodiments, the solid surface is nanoparticles. In certain embodiments, the size of the nanoparticles ranges from 1 nm to 500 nm in diameter, for example, in the ranges of 1 nm to 20 nm, 1 nm to 50 nm, 1 nm to 100 nm, 10 nm to 50 nm, 10 nm to 100 nm, 10 nm to 200 nm, 50 nm to 100 nm, 50 nm to 150 nm, 50 nm to 200 nm, 100 nm to 200 nm, or 200 nm to 500 nm. In some embodiments, the diameter of the nanoparticles may be 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 300 nm, or 500 nm. In some embodiments, the diameter of the nanoparticles is less than 200 nm.

[0094] The terms “nucleic acid molecule” or “polynucleotide” refer to single- or double-stranded polynucleotides, and polynucleotide analogs, that contain deoxyribonucleotides or ribonucleotides linked by 3'-5' phosphodiester bonds. Nucleic acid molecules include DNA, RNA, and cDNA. Polynucleotide analogs may have a backbone other than the standard phosphodiester bond found in natural polynucleotides, and may optionally contain modified sugar moieties or moieties other than ribose or deoxyribose. Polynucleotide analogs contain bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner that enables such hydrogen bonding between the oligonucleotide analog molecule and the bases in the standard polynucleotide in a sequence-specific manner. Examples of polynucleotide analogs include xeno nucleic acids (XNA), cross-linked nucleic acids (BNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), γPNA, morpholino polynucleotides, locked nucleic acids (LNA), threose nucleic acids (TNA), 2'-O-methyl polynucleotides, 2'-O-alkylribosyl-substituted polynucleotides, phosphorothioate polynucleotides, and boronophosphate polynucleotides. Polynucleotide analogs may contain purine or pyrimidine analogs, such as 7-deazapurine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, or general-purpose base analogs that can pair with any base, including hypoxanthine, nitroazole, isocarbostyryl analogs, azolecarboxamide, and aromatic triazole analogs, or base analogs having additional functionalities such as a biotin moiety for affinity binding. In some embodiments, the nucleic acid molecule or oligonucleotide is a modified oligonucleotide. In some embodiments, nucleic acid molecules or oligonucleotides are DNA with pseudocomplementary bases, DNA with protective bases, RNA molecules, BNA molecules, XNA molecules, LNA molecules, PNA molecules, γPNA molecules, or morpholino DNA, or combinations thereof.In some embodiments, nucleic acid molecules or oligonucleotides are skeletally modified, sugar-modified, or nucleic acid-base modified. In some embodiments, nucleic acid molecules or oligonucleotides have nucleic acid-base protecting groups such as Alloc, electrophilic protecting groups such as tilane, acetyl protecting groups, nitrobenzyl protecting groups, sulfonate protecting groups, or conventional base-instability protecting groups.

[0095] As used herein, a captured probe is an orthogonal ligation probe covalently linked to an OCLS barcode by a ligation event. While enzymatic ligation is illustrated herein, the use of chemical ligation to capture probes on OCLS barcodes is also intended. For example, there are various chemical reactions known in the art that can be used for the chemical ligation of DNA. Each of the two molecules linked together by chemical ligation must carry one of the reaction components for chemical ligation, and the reaction must occur only in very high proximity (the reaction proceeds only when the overhang of the orthogonal ligation probe binds to the overhang on the stick end of the OCLS barcode by a specific base-pair-mediated hydrogen bonding, provided that the sequence of the overhang of the orthogonal ligation probe is the reverse complement of the sequence of the overhang of the OCLS barcode). This can be adjusted by modifying the reaction conditions so that chemical ligation occurs only when two different DNA molecules (like the probe and barcode described here) are in very close proximity to each other, which occurs when correct base complementarity exists between the two matching ends (in this case, the matching probe and overhang).

[0096] In several embodiments, binding of a partially single-stranded probe to a partially single-stranded OCLS barcode, where at least a portion of the single-stranded elements are sticky-end overhangs produced by restriction enzyme digestion, occurs when the two overhangs have matching base complementarity. For two nucleic acid molecules to ligate each other, the homogeneous single-stranded sequences must be their reverse complements. Correct base complementarity here literally means that one is the reverse complement of the other. For ligation to occur when an overhang on the probe comes into contact with a correctly matching overhang on the cleaved barcode, they must be and must be their reverse complements—therefore, the association between the "correct" probe and the OCLS barcode is "solidified." This is because the initial interaction is likely to be a very "touchdown" transient type of interaction between the correct matching sequences; chemical ligation, the action of ligase enzymes, is employed to seal the probe and its homogeneous barcode.

[0097] Multiple embodiments provide methods for orthogonal cleavage-ligation sequencing (OCLS) of visual barcodes. These methods are disclosed herein, including with accompanying drawings. The identimere strands (such as those employed in OCS, as described in WO2022 / 187719) may consist of non-nucleic acid polymers (such as peptide linkers using orthogonal proteases or chemical linkers using orthogonal chemical cleavage agents), but the barcodes used for the OCLS described herein consist of dsDNA. Visually decoded barcodes used in the OCLS workflow are also constructed across multiple rounds of splitting and pooling, but the segments constituting the OCLS barcode are not labeled (because they have identimeres). Each segment of the OCLS library is ligated with the previous segment to construct a strand, and these segments can be co-coded with NGS-captured barcodes as outlined above. In a manner similar to the split-and-pool method outlined in WO2022 / 187719, more than 100 different wells can be used in each round to build a bead library across several rounds of splitting and pooling.

[0098] For decoding, the OCLS method involves the use of at least one but optionally two or more orthogonal restriction enzymes (REs), such as IIS-type enzymes (e.g., those used in Figures 4A-4E and 5A-5E, as well as the corresponding explanatory text provided herein), in a single reaction mixture. This step is followed by specific orthogonal ligation of differentially labeled probes, which enables visual differentiation of barcodes at each feature. This process is repeated over multiple cycles, as outlined in the drawings and explanatory text.

[0099] For comparison with orthogonal ligation sequencing (OCS; described in WO2022 / 187719), the embodiments of OCLS described herein are described: using a visual barcode that does not include any visual markers before the introduction of the cutting agent; involving the introduction of more than one cutting agent at a time; involving the introduction of a detectable marker during each cycle, determined by an increase in signal; and including both orthogonal ligation and orthogonal ligation steps.

[0100] The primary advantage provided by the embodiments of the method described herein is the use of an IIS-type enzyme (which recognizes an asymmetric DNA sequence and cleaves outside its recognition sequence). If conventional restriction enzymes were used instead, they would cleave at their recognition site, which would always leave the same overhang (i.e., the same RE always leaves the same overhang). This means that to multiplex with conventional REs, a different RE would have to be used for each "orthogonal" sticky end for ligation. For example, to decode 10 different sequences by orthogonal ligation, 10 different REs might be required in each cycle. By using an IIS-type enzyme instead, the enzyme's recognition site is a known number of bases away from the RE's cleavage site, which allows for the design of oligonucleotides so that a single (IIS-type) enzyme can produce many different "orthogonal" cleavage / ligation sites. The labels associated with each labeled ligation step are predetermined (it is known in advance which label corresponds to which ligation site), which allows for the determination of a code or "set of designed ligation sites" as consistent with the color after ligation and imaging. IIS-type Restriction Enzymes are well known in this field and include over 50 listed online at neb.com / tools-and-resources / selection-charts / type-iis-restriction-enzymes.This online resource provides recognition sequences and other characteristics for each of the listed IIS-type REs, including: AcuI, AlwI, BaeI, BbsI, BbsI-HF, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI-HF (registered trademarks). v2, BsaXI, BseRI, BsgI, BsmAI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI-v2, BtsIMut I, CspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MlyI, MmeI, MnlI, NmeAIII, PaqCI, PleI, SapI, and SfaNI.

[0101] Visually decoded barcodes used in the OCLS workflow are constructed across multiple rounds of splitting and pooling, similar to OCS barcodes (see WO2022 / 187719). However, the segments that make up the OCLS barcode are not labeled (as they have OCS identifiers). Each segment of the OCLS library is either joined to a bead or ligated to the previous segment to form a chain, and these segments can optionally be co-coded with NGS-captured barcodes, as described for OCS (WO2022 / 187719).

[0102] With regard to decoding, an embodiment of the OCLS method uses two or more orthogonal REs, such as IIS-type enzymes (e.g., those described herein), in a single reaction mixture (e.g., simultaneously). Following this step, specific orthogonal ligation of differentially labeled probes is performed, thereby enabling visual distinction of barcodes at each feature. This process is repeated over multiple cycles, as described in the drawings and description.

[0103] In an example where only one enzyme is used to decode the OCLS barcode in each cycle, each cycle would simply involve cleavage, ligation, and imaging. However, if two different stems are used (as shown in Figures 5A-5E), they are recognized by two different IIS-type enzymes, and each cycle involves: cleavage with the two enzymes, ligation to the first stem type (introducing all labeled ligation probes that recognize the first overhang type, as illustrated), an imaging step to determine the label associated with the first overhang type, followed by a second ligation step (introducing all labeled ligation probes that recognize the second overhang type), during which the second imaging step allows for visual determination of the second overhang type. Since the ligation probe (currently the product) contains another IIS-type enzyme site, the next cleavage cycle removes the previously ligated label before a new label is added in the next ligation cycle.

[0104] In this way, the barcode is ratcheted down by alternating between cleavage, ligation, and imaging. The ligation and imaging steps are repeated (within each cycle) in accordance with the number of different stem types used. This is because a ligation probe introduced after each cleavage event (if more than one enzyme is used during the cleavage step) is connected to the imaging step to enable deconvolution. In other words, this creates a series of downstream approaches: cleavage, ligation, imaging, ligation, imaging.

[0105] During the procedure, the system performs ligation and imaging for each different stem used, because the ligation probe contains redundant labels between two (or many) stem types. To determine the overhang sequence, images are taken between each ligation event. The labels on the ligation probe reveal what the overhang sequence was.

[0106] How to use The visual barcoding techniques provided herein have a wide variety of applications, such as for the visual identification of beads or molecules tagged with the barcodes described herein. For example, different antibodies tagged using this approach can be used in a visual cyclic IF-like workflow. Similarly, in high-throughput biochemical and cell-based screening for rapid visual identification of drugs, these molecular barcodes can be used on beads carrying drugs or other libraries, thereby enabling their correlation with visual reporter activation events and / or omics-based readouts. Other applications include the creation of spatially encoded arrays when attached to beads for encoding NGS-captured oligos (see a similar system described in the shared international application PCT / US24 / 17772, “COMPOSITIONS AND METHODS FOR MOLECULAR BARCODING,” whose entire disclosure is incorporated herein by reference). These are not intended to be limited use cases, and those skilled in the art will recognize that the OCLS barcodes provided, beads or other solid supports to which such barcodes are loaded, and labeled orthogonal ligation probes for use with such barcodes are suitable for many other uses and applications.

[0107] Kits and manufactured products Furthermore, this specification also provides kits and products comprising components for OCLS analysis using the methods and / or components described herein. In some embodiments, the kit further comprises other reagents for processing and analyzing biological samples and / or molecules such as nucleic acids, polynucleotides, proteins, polypeptides, or peptides. The kits and products may comprise any one or more of the reagents and components used in the provided methods. In some embodiments, the kit comprises one or more of beads or other support surfaces, OCLS barcodes (optionally already attached to the beads or other support surfaces), individual orthogonal ligation probes or sets of such probes (which may optionally be designed to pair with and package together with a homologous OCLS barcode), and one or more processing compounds or reaction compounds or processing solutions or reaction solutions for use in methods of encoding or decoding OCLS barcodes.

[0108] In some embodiments, the kit also includes one or more buffers or reaction solutions that are useful or necessary for any of the reactions to occur. Buffers such as washing buffers, reaction buffers, binding buffers, and elution buffers are known to those skilled in the art. In some embodiments, the kit further includes buffers for adding other reagents described herein and one or more additional components. Reagents, buffers, and other components may be supplied in vials (such as sealed vials), containers, ampoules, bottles, wide-mouthed bottles, and flexible packaging (e.g., sealed Mylar bags or plastic bags). Any of the components of the kit may be sterile and / or sealed.

[0109] In addition to the components described above, the kit may further include instructions for carrying out the method using the components of the kit, such as instructions for sample preparation, visual barcode coding, visual barcode decoding, signal detection and acquisition, and / or analysis of data obtained from the method. The kit described herein may also include other materials, such as other buffers, diluents, filters, syringes, and / or accompanying documentation with instructions for carrying out at least one of the methods described herein, which may be considered desirable from a commercial and user perspective.

[0110] Any of the kit components described above, and any molecules, molecular complexes or conjugates, reagents (e.g., chemical or biological reagents), agents, structures (e.g., supports, surfaces, particles, or beads), reaction intermediates, reaction products, binding complexes, or any other products disclosed and / or used in exemplary kits and methods, may be provided separately or in any suitable combination to form a kit.

[0111] Furthermore, devices useful for applying chemicals or compositions or cleaning fluids / solutions to fixed visual barcodes, such as flow-slow fluid devices and microfluidic devices, are also intended, for example, when decrypting reactants and when analyzing and collecting data from such processes through the use of the provided methods (washing fluids, buffers, and reaction solutions, etc.).

[0112] Devices for detecting and measuring the spectral characteristics of labeled and captured orthogonal ligation probes are also intended, such as devices for detecting visually detectable labels.

[0113] Further embodiments include analytical software and amino acid deconvolution databases prepared using or intended to be used with analytical / detection / quantification / sequencing methods employing OCLS barcodes and / or orthogonal ligation probes provided herein.

[0114] The following exemplary embodiments and examples are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art should recognize that, in view of the Disclosure, many modifications can be made to the specific embodiments disclosed herein, and similar or comparable results can still be obtained without departing from the spirit and scope of the Disclosure.

[0115] Exemplary embodiments 1. Visual barcodes including the following: At least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, wherein each of the dsDNA segments contains, Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The dsDNA oligonucleotide segment (cassette) including the said; Furthermore, The unfixed end of one end of the visual barcode. 2. The visual barcode according to Embodiment 1 or any other barcode embodiment, which is fixed to a solid substrate by a flexible linker attached to or near the end of the dsDNA of the visual barcode that is not the unfixed end. 3. A visual barcode according to Embodiment 1, or any other barcode embodiment, that does not include visually detectable markers. 4. The visual barcode according to Embodiment 1 or any other barcode embodiment, wherein the RE is an IIS-type restricted endonuclease and the CS does not overlap with the corresponding RS. 5. The visual barcode according to Embodiment 1 or any other barcode embodiment, wherein at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to the IIS type RE, which is appropriately positioned so that the cognate IIS type RE can cut the designed CS based on its position relative to the RS. 6. A visual barcode as shown in Figure 1A or Figure 1B, or as described in any one of embodiments 1 to 5 herein. 7. A set of visual barcodes according to any one of embodiments 1 to 6, wherein the set comprises a plurality of visual barcodes, each comprising a different set of dsDNA segments having different recognition sites (RS), designed cleavage sites (CS), or both for specific restriction endonucleases (REs), or any other barcode embodiment. 8. The collection of visual barcodes according to embodiment 7, wherein at least two different visual barcodes are fixed to the same solid substrate. 9. An orthogonal cleavage-ligation sequencing (OCLS) barcode comprising two or more dsDNA segments (cassettes), wherein each dsDNA segment comprises a recognition site (RS) for a specific restriction endonuclease (RE) and one or more overlapping regions configured to allow ligation to adjacent dsDNA segments to form a segmental strand, and the strand of the segment constitutes the OCLS barcode. 10. Visually detectable orthogonal ligation probes, including the following: A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein its sequence is Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The complete or partial double-stranded DNA oligonucleotide containing; Furthermore, A visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide. 11. The complete or partially double-stranded DNA oligonucleotide comprises the visually detectable orthogonal ligation probe according to embodiment 10 or any other probe embodiment: Linear double-stranded DNA oligonucleotides with a 5' overhang; Linear double-stranded DNA oligonucleotides with a 3' overhang; A single-stranded DNA oligonucleotide with a 5' overhang and a hairpin stem-loop configuration; or A single-stranded DNA oligonucleotide with a hairpin stem-loop structure and a 3' overhang. 12. The visually detectable orthogonal ligation probe according to embodiment 10 or any other probe embodiment, wherein the visually detectable label is attached to the oligonucleotide via a flexible linker. 13. The visually detectable orthogonal ligation probe according to embodiment 10 or any other probe embodiment, wherein the visually detectable label comprises one or more fluorescent labels, bioluminescent labels, chemiluminescent labels, chromophores, quantum dots, Raman labels, biotin moieties, or radioisotopes. 14. The visually detectable orthogonal ligation probe according to embodiment 10 or any other probe embodiment, wherein the RE is an IIS-type restricted endonuclease and the RS is specific to the IIS-type RE. 15. A visually detectable orthogonal ligation probe according to Embodiment 10 or any other probe embodiment, comprising an RS specific to an IIS type RE, which is appropriately positioned so that a cognate IIS type RE can cut a designed CS based on its position relative to the RS. 16. A visually detectable orthogonal ligation probe or any other probe configuration as shown in Figure 3A or Figure 3B or as essentially described herein, according to any one of embodiments 10 to 15. 17. A set of visually detectable orthogonal ligation probes or any other probe configuration described in any one of embodiments 10 to 16, wherein the set comprises a plurality of visually detectable orthogonal ligation probes, each of which comprises a different recognition site (RS) for specific restriction endonucleases (REs), or both a different RS and a different cleavage site (CS). 18. The set of visually detectable orthogonal ligation probes according to embodiment 17, or any other set of embodiments, wherein at least two different detectable orthogonal ligation probes include visually distinguishable detectable markers. 19. A set of visually detectable orthogonal ligation probes according to Embodiment 18, or any other set embodiment, comprising a plurality of different probes, each configured such that the cutting of the probe by the RE generates an overhang having a different arrangement from at least 5, at least 7, at least 10, at least 12, at least 15, or more than 15 other probes in the set. 20. Orthogonal cleavage-ligation sequencing (OCLS) oligonucleotide pairs, the following: At least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, wherein each of the dsDNA segments contains, Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The dsDNA oligonucleotide segment (cassette) including, An unfixed end at one end of a visual barcode. The visual barcode includes, A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein its sequence is Recognition sites (RS) for specific restriction endonucleases (REs), or Both RS and CS Including the complete or partial double-stranded DNA oligonucleotide; and, A visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide. This includes a visually detectable orthogonal ligation probe and Includes, The cutting of the RS or CS in the visual barcode generates a single-stranded "sticky end" overhang having an arrangement that is perfectly complementary to the sticky end (overhang) generated by the cutting of the RS in the visually detectable orthogonal ligation probe. The aforementioned OCLS oligonucleotide pair. twenty one. A set of OCLS oligonucleotide pairs according to embodiment 20, wherein each pair of visual barcodes and visually detectable orthogonal ligation probes has a different, fully complementary sequence overlap, and each visually detectable orthogonal ligation probe includes a different, visually distinguishable, detectable label. twenty two. How to encode a visual barcode, including the following: A double-stranded DNA (dsDNA) oligonucleotide, which is fixed to a solid support at its first end and has a single-stranded overhang at its unfixed second end, It has a first overhanging end and a second overhanging end that are suitable for binding the immobilized dsDNA oligonucleotide to the single-stranded overhang, and Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The first dsDNA segment contains within its sequence the first dsDNA segment The process of bringing it into contact with, The contact step occurs under conditions sufficient to allow specific hybridization of the single-stranded overhang of the first dsDNA segment to the single-stranded overhang of the immobilized dsDNA oligonucleotide; A step of ligating the first dsDNA segment to the immobilized dsDNA oligonucleotide in order to form a first captured barcode segment including the second overhanging end of the first dsDNA segment; The first captured barcode segment is It has a first overhanging end and a second overhanging end that are suitable for binding the first captured barcode segment oligonucleotide to the single-strand overhang, and RS for a specific RE different from the RS / RE in the first dsDNA segment, A different designed CS in the first dsDNA segment, or Both The second dsDNA segment contains within its sequence the second dsDNA segment The process of bringing it into contact with; and, A step of ligating a second dsDNA segment to a first captured barcode segment in order to form a second captured barcode segment including the second overhanging end of the second dsDNA segment, wherein the first captured barcode segment and the second captured barcode segment constitute the visual barcode. twenty three. The method according to embodiment 22 or any other method embodiment, further comprising the step of repeating the contacting step and the ligating step one or more additional times in order to form the visual barcode, each time wherein an additional dsDNA segment is attached to the captured barcode segment. twenty four. The method according to Embodiment 22 or Embodiment 23 or any other method embodiment, wherein at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to the IIS type RE, which is appropriately positioned so that the homologous RE can cut the designed CS based on its position relative to the RS. twenty five. The method as shown in Figure 1A or Figure 1B or described herein, or any other method or method. 26. The method according to embodiment 24 or any other method embodiment, wherein the visual barcode is constructed using multiple rounds of splitting and pooling using unlabeled DNA segments. 27. The splitting and pooling of one or more rounds is carried out according to the method of embodiment 26 or any other method embodiment, including: The process of ligating one barcode cassette onto beads at once; The resulting beads are then divided into individual compartments, or optionally into wells on a plate; The process of ligating different first compartment-specific barcode cassettes onto the beads in each individual compartment in order to obtain a collection of beads containing two different pairs of barcode cassettes; A step of cleaning the beads containing two barcode cassettes; A process of pooling the beads containing two barcode cassettes; The process of dividing the pooled beads, which comprise two barcode cassettes, into individual compartments, optionally into wells of a plate; and A process of repeating the ligation process, the washing process, the pooling process, and the division process in order to increase the diversity of the barcode set. 28. The method according to any one of embodiments 22 to 27 or any other method embodiment, wherein the visual barcode includes a continuous strand of dsDNA segments (cassettes), or the visual barcode includes at least two separate cassettes attached directly and separately to the solid support. 29. The method according to any one of embodiments 22 to 28 or any other embodiment of the method, wherein the segment is co-coded together with a next-generation sequencing (NGS) capture barcode. 30. A visual barcode produced by the method described in any one of embodiments 22 to 29. 31. How to decrypt a visual barcode, including the following: A step of bringing at least one double-stranded DNA (dsDNA) orthogonal cleavage-ligation sequencing (OCLS) barcode containing at least one restriction site (RS) into contact with a restriction endonuclease (RE) that recognizes the RS, under conditions sufficient to cause the RE to cleave the dsDNA OCLS barcode, wherein the RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode; The step of bringing a partial ssDNA-partial dsDNA OCLS barcode into contact with at least one orthogonal ligation probe comprising a first terminal overhang and a dsDNA oligonucleotide having a visually detectable label, under conditions sufficient to allow the overhang of the orthogonal ligation probe to bond to the overhang on the partial ssDNA-partial dsDNA OCLS barcode by base-pair-mediated hydrogen bonding when the sequence of the overhang of the orthogonal ligation probe is the reverse complement of the sequence of the overhang of the partial ssDNA-partial dsDNA OCLS barcode; If base pair-mediated binding occurs, the orthogonal ligation probe is ligated to the partially single-stranded (ss)DNA-partial dsDNA OCLS barcode to generate a captured probe; and, A step of detecting the presence, absence, and / or quantity of a captured probe by imaging the visually detectable label. 32. The method according to embodiment 31 or any other embodiment of the method, further comprising repeating the cycle of the steps of contacting / cutting, contacting / base-pair-mediated binding, ligating, and detecting, one or more times, wherein each additional cycle involves cutting the dsDNA OCLS barcode at different cleavage sites (CS), base-pair-mediated binding of different orthogonal ligation probes, and / or detection of the presence, absence, and / or quantity of different labeled and captured probes. 33. One or more of the following are the method described in aspect 31 or any other method: The dsDNA OCLS barcode is attached to a bead or other solid surface; Multiple different dsDNA OCLS barcodes are attached to a single bead or to a single address on another solid surface; The dsDNA OCLS barcode contains more than one non-overlapping RS; At least one RS in the dsDNA OCLS barcode is recognized by an IIS-type RE, and a break occurs at a predetermined location outside the RS; At least one RS in the dsDNA OCLS barcode is recognized by an RE that is cut within the RS; or The visually detectable label includes at least one of the following: a fluorescent label, a bioluminescent label, a chemiluminescent label, a chromophore, a quantum dot, a Raman label, or a radioisotope. 34. One or more of the following are the method described in aspect 32 or any other method aspect: The orthogonal ligation probe comprises a single-stranded DNA oligonucleotide having a stem-loop hairpin structure, wherein the overhang is at the end of the stem of the hairpin. 35. The method according to embodiment 34 or any other method embodiment, further comprising the step of contacting the environment containing the captured probe with a 5'-exonuclease after the ligation step. 36. The method described in aspect 32 or any other method embodiment, including one or more of the following: A step of bringing at least one dsDNA OCLS barcode into continuous contact with two or more REs, each recognizing a different non-overlapping RS within the dsDNA OCLS, under conditions sufficient to cause each RE to cleave the dsDNA OCLS barcode, wherein each RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode; The ligation process includes chemical ligation; or The ligation step includes enzyme-mediated ligation. 37. The method according to embodiment 31 or any other method embodiment, including the following: A step of bringing two or more dsDNA OCLS barcodes, each containing at least one RS, into contact with an RE that recognizes the RS, under conditions sufficient to cause the RE to cleave the dsDNA OCLS barcode, wherein the RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode, and the RS / RE differs for each dsDNA OCLS barcode. 38. The method according to aspect 37 or any other method, comprising the step of bringing two or more dsDNA OCLS barcodes into contact with two or more orthogonal REs. 39. The method according to embodiment 38, wherein the step of bringing the two or more orthogonal REs into contact is performed simultaneously or sequentially. 40. The method according to aspect 39 or any other method, wherein the differentially labeled probe is ligated using a specific orthogonal reaction, following the step of contacting one or more of the orthogonal REs. 41. The method according to aspect 40 or any other method, wherein the specific orthogonal ligation of the differentially labeled probes enables visual distinction of the barcodes in each feature of the array of visual barcodes. 42. The method according to embodiment 31 or any other method embodiment, wherein the step of ligating the orthogonal ligation probe to the partial ssDNA-partial dsDNA OCLS barcode is to add a new RS to the resulting captured probe. 43. A method for decoding a visual barcode using orthogonal section-ligation sequencing (OCLS) as described herein. 44. An improved system for molecular barcoding, comprising repeated cycles of labeling, orthogonal cleavage, ligation, and imaging for identifying individual features, wherein the orthogonal cleavage comprises cleaving a double-stranded DNA barcode having an IIS-type restriction endonuclease. 45. The system according to embodiment 44, wherein two or more signation and orthogonal probeligation specific cycles occur consecutively or simultaneously. 46. The orthogonal section-ligation sequencing (OCLS) system is shown in Figures 5A-5E. [Examples]

[0116] Example 1: Orthogonal section-ligation sequencing (OCLS) using labeled ssDNA hairpin probes This embodiment describes an exemplary system and method of OCLS using labeled single-stranded DNA forming a hairpin structure as a probe.

[0117] To demonstrate a single-cycle OCLS (cleavage, ligation, cleavage), the oligo pairs TIIS stem 1 (BsmFI) and TIIS stem 1 comp were first annealed in 1×NHS-conjugated conjugation buffer (NCB: 100 mM NaPO4, pH 8.5). Annealing was performed by mixing these oligos equimolarly (to a final concentration of 200 μM), subjecting the mixture to 95°C for 5 minutes on a heat block, and then allowing the block to cool to room temperature for at least 1 hour. Next, the annealed dsDNA was biotin-added (via the primary amino group provided to the 5' end of the TIIS stem oligo) by adding 2 mM NHS-LC-biotin (APExBIO A8004) to a solution containing 200 μM double-stranded oligos. After allowing the biotin-addition reaction to proceed overnight at room temperature, the reaction was quenched by adding 1 M tris buffer (pH 7.5) to a final concentration of 50 mM. The quenched reaction solution was desalted using two consecutive Zeba® spin desalting columns, 7K MWCO (Thermo Fisher 89882), and the buffer was exchanged with 10 mM Tris pH 7.5 buffer containing 80 mM NaCl. The biotin-added and annealed oligonucleotides were then conjugated to MyOne T1 streptavidin 42 iNa beads (Thermo Fisher 65601) at a concentration of 200 nM of annealed oligonucleotides mixed with 0.1 mg / ml MyOne T1 beads in 1× streptavidin-conjugated buffer (SBB: 10 mM Tris pH 7.5, 500 mM NaCl). DNA hairpin ligation probes (TIIS s1hp1 and s1hp3) were labeled with Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific A20000) and Alexa Fluor 555 NHS Ester (Thermo Fisher Scientific A20009) using the same method as described above, but prior annealing was not required.

[0118] MyOne T1 beads coated with TIIS Stem 1 oligo (0.25 mg / ml beads in a 200 μl volume) were incubated with BsmFI (NEB R0572L) desalted using a Zeba® spin desalting column and 7K MWCO (Thermo Fisher 89882). Glycerol was removed by elution in 1×rCutSmart® buffer before use. BsmFI enzyme (40 μl) was added to 200 μl of 0.25 mg / ml MyOne T1 beads and incubated at 37°C for 40 minutes (first round digestion). After 40 minutes, the samples were subjected to six washes (twice with 1×rCutSmart® buffer, twice with 10 mM Tris-HCl pH7.5 / 500 mM NaCl, and twice with 1×T4 DNA ligase buffer (NEB M0202S)). During one of the 200 μl Tris-NaCl washes, 30 μl of the suspended beads sample was collected and set aside (Sample 1). After these washes, the BsmFI digested stem 1 oligo-coated beads were subjected to a ligation reaction containing 1 μM TIIS s1hp1 labeled with Alexa-488. In this ligation reaction, 10 units / μl of T4 DNA ligase (NEB M0202S) and 0.5 units / μl of T4 PNK (NEB M0201S) were added. This ligation was performed after incubation at room temperature for 40 minutes. At this point, the reaction product was subjected to the same six washes as described above, but during one of the 170 μl Tris-NaCl washes, a 30 μl sample of the suspended beads was collected and set aside (Sample 2). After these washes, the beads were then subjected to a second round of digestion with BsmFI, and the beads and enzyme were maintained at the same concentrations as during the first digestion described above. After two washes in 1×rCutSmart® buffer, two more washes were performed using 140 μl of 10 mM Tris-HCl pH7.5 / 500 mM NaCl. During the second and final wash, a 30 μl sample of the suspended beads was collected (Sample 3).

[0119] The three bead samples were analyzed using two separate methods. In the first method, 2.5 μl of each bead sample was bonded to a biotin-coated glass slide and then mounted on a Leica THUNDER wide-field microscope equipped with a 20×, 0.8NA PLAN-apo objective lens for fluorescence microscopy. Using the exact same imaging parameters, images of the three bead samples were acquired using a 488 LED light source, and the fluorescence intensity of the A488 beads was quantified using the Volocity® image analysis package from Qurom Technologies Inc. This analysis showed that after ligation with Alexa-488-labeled TIIS s1hp1, the total intensity of the beads, after subtracting background, increased 42-fold (p<0.0001) (Figure 6 graph) compared to the BsmFI-digested stem 1 oligo-supported beads. This demonstrates that the stem 1 oligo was digested by BsmFI on the beads and that the Alexa-488-labeled s1hp1 probe, which contained a congeneral overhang, was able to ligate this oligo. The Alexa-488-labeled s1hp1 probe introduced a new BsmFI site on the stem 1 oligo after ligation, which was at a suitable distance from the encoded cleavage site downstream of the A488 label, so a second productive cleavage event would remove the label from the stem 1 oligo. After a second digestion by the BsmFI enzyme, the beads were found to have returned to baseline fluorescence levels, demonstrating that BsmFI recognized the BsmFI site introduced during ligation of the Alexa-488-labeled s1hp1 probe (p<0.0001) and cleaved the stem 1 oligo at the encoded “next correct” cleavage site. These results indicate that over one cycle, the enzyme “ratcheted” down the stem 1 oligo to the next designed sequence for ligation of the “next correct” labeled probe. These results demonstrate one round of OCLS performed on the OCLS barcoded oligo bound to the beads.

[0120] The second method employed to analyze these results was performed on a 10% TBE urea gel (Thermo Fisher). Briefly, 15 μl each of the above bead samples (Samples 1-3) was mixed with 15 μl of 2× formamide loading dye (95% formamide, 10 mM EDTA), heated to 95°C for 5 minutes, and then placed directly on wet ice. Furthermore, a fourth sample containing beads coated with undigested stem 1 oligo was also prepared and prepared for loading onto the denatured gel as described above. These four samples were electrophoresed on the gel in 1× TBE buffer at 150 volts for 1.5 hours, and then imaged on a ThermoFisher iBright system equipped with an illumination module capable of recording SYBR gold-stained gels and for imaging dyes 488, 550, 647, and 750. Analysis of this gel demonstrated that after digestion of beads coated with stem 1 oligo using the BsmFI enzyme (Sample 1), neither a band corresponding to the control oligo (undigested stem 1 oligo) nor a band corresponding to the expected ligation product was present. The stem 1 oligo was clearly digested by incubation with BsmFI, as evidenced by smaller fragments on the gel. In the lane corresponding to the bead sample (Sample 2), which was first cleaved with the BsmFI enzyme and subsequently ligated with the Alexa-488-labeled s1hp1 probe, a clear band that migrated for approximately 60 bases was present in the 488 imaging channel, consistent with the expected product size after ligation. This result demonstrates that the Alexa-488-labeled s1hp1 probe ligated the BsmFI-digested stem 1 oligo immobilized on the beads. Finally, this 488-positive ligation band disappeared in the lane containing the bead sample that underwent a second digestion with BsmFI (Sample 3). This indicates that a novel BsmFI site introduced by ligation with the Alexa-488-labeled s1hp1 probe was recognized by the enzyme, and that the stem 1 oligo was digested at the following correctly encoded cleavage site.Therefore, the enzyme "ratcheted" down the stem 1 oligo to the next design sequence for ligation of the next correctly labeled probe. These results are consistent with bead imaging analysis.

[0121] Figure 6 shows the OCLS method and workflow adopted in this embodiment, as well as the results obtained from the described method. An AF-488-labeled ssDNA hairpin probe of the type shown in Figure 3B (bottom) was used. The labeled ssDNA hairpin probe contained an encoded RS and was used for orthogonal ligation after cutting an OCLS barcode of the type shown in Figure 1B (Figure 2, right), which contained multiple encoded CSs. In the experiment outlined in Figure 6 (schematic diagram), an imaging step was performed in the 488 nm (luminescence) channel to determine the bead signal intensity after the initial OCLS barcode cutting with BsmFI, an IIS-type RE. After probe ligation and the subsequent washing step, the beads were imaged in the 488 nm channel to determine the signal obtained from the label and therefore the overhangs that appeared on the cut OCLS barcode. Since the same RS for the IIS-type RE(BsmFI) used in the first cleavage reaction was also coded in the ligated probe, this enzyme was added to the beads after the next wash to remove components from the previous reactant to digest the OCLS barcode for the second time. After the second digestion and washing step, an imaging step was performed in the 488 nm channel and the signal obtained from the beads was measured. The signals obtained over all three imaging steps in the 488 nm channel are plotted in the graph in Figure 6, with relative fluorescence units (RFU) on the y axis.

[0122] These experiments demonstrated successful probe ligation and subsequent cleavage. After the first cleavage with BsmFI (TIIS REcut 1), the beads contained approximately the same average intensity value (<1,000 RFU) as after the second cleavage with BsmFI (TIIS REcut 2), but after ligation of the AF-488-labeled ssDNA hairpin probe, the average intensity value of the beads was approximately 33,000 RFU. In the ligation reaction containing the labeled incorrect ssDNA hairpin probe (hairpin probe 3, containing an incorrect overhang relative to the OCLS barcode), no ligation product was produced (also determined by gel analysis).

[0123] The results reported in this embodiment suggest that many different beads in a library encoded with different combinations of OCLS barcodes containing different CSs can be decoded in a highly parallel manner using the OCLS workflow described herein. This method involves ratcheting down the OCLS barcode by adding a labeled orthogonal ligation probe containing the encoded RS for use with an IIS-type RE. In the shown embodiment, the RS corresponding to the IIS-type enzyme was used, and the individual probes used in each decoding cycle were labeled ssDNA hairpin oligos containing a specific overhang for recognition of the overhang appearing on the cleaved OCLS barcode, as shown in Figure 6.

[0124] Example 2: OCLS option using labeled ssDNA hairpin probes In the methods described in Example 1 and elsewhere in this specification, each subsequent probe used in each subsequent cycle optionally contains a different IIS type RS, which can beneficially reduce abnormal cleavage between cycles. This approach reduces the tendency for enzymes used in the previous cycle to cleave intact OCLS barcodes that may have escaped digestion (remained uncleaved) after the previous cleavage cycle.

[0125] In addition, using labeled ssDNA hairpin oligos as ligation probes enables a "cleanup" step after the ligation step, which can be performed before or after the imaging step. This cleanup step is released when using a 5'-exonuclease, such as a lambda exonuclease, which recognizes the 5'-phosphorylated end and digests and removes that strand. This optional cleanup step makes the digested barcode incompatible with any subsequent enzymes and probes used in future cycles (and therefore excluded from analysis from that point onward). Since the ligated product (containing labeled ssDNA hairpin oligos) can be designed not to contain open 5'-phosphorylated ends, the cleanup step removes any dsDNA remaining after the ligation step, which was not 100% efficient.

[0126] Example 3: Individual segments and chains of an OCLS barcode containing a standard RE site. In another embodiment, a standard set of orthogonal restriction enzymes can be used. In this case, OCLS barcodes can be purchased from a vendor (such as IDT) as fully constructed barcodes or barcode segments containing one or more orthogonal recognition sites (RSs). These barcodes can be constructed from segments as shown in Figure 1A, designed for specific cleavage at the RSs by site-specific restriction endonuclease enzymes (REs) as shown in Figure 2 (left), and used as shown in Figure 4A. In this case, decoding can be achieved by immobilizing beads on a surface such as a standard microscope slide or on another surface / application where feature identification and / or feature location determination is advantageous, and then introducing one or more orthogonal REs (in a single reaction mixture) and contacting the enzymes with one or more OCLS barcodes. As shown in Figure 4A, one or more OCLS barcodes are digested with an RE enzyme capable of expressing specific overhangs on the OCLS barcode, followed by a washing step (e.g., using 1×CutSmart buffer, New England Biolabs) to remove the RE enzyme, and then washed with 1×T4 DNA ligase buffer (e.g., supplied by New England Biolabs along with the T4 DNA ligase enzyme; M0202). The 1×T4 DNA ligase buffer contains ATP and Mg, which are important components for the successful ligation reaction. 2+The following is included: After equilibration in ligase buffer, T4 DNA ligase and polynucleotide kinase (PNK; this enzyme phosphorylates any unphosphorylated 5' end of the oligo, making them suitable substrates for T4 DNA ligase) enzymes are introduced into 1×T4 DNA ligase buffer containing the labeled probe, which can be achieved, for example, in a flow cell with microfluidics, or in a microfluidic apparatus under instrument control software to control both the fluid flow rate and timing to enable an in-line imaging process. This instrument configuration is known to be somewhat standard in the art and is used in methods and systems that require cycling of different solutions in conjunction with imaging, such as next-generation DNA sequencing (SBS) workflows by synthesis.

[0127] Here, each differentially labeled probe contains a specific overhang for one of the overhangs created on the OCLS barcode after the digestion step described above. Upon introduction of the enzymatic ligation solution containing the differentially labeled probes, ligation occurs between the correctly matching overhang (complete base complementarity) on the OCLS barcode and one of the probes in the enzymatic ligation solution. After incubation with the enzymatic ligation solution, a washing step is performed to remove ligase and PNK enzyme in order to carry out the imaging step. The imaging step identifies specific overhang sequences that appear on the OCLS barcode after the described orthogonal digestion and ligation steps. Due to the fact that each orthogonal ligation probe has a distinct label or combination of labels, distinction between different overhangs present on different OCLS barcodes is made possible by the imaging step or by a step that otherwise specifically identifies the detectable probe label associated with the OCLS barcode bead (see Figure 4B). Depending on the number of unique overhangs that appear on the OCLS barcodes bound to individual beads or features after the digestion process, the ligation and imaging steps may be performed multiple times (as shown in Figures 4C and 4D). This is because each orthogonal overhang created on the OCLS barcode after the digestion process needs to be individually distinguished (read individually) by a specific probe carrying a label or combination of labels corresponding to the matching overhang. It is also possible to distinguish multiple different overhangs on a single bead in this manner during a single ligation event, as shown in Figure 4E, and to create detectable combinations of labels on the OCLS barcode. The OCLS method relies on the creation of specific orthogonal overhangs on the dsDNA OCLS barcode (orthogonality arises from RE digestion of the OCLS barcode), as well as the use of labeled probes that encompass both predetermined labels and overhangs that correspond to the detected overhangs that appear on the OCLS barcode (resulting in specific base complementarity with the overhangs).In this way, many individual members of a library of beads or features can be distinguished over multiple cycles, similar to the cycles performed in a DNA sequencing workflow. In contrast, in an NGS workflow, nucleotides containing both a predetermined label and a base corresponding to the next correct base in the primed nucleic acid template (resulting in specific base complementarity with the base) are incorporated by polymerase and imaged to determine which bases were incorporated in each cycle. Many beads or features can be sequenced over multiple cycles and in a highly parallel manner by NGS or OCLS.

[0128] Example 4: Individual segments and chains of an OCLS barcode containing an IIS-type RE recognition area (RS) and / or cleavage area (CS). As described in Example 3, OCLS barcodes may contain one or more recognition sites (RSs) for cleavage by one or more standard REs, but in another embodiment, IIS-type RE enzymes can be used. IIS-type RE enzymes have RSs located a known number of bases away from each of their CSs. IIS-type RE enzymes bind to their cognate RSs, and then these enzymes cleave their respective CSs, leaving a known number of overhanging bases. The specificity of dsDNA recognition by IIS-type REs arises at the RSs, but the CSs can be any set of bases; these enzymes will cut any designed CS as long as it is at the correct distance from the RSs. Thus, it is possible to create OCLS barcodes with one or more CSs that do not contain RSs or contain one or more than one RSs. As shown in Figure 5A, experiments can be carried out to attach two different barcodes to individual beads that can be contained within a library of OCLS barcode-attached beads. The two different OCLS barcodes on the beads can each contain different combinations of RSs and CSs for compatibility with two different (orthogonal) IIS-type enzymes. In this embodiment, beads can be immobilized on a cycling surface as described in Example 2, and two different IIS-type enzymes (BsmFI and BpuEI) can be introduced into a single reaction mixture consisting of mutually compatible buffers such as NEB CutSmart buffer. As shown in Figure 5A, digestion by these enzymes removes dsDNA cleavage products by cleaving at their respective CSs rather than at the RS, which differs from standard REs (i.e., REs that cut within their RS). Therefore, as shown in Figure 5B, the CS can be designed within the barcode, provided that the RS is located within the OCLS barcode intended for use with the IIS-type RE at the correct distance for the specific enzyme used. Each IIS-type enzyme substrate (OCLS barcode) shown in Figure 5B (BsmFI-specific barcode above, BpuEI-specific barcode below) is cleaved at a known number of bases from the RS at the CS, thereby leaving a known number of bases in the overhang that will be left on the barcode after digestion.After cleavage with an IIS-type enzyme, a washing step can be performed (for example, using 1×CutSmart buffer from NEB) to remove the enzyme and cleavage products, followed by the introduction of 1×T4 DNA ligase buffer for equilibration before the ligation step.

[0129] In this embodiment, and as shown in Figure 5C, two distinguishable overhangs on each feature require two cycles of ligation and imaging of a labeled probe for identification of the two distinct overhangs generated after digestion. However, in this embodiment, the probe not only includes distinguishable labels and corresponding overhangs as in Example 3, but also includes an additional RS for the IIS-type RE (as demonstrated in Example 1 using an ssDNA hairpin ligation probe). After the ligation and imaging steps, the newly introduced RS creates a new “cassette” for the IIS-type enzyme (an RS located at the appropriate distance from the next CS in the OCLS barcode) by ligating the probe to the correct OCLS barcode through the matching overhang. Before introducing the IIS-type enzyme for the next digestion cycle, a washing step can be performed using 1×CutSmart buffer. By encoding the new IIS-type enzyme RS within the introduced, labeled ligation probe, this process of “ratcheting” down the OCLS barcode can be achieved, which is a process of designing the CS for each cycle. This was demonstrated in Example 1 using a labeled ssDNA hairpin oligo containing the BsmFI site, which allowed the barcode to be cut a second time after the ligation process. Examples of oligos that can be used as barcodes in this manner include (TIIS stem 1; SEQ ID NO: 1 and 2, TIIS stem 2; SEQ ID NO: 12 and 13) and corresponding probe oligos that can be used to read OCLS barcodes in this manner (TIIS stem 1 probe; SEQ ID NO: 3-11, TIIS stem 2 probe; SEQ ID NO: 14-19).

[0130] The descriptions contained herein are included to demonstrate specific aspects of the disclosure. Those skilled in the art should recognize that, in view of the disclosure, many modifications can be made to the specific aspects disclosed herein, and similar or equivalent results can be obtained without departing from the spirit and scope of the disclosure.

[0131] knot As those skilled in the art will understand, each embodiment disclosed herein may include, be essentially composed of, or consist of its specific defined elements, processes, components, or constituents. Therefore, the terms “include” or “including” should be interpreted as “comprise, constitute of, or constitute essentially of.” The transitional terms “comprises” or “comprises” mean, but are not limited to, having, and may include even a large number of unspecified elements, processes, components, or constituents. The transitional term “consists of” also excludes any unspecified elements, processes, components, and constituents. The transitional term “consistently of” limits the scope of the embodiment to the specified elements, processes, components, or constituents, and only those that do not materially affect the embodiment.

[0132] Unless otherwise indicated, all numerical values ​​used in this specification and the appended claims, such as quantities of components, molecular weights and other properties, and reaction conditions, should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters shown in this specification and the appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention. Each numerical parameter should be interpreted, at least in light of the reported number of significant figures and by applying common rounding techniques, and not as an attempt to limit the application of the doctrine of equivalents to the appended claims. If further clarification is needed, the term “about” has a meaning that, when used with a specified number or range, is reasonably considered to be so by a person skilled in the art, namely, being somewhat more or somewhat less than the specified number or range by a range of ±20%; ±19%; ±18%; ±17%; ±16%; ±15%; ±14%; ±13%; ±12%; ±11%; ±10%; ±9%; ±8%; ±7%; ±6%; ±5%; ±4%; ±3%; ±2%; or ±1%.

[0133] Even though the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. However, any numerical value inherently contains a certain degree of error that may inevitably arise from the standard deviation observed in each test measurement.

[0134] In the context describing the present invention (particularly in the context of the appended claims), the terms “a,” “an,” “the,” and similar referent should be considered to cover both singular and plural forms unless otherwise indicated herein and unless clearly contradicted by the context. The descriptions of value ranges herein are merely intended to serve as abbreviations for individually referring to each individual value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually described herein. All methods described herein may be carried out in any suitable order unless otherwise indicated herein or unless clearly contradicted by the context. Any and all examples or illustrative language provided herein (e.g., “~, etc.”) are merely intended to better illustrate the present invention and not to limit the scope of the claimed invention. Nothing in this specification should be construed as indicating any unclaimed element essential to the practice of the present invention.

[0135] The grouping of alternative elements or embodiments of the Invention disclosed herein shall not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, it is anticipated that one or more members of a group may be included in or removed from a group. In the event of any such inclusion or removal, this specification shall be deemed to encompass the modified group to satisfy the description of all Markush groups used in the appended claims.

[0136] Specific embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the preceding description. The inventors anticipate that those skilled in the art will appropriately adopt such variations, and they intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations thereof is incorporated herein unless otherwise indicated herein and unless it is clearly inconsistent with the context.

[0137] Furthermore, numerous references are made throughout this specification to patents, print publications, journal articles, other documents, and website content (References in this Specification). Each reference is individually incorporated into this Specification by reference in its entirety at the filing date of the first application in the priority chain that includes the particular reference. For example, with respect to chemical compounds, nucleic acids, and amino acid sequences mentioned herein that are available in public databases, the information from the database entries is incorporated into this Specification by reference at the filing date of the application in the priority chain that first includes the database identifier for that compound or sequence in the text.

[0138] It should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be adopted are within the scope of the invention. Therefore, alternative configurations of the present invention can be used in accordance with the teachings herein, but not limited to them, as examples. Accordingly, the present invention is not limited to those precisely illustrated and described herein.

[0139] The specific details presented herein are for illustrative purposes only and for illustrative purposes of preferred embodiments of the invention, and are presented for the greater good of providing what is considered to be the most useful and easily understandable explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt has been made to provide structural details of the invention in more detail than is necessary for a basic understanding of the invention, and the descriptions and / or examples accompanying the drawings will be made clear to those skilled in the art how some embodiments of the invention can actually be embodied.

[0140] The definitions and descriptions used in this disclosure are intended to be dominant in any future interpretation unless expressly and unambiguously modified in the examples, or unless any interpretation becomes meaningless or essentially meaningless by application of the meaning. Where an interpretation of a term becomes meaningless or essentially meaningless, the definition shall be that of Webster's Dictionary, 11th Edition, or Oxford Dictionary of Biochemistry and Molecular Biology, 2 nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and / or A Dictionary of Chemistry, 8 th It should be cited from dictionaries known to those skilled in the art, such as Edition (Ed. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

1. Visual barcodes including the following: At least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, wherein each of the dsDNA segments contains, Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The dsDNA oligonucleotide segment (cassette) including; Furthermore, The unfixed end of one end of the visual barcode.

2. The visual barcode according to claim 1, wherein the visual barcode is fixed to a solid substrate by a flexible linker attached to or near the end of the dsDNA of the visual barcode, which is not the unfixed end.

3. A visual barcode according to claim 1, which does not include visually detectable markers.

4. The visual barcode according to claim 1, wherein the RE is an IIS-type restricted endonuclease, and the CS does not overlap with the corresponding RS.

5. The visual barcode according to claim 1, wherein at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to the IIS type RE, which is appropriately positioned so that the homologous IIS type RE can cut the designed CS based on its position relative to the RS.

6. A visual barcode as described in any one of claims 1 to 5, as shown in Figure 1A or Figure 1B or essentially as described herein.

7. A set of visual barcodes according to any one of claims 1 to 6, wherein the set comprises a plurality of visual barcodes, each comprising a different set of dsDNA segments having different recognition sites (RS), designed cleavage sites (CS), or both for specific restriction endonucleases (REs).

8. The collection of visual barcodes according to claim 7, wherein at least two different visual barcodes are fixed to the same solid substrate.

9. An orthogonal cleavage-ligation sequencing (OCLS) barcode comprising two or more dsDNA segments (cassettes), wherein each dsDNA segment comprises a recognition site (RS) for a specific restriction endonuclease (RE) and one or more overlapping regions configured to allow ligation to adjacent dsDNA segments to form a segmental chain, and the chain of the segments constitutes the OCLS barcode.

10. Visually detectable orthogonal ligation probes, including the following: A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein its sequence is Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The complete or partial double-stranded DNA oligonucleotide containing; Furthermore, A visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide.

11. The visually detectable orthogonal ligation probe according to claim 10, wherein the complete or partially double-stranded DNA oligonucleotide comprises: Linear double-stranded DNA oligonucleotides with a 5' overhang; Linear double-stranded DNA oligonucleotides with a 3' overhang; A single-stranded DNA oligonucleotide with a 5' overhang and a hairpin stem-loop configuration; or A single-stranded DNA oligonucleotide with a hairpin stem-loop structure and a 3' overhang.

12. The visually detectable orthogonal ligation probe according to claim 10, wherein the visually detectable label is attached to the oligonucleotide via a flexible linker.

13. The visually detectable orthogonal ligation probe according to claim 10, wherein the visually detectable label comprises one or more fluorescent labels, bioluminescent labels, chemiluminescent labels, chromophores, quantum dots, Raman labels, biotin moieties, or radioisotopes.

14. The visually detectable orthogonal ligation probe according to claim 10, wherein the RE is an IIS-type restriction endonuclease and the RS is specific to the IIS-type RE.

15. A visually detectable orthogonal ligation probe according to claim 10, comprising an RS specific to an IIS type RE, which is appropriately positioned so that a cognate IIS type RE can cut a designed CS based on its position relative to the RS.

16. A visually detectable orthogonal ligation probe as described in any one of claims 10 to 15, as shown in Figure 3A or Figure 3B or essentially as described herein.

17. A set of visually detectable orthogonal ligation probes according to any one of claims 10 to 16, wherein the set comprises a plurality of visually detectable orthogonal ligation probes, each comprising a different recognition site (RS) for a specific restriction endonuclease (RE), or both a different RS and a different cleavage site (CS).

18. The set of visually detectable orthogonal ligation probes according to claim 17, wherein at least two different detectable orthogonal ligation probes include visually distinguishable detectable labels.

19. A set of visually detectable orthogonal ligation probes according to claim 18, comprising a plurality of different probes, each configured such that the cutting of the probe by the RE produces an overhang having a different arrangement from at least 5, at least 7, at least 10, at least 12, at least 15, or more than 15 other probes in the set.

20. Orthogonal cleavage-ligation sequencing (OCLS) oligonucleotide pairs, the following: At least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, wherein each of the dsDNA segments contains, Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The dsDNA oligonucleotide segment (cassette) including, An unfixed end at one end of a visual barcode. The visual barcode includes, A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein its sequence is Recognition sites (RS) for specific restriction endonucleases (REs), or Both RS and cleavage site (CS) A complete or partial double-stranded DNA oligonucleotide containing; and, A visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide. This includes a visually detectable orthogonal ligation probe and Includes, The cutting of the RS or CS in the visual barcode generates a single-stranded "sticky end" overhang having an arrangement that is perfectly complementary to the sticky end (overhang) generated by the cutting of the RS in the visually detectable orthogonal ligation probe. The aforementioned OCLS oligonucleotide pair.

21. A set of OCLS oligonucleotide pairs according to claim 20, wherein each pair of visual barcodes and visually detectable orthogonal ligation probes has a different, fully complementary sequence overlap, and each visually detectable orthogonal ligation probe includes a different, visually distinguishable, detectable label.

22. How to encode a visual barcode, including the following: A double-stranded DNA (dsDNA) oligonucleotide, which is fixed to a solid support at its first end and has a single-stranded overhang at its unfixed second end, It has a first overhanging end and a second overhanging end that are suitable for binding the immobilized dsDNA oligonucleotide to the single-stranded overhang, and Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or Both RS and CS The first dsDNA segment contains within its sequence the first dsDNA segment The process of bringing it into contact with, The contact step occurs under conditions sufficient to allow specific hybridization of the single-stranded overhang of the first dsDNA segment to the single-stranded overhang of the immobilized dsDNA oligonucleotide; A step of ligating the first dsDNA segment to the immobilized dsDNA oligonucleotide in order to form a first captured barcode segment including the second overhanging end of the first dsDNA segment; The first captured barcode segment is It has a first overhanging end and a second overhanging end that are suitable for binding the first captured barcode segment oligonucleotide to the single-strand overhang, and RS for a specific RE different from the RS / RE in the first dsDNA segment, A different designed CS in the first dsDNA segment, or Both The second dsDNA segment contains within its sequence the second dsDNA segment The process of bringing it into contact with; and, A step of ligating a second dsDNA segment to a first captured barcode segment in order to form a second captured barcode segment including the second overhanging end of the second dsDNA segment, wherein the first captured barcode segment and the second captured barcode segment constitute the visual barcode.

23. The method according to claim 22, further comprising the step of repeating the contact step and the ligation step one or more times in order to form the visual barcode, each time by attaching an additional dsDNA segment to the captured barcode segment.

24. The method according to claim 22 or 23, wherein at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to the IIS type RE, which is appropriately positioned, so that the homologous RE can cut the designed CS based on its position relative to the RS.

25. The method according to any one of claims 22 to 24, as shown in Figure 1A or Figure 1B or described herein.

26. The method according to claim 24, wherein the visual barcode is constructed using multiple rounds of splitting and pooling using unlabeled DNA segments.

27. The method according to claim 26, wherein the division and pooling of one or more rounds includes: The process of ligating one barcode cassette onto beads at once; The resulting beads are then divided into individual compartments, or optionally into wells on a plate; The process of ligating different first compartment-specific barcode cassettes onto the beads in each individual compartment in order to obtain a collection of beads containing two different pairs of barcode cassettes; A step of washing the beads containing two barcode cassettes; A step of pooling the beads containing two barcode cassettes; The process of dividing the pooled beads, which include two barcode cassettes, into individual compartments, optionally into wells of a plate; and A process of repeating the ligation process, the washing process, the pooling process, and the division process in order to increase the diversity of the barcode set.

28. The method according to any one of claims 22 to 27, wherein the visual barcode comprises a continuous strand of dsDNA segments (cassettes), or the visual barcode comprises at least two separate cassettes attached directly and separately to the solid support.

29. The method according to any one of claims 22 to 28, wherein the segment is co-coded with a next-generation sequencing (NGS) capture barcode.

30. A visual barcode produced by the method described in any one of claims 22 to 29.

31. How to decrypt a visual barcode, including the following: A step of bringing at least one double-stranded DNA (dsDNA) orthogonal cleavage-ligation sequencing (OCLS) barcode containing at least one restriction site (RS) into contact with a restriction endonuclease (RE) that recognizes the RS, under conditions sufficient to cause the RE to cleave the dsDNA OCLS barcode, wherein the RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss)DNA-partial dsDNA OCLS barcode; The step of bringing the partial ssDNA-partial dsDNA OCLS barcode into contact with at least one orthogonal ligation probe comprising a first terminal overhang and a dsDNA oligonucleotide having a visually detectable label, under conditions sufficient to allow the overhang of the orthogonal ligation probe to bond to the overhang on the partial ssDNA-partial dsDNA OCLS barcode by base-pair-mediated hydrogen bonding when the sequence of the overhang of the orthogonal ligation probe is the reverse complement of the sequence of the overhang of the partial ssDNA-partial dsDNA OCLS barcode; If base pair-mediated binding occurs, the orthogonal ligation probe is ligated to the partially single-stranded (ss) DNA-partial dsDNA OCLS barcode to generate a captured probe; and, A step of detecting the presence, absence, and / or quantity of a captured probe by imaging the visually detectable label.

32. The method according to claim 31, further comprising a step of repeating the cycle of the steps of contacting / cutting, contacting / base-pair-mediated binding, ligating, and detecting, one or more times, wherein each additional cycle involves cutting the dsDNA OCLS barcode at different cleavage sites (CS), base-pair-mediated binding of different orthogonal ligation probes, and / or detection of the presence, absence, and / or quantity of different labeled and captured probes.

33. The method according to claim 31, which is one or more of the following: The dsDNA OCLS barcode is attached to a bead or other solid surface; Multiple different dsDNA OCLS barcodes are attached to a single bead or to a single address on another solid surface; The dsDNA OCLS barcode includes one or more non-overlapping RSs; At least one RS in the dsDNA OCLS barcode is recognized by an IIS-type RE, and a break occurs outside the RS at a predetermined location; At least one RS in the dsDNA OCLS barcode is recognized by an RE that cuts within the RS; or The visually detectable label includes at least one of the following: a fluorescent label, a bioluminescent label, a chemiluminescent label, a chromophore, a quantum dot, a Raman label, or a radioisotope.

34. The method according to claim 32, which is one or more of the following: The orthogonal ligation probe comprises a single-stranded DNA oligonucleotide having a stem-loop hairpin structure, wherein the overhang is at the end of the stem of the hairpin.

35. The method according to claim 34, further comprising the step of contacting the environment containing the captured probe with a 5'-exonuclease after the ligation step.

36. The method according to claim 32, comprising one or more of the following: A step of bringing at least one dsDNA OCLS barcode into continuous contact with two or more REs, each recognizing a different non-overlapping RS within the dsDNA OCLS, under conditions sufficient to cause each RE to cleave the dsDNA OCLS barcode, wherein each RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss) DNA-partial dsDNA OCLS barcode; The ligation process includes chemical ligation; or The ligation step includes enzyme-mediated ligation.

37. The method according to claim 31, including the following: A step of bringing two or more dsDNA OCLS barcodes, each containing at least one RS, into contact with an RE that recognizes the RS, under conditions sufficient to cause the RE to cleave the dsDNA OCLS barcode, wherein the RS / RE cleavage results in a single-stranded overhang for generating a partially single-stranded (ss) DNA-partial dsDNA OCLS barcode, and the RS / RE differs for each dsDNA OCLS barcode.

38. The method according to claim 37, comprising the step of bringing two or more dsDNA OCLS barcodes into contact with two or more orthogonal REs.

39. The method according to claim 38, wherein the step of bringing the two or more orthogonal REs into contact is performed simultaneously or sequentially.

40. The method according to claim 39, wherein the differentially labeled probe is ligated using a specific orthogonal reaction, following the step of contacting one or more of the orthogonal REs.

41. The method according to claim 40, wherein the specific orthogonal ligation of the differentially labeled probes enables visual distinction of the barcodes in each feature in an array of visual barcodes.

42. The method according to claim 31, wherein the step of ligating the orthogonal ligation probe to the partial ssDNA-partial dsDNA OCLS barcode is to add a new RS to the resulting captured probe.

43. A method for decoding a visual barcode using orthogonal section-ligation sequencing (OCLS) as described herein.

44. An improved system for molecular barcoding, comprising repeated cycles of labeling, orthogonal cleavage, ligation, and imaging for identifying individual features, wherein the orthogonal cleavage comprises cleaving a double-stranded DNA barcode having an IIS-type restriction endonuclease.

45. The system according to claim 44, wherein two or more marking and orthogonal probeligation specific cycles occur consecutively or simultaneously.

46. The orthogonal section-ligation sequencing (OCLS) system is shown in Figures 5A to 5E.

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