Target probe complexes and analyte detection complexes for in situ detecting and identifying target analytes

Abstract

The present disclosure provides compositions and methods for detecting a plurality target analytes, including nucleic acids and proteins, in a cellular sample by conducting sequencing inside the cellular sample.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 618,089, filed January 5, 2024, 63 / 549,268, filed on February 2, 2024, 63 / 682,580, filed on August 13, 2024, and 63 / 704,218, filed on October 7, 2024, the entire contents of each of which are incorporated herein by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (ELEM_024_001WO_SeqList_ST26.xml; Size 12,574 bytes; and Date of Creation: December 19, 2024) are herein incorporated by reference in their entireties. TECHNICAL FIELD

[0003] The present disclosure provides compositions, apparatuses and methods for generating concatemer molecules inside a cellular sample using probes that detect target analytes . In some embodiments, the concatemer molecules generated inside a cellular sample can be used for detecting the presence of target analytes (proteins, polynucleotides, and the like), and identifying and imaging the target analytes. BACKGROUND

[0004] Cells within a tissue of a subject exhibit differences in composition, morphology and function due to varied analyte levels (e.g., gene and / or protein expression) within the cells. The specific position of a cell within a tissue (e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment), developmental stage, and pathological conditions can affect, e.g., cell morphology, differentiation, fate, viability, proliferation, signaling, and crosstalk with other cells in the tissue, and other cellular behaviors. Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes such as proteins, nucleic acids, polysaccharides or lipids, in the context of an intact tissue, a portion of a tissue or cultured cells. However, previous methods are limited by the number of analytes that can be identified and imaged, and the amount of time and labor involved. Thus, there exists a need for improved spatially resolved analysis methods which can be used to image multiple analytes in a cell or tissue. SUMMARY

[0005] The disclosure provides a multivalent analyte detection reagent, comprising: a multivalent affinity reagent (1900) comprising a core attached to a plurality of polymer arms, wherein individual polymer arms are attached to affinity moieties that can bind a target analyte, wherein at least one of the polymer arms attached to the affinity moieties comprises a linear polymer arm, a forked polymer arm or a branched polymer arm, and optionally wherein the multivalent affinity reagent (1900) comprises at least one fluorophore.

[0006] The disclosure provides a multivalent analyte detection reagent, comprising: a multivalent oligo reagent (2000) comprising a core attached to a plurality of polymer arms, wherein individual polymer arms are attached to affinity moieties that can bind a target analyte and at least one polymer arm is attached to an oligonucleotide, wherein at least one of the polymer arms attached to the affinity moieties comprises a linear polymer arm, a forked polymer arm or a branched polymer arm, wherein the at least one polymer arm attached to the oligonucleotide comprises a linear polymer arm, a forked polymer arm or a branched polymer arm, wherein the oligonucleotide comprises a sequencing primer binding site and a target barcode sequence corresponding to the affinity moieties attached to individual polymer arms, and optionally wherein the multivalent oligo reagent (2000) comprises at least one fluorophore.

[0007] The disclosure provides a multivalent analyte detection reagent, comprising: a multivalent heterofunctional reagent (2500) comprising a multi-arm polymer attached to at least one oligonucleotide and a plurality of affinity moieties that can bind a target analyte, wherein the multi-arm polymer comprises a forked multi-arm polymer or a branched multiarm polymer, wherein the oligonucleotide comprises a sequencing primer binding site and a target barcode sequence corresponding to the plurality of affinity moieties attached to the multi-arm polymer, and optionally wherein the multivalent heterofunctional reagent (2500) comprises at least one fluorophore.

[0008] The disclosure provides plurality of multivalent analyte detection reagents, comprising: a plurality of multivalent oligo reagents (2000) of the disclosure, wherein the plurality of multivalent oligo reagents comprises (a) a first sub-population of multivalent oligo reagents (2000-1), and (b) a second sub-population of multivalent oligo reagents (20002). In some embodiments, individual multivalent oligo reagents in the first sub-population comprise a core attached to a plurality of polymer arms, wherein individual polymer arms are attached to first affinity moieties that can bind a first target analyte, and at least one of the polymer arms is attached to an oligonucleotide comprising a first sequencing primer binding site (2010-1) and a first target barcode sequence (2020-1) corresponding to the first affinity moieties that is at least 2 nucleotides in length. In some embodiments, individual multivalent oligo reagents in the second sub-population comprise a core attached to a plurality of polymer arms, wherein individual polymer arms are attached to second affinity moieties that can bind a second target analyte, and at least one of the polymer arms is attached to an oligonucleotide comprising a second sequencing primer binding site (2010-2) and a second target barcode sequence (2020-2) corresponding to the second affinity moieties that is at least 2 nucleotides in length. In some embodiments, the first and second target barcode sequences are different. In some embodiments, the first and second affinity moieties are different. In some embodiments, the first and second sequencing primer binding sites are the same sequence or different sequences. In some embodiments, wherein one nucleo-base in a first position in the first target barcode sequence (2020-1) generates a first color signal in a first sequencing cycle, and one nucleo-base in a first position in the second target barcode sequence (2020-2) generates a second color signal in the same first sequencing cycle, and wherein the first and second color signals are different. In some embodiments, one nucleo-base in a second position in the first target barcode sequence (2020-1) generates a third color signal in a second sequencing cycle, and one nucleo-base in a second position in the second target barcode sequence (2020-2) generates a fourth color signal in the same second sequencing cycle, and wherein the third and fourth color signals are different, In some embodiments, the first color signal identifies the first target analyte, In some embodiments, the fourth color signal identifies the second target analyte. In some embodiments, the first and third color signal can be the same color signal or the different color signals. In some embodiments, the second and fourth color signals can be the same color signal or different color signals.

[0009] The disclosure provides a plurality of multivalent analyte detection reagents, comprising: a plurality of multivalent heterofunctional reagents (2500) of the disclosure, wherein the plurality of multivalent heterofunctional reagents comprises (a) a first subpopulation of multivalent heterofunctional reagents (2500-1) and (b) a second sub-population of multivalent heterofunctional reagents (2500-2). In some embodiments, individual multivalent heterofunctional reagents in the first sub-population comprise a multi-arm polymer attached to (1) at least one oligonucleotide comprising a first sequencing primer binding site (2510-1) and a first target barcode sequence (2520-1) at least 2 nucleotides in length and (2) a plurality of affinity moieties that can bind a first target analyte, wherein the first target barcode sequence (2520-1) corresponds to the first affinity moieties. In some embodiments, individual multivalent heterofunctional reagents in the second sub-population comprise a multi-arm polymer attached to (1) at least one oligonucleotide comprising a second sequencing primer binding site (2510-2) and a second target barcode sequence (25202) at least 2 nucleotides in length and (2) a plurality of affinity moieties that can bind a second target analyte, wherein the second target barcode sequence (2520-2) corresponds to the second affinity moieties. In some embodiments, the first and second target barcode sequences are different. In some embodiments, the first and second affinity moieties are different. In some embodiments, the first and second sequencing primer binding sites are the same sequence or different sequences. In some embodiments, one nucleo-base in a first position in the first target barcode sequence (2520-1) generates a first color signal in a first sequencing cycle, and one nucleo-base in a first position in the second target barcode sequence (2520-2) generates a second color signal in the same first sequencing cycle. In some embodiments, the first and second color signals are different. In some embodiments, one nucleo-base in a second position in the first target barcode sequence (2520-1) generates a third color signal in a second sequencing cycle, and one nucleo-base in a second position in the second target barcode sequence (2520-2) generates a fourth color signal in the same second sequencing cycle. In some embodiments, the third and fourth color signals are different. In some embodiments, the first color signal identifies the first target analyte, and wherein the fourth color signal identifies the second target analyte. In some embodiments, the first and third color signal can be the same color signal or the different color signals. In some embodiments, the second and fourth color signals can be the same color signal or different color signals.

[0010] In some embodiments of the multivalent analyte detection reagent or pluralities of multivalent analyte detection reagents of the disclosure, the target analyte is located on the exterior of a cell, is embedded in a cell membrane, is located in the cytoplasm, is located on an cell organelle, is located inside a cell, or a combination thereof. In some embodiments, the target analyte is located on a cellular organelle or structure or inside a cellular organelle or structure, optionally wherein the cellular organelle or structure comprises a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a microtubule, a centriole, a spindle, an actin filament, a flagellum, a cilium, a peroxisome, a lysosome, a chloroplast or a combination thereof. In some embodiments, the target analyte comprises a polypeptide, a protein, a protein fragment, an enzyme or an antibody. In some embodiments, the target analyte comprises a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, a homopolysaccharide or a heteropolysaccharide. In some embodiments, the target analyte comprises a lipid, a triglyceride, a phospholipid, a steroid, a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a saccharolipid, a polyketide, a sterol lipid or a prenol lipid. In some embodiments, the target analyte comprises an oligonucleotide, a polynucleotide, DNA, cDNA or RNA. In some embodiments, the target analyte comprises a cell surface receptor comprising an ion channel receptor, a G-protein coupled receptor or an enzyme-linked receptor. In some embodiments, the target analyte comprises a cluster of differentiation (CD) comprising a cell receptor CD, a cell ligand CD, a cell signaling CD, or a cell adhesion CD. In some embodiments, the target analyte comprises a cytokine, an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor, a chemokine, a vascular growth factor, a platelet derived growth factor, a lymphokine, a monokine or a colony stimulating factor. In some embodiments, the target analyte comprises a peptide hormone.

[0011] The disclosure provides a method for detecting a plurality of target analytes inside a cellular sample, comprising: (a) providing a cellular sample deposited on a support, wherein the cellular sample comprises a plurality of analytes including at least one target analyte; (b) providing a multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of the disclosure, wherein individual multivalent analyte detection reagents comprise a fluorophore; (c) contacting the cellular sample with the plurality of multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for moving the plurality of multivalent analyte detection reagents into the cellular sample and suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes inside the cellular sample to form a plurality of multivalent reagent-analyte complexes inside the cellular sample; (d) washing the cellular sample to remove multivalent analyte detection reagents that are not bound to a cognate target analyte, under a condition suitable to retain the plurality of f multivalent reagent-analyte complexes inside the cellular sample; and (e) detecting the plurality of multivalent reagent-analyte complexes inside the cellular sample by imaging fluorescent signals emitted from the plurality of multivalent reagent-analyte complexes inside the cellular sample, thereby detecting the plurality of target analytes inside a cellular sample.

[0012] In some embodiments of the methods of the disclosure, the multivalent analyte detection reagents comprise fluorophore-labeled multivalent affinity reagents comprising at least a first and second sub-population of fluorophore-labeled multivalent affinity reagents. In some embodiments, individual fluorophore-labeled multivalent affinity reagents in the first sub-population (1900-1) bind a first target analyte and comprise a first fluorophore. In some embodiments, individual fluorophore-labeled multivalent affinity reagents in the second subpopulation (1900-2) bind a second target analyte and comprise a second fluorophore. In some embodiments, the first and second target analytes are different target analytes. In some embodiments, the first and second fluorophores emit fluorescent signals that are distinguishable from each other.

[0013] In some embodiments of the methods of the disclosure, the multivalent analyte detection reagents comprise fluorophore-labeled multivalent oligo reagents comprising at least a first and second sub-population of fluorophore-labeled multivalent oligo reagents. In some embodiments, individual fluorophore-labeled multivalent oligo reagents in the first subpopulation (2000-1) bind a first target analyte and comprise a first fluorophore. In some embodiments, individual fluorophore-labeled multivalent oligo reagents in the second subpopulation (2000-2) bind a second target analyte and comprise a second fluorophore. In some embodiments, the first and second target analytes are different target analytes. In some embodiments, the first and second fluorophore emit fluorescent signals that are distinguishable from each other.

[0014] In some embodiments of the methods of the disclosure, the multivalent analyte detection reagents comprise fluorophore-labeled multivalent heterofunctional reagents comprising at least a first and second sub-population of fluorophore-labeled multivalent heterofunctional reagents. In some embodiments, individual fluorophore-labeled multivalent heterofunctional reagents in the first sub-population (2500-1) bind a first target analyte and comprise a first fluorophore. In some embodiments, individual fluorophore-labeled multivalent heterofunctional reagents in the second sub-population (2500-2) bind a second target analyte and comprise a second fluorophore. In some embodiments, the first and second target analytes are different target analytes. In some embodiments, the first and second fluorophore emit fluorescent signals that are distinguishable from each other.

[0015] The disclosure provides a method for conducting a sequencing-based workflow for detecting a plurality of target analytes inside a cellular sample, comprising: (a) providing a cellular sample deposited on a support, wherein the cellular sample harbors a plurality of analytes including at least one target analyte; (b) providing the multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of the disclosure; (c) contacting the cellular sample with a plurality of the multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for moving the plurality of multivalent analyte detection reagents into the cellular sample and suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes inside the cellular sample to form a plurality of multivalent reagent-analyte complexes, wherein individual multivalent reagent-analyte complexes comprise affinity moieties of an individual multivalent analyte detection reagent bound to a plurality of cognate target analytes, and wherein individual multivalent reagent-analyte complexes comprise a sequencing primer binding site and a target barcode sequence; (d) washing the cellular sample to remove multivalent analyte detection reagents inside the cellular sample that are not bound to a cognate target analyte, under a condition suitable to retain the plurality of multivalent reagent-analyte complexes inside the cellular sample; and (e) sequencing the target barcodes of the plurality of multivalent reagent-analyte complexes, wherein the sequencing is conducted inside the cellular sample.

[0016] In some embodiments of the method for conducting a sequencing-based workflow for detecting a plurality of target analytes inside a cellular sample of the disclosure, the sequencing of step e) comprises: (a) hybridizing the sequencing primer binding sites with a plurality of sequencing primers, thereby forming a plurality of nucleic acid duplexes, and contacting the plurality of nucleic acid duplexes with a first plurality of sequencing polymerases to form a first plurality of complexed polymerases, wherein individual first complexed polymerases comprise a duplex bound with a first sequencing polymerase; (b) contacting the first complexed polymerases with a plurality of first fluorophore-labeled multivalent molecules, wherein individual first fluorophore-labeled multivalent molecules comprise a core attached to a plurality of nucleotide-arms attached to nucleotide moieties, and wherein the contacting is conducted under a condition suitable for binding a complementary nucleotide moiety of one of the first fluorophore-labeled multivalent molecules to individual first complexed polymerases at a position that is opposite of a nucleotide in the target barcode region thereby forming a plurality of first multivalent-binding complexes, wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer; (c) detecting a color signal emitted by the first fluorophore-labeled multivalent molecules that are bound to the first complexed polymerases; (d) removing the first sequencing polymerases and the first fluorophore-labeled multivalent molecules and retaining the nucleic acid duplexes; (e) contacting the nucleic acid duplexes retained at step (d) with a second plurality of sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers thereby extending the sequencing primers by one nucleotide; (f) removing the chain terminating moieties from the chain terminating nucleotides incorporated at step (e) and retaining the nucleic acid duplexes; and (g) contacting the nucleic acid duplexes of step (f) with a plurality of second fluorophore-labeled multivalent molecules and a third plurality of sequencing polymerases to form a plurality of second multivalent-binding complexes wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer, and repeating steps (c) - (f) at least once.

[0017] In some embodiments of the method, the plurality of multivalent analyte detection reagents comprise at least a first and second sub-population of multivalent oligo reagents. In some embodiments, individual multivalent oligo reagents in the first sub-population (2000-1) bind a first target analyte and comprise a first sequencing primer binding site and a first target barcode. In some embodiments, individual multivalent oligo reagents in the second subpopulation (2000-2) bind a second target analyte and comprise a second sequencing primer binding site and a second target barcode. In some embodiments, the first and second target analytes are different target analytes. In some embodiments, the first and second sequencing primer binding sites have different sequences. In some embodiments, the first and second target barcodes have different sequences.

[0018] In some embodiments of the method, the plurality of multivalent analyte detection reagents comprise multivalent heterofunctional reagents comprising at least a first and second sub-population of multivalent oligo reagents. In some embodiments, individual multivalent heterofunctional reagents in the first sub-population (2500-1) bind a first target analyte and comprise a first sequencing primer binding site and a first target barcode. In some embodiments, individual multivalent heterofunctional reagents in the second sub-population (2500-2) bind a second target analyte and comprise a second sequencing primer binding site and a second target barcode. In some embodiments, the first and second target analytes are different target analytes. In some embodiments, the first and second sequencing primer binding sites have different sequences. In some embodiments, the first and second target barcodes have different sequences.

[0019] In some embodiments of the method, sequencing the target barcodes comprises sequencing the first sub-population of target barcodes while inhibiting sequencing the second sub-population of target barcodes by: (a) contacting the first sub-population of sequencing primer binding sites inside the cellular sample with a plurality of first batch sequencing primers under conditions suitable to hybridize individual first batch sequencing primers to the first sub-population of sequencing primer binding sites, wherein individual soluble first batch sequencing primers comprise an extendible terminal 3’ end which permits polymerase-catalyzed extension of the first batch sequencing primers that are hybridized to the first subpopulation of sequencing primer binding sites; (b) contacting the second sub-population of sequencing primer binding sites inside the same cellular sample with a plurality of second batch sequencing primers under conditions suitable to hybridize individual second batch sequencing primers to the second sub-population of sequencing primer binding sites, wherein individual soluble second batch sequencing primers comprise a terminal 3’ reversible blocking moiety which blocks polymerase-catalyzed extension of the second batch sequencing primers that are hybridized to the second sub-population of sequencing primer binding sites; and (c) conducting a first plurality of sequencing cycles inside the cellular sample using a sequencing polymerase thereby generating a first plurality of sequencing read products of the first target barcodes, wherein the first and second batch sequencing primers are soluble.

[0020] In some embodiments of the method, sequencing the target barcodes comprises conducting batch sequencing of the first and second target barcodes by: (a) contacting the first sub-population of sequencing primer binding sites inside the cellular sample with a plurality of first batch sequencing primers having terminal 3’ extendible ends and a plurality of nucleotide reagents, and conducting a first plurality of polymerase-catalyzed sequencing cycles thereby generating a plurality of first batch sequencing read products inside the cellular sample; (b) removing the plurality of first batch sequencing read products from the first sub-population of sequencing primer binding sites while retaining the first subpopulation of multivalent reagent-analyte complexes inside the cellular sample, wherein the plurality of first batch sequencing read products are removed by enzymatic degradation or a chemical de-hybridization reagent; (c) contacting the second sub-population of sequencing primer binding sites inside the same cellular sample with a plurality of second batch sequencing primers and a plurality of nucleotide reagents, and conducting a second plurality of polymerase-catalyzed sequencing cycles thereby generating a plurality of second batch sequencing read products inside the cellular sample; and (d) optionally removing the plurality of second batch sequencing read products from the second sub-population of sequencing primer binding sites while retaining the second sub-population of multivalent reagent-analyte complexes inside the cellular sample, wherein the plurality of second batch sequencing read products are removed by enzymatic degradation or de-hybridization reagent.

[0021] The disclosure provides a method for conducting cell painting, comprising: (a) providing a plurality of barcoded multivalent reagent-analyte complexes inside a cellular sample, wherein the plurality of barcoded multivalent reagent-analyte complexes comprises the plurality of multivalent analyte detection reagents of the disclosure, wherein individual barcoded multivalent reagent-analyte complexes comprise individual multivalent oligo reagents from either the first sub-population of multivalent oligo reagents (2000-1) or the second sub-population of multivalent oligo reagents (2000-2), thereby generating a first and second sub-population of barcoded multivalent reagent-analyte complexes; (b) conducting a first sequencing cycle inside the cellular sample, wherein the first sequencing cycle comprises sequencing essentially simultaneously a first nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagent-analyte complexes using a plurality of sequencing primers having the same sequence or different sequences, wherein the first nucleo-base position of the first target barcode generates a first color signal and the first nucleo-base position of the second barcode generates a second color signal, wherein the first and second color signals are distinguishable from each other in the first sequencing cycle, and wherein the first color signal identifies the first target analyte; (c) conducting a second sequencing cycle inside the cellular sample, wherein the second sequencing cycle comprises sequencing essentially simultaneously the second nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagent-analyte complexes, wherein the second nucleo-base position of the first target barcode generates the second color signal, wherein the second nucleo-base position of the second barcode generates the first color signal, wherein the first and second color signals are distinguishable from each other in the second sequencing cycle, and wherein the first color signal identifies the second target analyte; (d) imaging the first and second color signals generated inside the cellular sample in the first sequencing cycle at step (b) and identifying the first target analyte wherein the imaging of step (d) can be conducted essentially simultaneously with the sequencing of step (b); and (e) imaging the first and second color signals generated inside the cellular sample in the second sequencing cycle at step (c) and identifying the second target analyte. In some embodiments, the imaging of step (e) can be conducted essentially simultaneously with the sequencing of step (c).

[0022] The disclosure provides a method for conducting cell painting, comprising: (a) providing a plurality of barcoded multivalent reagent-analyte complexes inside a cellular sample, wherein the plurality of barcoded multivalent reagent-analyte complexes comprises the plurality of multivalent analyte detection reagents of the disclosure, wherein individual barcoded multivalent reagent-analyte complexes comprise individual multivalent heterofunctional reagents from either the first sub-population of multivalent heterofunctional reagents (2500-1) or the second sub-population of multivalent heterofunctional reagents (2500-2), thereby generating a first and second sub-population of barcoded multivalent reagent-analyte complexes; (b) conducting a first sequencing cycle inside the cellular sample, wherein the first sequencing cycle comprises sequencing essentially simultaneously a first nucleo-base position of the first and second target barcodes in the first and second subpopulations of barcoded multivalent reagent-analyte complexes using a plurality of sequencing primers having the same sequence or different sequences, wherein the first nucleo-base position of the first target barcode generates a first color signal and the first nucleo-base position of the second barcode generates a second color signal, wherein the first and second color signals are distinguishable from each other in the first sequencing cycle, and wherein the first color signal identifies the first target analyte; (c) conducting a second sequencing cycle inside the cellular sample, wherein the second sequencing cycle comprises sequencing essentially simultaneously the second nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagentanalyte complexes, wherein the second nucleo-base position of the first target barcode generates the second color signal, wherein the second nucleo-base position of the second barcode generates the first color signal, wherein the first and second color signals are distinguishable from each other in the second sequencing cycle, and wherein the first color signal identifies the second target analyte; (d) imaging the first and second color signals generated inside the cellular sample in the first sequencing cycle at step (b) and identifying the first target analyte wherein the imaging of step (d) can be conducted essentially simultaneously with the sequencing of step (b); and (e) imaging the first and second color signals generated inside the cellular sample in the second sequencing cycle at step (c) and identifying the second target analyte. In some embodiments, the imaging of step (e) can be conducted essentially simultaneously with the sequencing of step (c).

[0023] In some embodiments of the method for conducting cell painting, the first sequencing cycle comprises: (a) hybridizing the first sequencing primer binding sites with a plurality of first sequencing primers thereby forming a plurality of nucleic acid duplexes, or hybridizing the second sequencing primer binding sites with a plurality of second sequencing primers, thereby forming a plurality of nucleic acid duplexes, and contacting the plurality of nucleic acid duplexes with a first plurality of sequencing polymerases to form a first plurality of complexed polymerases, wherein individual complexed polymerases comprise a duplex bound with a first sequencing polymerase; (b) contacting the first complexed polymerases with a plurality of first fluorophore-labeled multivalent molecules, wherein individual fluorophore-labeled multivalent molecules comprise a core attached to a plurality of nucleotide-arms attached to a nucleotide moieties, and wherein the contacting is conducted under a condition suitable for binding a complementary nucleotide moiety of one of the first fluorophore-labeled multivalent molecules to individual first complexed polymerases at a position that is opposite of a nucleotide in the target barcode region thereby forming a plurality of first multivalent-binding complexes, wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer; (c) detecting a color signal emitted by the first fluorophore-labeled multivalent molecules that are bound to the first complexed polymerases; (d) removing the first sequencing polymerases and the first fluorophore-labeled multivalent molecules and retaining the nucleic acid duplexes; (e) contacting the nucleic acid duplexes retained at step (d) with a second plurality of sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers thereby extending the sequencing primers by one nucleotide; and (f) removing the chain terminating moieties from the incorporated chain terminating nucleotides and retaining the nucleic acid duplexes.

[0024] In some embodiments of the method for conducting cell painting, the second sequencing cycle comprises: (a) contacting the plurality of nucleic acid duplexes with a plurality of first sequencing polymerases to form a first plurality of complexed polymerases, wherein individual complexed polymerases comprise a duplex bound with a first sequencing polymerase; (b) contacting the first complexed polymerases with a plurality of fluorophore-labeled multivalent molecules, wherein individual fluorophore-labeled multivalent molecules comprise a core attached to a plurality of nucleotide-arms attached to a nucleotide moieties, and wherein the contacting is conducted under a condition suitable for binding a complementary nucleotide moiety of one of the fluorophore-labeled multivalent molecules to individual first complexed polymerases at a position that is opposite of a nucleotide in the target barcode region thereby forming individual multivalent-binding complexes, wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer; (c) detecting a color signal emitted by the fluorophore-labeled multivalent molecules that are bound to the first complexed polymerases; (d) removing the first sequencing polymerases and the bound fluorophore-labeled multivalent molecules and retaining the nucleic acid duplexes; (e) contacting the nucleic acid duplexes retained at step (d) with a plurality of second sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers thereby extending the sequencing primers by one nucleotide; and (f) removing the chain terminating moieties from the chain terminating nucleotides incorporated at step (e) and retaining the nucleic acid duplexes. In some embodiments, the method comprises repeating steps (a)-(f) at least once.

[0025] The disclosure provides a method for detecting a plurality of target analytes in a sample, comprising: (a) providing a sample comprising a plurality of analytes including at least one target analyte; (b) providing a multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of the disclosure, wherein individual multivalent analyte detection reagents comprise a fluorophore; (c) contacting the sample with the plurality of multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes in the sample to form a plurality of multivalent reagent-analyte complexes, wherein the multivalent reagent-analyte complex exhibits physical, chemical and / or optical characteristics that are distinguishable from the multivalent analyte detection reagent that is not bound to its cognate target analyte; (d) removing the multivalent analyte detection reagents that are not bound to a cognate target analyte, under a condition suitable to retain the plurality of multivalent reagent-analyte complexes; and (e) detecting the plurality of the retained multivalent reagent-analyte complexes.

[0026] In some embodiments of the method for detecting a plurality of target analytes in a sample, in step (a) the target analyte in the sample is in solution, embedded in a gel or immobilized to a support.

[0027] In some embodiments of the method for detecting a plurality of target analytes in a sample, the detecting of step (e) comprises: measuring a change in optical properties of the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; measuring a color change of the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; or measuring an increased fluorescence intensity of the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents. In some embodiments, the detecting of step (e) comprises: using atomic mass spectrometry to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; using flow cytometry to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; using capillary electrophoresis to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; using capillary electrochromatography to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; using gel electrophoresis to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; or using a microarray to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents.

[0028] The disclosure provides a method for conducting a sequencing-based workflow for detecting a plurality of target analytes in a sample, comprising (a) providing a sample comprising a plurality of analytes including at least one target analyte; (b) providing a plurality of the multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of the disclosure, wherein multivalent oligo reagents or the multivalent heterofunctional reagents comprise a fluorophore; (c) contacting the sample with a plurality of the multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes in the sample to form a plurality of multivalent reagent-analyte complexes; (d) removing the multivalent analyte detection reagents that are not bound to a cognate target analyte, under a condition suitable to retain the plurality of multivalent reagent-analyte complexes; and (e) sequencing the target barcodes of the plurality of multivalent reagent-analyte complexes thereby identifying the target analyte that was bound to the multivalent analyte detection reagent.

[0029] In some embodiments of the method or conducting a sequencing-based workflow for detecting a plurality of target analytes in a sample, the at least one target analyte comprises molecules from air, water, soil or food. In some embodiments, the at least one target analyte comprises molecules isolated from viruses, fungi, prokaryotes or eukaryotes. In some embodiments, the at least one target analyte comprises molecules isolated from human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect or bacteria. In some embodiments, the at least one target analyte comprises molecules isolated from blood, urine, serum, lymph, tumor, saliva, anal secretions, vaginal secretions, amniotic samples, perspiration, semen, environmental samples or culture samples. In some embodiments, the at least one target analyte comprises molecules isolated from head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

[0030] In some embodiments of the methods of the disclosure, individual fluorophore-labeled multivalent molecules comprise: (a) a core, (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide moiety, and (c) a fluorophore, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits. In some embodiments, the plurality of nucleotide arms attached to a given core have the same type of nucleotide moiety, and wherein the nucleotide moiety comprises dATP, dGTP, dCTP, dTTP or dUTP. In some embodiments, the plurality of multivalent molecules comprise one type of a multivalent molecule wherein each multivalent molecule in the plurality has the same type of nucleotide moiety selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules each type having nucleotide moieties selected from a group consisting of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, the individual fluorophore-labeled multivalent molecules comprise a fluorophore that corresponds to the nucleotide moiety.

[0031] The disclosure provides a method for conducting multi-omic detection and identification, comprising: (a) providing a cellular sample deposited on a support, wherein the cellular sample harbors a plurality of target polynucleotides and a plurality of target analytes; (b) contacting the cellular sample with a plurality of barcoded target probe complexes (900), wherein the contacting is conducted under a condition suitable for moving the plurality of target probe complexes (900) into the cellular sample and for selectively binding the plurality of target probe complexes (900) to their corresponding target polynucleotides inside the cellular sample, wherein individual barcoded target probe complexes (900) in the plurality comprise a circularized barcoded oligonucleotide (500) hybridized to a linear target probe (600), wherein the circularized barcoded oligonucleotide (500) comprises (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300), and (iii) a universal circularized region (400), wherein the linear target probe (600) comprises an oligonucleotide having (i) a target binding moiety (700) which can selectively bind a target polynucleotide and (ii) a universal probe region (800), wherein the 3’ end of the linear target probe (600) is extendible, wherein the universal circularized region (400) is hybridized to the universal probe region (800) of the target probe (600), and wherein target barcode sequence (300) corresponds to the target binding moiety (700) of the linear target probe (600); (c) removing an excess of target probe complexes (900) that are not bound to their corresponding target polynucleotides and retaining the plurality of target probe complexes (900) bound to their corresponding target polynucleotides; (d) contacting the cellular sample with a plurality of barcoded bipartite complexes , wherein the contacting is conducted under a condition suitable for moving the plurality of barcoded bipartite complexes into the cellular sample and for binding the plurality of barcoded bipartite complexes to their corresponding target analytes inside the cellular sample, wherein individual barcoded bipartite complexes comprise a primary antibody bound to a secondary antibody, wherein the primary antibody selectively binds a target analyte, wherein the secondary antibody is attached to a bridge circle complex, the bridge circle complex comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500), wherein the circularized barcoded oligonucleotide (1400) comprises: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte and (iii) a universal circularized region (1300), wherein the linear bridge oligonucleotide (1500) comprises a universal sequence region, wherein the 5’ end of the linear bridge oligonucleotide (1500) is attached to the secondary antibody, wherein the 3’ end of the linear bridge oligonucleotide (1500) is extendible, and wherein the universal sequence region of the bridge oligonucleotide (1500) is hybridized to the universal circularized region (1300) of the circularized barcoded oligonucleotide (1400); (e) removing an excess of barcoded bipartite complexes that are not bound to their corresponding target analytes and retaining the plurality of bipartite complexes bound to their corresponding target analytes; (f) contacting the cellular sample with a rolling circle amplification reagent comprising a plurality of strand-displacing DNA polymerases and a plurality of nucleotides, under a condition suitable for moving the rolling circle amplification reagent into the cellular sample; (g) conducting a rolling circle amplification reaction inside the cellular sample thereby generating a plurality of barcoded concatemer molecules comprising (i) a first sub-population of barcoded concatemer molecules generated from the plurality of barcoded target probe complexes (900) wherein individual barcoded concatemer molecules of the first sub-population comprise a plurality of the target barcode sequence (300), and (ii) a second sub-population of barcoded concatemer molecules generated from the plurality of barcoded bipartite complexes wherein individual barcoded concatemer molecules of the second sub-population comprise a plurality of the target barcode sequence (1200); and (h) sequencing the target barcode sequence (300) of the first subpopulations of barcoded concatemer molecules and sequencing the target barcode sequence (1200) of the second sub-populations of barcoded concatemer molecules wherein the sequencing is conducted inside the cellular sample.

[0032] In some embodiments of the method for conducting multi-omic detection and identification of the disclosure, the cellular sample comprises a whole single cell, a plurality of whole cells, an intact tissue, sectioned cell or a sectioned tissue sample. In some embodiments, the cellular sample comprises a fresh cellular sample, a freshly-frozen cellular sample, or a formalin-fixed and paraffin-embedded (FFPE) cellular sample. In some embodiments, the cellular sample comprises a fixed and permeabilized cellular sample.

[0033] In some embodiments of the method for conducting multi-omic detection and identification of the disclosure, the plurality of target polynucleotides comprise DNA, cDNA, RNA, or a mixture thereof. In some embodiments, the plurality of target analytes comprises polypeptides, lipids, nucleic acids, polysaccharides or a combination thereof.

[0034] In some embodiments, the plurality of nucleotides in the rolling circle amplification reagent comprises dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, the rolling circle amplification reagent further comprises a plurality of compaction oligonucleotides, wherein individual compaction oligonucleotides comprise a 5' region that binds a first portion of one of the concatemer molecules and a 3' region that binds a second portion of the same concatemer molecule to pull together distal portions of the concatemer molecule thereby causing compaction of the concatemer molecule.

[0035] In some embodiments, the sequencing comprises sequencing the first and second sub-population of barcoded concatemer molecules essentially simultaneously. In some embodiments, the sequencing comprises sequencing the first and second sub-population of barcoded concatemer molecules in separate batches.

[0036] In some embodiments, the sequencing comprises (a) contacting the first subpopulation of barcoded concatemer molecules with a plurality of a first sequencing primer, a plurality of sequencing polymerases, and a plurality of detectably labeled multivalent molecules, and (b) contacting the second sub-population of barcoded concatemer molecules with a plurality of a second sequencing primer, a plurality of sequencing polymerases, and a plurality of detectably labeled multivalent molecules, wherein individual multivalent molecules comprise a core attached to a plurality of nucleotide-arms where individual nucleotide arms comprise a core attachment moiety, a spacer, a linker, and a nucleotide moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0038] FIG. 1 is a schematic of various exemplary configurations of multivalent molecules. Left (Class I): schematics of multivalent molecules having a “starburst” or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide moieties are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’.

[0039] FIG. 2 is a schematic of an exemplary multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0040] FIG. 3 is a schematic of an exemplary multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0041] FIG. 4A shows a schematic of an exemplary multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms comprise a core attachment moiety, a spacer, a linker, and a nucleotide moiety.

[0042] FIG. 4B is a schematic of an exemplary nucleotide-arm of a multivalent molecule, wherein the nucleotide arm comprises a core attachment moiety, a spacer, a linker, and a nucleotide moiety (e.g., nucleotide unit).

[0043] FIG. 5A shows a schematic of an exemplary multivalent probe comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms comprise a core attachment moiety, a spacer, a linker, and a target-specific oligonucleotide probe.

[0044] FIG. 5B is a schematic of an exemplary nucleotide-arm of a multivalent probe comprising a core attachment moiety, a spacer, a linker, and a target-specific oligonucleotide probe.

[0045] FIG. 6 shows the chemical structure of an exemplary spacer (Top), and the chemical structures of various exemplary linkers, including an 11-atom Linker, 16-atom Linker, 23-atom Linker and an N3 Linker (Bottom).

[0046] FIG. 7 shows the chemical structures of various exemplary linkers, including Linkers 1-9.

[0047] FIG. 8 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0048] FIG. 9 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0049] FIG. 10 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0050] FIG. 11 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0051] FIG. 12 shows the chemical structure of an exemplary biotinylated nucleotide-arm. In this example, the nucleotide moiety is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0052] FIG. 13 is a schematic of a guanine tetrad (e.g., G-tetrad).

[0053] FIG. 14 is a schematic of an exemplary intramolecular G-quadruplex structure.

[0054] FIG. 15 is a schematic showing an embodiment of a circularized barcoded oligonucleotide (500) hybridizing to a target probe (600) to generate a target probe complex (900). The circularized barcoded oligonucleotide (500) can comprise (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe (600), and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (400) that binds a universal probe region (800) of the target probe (600). The target probe (600) can comprise an oligonucleotide having (i) a target binding moiety (700) which selectively binds a target polynucleotide; and (ii) a universal probe region (800) that binds the universal circularized region (400).

[0055] FIG. 16 is a schematic showing an embodiment of a circularized barcoded oligonucleotide (500) hybridizing to a target probe (600) to generate a target probe complex (900). The circularized barcoded oligonucleotide (500) can comprise (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); and (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe (600), and optionally comprises a short random sequence (NNN). The circularized barcoded oligonucleotide (500) can further comprise (iii) a universal circularized region which includes a first sub-region (410) and a second sub-region (420). The universal probe region can also be divided into two sub-regions, wherein the first sub-region of the universal circularized region (410) binds the second sub-region of the universal probe region (820), and the second subregion of the universal circularized region (420) binds the first sub-region of the universal probe region (810). The target probe (600) can comprise an oligonucleotide having (i) a target binding moiety (700) which selectively binds a target polynucleotide; and (ii) a universal probe region which includes the first sub-region (810) and a second sub-region (820).

[0056] FIG. 17 is a schematic showing an embodiment of a circularized barcoded oligonucleotide (500) hybridizing to a target probe (600) to generate a target probe complex (900). The circularized barcoded oligonucleotide (500) can comprise (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe (600); (iii) a sample index sequence (310); (iv) a batch barcode sequence (320); (v) a universal first sub-region (410); (vi) a compaction oligonucleotide binding site sequence (415) (or a complementary sequence thereof); and (vii) a universal second sub-region (420). The target probe (600) can comprise an oligonucleotide having (i) a target binding moiety (700) which selectively binds a target polynucleotide; (ii) an optional linker region (710); (iii) a universal probe first sub-region (810); (iv) a compaction oligonucleotide binding site sequence (415) (or a complementary sequence thereof); and (v) a universal probe second sub-region (820).

[0057] FIG. 18 is a schematic showing several embodiments of linear barcoded oligonucleotides (100) with the features shown in FIGS. 15-17.

[0058] FIG. 19 is a schematic showing one embodiment of a linear barcoded oligonucleotide (100) with the features shown in FIG. 15 undergoing an intramolecular ligation reaction to generate a circularized barcoded oligonucleotide (500).

[0059] FIG. 20 is a schematic showing one embodiment of a linear barcoded oligonucleotide (100) with the features shown in FIG. 16 undergoing an intramolecular ligation reaction to generate a circularized barcoded oligonucleotide (500).

[0060] FIG. 21 is a schematic showing one embodiment of a linear barcoded oligonucleotide (100) with the features shown in FIG. 17 undergoing an intramolecular ligation reaction to generate a circularized barcoded oligonucleotide (500).

[0061] FIG. 22 is a schematic showing several embodiments of target probes (600) with the features shown in FIGS. 15-17.

[0062] FIG. 23A is a schematic showing an embodiment of a bridge circle complex (1600) comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500). The circularized barcoded oligonucleotide (1400) can comprise: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (1300) that binds a universal sequence region of the bridge oligonucleotide (1500). The bridge oligonucleotide (1500) can comprise an oligonucleotide having a universal sequence region that binds the universal circularized region (1300) of a circularized barcoded oligonucleotide (1400).

[0063] FIG. 23B is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which includes an antibody attached to the bridge circle complex (1600) which is shown in FIG. 23 A.

[0064] FIG. 24A is a schematic showing an embodiment of a bridge circle complex (1600) comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500). The circularized barcoded oligonucleotide (1400) can comprise: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (1300) that binds a universal sequence region of the bridge oligonucleotide (1500). The bridge oligonucleotide (1500) can comprise an oligonucleotide having a universal sequence region that binds the universal circularized region (1300) of a circularized barcoded oligonucleotide (1400), and a linker region (1505).

[0065] FIG. 24B is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which includes an antibody attached to the bridge circle complex (1600) which is shown in FIG. 24A.

[0066] FIG. 25A is a schematic showing an embodiment of a bridge circle complex (1600) comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500). The circularized barcoded oligonucleotide (1400) can comprise: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (1300) sub-divided into two sub-regions, such as for example a first sub-region (1310) and a second sub-region (1320). The first sub-region (1310) binds a portion of the bridge oligonucleotide (1520), and the second sub-region (1320) binds another portion of the bridge oligonucleotide (1510). The universal sequence region of the bridge oligonucleotide can be sub-divided into a first subregion (1510) and a second sub-region (1520).

[0067] FIG. 25B is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which includes an antibody attached to the bridge circle complex (1600) which is shown in FIG. 25A.

[0068] FIG. 26A is a schematic showing an embodiment of a bridge circle complex (1600) comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500). The circularized barcoded oligonucleotide (1400) can comprise: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (1300) which can be sub-divided into two sub-regions, such as for example a first sub-region (1310) and a second sub-region (1320). The first sub-region (1310) binds a portion of the bridge oligonucleotide (1520), and the second sub-region (1320) binds another portion of the bridge oligonucleotide (1510). The bridge oligonucleotide (1500) can comprise a linker region (1505) and a universal sequence region which can be sub-divided into a first sub-region (1510) and a second sub-region (1520).

[0069] FIG. 26B is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which includes an antibody attached to the bridge circle complex (1600) which is shown in FIG. 26A.

[0070] FIG. 27A is a schematic showing an embodiment of a bridge circle complex (1600) comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500). The circularized barcoded oligonucleotide (1400) can comprise: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte; (iii) a sample index sequence (1210); (iv) a batch barcode sequence (1220); (v) a universal circularized first subregion (1310) which binds a portion of a bridge oligonucleotide (1500); (vi) a compaction oligonucleotide binding site (1315) (or complementary sequence thereof); and (vii) a universal circularized region second sub-region (1320) which binds another portion (1510) of the same bridge oligonucleotide. The bridge oligonucleotide (1500) can comprise a linker region (1505), first sub-region (1510) of a universal sequence region, a compaction oligonucleotide binding site (1515) (or complementary sequence thereof), and a second subregion (1520) of the universal sequence region.

[0071] FIG. 27B is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which includes an antibody attached to the bridge circle complex (1600) which is shown in FIG. 27A.

[0072] FIG. 28 is a schematic showing different embodiments of linear barcoded oligonucleotides (1000) comprising in any arrangement: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (1300) which can be sub-divided into two sub-regions, such as for example a first sub-region (1310) and a second sub-region (1320).

[0073] FIG. 29 is a schematic showing an embodiment of intramolecular ligation of the ends of a linear barcoded oligonucleotide (1000) with the features depicted in FIG. 24A.

[0074] FIG. 30 is a schematic showing an embodiment of intramolecular ligation of the ends of a linear barcoded oligonucleotide (1000) with the features depicted in FIG. 25 A.

[0075] FIG. 31 is a schematic showing an embodiment of intramolecular ligation of the ends of a linear barcoded oligonucleotide (1000) with the features depicted in FIG. 27A.

[0076] FIG. 32 is a schematic showing different embodiments of linear bridge oligonucleotides (1500) comprising in any arrangement: (i) a linker region (1505); (ii) a universal sequence region; (iii) a first sub-region (1510) of a universal sequence region; (iv) a compaction oligonucleotide binding site (1515) (or complementary sequence thereof); and (v) a second sub-region (1520) of a universal sequence region.

[0077] FIG. 33A is a schematic showing an embodiment of a target analyte comprising a first and second epitope.

[0078] FIG. 33B is a schematic showing an embodiment of an antibody bridge circle complex (1700) binding directly to a target analyte. The antibody bridge circle complex (1700) can comprise an antibody having an antigen binding domain that binds a first epitope of a first target analyte.

[0079] FIG. 34A is a schematic showing an embodiment of a first antibody bridge circle complex (1700-1) binding directly to a first target analyte. The first antibody bridge circle complex (1700-1) can comprise a first antibody having an antigen binding domain that binds an epitope of a first target analyte. The first antibody bridge circle complex (1700-1) comprises a first bridge circle complex (1600-1) attached to the first antibody.

[0080] FIG. 34B is a schematic showing an embodiment of a second antibody bridge circle complex (1700-2) binding directly to a second target analyte. The second antibody bridge circle complex (1700-2) can comprise a second antibody having an antigen binding domain that binds an epitope of a second target analyte. The second antibody bridge circle complex (1700-2) comprises a second bridge circle complex (1600-2) attached to the second antibody. In FIGS. 34A and 34B, the first and second target analytes are different target analytes, and the first and second antibodies comprise different antibodies. In FIGS. 34A and 34B, the first and second bridge circle complexes ((1600-1) and (1600-2)) can also comprise different bridge circle complexes.

[0081] FIG. 35 is a schematic showing an embodiment of a first antibody bridge circle complex (1700-1) and a second antibody bridge circle complex (1700-2) binding different epitopes of the same target analyte. The first antibody bridge circle complex (1700-1) can comprise a first antibody having an antigen binding domain that binds a first epitope of the target analyte. The first antibody bridge circle complex (1700-1) comprises a first bridge circle complex (1600-1) attached to the first antibody. The second antibody bridge circle complex (1700-2) can comprise a second antibody having an antigen binding domain that binds a different epitope of the same target analyte. The second antibody bridge circle complex (1700-2) comprises a second bridge circle complex (1600-2) attached to the second antibody. The first and second antibodies can comprise different antibodies. The first and second bridge circle complexes ((1600-1) and (1600-2)) can also comprise different bridge circle complexes.

[0082] FIG. 36 is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which is bound to its cognate target analyte inside a cellular sample, wherein the antibody bridge circle complex (1700) is undergoing a rolling circle amplification (RCA) reaction to generate a concatemer molecule. The concatemer molecule comprises repeat tandem polynucleotide units where individual polynucleotide units can comprise: (i) a universal circularized region (1300); (ii) a target barcode sequence (1200) that corresponds to a target analyte; and (iii) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof). The concatemer molecule can be subjected to reiterative sequencing inside the cellular sample.

[0083] FIG. 37 is a schematic showing an embodiment of an analyte detection complex comprising an antibody bridge circle complex (1700) which is bound to its cognate target analyte inside a cellular sample, wherein the antibody bridge circle complex (1700) is undergoing a rolling circle amplification (RCA) reaction to generate a concatemer molecule. In some embodiments, the concatemer molecule comprises repeat tandem polynucleotide units where individual polynucleotide units comprise: (i) a first sub-region of a universal circularized region (1320); (ii) a second sub-region of a universal circularized region (1310); (iii) a target barcode sequence (1200) that corresponds to a target analyte; and (iv) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof). In some embodiments, the concatemer is subjected to reiterative sequencing inside the cellular sample.

[0084] FIG. 38 is a schematic showing an embodiment of two analyte detection complexes comprising first and second antibody bridge circle complexes ((1700-1) and (1700-2)) which are bound to their cognate target analytes inside a cellular sample, wherein the two antibody bridge circle complexes are undergoing rolling circle amplifications (RCA) reaction to generate first and second concatemer molecules. The first concatemer molecule can comprise repeat tandem polynucleotide units where individual polynucleotide units comprise: (i) a first universal circularized region (1300-1); (ii) a first target barcode sequence (1200-1) that corresponds to a first target analyte; and (iii) a first sequencing primer binding site sequence (1100-1) (or a complementary sequence thereof). The second concatemer molecule can comprise repeat tandem polynucleotide units where individual polynucleotide units comprise: (i) a second universal circularized region (1300-2); (ii) a second target barcode sequence (1200-2) that corresponds to a second target analyte; and (iii) a second sequencing primer binding site sequence (1100-2) (or a complementary sequence thereof). The first concatemer molecule represents a first sub-population of concatemer molecules. The second concatemer molecule represents a second sub-population of concatemer molecules. The first and second concatemer molecules can be subjected to batch sequencing inside the cellular sample.

[0085] FIG. 39 is a schematic showing an embodiment of a plurality of target probe complexes ((900-1) and (900-2)) and a plurality of analyte detection complexes (e.g., bipartite complexes ((1800-1) and (1800-2)) located inside a cellular sample. The target probe complexes and analyte detection complexes can be used to generate a plurality of barcoded concatemer molecules which can be sequenced for multi-omic detection and identification of cellular polynucleotides and target analytes.

[0086] FIG. 40 is a table showing several embodiments of target barcode sequences that can be employed for simultaneously detecting and identifying two or more cellular target analytes (e.g., cellular structures) by conducting a single sequencing cycle and employing multi-color imaging (e.g., fluorescent imaging). The target barcode sequences listed in the table in FIG. 40 can be used for cell painting. Target barcode sequences, from top to bottom, are: CTACCCGTGGTG (SEQ ID NO: 1); TCAAAATGGGGT (SEQ ID NO: 2); CATCACTGTGGG (SEQ ID NO: 3); CCCTCAGTGTGG (SEQ ID NO: 4); and AAACCTTGGTTT (SEQ ID NO: 5).

[0087] FIG. 41 shows images of fluorescent signals emitted from sequencing the barcode regions of concatemer molecules inside a cellular sample wherein the concatemer molecules were generated from bipartite complexes . Cells were permeabilized and fixed, and reacted with barcoded bipartite complexes such that the bipartite complexes bound their cognate target analytes, for example C-myc or tubulin. The analyte-bipartite complexes were subjected to rolling circle amplification to generate barcoded concatemer molecules corresponding to tubulin or c-myc. The barcoded concatemer molecules were sequenced using sequencing primers specific for the tubulin concatemer molecules or the c-myc concatemer molecules, and a two-stage sequencing workflow employing labeled multivalent molecules and non-labeled nucleotide analogs. The images shown in FIG. 41 represent five consecutive sequencing cycles of the same cells and the same field-of-view using sequencing primers specific for the tubulin barcoded concatemer molecules. The images are rendered in false color. The fluorescent signals emitted during the five sequencing cycles detect and identify tubulin structures inside the cells. The target barcode sequences are listed in the table at top: C-myc, TCAAAATGGGGT (SEQ ID NO: 2); Tubulin, CTACCCGTGGTG (SEQ ID NO: 1).

[0088] FIG. 42A shows images of fluorescent signal emitted from sequencing the barcode regions of concatemer molecules inside a cellular sample wherein the concatemer molecules were generated from bipartite complexes as described for FIG. 41. TOP: The barcoded concatemer molecules were simultaneously sequenced using a mixture of sequencing primers specific for the histone concatemer molecules or the tubulin concatemer molecules and a two-stage sequencing workflow employing labeled multivalent molecules and non-labeled nucleotide analogs. The top image shows fluorescent signals emitted from a single sequencing cycle in which the tubulin barcode emits a green signal and the histone barcode emits a red signal. The top image shows histone (red) and tubulin (green) structures inside the cells. The top image is not a merged image. BOTTOM: The sequencing read products generated from sequencing the tubulin concatemer molecules and histone concatemer molecules were removed from the concatemer molecules by extensive washing. The histone concatemer molecules were sequenced using sequencing primers specific for the histone concatemer molecules and a two-stage sequencing workflow employing labeled multivalent molecules. The bottom image shows fluorescent signals emitted from a single sequencing cycle in which the histone barcode emits a red signal. The bottom image shows histone (red) structures inside the cells. The top and bottom images represent the same cells and the same field-of-view.

[0089] FIG. 42B TOP is the same fluorescent image shown in the top image of FIG. 42A. FIG. 42B BOTTOM: The sequencing read products generated from sequencing the tubulin concatemer molecules and histone concatemer molecules (see FIG. 42A TOP) were removed from the concatemer molecules by extensive washing. The tubulin concatemer molecules were sequenced using sequencing primers specific for the tubulin concatemer molecules and a two-stage sequencing workflow employing labeled multivalent molecules and non-labeled nucleotide analogs. The bottom image shows fluorescent signals emitted from a single sequencing cycle in which the tubulin barcode emits a green signal. The bottom image shows tubulin (green) structures inside the cells. The top and bottom images represent the same cells and the same field-of-view.

[0090] FIG. 43 shows an image of a dividing cell. The image was generated by fluorescent signals emitted from sequencing the barcode regions of concatemer molecules inside a cellular sample wherein the concatemer molecules were generated from bipartite complexes and sequenced as described for FIG. 41. The image shown in FIG. 43 is not a merged image.

[0091] FIG. 44A is a schematic showing a multivalent affinity reagent (1900) comprising (1) a core and (2) a plurality of polymer arms attached to the core wherein individual polymer arms comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) an affinity moiety that can bind a target analyte. The schematic shown in FIG. 44A is an embodiment of a multivalent analyte detection reagent.

[0092] FIG. 44B is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a spacer, a linker, and an affinity moiety that can bind a target analyte.

[0093] FIG. 44C is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a spacer and an affinity moiety that can bind a target analyte.

[0094] FIG. 44D is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a linker and an affinity moiety that can bind a target analyte.

[0095] FIG. 44E is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a spacer, a forked linker, and a plurality of affinity moieties that can bind a target analyte.

[0096] FIG. 44F is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a spacer, a branched linker, and a plurality of affinity moieties that can bind a target analyte.

[0097] FIG. 44G is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a spacer, a forked spacer, and a plurality of affinity moieties that can bind a target analyte.

[0098] FIG. 44H is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a spacer, a branched spacer, and a plurality of affinity moi eties that can bind a target analyte.

[0099] FIG. 441 is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a linker, a forked linker, and a plurality of affinity moieties that can bind a target analyte.

[00100] FIG. 44J is a schematic of an embodiment of a polymer arm comprising a core attachment moiety, a linker, a branched linker, and a plurality of affinity moieties that can bind a target analyte.

[00101] FIG. 45A is a schematic showing a multivalent oligo (oligonucleotide) reagent (2000) comprising (1) a core and (2) a plurality of polymer arms wherein at least one of the polymer arms comprises an oligonucleotide and the remainder of the polymer arms comprise an affinity moiety that can bind a target analyte. In some embodiments, individual polymer arms comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) an oligonucleotide or an affinity moiety that can bind a target analyte. The schematic shown in FIG. 45A is an embodiment of a multivalent analyte detection reagent.

[00102] FIG. 45B is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, a linker, and an oligonucleotide. One end of the oligonucleotide can be attached to a multiatom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). The oligonucleotide can comprise a sequencing primer binding site ((2010) seq p.b.s.). The oligonucleotide can comprise a target barcode sequence ((2020) BC).

[00103] FIG. 45C is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, and an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). The oligonucleotide can comprise a sequencing primer binding site ((2010) seq p.b.s.). The oligonucleotide can comprise a target barcode sequence ((2020) BC).

[00104] FIG. 45D is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a linker, and an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). The oligonucleotide can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00105] FIG. 45E is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, and a forked linker. At least one prong of the forked linker can be attached to an oligonucleotide. One end of individual oligonucleotide(s) can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). Individual oligonucleotides can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00106] FIG. 45F is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, and a branched linker. At least one branch of the branched linker can be attached to an oligonucleotide. One end of individual oligonucleotides can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). Individual oligonucleotide(s) can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00107] FIG. 45G is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, and a forked spacer. At least one prong of the forked spacer can be attached to an oligonucleotide. One end of individual oligonucleotides can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). Individual oligonucleotides can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00108] FIG. 45H is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, and a branched spacer. At least one branch of the branched spacer can be attached to an oligonucleotide. One end of individual oligonucleotides can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). Individual oligonucleotides can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00109] FIG. 451 is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a linker, and a forked linker. At least one prong of the forked linker can be attached to an oligonucleotide. One end of individual oligonucleotides can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). Individual oligonucleotides can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00110] FIG. 45J is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a linker, and a branched linker. At least one branch of the branched linker can be attached to an oligonucleotide. One end of individual oligonucleotides can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). Individual oligonucleotides can comprise a sequencing primer binding site ((2010) seq p.b.s.) and / or a target barcode sequence ((2020) BC).

[00111] FIG. 45K is a schematic of a polymer arm of a multivalent oligo reagent (2000) undergoing a sequencing reaction using a detectably labeled multivalent molecule. Various embodiments of multivalent molecules are shown in FIGS. 1-3, 4A and 4B. The polymer arm shown in FIG. 45K comprises a core attachment moiety, a spacer, a linker, a modifier moiety (mod), a multi-atom spacer, and an oligonucleotide having a sequencing primer binding site ((2010) seq p.b.s.) and a barcode sequence ((2020) BC). In this embodiment, the core of the multivalent oligo reagent (2000) is attached to at least one additional polymer arm which is attached to an affinity moiety wherein the affinity moiety is bound to its cognate target analyte (not shown). The multivalent oligo reagent that is bound to its cognate target analyte forms a multivalent reagent-analyte complex. A sequencing primer can hybridize to the oligonucleotide at the sequencing primer binding site (2010) which is adjacent to the barcode sequence (2020). Hybridization between the sequencing primer and the sequencing primer binding site forms a duplex region. In a sequencing reaction, a sequencing polymerase (not shown) can bind the duplex region, and a complementary nucleotide moiety of the detectably labeled multivalent molecule binds the oligonucleotide at a position that is opposite of a nucleotide in the barcode region to form a binding complex which can be detected in a sequencing reaction.

[00112] FIG. 45L is a schematic of an embodiment of a polymer arm of a multivalent oligo reagent (2000) wherein the polymer arm comprises a core attachment moiety, a spacer, a linker, and a bridge oligonucleotide (2050). The 5’ end of the bridge oligonucleotide (2050) can be attached to a multi-atom spacer (internal spacer). The 3’ end of the bridge oligonucleotide can comprise a 3’ extendible moiety. One end of the multi-atom spacer can be attached to a modifier moiety (mod). The bridge oligonucleotide (2050) can be hybridized to a circularized barcoded oligonucleotide (2040). The circularized barcoded oligonucleotide (2040) can comprise: (i) a sequencing primer binding site sequence (2010) (or a complementary sequence thereof); (ii) a target barcode sequence (2020) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (2030) that binds a universal sequence region of the bridge oligonucleotide (2050). The bridge oligonucleotide (2050) can comprise an oligonucleotide having a universal sequence region that binds the universal circularized region (2030) of a circularized barcoded oligonucleotide (2040).

[00113] FIG. 46 is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a polymer arm attached to an affinity moiety and an oligonucleotide. The oligonucleotide can comprise a sequencing primer binding site, and optionally a target barcode sequence. The schematic shown in FIG. 46 is an embodiment of a multivalent analyte detection reagent.

[00114] FIG. 47A is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a forked spacer having a multi-prong portion and a linear portion. Individual prong portions can be linked to an affinity moiety that can bind a target analyte. The linear portion can be linked to an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). In some embodiments, the oligonucleotide comprises a sequencing primer binding site ((2510) seq p.b.s.) and / or a target barcode sequence ((2520) BC). The schematic shown in FIG. 47A is an embodiment of a multivalent analyte detection reagent.

[00115] FIG. 47B is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a forked spacer having a multi-prong portion and a linear portion. Individual prong portions can be linked to an affinity moiety that can bind a target analyte. The linear portion can be linked to a bridge oligonucleotide (2550). The 5’ end of the bridge oligonucleotide (2550) can be attached to a multi-atom spacer (internal spacer). The 3’ end of the bridge oligonucleotide comprises a 3’ extendible moiety. One end of the multi-atom spacer can be attached to a modifier moiety (mod). The bridge oligonucleotide (2050) can be hybridized to a circularized barcoded oligonucleotide (2540). The circularized barcoded oligonucleotide (2540) can comprise: (i) a sequencing primer binding site sequence (2510) (or a complementary sequence thereof); (ii) a target barcode sequence (2520) that corresponds to a target analyte, and optionally comprises a short random sequence (NNN); and (iii) a universal circularized region (2530) that binds a universal sequence region of the bridge oligonucleotide (2550). The bridge oligonucleotide (2550) can comprise an oligonucleotide having a universal sequence region that binds the universal circularized region (2330) of a circularized barcoded oligonucleotide (2540). The schematic shown in FIG. 47B is an embodiment of a multivalent analyte detection reagent.

[00116] FIG. 48 is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a branched spacer having a cruciform structure. Individual cruciform arm portions can be linked to an affinity moiety that can bind a target analyte. At least one cruciform arm portion can be linked to an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). The oligonucleotide can comprises a sequencing primer binding site ((2510) seq p.b.s.) and / or a target barcode sequence ((2520) BC). The schematic shown in FIG. 48 is an embodiment of a multivalent analyte detection reagent.

[00117] FIG. 49 is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a forked spacer having a multi-prong portion and a linear portion. Individual prong portions can be linked to an oligonucleotide or an affinity moiety that can bind a target analyte. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). In some embodiments, the oligonucleotide comprises a sequencing primer binding site ((2510) seq p.b.s.). In some embodiments, the oligonucleotide comprises a target barcode sequence ((2520) BC). The schematic shown in FIG. 49 is an embodiment of a multivalent analyte detection reagent.

[00118] FIG. 50 is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a branched spacer having multiple branches and a linear portion. Individual branches can be linked to an affinity moiety that can bind a target analyte. The linear portion can be linked to an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). In some embodiments, the oligonucleotide comprises a sequencing primer binding site ((2510) seq p.b.s.). In some embodiments, the oligonucleotide comprises a target barcode sequence ((2520) BC). The schematic shown in FIG. 50 is an embodiment of a multivalent analyte detection reagent.

[00119] FIG. 51 is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising a branched spacer having multiple branches and a linear portion. Individual branches can be linked to an affinity moiety that can bind a target analyte. The linear portion can be linked to an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). The oligonucleotide can comprise a sequencing primer binding site ((2510) seq p.b.s.) and / or a target barcode sequence ((2520) BC). The schematic shown in FIG. 51 is an embodiment of a multivalent analyte detection reagent.

[00120] FIG. 52 is a schematic of an embodiment of a multivalent heterofunctional reagent (2500) comprising an H-shaped spacer comprising four arms. Individual arms can be linked to an affinity moiety that can bind a target analyte. One of the arms can be linked to an oligonucleotide. One end of the oligonucleotide can be attached to a multi-atom spacer (internal spacer). One end of the multi-atom spacer can be attached to a modifier moiety (mod). The oligonucleotide can comprise a sequencing primer binding site ((2510) seq p.b.s.) and / or a target barcode sequence ((2520) BC). The schematic shown in FIG. 52 is an embodiment of a multivalent analyte detection reagent.

[00121] FIG. 53 is a schematic of a multivalent heterofunctional reagent (2500) undergoing a sequencing reaction using a detectably labeled multivalent molecule. Various embodiments of multivalent molecules are shown in FIGS. 1-3, 4A and 4B. The multivalent heterofunctional reagent (2500) shown in FIG. 53 comprises a forked spacer having a multiprong portion and a linear portion. Individual prong portions can be linked to an affinity moiety that can bind a target analyte. The linear portion can be linked to an oligonucleotide having a sequencing primer binding site ((2010) seq p.b.s.) and a barcode sequence ((2020) BC). The multivalent heterofunctional reagent can bind to its cognate target analyte thereby forming a multivalent reagent-analyte complex. A sequencing primer hybridizes to the oligonucleotide at the sequencing primer binding site (2510) which is adjacent to the barcode sequence (2520). Hybridization between the sequencing primer and the sequencing primer binding site forms a duplex region. In a sequencing reaction, a sequencing polymerase (not shown) can bind the duplex region, and a complementary nucleotide moiety of the detectably labeled multivalent molecule binds the oligonucleotide at a position that is opposite of a nucleotide in the barcode region to form a binding complex which can be detected in the sequencing reaction.

[00122] FIG. 54 shows an image of fluorescence imaging of actin detected inside a cellular sample using fluorophore-labeled multivalent affinity reagents (1900) by conducting a binding-based detection method. Individual multivalent affinity reagents (1900) comprising a core and a plurality of polymer arms attached to the core and to phalloidin. An exemplary multivalent affinity reagent (1900) is shown in FIG. 44A.

[00123] FIG. 55 shows an image of fluorescence imaging of actin detected inside a cellular sample using a plurality of non-labeled multivalent oligo reagents (2000) by conducting a sequencing-based workflow. Individual multivalent oligo reagents (2000) comprise a core and a plurality of polymer arms attached to the core, and at least one of the polymer arms comprises an oligonucleotide and the remainder of the polymer arms comprise phalloidin. An exemplary multivalent oligo reagent (2000) is shown in FIG. 45A.

[00124] FIG. 56 shows an image of florescence imaging of actin detected inside a cellular sample using non-labeled multivalent heterofunctional reagents (2500). Individual multivalent heterofunctional reagents (2500) comprise a multi-arm polymer, and at least one of the polymer arms comprises an oligonucleotide and the remainder of the polymer arms comprise phalloidin. Exemplary multivalent heterofunctional reagents (2500) are shown in FIGS. 46-52.

[00125] FIG. 57 is a schematic showing an embodiment of an analyte detection complex (a bipartite complex, ) comprising bridge circle complex (an antibody bridge circle complex (ABCC) (1700)) comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500). The circularized barcoded oligonucleotide (1400) comprises (i) two sequencing primer binding site sequences ((1100-1) and (1100-2)), (ii) two target barcode sequences ((1200-1) and (1200-2)), and a universal circularized region (1300). The two target barcode sequences can identify the same target analyte. The bridge circle complex can be attached to a primary or secondary antibody.

[00126] FIG. 58 is a schematic of an embodiment of an amplification-free probe complex. Open triangles indicate abasic sites, and solid arrows indicate canonical nucleotides. XXX designates one or more modified nucleotides or modified nucleotide linkages.

[00127] FIG. 59A is a schematic of an embodiment of a portion of a modified oligonucleotide comprising at least one sequencing primer binding site, a canonical nucleo-base and an abasic site. The open triangle indicates an abasic site, while the solid arrow indicates a canonical nucleotide. XXX designates one or more modified nucleotides or modified nucleotide linkages.

[00128] FIG. 59B is a schematic of the same modified oligonucleotide and sequencing primer as shown in FIG. 59A, with a sequencing polymerase and a detectably labeled multivalent molecule. The open triangle indicates an abasic site, while the solid arrow indicates a canonical nucleotide. XXX designates one or more modified nucleotides or modified nucleotide linkages.

[00129] FIG. 60 a schematic of an embodiment of a portion of a modified oligonucleotide comprising at least one sequencing primer binding site and a target barcode sequence. XXX designates one or more modified nucleotides or modified nucleotide linkages. DETAILED DESCRIPTION Definitions

[00130] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[00131] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. Techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. For example, see Sambrook etaL, Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[00132] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[00133] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[00134] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[00135] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[00136] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Also, where ranges and / or subranges of values are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[00137] As used herein, “corresponding to” or “corresponds to” refers to two or more entities whose identities are sufficiently related such that the identity of one entity can be used to determine the identity, position and / or other properties of the other entity. As nonlimiting example, a target barcode sequence can be said to correspond to a particular target analyte or fluorophore color if the fluorophore color can be used to determine the identity of the barcode sequence, and similarly, the barcode sequence can be used to determine the identity of the target analyte.

[00138] The term “polymerase” and its variants, as used herein, comprises an enzyme comprising a domain that binds a nucleotide (or nucleoside) where the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. A polymerase can be any enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include, without limitation, naturally occurring polymerases and any functional subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). Polymerases can include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes comprising a nucleotide binding domain. In some embodiments, a polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. In some embodiments, a polymerase can be post-translationally modified proteins, or functional fragments thereof. A polymerase can be derived from a prokaryote, eukaryote, virus, or phage. A polymerase can comprise a DNA-directed DNA polymerase and RNA-directed DNA polymerase.

[00139] As used herein, the term “strand displacing” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize a new strand in a template-based manner. Strand displacing polymerases displace a complementary strand from a template strand and catalyze new strand synthesis. During synthesis, nucleotides complementary to the template strand can be incorporated into the 3’ end of the new strand. Strand displacing polymerases include, for example and without limitation, mesophilic and thermophilic polymerases. Strand displacing polymerases include, for example and without limitation, wild type enzymes, and variants including exonuclease minus mutants, mutant versions, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include, for example and without limitation, phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent® DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi™ from Expedeon™), or variant EquiPhi29™ DNA polymerase (e.g., from Thermo Fisher Scientific®), or chimeric QualiPhi™ DNA polymerase (e.g., from 4basebio™).

[00140] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids can include recombinant and chemically-synthesized forms. Nucleic acids can be isolated. Nucleic acids can include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), chimeric forms containing DNA and RNA, and combinations thereof. Nucleic acids can be single-stranded (ss) or double-stranded (ds). Nucleic acids can comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids comprise naturally-occurring internucleosidic linkages, for example and without limitation, phosphodiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids can comprise non-natural intemucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise one type of polynucleotide, or a mixture of two or more different types of polynucleotides.

[00141] The term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtapositioned components can be linked together covalently. For example, and without limitation, two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component. For example, and without limitation, linkage between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another non-limiting example, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be ligated to a vector where the linkage permits expression or functioning of the transgene sequence contained in the vector. In some embodiments, a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and / or translation initiation sequence, transcription and / or translation termination sequence, polypeptide secretion signal sequences, and the like. In some embodiments, the host cell regulatory sequence controls expression of the level, timing and / or location of the transgene.

[00142] The terms “linked”, “joined”, “attached”, “appended” and variants thereof as used herein comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in the particular procedure. The procedures can include but are not limited to: nucleotide binding; nucleotide incorporation; de-blocking (e.g, removal of chain-terminating moiety); washing; removing; flowing; detecting; imaging and / or identifying. Such linkage can comprise, for example, and without limitation, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such linkage occurs intramolecularly, for example, by linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. In some embodiments, such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Suitable linkages are known in the art and some examples of linkages can be found, for example and without limitation, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[00143] The term “primer” and related terms used herein refers to an oligonucleotide that is capable of hybridizing with a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers can comprise natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers may have any length, but typically range from about 4-50 nucleotides. A typical primer comprises a 5' end and 3' end. The 3' end of the primer can include a 3' OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of the primer can lack a 3' OH moiety, or can include a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety (e.g., a fluorophore). A primer can be in solution (e.g., a soluble primer) or can be immobilized to a support (e.g., a capture primer).

[00144] The term “template nucleic acid”, “template polynucleotide”, “template strand” and other variations as used herein refers to a nucleic acid strand that serves as the basis nucleic acid molecule for any of the sequencing-based imaging methods describe herein. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from a naturally-occurring source, recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other suitable forms. In some embodiments, the template nucleic acid can be in a concatemer form. The template nucleic acids can include an insert portion having an insert sequence. The template nucleic acids can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant or synthetic molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue (FFPE), needle biopsies, circulating tumor cells, cell free circulating DNA, or any type of nucleic acid library. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and composition analysis.

[00145] The term “adaptor” and related terms as used herein refers to oligonucleotides that can be operably linked to a target polynucleotide, where the adaptor confers a function to the co-joined adaptor-target molecule. Adaptors can comprise DNA, RNA, chimeric DNA / RNA, analogs thereof, or any combination thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adaptors can be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length, including 4-100 nucleotides, or longer. Adaptors can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5' overhang and 3' overhang ends. In some embodiments, the 5' end of a singlestranded adaptor, or one strand of a double-stranded adaptor, can have a 5' phosphate group. In some embodiments, the 5' end of a single-stranded adaptor, or one strand of a doublestranded adaptor, can lack a 5' phosphate group. Adaptors can include a 5' tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed (e.g., do not include a 5' tail). An adaptor can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized capture primers). Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and / or phosphoramidate linkage. Adaptors can include a barcode sequence which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and / or increase sensitivity of variant detection. Adaptors can include at least one (e.g., one, two, three, four, five or more) restriction enzyme recognition sequences, including recognition sequences for any one or any combination of two or more restriction enzymes selected from the group consisting of type I, type II, type III, type IV, type Hs type IIB, and combinations thereof.

[00146] In some embodiments, any of the amplification primer sequences, sequencing primer sequences, capture primer sequences, target capture sequences, circularization anchor sequences, target barcode sequences, spatial barcode sequences, or anchor region sequences can be about 3-50 nucleotides in length, or about 5-40 nucleotides in length, or about 5-25 nucleotides in length, or any range therebetween. In some embodiments, any of the aforementioned sequences may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.

[00147] The term “universal sequence” and related terms as used herein refers to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules. For example, and without limitation, an adaptor having a universal sequence can be operably joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal adaptor sequence. Non-limiting examples of universal adaptor sequences can include an amplification primer sequence, a sequencing primer sequence or a capture primer sequence (e.g., soluble primers, or immobilized capture primers).

[00148] As used herein, the term “selectively binds” in the context of any binding agent, e.g., an oligonucleotide or oligonucleotide complex of the disclosure, refers to a binding agent that binds specifically to a target, e.g., a target sequence, such as with a high affinity, and does not significantly bind other unrelated targets or sequences. The person of ordinary skill in the art will appreciate that a binding agent that binds specifically to a target with high affinity, but binds to non-targets (off-targets) with suitably low affinity can still be said to selectively bind to the target.

[00149] As used herein, the term “target polynucleotide” refers to any nucleic acid inside a cellular sample having a sequence that can bind at least one target probe complex (900). Target polynucleotides include without limitation RNA, cDNA and DNA. In some embodiments, target polynucleotides comprise polynucleotides expressed inside a cellular sample including, without limitation, naturally occurring polynucleotides and recombinant polynucleotides.

[00150] As used herein, the term “target analyte” refers to any analyte that can be bound by an affinity moiety of any of the multivalent analyte detection reagents disclosed herein. Exemplary target analytes can be on the surface of or inside a cell. Exemplary target analytes include, but are not limited to polynucleotides, proteins, lipids, polysaccharides and the like. When used with respect to an antibody of an antibody bridge circle or bipartite complex, the term “target analyte” refers to any analyte that can be bound by the antibody of an antibody bridge circle complex or a bipartite complex disclosed herein. Exemplary target analytes include, but are not limited to polypeptides, lipids, nucleic acids, polysaccharides or a combination thereof.

[00151] As used herein, the term “target sequence” refers to a sequence in a concatemer molecule which can bind a probe arm of a multivalent probe. A multivalent probe can comprise a core attached to a plurality of probe arms, and individual probe arms can comprise a polymer linked to a target-specific oligonucleotide probe (see FIG. 5A). Exemplary a target sequences comprise a target barcode sequence, a sample index sequence and / or a batch barcode sequence.

[00152] A target probe complex (900) can be used to detect a target polynucleotide inside a cellular sample. In some embodiments, a target probe complex (900) comprises two oligonucleotide molecules including a circularized barcoded oligonucleotide (500) and a linear target probe (600) (e.g., see FIG. 15). In some embodiments, the linear target probe (600) comprises a target binding moiety (700) and the circularized barcoded oligonucleotide (500) comprises a target barcode sequence (300). In some embodiments, the target binding moiety (700) comprises a moiety that can bind a target analyte including, but not limited to, a polynucleotide, protein, lipid, polysaccharide and the like. In some embodiments, the target binding moiety (700) comprises a nucleic acid sequence that can selectively bind to a portion of a target polynucleotide. Thus, the target binding moiety (700) and the target barcode sequence (300) are placed on two separate oligonucleotides. In some embodiments, the target probe complex (900) can be generated by hybridizing a circularized barcoded oligonucleotide (500) with the target probe (600) which pairs together the target binding moiety (700) and the target barcode sequence (300) in the same complex (900). With respect to the target barcode sequence (300) and the target binding moiety (700), the term “corresponds to” as used herein refers to the pairing of the target barcode sequence (300) and the target binding moiety (700) in the same target probe complex (900).

[00153] A “antibody bridge circle complex (1700)” refers to a complex (FIGS. 23-27) that can be used to detect a target polypeptide inside a cellular sample. The antibody bridge circle complex (1700) can comprise a bridge circle complex (1600) attached to an antibody.

[00154] A “bridge circle complex (1600)” refers to a complex that comprises two oligonucleotide molecules including a circularized barcoded oligonucleotide (1400) and a linear bridge oligonucleotide (1500) (e.g., see FIGS. 23-27).The circularized barcoded oligonucleotide (1400) can comprise a target barcode sequence (1200). The bridge circle complex (1600) can be generated by hybridizing a circularized barcoded oligonucleotide (1400) with the bridge oligonucleotide (1500). One end of the linear bridge oligonucleotide (1500) can be attached to an antibody which can selectively bind to a target analyte. The bridge circle complex (1600) can be attached to an antibody which pairs together the target analyte binding capability of the antibody and the target barcode sequence (1200). With respect to the target barcode sequence (1200) and the polypeptide binding capability of the antibody, the term “corresponds to” as used herein refers to the pairing of the target barcode sequence (1200) and the polypeptide binding capability in the same antibody bridge circle complex (1700).

[00155] As used herein, the term “sequencing read product” refers to a primer extension product generated by conducting a sequencing reaction using a sequencing primer hybridized to a template molecule to be sequenced (e.g., a concatemer molecule), a sequencing polymerase and a plurality of nucleotides. In some embodiments, the sequencing polymerase catalyzes nucleotide incorporation using the 3’ end of the sequencing primer as an initiation site and generates an extension product comprising a sequence that is complementary to the template molecule. In some embodiments, a nucleotide incorporation reaction extends the sequencing primer by one nucleotide. In some embodiments, the number of nucleotide incorporation reactions that are conducted will dictate the length of the sequencing read product. For example, conducting eleven nucleotide incorporation reactions will generate a sequencing primer that is extended by eleven nucleotides. In some embodiments, one cycle of a sequencing reaction comprises: conducting one nucleotide incorporation reaction using a sequencing polymerase and a nucleotide thereby extending the sequencing primer by one nucleotide. In some embodiments, one cycle of a sequencing reaction comprises: (i) binding a multivalent molecule to the 3’ end of a first sequencing primer and a sequencing polymerase under a condition that inhibits polymerase-catalyzed nucleotide incorporation where the multivalent molecule comprises a plurality of nucleotide arms attached to a core; (ii) removing the multivalent molecule and the sequencing polymerase while retaining the template molecule hybridized to the sequencing primer; and (iii) conducting one nucleotide incorporation reaction using a second sequencing polymerase and a nucleotide thereby extending the sequencing primer by one nucleotide.

[00156] As used herein the term “batch sequencing” refers to a method which generally comprises separately sequencing two or more sub-populations (e.g., batches) of template molecules from a larger population of template molecules In a non-limiting example, a cellular sample harbors a plurality of concatemer template molecules comprising at least a first and second sub-population of concatemer template molecules. The first sub-population of concatemer template molecules can undergo a first batch sequencing workflow using a plurality of first batch sequencing primers which selectively hybridize to the first subpopulation of concatemer template molecules, wherein the first batch sequencing workflow comprises conducting a first plurality of sequencing cycles thereby generating a first plurality of sequencing read products, and the second sub-population of concatemer template molecules can undergo a second batch sequencing workflow using a plurality of second batch sequencing primers which selectively hybridize to the second sub-population of concatemer template molecules wherein the second batch sequencing workflow comprises conducting a second plurality of sequencing cycles thereby generating a second plurality of sequencing read products, wherein the first and second sequencing primers have different sequences.

[00157] When used herein in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also can include hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a duplex region. Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be wholly complementary, or may be partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[00158] When used herein in reference to nucleic acids, the terms “extend”, “extending”, “extension” and other variants, refer to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3' OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule known in the art may be used, including, but not limited to, primer extension catalyzed by a DNA polymerase or RNA polymerase.

[00159] As used herein, the term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical or non-canonical nucleotides are consistent with use of the term. The phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. The term “nucleoside” as used herein refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a detectable reporter moiety.

[00160] Nucleotides (and nucleosides) typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2-isopentenyladenine (6iA), N6-A2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moi eties; and the like. Additional exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[00161] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chern. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). In some embodiments, the sugar moiety comprises ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3 'dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[00162] In some embodiments, nucleotides comprise a chain of one, two or three phosphorus atoms, where the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.

[00163] The term “reporter moiety”, “reporter moieties” or related terms as used herein refers to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label”. Any suitable reporter moiety known in the art may be used, including, but not limited to, luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme reporter moiety. A reporter moiety can generate a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event can include two reporter moieties approaching each other, or associating with each other, or binding to each other. It is well known to one skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or supports (e.g., surfaces).

[00164] In some embodiments, a reporter moiety (or label) comprises a fluorescent label or a fluorophore. Exemplary fluorescent moieties which may serve as fluorescent labels or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY® and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor® dyes, DyLight® dyes, Atto™ dyes, LightCycler® Red dyes, CAL Fluor dyes, JOE and derivatives thereof, Oregon Green™ dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, nearinfrared dyes and others known in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and may consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-1 -yloxy)-6-oxohexyl]-2-(3 - {1 -[6-(2,5-dioxopyrrolidin-1 -yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3- dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise 1-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium or 1-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may comprise l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule. Additional dyes are described, for example in U.S. Patent Application Publication No. 2024 / 0240249, the contents of which are incorporated by reference in their entirety herein.

[00165] In some embodiments, the reporter moiety can be a fluorescence resonance energy transfer (FRET) pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[00166] An “affinity moiety” is any moiety known in the art that can bind to a target analyte with sufficient affinity and specificity to be used with the methods and compositions described herein. Exemplary affinity moieties include, but are not limited to, antibodies and antibody derivatives as described herein, protein domains capable of binding target analytes and the like. Other embodiments of affinity moieties include, but are not limited to, nonantibody molecules that can bind a target analyte, including but not limited to, phalloidin and wheat germ agglutinin.

[00167] The term “binds specifically” or “specific binding activity” and related terms, refers to two molecules, such as an affinity moiety and target analyte, that form a complex that is relatively stable under assay conditions. The term is also applicable where an antigenbinding domain is specific for a particular epitope, which is carried by a number of antigens, in which case the antibody carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope. Specific binding is characterized by a high affinity and a low to moderate capacity. As an example, the binding is considered specific when the affinity constant is about 1 * 10 6 M, generally at least about 1 * 10 7 M, usually at least about 1 x 10 8 M, and preferably at least about 1 * ICT9 M or 1 x 10 10 M or less.

[00168] When used herein in reference to nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is the same as the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to a template sequence, or is substantially identical to a sequence that is complementary to the template sequence.

[00169] The term “support” as used herein refers to a substrate that is designed for deposition of biological molecules or cellular samples for assays and / or analyses. Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), polypeptides, saccharides, lipids, a single cell or multiple cells. Examples of cellular samples include, but are not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool, sweat, tears, smears, fluids from tissues or organs, as well as tissue samples (e.g., biopsy samples).

[00170] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example comprising a capillary or interior surface of a capillary.

[00171] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof.

[00172] In some embodiments, the support comprises a bead having any shape, including spherical, hemi-spherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like.

[00173] The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[00174] The term “persistence time” and related terms as used herein refers to the length of time that a binding complex remains stable without any binding component dissociating from the binding complex. An exemplary binding complex comprises a nucleic acid template and nucleic acid primer, a polymerase, a nucleotide unit of a multivalent molecule or a free (e.g., unconjugated) nucleotide. The persistence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measured by observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, and without limitation, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One exemplary non-limiting label is a fluorescent label. Such labels for detection may also be referred to as “detectable labels” herein. Introduction - Nucleic Acid Target Probe Complexes

[00175] The present disclosure provides nucleic acid target probe complexes and methods that employ target probe complexes for in situ detection of target polynucleotides. Target polynucleotides may be inside a cellular sample, including, for example and without limitation, a single cell, multiple cells, a tissue, an organ, a tumor, or portions thereof. In some embodiments, individual target probe complexes (900) comprise two oligonucleotides including a circularized barcoded oligonucleotide (500) and a target probe (600) (e.g., FIGS. 15, 16 and 17).

[00176] In some embodiments, the target probe (600) comprises a linear oligonucleotide having a first region that selectively binds to a portion of a target polynucleotide (target binding moiety, 700) and a second region that binds to a universal sequence in a circularized barcoded oligonucleotide (500) (the universal probe region, 800). Thus, the target probe can include a target-specific sequence and a universal sequence. Different sets (e.g., subpopulations) of target probes (600) can be designed to include different target-specific sequences and different universal sequences, or to include different target-specific sequences and the same universal sequences.

[00177] In some embodiments, the circularized barcoded oligonucleotide (500) comprises a covalently closed circular oligonucleotide harboring a sequencing primer binding site sequence (200), a target barcode sequence (300) that corresponds to the target binding moiety (700) of the target probe, and a universal sequence (400) that binds the universal sequence of the target probe (600). The sequencing primer binding site sequence (200) can be a universal sequence or a batch-specific sequence. Thus, the circularized barcoded oligonucleotide can include a target barcode sequence and a universal sequence. Different sets (e.g, subpopulations) of circularized barcoded oligonucleotides (500) can be designed to include different target barcode sequences and different universal sequences, or to include different target barcode sequences and the same universal sequence.

[00178] In some embodiments of the assembled target probe complex (900), the target binding moiety and the target barcode sequence are placed on separate oligonucleotides. The modular designs of the circularized barcoded oligonucleotide (500) and the target probe (600) can offer flexibility for preparing different sets of target probe complexes (900) where each set of target probe complexes (900) can selectively bind a target polynucleotide. In some embodiments, detection of different target polynucleotides inside a cellular sample relies on different target probe complexes (900) carrying different sequence primer binding site sequences and the different target barcode sequences. The target probe complexes (900) can have many uses, including but not limited to co-localized detection, batch sequencing, reiterative sequencing and multiplex workflows.

[00179] The target probe complexes (900) of the instant disclosure can offer the advantage of employing circularized barcoded oligonucleotides, rather than existing padlock probe based technologies that require enzymatic ligation or polymerase-catalyzed gap fill-in reaction. The circularized barcoded oligonucleotides are already covalently closed circular oligonucleotides (e.g., pre-circularized), which obviates the requirement for enzymatic closure of a nick or gap. This circularized barcoded oligonucleotide thereby reduces the number of enzymatic steps in the reaction, reducing cost of the workflow, and simplifying the workflow.

[00180] The target probe complexes (900) can bind selectively to any type of target polynucleotide, including RNA, cDNA, DNA, or a combination thereof, to enable high target specificity and sensitivity. The target probe complexes (900) can be used to detect and sequence target polynucleotides inside a cellular sample. Nucleic Acid Target Probe Complexes

[00181] The present disclosure provides a plurality of nucleic acid target probe complexes (900) wherein individual complexes comprise two oligonucleotides including a target probe (600) and a circularized barcoded oligonucleotide (500) which are bound together (e.g., FIGS. 15, 16 and 17).

[00182] In some embodiments, the circularized barcoded oligonucleotide (500) comprises (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; and (iii) a universal circularized region (400) that binds a universal probe region of the target probe (600).

[00183] In some embodiments, the target probe (600) comprises an oligonucleotide and a target binding moiety (700), wherein the target binding moiety (700) comprises a moiety that can bind a target analyte including, but not limited to, a polynucleotide, protein, lipid, or polysaccharide. In some embodiments, the target probe (600) comprises an oligonucleotide having (i) a target binding moiety (700) which selectively binds a target polynucleotide; and (ii) a universal probe region (800) that binds the universal circularized region (400).

[00184] In some embodiments, in an assembled target probe complex (900), the universal circularized region (400) of the circularized barcoded oligonucleotide is hybridized to the universal probe region (800) of the target probe (600). In some embodiments, the duplex region formed by hybridization between the universal circularized region (400) and the universal probe region (800) is fully or partially complementary.

[00185] In some embodiments, a target barcode sequence (300) can be linked to a given universal circularized region (400), and the given universal circularized region (400) hybridizes to the universal probe region (800) which is directly linked to the target binding moiety (700). Thus, in an assembled target probe complex, the target barcode sequence (300) corresponds to a given target binding moiety (700). Circularized Barcoded Oligonucleotides

[00186] In some embodiments, individual circularized barcoded oligonucleotides (500) comprise single-stranded covalently closed oligonucleotides, which comprise: (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; and (iii) a universal circularized region (400) that binds a universal probe region of the target probe (600) (e.g., FIG. 15).

[00187] In some embodiments, individual circularized barcoded oligonucleotides (500) comprise a single-stranded covalently closed oligonucleotide which comprises DNA, RNA or chimeric DNA / RNA. In some embodiments, individual circularized barcoded oligonucleotides (500) comprise canonical nucleotides or nucleotide analogs or a combination of both.

[00188] In some embodiments, the sequencing primer binding site sequence (200) can be 10-50 nucleotides in length, or any range therebetween. In some embodiments, the target barcode sequence (300) can be 3-20 nucleotides in length, or any range therebetween. In some embodiments, the universal circularized region (400) can be 10-200 nucleotides in length, or any range therebetween. In some embodiments, the universal circularized region (400) can be 20-75 nucleotides in length.

[00189] In some embodiments, the circularized barcoded oligonucleotide (500) comprises a universal circularized region (400) which includes a first sub-region (410) and a second sub-region (420). In some embodiments, the first sub-region (410) binds a portion of the universal probe region (800), and the second sub-region (420) binds a portion of the universal probe region (800). In some embodiments, the first and second sub-regions of the universal probe region are not the same region. In some embodiments, the universal probe region (800) includes a first sub-region (810) and a second sub-region (820), which binds the second subregion and first sub-region of the circularized barcoded oligonucleotide (e.g., FIG. 16).

[00190] In some embodiments, the circularized barcoded oligonucleotide (500) further comprises any combination of (iv) a sample index sequence (310); (v) a batch barcode sequence (320); and / or (vi) a compaction oligonucleotide binding site sequence (415) (or a complementary sequence thereof). In some embodiments, the circularized barcoded oligonucleotide (500) comprises, in any combination and arranged in any order: (iv) a sample index sequence (310); (v) a batch barcode sequence (320), and / or (vii) a compaction oligonucleotide binding site sequence (415) (e.g., FIG. 17). In some embodiments, the sample index sequence (310) can be used to distinguish cellular samples, e.g., from different sources, e.g., in a multiplex assay. In some embodiments, a batch barcode sequence (320) can be used for batch sequencing. In some embodiments, the target barcode sequence (300) of any of the circularized barcoded oligonucleotides (500) comprise a short random sequence comprising 3-20 nucleotides in length (e.g., NNN or NNNN) , or any range therebetween. The short random sequence is designed, for example, to provide nucleotide diversity and color balance generated by the detectable signals when sequencing concatemers generated by amplifying the circularized barcoded oligonucleotides (500).

[00191] In some embodiments, in the short random sequence each base “N” at a given position is independently selected from A, G, C, T or U. In some embodiments, the random sequence lacks consecutive repeat sequences having 2 or 3 of the same nucleo-base, for example, AA, TT, CC, GG, UU, AAA, TTT, CCC, GGG or UUU.

[00192] In some embodiments, in a population of target probe complexes (900), the short random sequence comprises a high diversity sequence which includes approximately equal proportions of all four nucleotides (e.g., A, G, C, T and / or U) that will be represented in each cycle of a sequencing run.

[00193] In some embodiments, the short random sequence (e.g., NNN) includes, but is not limited to, AGC, AGT, GAC, GAT, CAT, CAG, TAG, TAC. The skilled artisan will recognize that many more random sequences can be prepared (e.g., 64 possible combinations) where each base “N” at a given position in the random sequence is independently selected from A, G, C, T or U.

[00194] In some embodiments, the circularized barcoded oligonucleotides (500) can be prepared by conducting intramolecular ligation of the ends of linear barcoded oligonucleotides (100) having a 3' OH end and a 5' phosphorylated end. For example, CIRCLIGASE™ OR CIRCLIGASE II™ can be used to generate the circularized barcoded oligonucleotides (500).

[00195] In some embodiments, the linear barcoded oligonucleotides (100) can be chemically synthesized, or the linear barcoded oligonucleotides (100) can be prepared using recombinant nucleic acid workflows that employ adaptors with enzymatic ligation and / or PCR, or the linear barcoded oligonucleotides (100) can be prepared using a combination of both technologies. Some exemplary embodiments of barcoded oligonucleotides (100) are shown in FIG. 18.

[00196] In some embodiments, a single-stranded splint can be used to conduct intramolecular ligation of the ends of linear barcoded oligonucleotides. The single-stranded splint can include a first binding arm that hybridizes to a portion of the 5' end of a linear barcoded oligonucleotide (100). The single-stranded splint can include a second binding arm that hybridizes to a portion of the 3' end of a linear barcoded oligonucleotide (100). In some embodiments, the 5' and 3' end portions of the linear barcoded oligonucleotide (100) hybridize to a splint, thereby forming a nick between the terminal 5' and 3' ends. The nick can be closed by conducting a ligation reaction using a ligase, for example and without limitation, a T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. The ligation reaction generates a circularized barcoded oligonucleotide (500). Accordingly, a “circularized barcoded oligonucleotide” or “circularized barcoded oligonucleotide (500)” as used herein refers to a linear barcoded oligonucleotide, e.g., a linear barcoded oligonucleotide (100), that has been covalently closed at its 5' and 3' ends by hybridization and / or ligation (see, for example, FIGS. 19-21). Exemplary single-stranded splints and methods of using are disclosed in WO2023168444, the contents of which are incorporated by reference in their entirety herein.

[00197] In some embodiments, the linear barcoded oligonucleotides (100) comprise, in any arrangement: (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; and (iii) a universal circularized region (400) that binds a universal probe region of the target probe (600).

[00198] In some embodiments, the linear barcoded oligonucleotides (100) comprise: (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; and (iii) a universal circularized region (400) which can be sub-divided into two subregions, such as for example a first sub-region (410) and a second sub-region (420). In some embodiments, the first sub-region (410) binds a portion of the universal probe region (800), and the second sub-region (420) binds a portion of the universal probe region (800). In some embodiments, the first and second sub-regions of the universal probe region are not the same region.

[00199] In some embodiments, the linear barcoded oligonucleotides (100) comprise, in any arrangement: (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; (iii) universal circularized first sub-region (410); and (iv) a universal circularized region second sub-region (420). Exemplary barcoded oligonucleotides (100) are shown in FIG. 18. The skilled artisan will realize that many other arrangements of the linear barcoded oligonucleotides (100) are possible. In some embodiments, any of these linear barcoded oligonucleotides can be subjected to an intramolecular ligation to generate circularized barcoded oligonucleotides (500). Exemplary intramolecular ligation reactions are shown in FIGS. 19, 20 and 21. In some embodiments, the ligation reactions can be conducted with CIRCLIGASE™ or CIRCLIGASE II™. In some embodiments, the ligation reaction can be conducted with a single-stranded splint and a ligase enzyme, for example and without limitation, a T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. Target Probes

[00200] In some embodiments, individual target probes (600) comprise single-stranded oligonucleotides. In some embodiments, individual target probes (600) comprise DNA, RNA or chimeric DNA / RNA. In some embodiments, individual target probes (600) comprise canonical nucleotides, or nucleotide analogs, or a combination thereof.

[00201] In some embodiments, the target probes (600) can be chemically synthesized, or the target probes (600) can be prepared using recombinant nucleic acid workflows, e.g., that employ adaptors with enzymatic ligation and / or PCR, or the target probes (600) can be prepared using a combination of both technologies.

[00202] In some embodiments, individual target probes (600) comprise a target binding moiety (700) and a universal probe region (800). In some embodiments, individual target probes (600) comprise a target binding moiety which selectively binds a target polynucleotide. In some embodiments, the target binding moiety (700) is joined directly to a universal probe region (800) which is designed to bind to the universal circularized region (400) of a circularized barcoded oligonucleotide (500). In some embodiments, the target binding moiety (700) is joined indirectly, e.g., via an adaptor, to a universal probe region (800) which is designed to bind to the universal circularized region (400) of a circularized barcoded oligonucleotide (500) (e.g., FIGS. 15-17).

[00203] In some embodiments, the target binding moiety (700) can be 5-100 nucleotides in length, or any range therebetween. In some embodiments, the target binding moiety (700) can be 15-75 nucleotides in length, or any range therebetween. In some embodiments, the target binding moiety (700) comprises a poly-T sequence having 3-50 consecutive thymine bases, or any range therebetween.

[00204] In some embodiments, the universal probe region (800) can be 5-100 nucleotides in length, or any range therebetween. In some embodiments, the universal probe region (800) can be 15-75 nucleotides in length, or any range therebetween.

[00205] In some embodiments, the universal probe region comprises a first sub-region (810) and a second sub-region (820). In some embodiments, the first sub-region (810) of the target probe (600) binds the second sub-region of the universal circularized region (420). In some embodiments, the second sub-region (820) of the target probe (600) binds the first subregion of the universal circularized region (410). See, for an example embodiment, FIG. 16.

[00206] In some embodiments, the target probes (600) comprise a 3' OH extendible end, or a 3' non-extendible end that can be converted into a 3' OH extendible end. In some embodiments, the target probes (600) comprise a 5' end that inhibits ligation.

[00207] In some embodiments, the target probes (600) comprise one or more phosphorothioate linkage(s) at their 5' and / or 3' ends, e.g., to confer exonuclease resistance. In some embodiments, the target probes (600) comprise one or more phosphorothioate linkage(s) at an internal position, e.g., to confer endonuclease resistance. In some embodiments, the target probes (600) comprise one or more 2’-O-methylcytosine bases at their 5' and / or 3' ends. . In some embodiments, the target probes (600) comprise one or more 2’-O-methylcytosine bases at an internal position. In some embodiments, the 5' end of each of the target probes (600) is phosphorylated. In some embodiments, the 5' end of each of the target probes (600) is non-phosphorylated. In some embodiments, the 3' end of each of the target probes (600) comprises a terminal 3' OH group or a terminal 3' blocking group. In some embodiments, individual target probes (600) comprise a terminal 3’ extendible end.

[00208] In some embodiments, the target probe (600) further comprises an optional linker region (710). In some embodiments, the optional linker region (710) is located proximal to the target binding moiety (700) (e.g., FIG. 17). In some embodiments, the linker region (710) comprises a polynucleotide having a sequence that does not hybridize to the target polynucleotide. In some embodiments, the linker region (710) comprises a polynucleotide having a sequence that does not hybridize to any portion of the circularized barcoded oligonucleotide (500). In some embodiments, the linker region (710) comprises a spacer, e.g., an 18-carbon spacer (e.g., a hexa-ethyleneglycol spacer), one or more C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the linker region (710) comprises a polyethylene glycol moiety, including, but not limited to, a PEG2, a PEG3 or a PEG4 spacer. In some embodiments, a PEG2 spacer comprises two polyethylene glycol units, a PEG3 spacer comprises three polyethylene glycol units, and a PEG4 spacer comprises four polyethylene glycol units.

[00209] In some embodiments, the target probe (600) further comprises a compaction oligonucleotide binding region (815), or a complementary sequence thereof, which is located between the first sub-region (810) and the second sub-region (820). Exemplary embodiments of various target probes (600) are shown in FIG. 22. A Plurality of Target Probe Complexes

[00210] The present disclosure provides compositions comprising a plurality of target probe complexes (900). In some embodiments, the plurality of target probe complexes comprises at least a first and second sub-population of target probe complexes (900-1 and 900-2). In some embodiments, the first sub-population comprises a plurality of first target probe complexes. In some embodiments, individual first target probe complexes in the first sub-population are the same, or substantially the same. In some embodiments, individual first target probe complexes in the first sub-population are not the same. In some embodiments, the second sub-population comprises a plurality of second target probe complexes. In some embodiments, individual second target probe complexes in the second sub-population are the same, or substantially the same. In some embodiments, individual second target probe complexes in the second sub-population are not the same.

[00211] In some embodiments, individual first target probe complexes in the first subpopulation comprise a first circularized barcoded oligonucleotide (500-1) and a first target probe (600-1). In some embodiments, the first circularized barcoded oligonucleotide (500-1) comprises: (i) a first sequencing primer binding site sequence (200-1) (or a complementary sequence thereof), (ii) a first target barcode sequence (300-1) that corresponds to a target binding moiety of the first target probe (600-1), and (iii) a first universal circularized region (400-1) that binds a universal probe region of the first target probe (600-1). In some embodiments, the first target probe (600-1) comprises: an oligonucleotide having (i) a first target binding moiety (700-1) which selectively binds at least a portion of a first target polynucleotide, and (ii) a first universal probe region (800-1) that binds the first universal circularized region (400-1). In some embodiments, individual target probe complexes in the first sub-population comprise a first universal circularized region (400-1) hybridized to a first universal probe region (800-1), thereby forming a first target probe complex (900-1).

[00212] In some embodiments, individual second target probe complexes in the second sub-population comprise a second circularized barcoded oligonucleotide (500-2) and a second target probe (600-2). In some embodiments, the second circularized barcoded oligonucleotide (500-2) comprises: (i) a second sequencing primer binding site sequence (200-2) (or a complementary sequence thereof); (ii) a second target barcode sequence (300-2) that corresponds to a target binding moiety of the second target probe (600-2), and (iii) a second universal circularized region (400-2) that binds a universal probe region of the second target probe (600-2). In some embodiments, the second target probe (600-2) comprises: an oligonucleotide having (i) a second target binding moiety (700-2) which selectively binds at least a portion of a second target polynucleotide, and (ii) a second universal probe region (800-2) that binds the second universal circularized region (400-2). In some embodiments, individual target probe complexes in the second sub-population comprise a second universal circularized region (400-2) hybridized to a second universal probe region (800-2), thereby forming a second target probe complex (900-2).

[00213] In some embodiments, the first target probe complex (900-1) comprises a first sequencing primer binding site sequence (200-1). In some embodiments, the first sequencing primer binding site sequence (200-1) may be the same or different from the second sequencing primer binding site sequences (200-2) of the second target probe complex (9002).

[00214] In some embodiments, the first target probe complex (900-1) comprises a first barcode sequence (300-1) having the same or a different sequence from the second barcode sequences (300-2) of the second target probe complex (900-2).

[00215] In some embodiments, the first target probe complex (900-1) comprises a first universal circularized region (400-1) having the same or a different sequence from the second universal circularized region (400-2) of the second target probe complex (900-2).

[00216] In some embodiments, the first target probe complex (900-1) comprises a first target binding moiety (700-1) having the same or different sequence from the second target binding moiety (700-2) of the second target probe complex (900-2).

[00217] In some embodiments, the first target probe complex (900-1) comprises a first universal probe region (800-1) having the same or a different sequence from the second universal probe region (800-2) of the second target probe complex (900-2).

[00218] In some embodiments, the first sub-population of target probe complexes (900-1) and the second sub-population of target probe complexes (900-2) comprise: (i) a first target binding moiety (700-1) and a second target binding moiety (700-2) having different sequences; (ii) a first universal probe region (800-1) and a second universal probe region (800-2) having different sequences; (iii) a first target barcode sequence (300-1) and a second target barcode sequences (300-2) having different sequences; and (iv) a first sequencing primer binding site sequence (200-1) and a second sequencing primer binding site sequences (200-2) having the same sequence. In some embodiments, the target probe complexes in the first sub-population (900-1) and the target probe complexes in the second sub-population (900-2) can selectively hybridize to different portions of the same target polynucleotide. In some embodiments, the target probe complexes in the first sub-population (900-1) can selectively hybridize to at least a portion of a first target polynucleotide, and the target probe complexes in the second sub-population (900-2) can selectively hybridize to at least a portion of a second target polynucleotide, wherein the first and second target polynucleotides are different polynucleotides.

[00219] In some embodiments, the first sub-population of target probe complexes (900-1) and the second sub-population of target probe complexes (900-2) comprise: (i) a first target binding moiety (700-1) and a second target binding moiety (700-2) having different sequences; (ii) a first universal probe region (800-1) and a second universal probe region (800-2) having different sequences; (iii) a first target barcode sequence (300-1) and a second target barcode sequences (300-2) having different sequences; and (iv) a first sequencing primer binding site sequence (200-1) and a second sequencing primer binding site sequences (200-2) having different sequences. In some embodiments, the target probe complexes in the first sub-population (900-1) and the target probe complexes in the second sub-population (900-2) can selectively hybridize to different portions of the same target polynucleotide. In some embodiments, the target probe complexes in the first sub-population (900-1) can selectively hybridize to at least a portion of a first target polynucleotide, and the target probe complexes in the second sub-population (900-2) can selectively hybridize to at least a portion of a second target polynucleotide, wherein the first and second target polynucleotides are different polynucleotides.

[00220] In some embodiments, the first sub-population of target probe complexes (900-1) and the second sub-population of target probe complexes (900-2) comprise: (i) a first target binding moiety (700-1) and a second target binding moiety (700-2) have the same sequence or have a sequence that binds the same target polynucleotide; (ii) a first universal probe region (800-1) and a second universal probe region (800-2) having different sequences; (iii) a first target barcode sequence (300-1) and a second target barcode sequences (300-2) having different sequences; and (iv) a first sequencing primer binding site sequence (200-1) and a second sequencing primer binding site sequences (200-2) having different sequences. In some embodiments, the target probe complexes in the first sub-population (900-1) and the target probe complexes in the second sub-population (900-2) can selectively hybridize to target polynucleotides having the same sequence. Target Probe Complexes and Target Polynucleotides

[00221] In some embodiments, at least one target analyte analyzed by the compositions and methods described herein comprises a target polynucleotide. The present disclosure provides compositions comprising a plurality of target probe complexes (900) and a plurality of target polynucleotides. In some embodiments, the plurality of target polynucleotides comprise RNA, cDNA, DNA, or a combination thereof.

[00222] In some embodiments, the plurality of target probe complexes (900) and the plurality of target polynucleotides are located inside a cellular sample. In some embodiments, the plurality of target probe complexes (900) and the plurality of target polynucleotides are located in a container (e.g., a flowcell) or a well of a multi-well plate.

[00223] In some embodiments, individual target probe complexes (900) in the plurality are bound to a target polynucleotide.

[00224] In some embodiments, a target binding moiety (700) of an individual target probe complex (900) binds selectively to a portion of a target polynucleotide.

[00225] In some embodiments, a first target probe complex (900-1) selectively binds a first portion of a first target polynucleotide and a second target probe complex (900-2) selectively binds a second portion of the same target polynucleotide.

[00226] In some embodiments, a first target probe complex (900-1) selectively binds a first target polynucleotide and a second target probe complex (900-2) selectively binds a second target polynucleotide where the first and second target polynucleotides are different polynucleotides. Target Probe Complexes and RCA Reagents

[00227] The present disclosure provides compositions comprising a plurality of target probe complexes (900), a plurality of target polynucleotides, and rolling circle amplification reagents. In some embodiments, the rolling circle amplification reagents comprise a plurality of strand-displacing DNA polymerases and a plurality of nucleotides.

[00228] In some embodiments, the plurality of target probe complexes (900), the plurality of target polynucleotides, and the rolling circle amplification reagents are located inside a cellular sample. In some embodiments, the plurality of target probe complexes (900), the plurality of target polynucleotides, and the rolling circle amplification reagents are located in a container (e.g., a flowcell) or a well of a multi-well plate.

[00229] In some embodiments, the plurality of strand-displacing DNA polymerases comprises polymerases that exhibit the ability to displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include, for example and without limitation, mesophilic and thermophilic polymerases. Strand displacing polymerases include, for example and without limitation, wild type enzymes, and variant enzymes including, for example and without limitation, exonuclease minus mutants, mutant versions thereof, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include, for example and without limitation, phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent® DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be a wild type phi29 DNA polymerase (e.g., MagniPhi™ from Expedeon™), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific®), or a chimeric QualiPhi™ DNA polymerase (e.g., from 4basebio™).

[00230] In some embodiments, the plurality of nucleotides comprises any combination of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, at least one of the nucleotides comprises a scissile moiety. For example, a nucleotide having a scissile moiety can be incorporated in a primer extension reaction and the incorporated nucleotide can be enzymatically converted into an abasic site. In some embodiments, nucleotides comprising a scissile moiety include uridine, 8-oxo-7,8-dihydroguanine and deoxyinosine. In some embodiments, uridine can be converted to an abasic site, e.g., using uracil DNA glycosylase (UDG). In some embodiments, 8-Oxoguanine (8oxoG) can be converted to an abasic site using formamidopyrimidine [fapy]-DNA glycosylase (FPG glycosylase). In some embodiments, deoxyinosine can be converted to an abasic site using 3-Methyladenine DNA glycosylase II (AlkA glycosylase).

[00231] In some embodiments, the plurality of nucleotides comprises at least one nucleotide that is labeled with a detectable reporter moiety. For example, a detectable reporter moiety may comprise a fluorophore. Target Probe Complexes and Nascent Extension Product or Concatemer Molecules

[00232] The present disclosure provides compositions comprising a plurality of target probe complexes (900), wherein individual target probe complexes have a target probe (600) attached at their terminal 3' ends with a nascent polymerase-catalyzed extension product. In some embodiments, the nascent extension product comprises a sequence that is complementary to at least a portion of the circularized barcoded oligonucleotide (500). In some embodiments, the nascent extension product is labeled with a detectable reporter moiety. In some embodiments, the nascent extension product is unlabeled.

[00233] In some embodiments, the plurality of target probe complexes (900) are attached at their terminal 3' ends to a nascent polymerase-catalyzed extension product. In some embodiments, the nascent polymerase-catalyzed extension product is a concatemer molecule or a portion thereof.

[00234] In some embodiments, the individual target probe complexes having a target probe (600) attached at their terminal 3' ends with a nascent polymerase-catalyzed extension product are located inside a cellular sample. In some embodiments, the individual target probe complexes having a target probe (600) attached at their terminal 3' ends with a nascent polymerase-catalyzed extension product comprising a concatemer molecule (or portion thereof) are located in a container (e.g., a flowcell) or a well of a multi-well plate.

[00235] In some embodiments, the concatemer molecule comprises a plurality of tandem repeat polynucleotide units wherein each unit comprises a sequence that is complementary to the circularized barcoded oligonucleotide (500). In some embodiments, a given concatemer molecule corresponds to a given target polynucleotide. In some embodiments, the concatemer molecule is labeled with a detectable reporter moiety, e.g., a fluorescent moiety. In some embodiments, the concatemer molecule is non-labeled.

[00236] In some embodiments, the concatemer molecule comprises any combination of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, the concatemer molecule comprises at least one nucleotide with a scissile moiety. For example, and without limitation, a nucleotide having a scissile moiety can be incorporated in a primer extension reaction (e.g., rolling circle amplification reaction) and the incorporated nucleotide can be enzymatically converted into an abasic site. In some embodiments, nucleotides comprising a scissile moiety include uridine, 8-oxo-7,8-dihydroguanine and deoxyinosine. In some embodiments, uridine can be converted to an abasic site using uracil DNA glycosylase (UDG). In some embodiments, 8oxoG can be converted to an abasic site using FPG glycosylase. In some embodiments, deoxyinosine can be converted to an abasic site using AlkA glycosylase. Target Probe Complexes, RCA Reagents and Compaction Oligonucleotides

[00237] The present disclosure provides compositions comprising a plurality of target probe complexes (900), a plurality of target polynucleotides, rolling circle amplification reagents, and a plurality of compaction oligonucleotides.

[00238] In some embodiments, the plurality of target probe complexes (900), the plurality of target polynucleotides, the rolling circle amplification reagents, and the plurality of compaction oligonucleotides are located inside a cellular sample. In some embodiments, the plurality of target probe complexes (900), the plurality of target polynucleotides, the rolling circle amplification reagents, and the plurality of compaction oligonucleotides are located in a contained or a well of a multi-well plate.

[00239] In some embodiments, the present disclosure provides compositions comprising individual target probe complexes (900) attached to individual concatemer molecules, a plurality of target polynucleotides, rolling circle amplification reagents, and a plurality of compaction oligonucleotides.

[00240] In some embodiments, rolling circle amplification reagents comprise a plurality of strand-displacing DNA polymerases and a plurality of nucleotides. In some embodiments, the plurality of nucleotides comprises any combination of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, at least one of the nucleotides comprises a scissile moiety. For example, a nucleotide having a scissile moiety can be incorporated in a primer extension reaction and the incorporated nucleotide can be enzymatically converted into an abasic site. In some embodiments, nucleotides comprising a scissile moiety include uridine, 8-oxo-7,8-dihydrogunine and deoxyinosine.

[00241] In some embodiments, the compaction oligonucleotides bind portions of concatemer molecules generated by conducting a rolling circle amplification region. In some embodiments, individual compaction oligonucleotides comprise a 5' region that binds a first portion of the concatemer molecule and a 3' region that binds a second portion of the same concatemer molecule. In some embodiments, the 5' and 3' regions of a compaction oligonucleotide can hybridize to binding sites in a concatemer molecule to pull together distal portions of the concatemer molecule, thereby causing compaction of the concatemer molecule, e.g., to form a DNA nanoball.

[00242] In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a universal probe region (800) sequence.

[00243] In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a universal circularized region (400).

[00244] In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a first sub-region (410) of a universal circularized region.

[00245] In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a second sub-region (420) of a universal circularized region.

[00246] In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a compaction oligonucleotide binding site sequence (415). Target Probe Complexes in a Reaction Mixture

[00247] The present disclosure provides compositions comprising a plurality of target probe complexes (900) in a reaction mixture (e.g., in solution). In some embodiments, the reaction mixture comprises any one, or any combination of two or more, of a plurality of target polynucleotides, rolling circle reagents and / or a plurality of compaction oligonucleotides. In some embodiments, the plurality of target probe complexes (900) and the reaction mixture are located in a container (e.g., a flowcell) or a well of a multi-well plate.

[00248] In some embodiments, the plurality of target polynucleotides comprises DNA (e.g., genomic DNA), cDNA, RNA, or a mixture thereof. In some embodiments, the plurality of target polynucleotides comprise target polynucleotides extracted from a cellular sample.

[00249] In some embodiments, individual target probe complexes (900) in the reaction mixture comprise target probes (600) attached at their terminal 3' ends to a nascent polymerase-catalyzed extension product. In some embodiments, the nascent extension product comprises a sequence that is complementary to at least a portion of the circularized barcoded oligonucleotide (500).

[00250] In some embodiments, individual target probe complexes (900) in the reaction mixture comprise a target probe (600) attached at their terminal 3' ends with a nucleic acid concatemer molecule. In some embodiments, the concatemer molecule comprises a plurality of tandem repeat polynucleotide units wherein each unit comprises a sequence that is complementary to the circularized barcoded oligonucleotide (500). Target Probe Complexes and a Hybridization Solution

[00251] In some embodiments, the present disclosure provides compositions comprising a plurality of target probe complexes (900) in a hybridization solution.

[00252] In some embodiments, the plurality of target probe complexes (900) and the hybridization solution are located inside a cellular sample. In some embodiments, the plurality of target probe complexes (900) and the hybridization solution are located in a container (e.g., a flowcell) or a well of a multi-well plate.

[00253] In some embodiments, the hybridization solution comprises a saline-sodium citrate (SSC) solution. In some embodiments, the hybridization solution comprises a 2X, 3X, 4X, 5X, 7X, 8X, 9X or 10X SSC solution. In some embodiments, the hybridization solution comprises a 2X, 3X or 5X SSC solution. In some embodiments, the hybridization solution comprises acetonitrile. In some embodiments, the hybridization solution comprises dimethylsulfoxide (DMSO). In some embodiments, the hybridization solution comprises Denhardt’s solution which includes Ficoll (e.g., type 400), polyvinylpyrrolidone and bovine serum albumin (BSA). In some embodiments, the hybridization solution comprises betaine. In some embodiments, the hybridization solution comprises trehalose. In some embodiments, the hybridization solution comprises guanidinium isothiocyanate (GITC). In some embodiments, the hybridization solution comprises any one or any combination of two or more of a pH buffering agent, a chelator, a detergent, a denaturing agent, a crowding agent and / or a blocking agent. In some embodiments, the hybridization solution comprises a sodium salt (e.g, NaCl). In some embodiments, the pH buffering agent comprises HEPES (e.g., 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) or MES (e.g., 2-(N-morpholino)ethanesulfonic acid). In some embodiments, the chelator comprises EDTA (ethylenediaminetetraacetic acid) and / or EGTA (ethylene glycol tetraacetic acid). In some embodiments, the detergent comprises Tween-20, Triton-X 100 and / or SDS. In some embodiments, the denaturing agent comprises formamide, 2-pyrollidone, urea and / or ethylene carbonate. In some embodiments, the crowding agent comprises dextran sulfate and / or PEG, for example and without limitation, 1KPEG, 2KPEG, 4KPEG, 5KPEG and / or PEG200. In some embodiments, the blocking agent comprises BSA. Target Probe Complexes Inside a Cellular Sample

[00254] In some embodiments, the present disclosure provides compositions comprising a plurality of target probe complexes (900) and a cellular sample. In some embodiments, the plurality of target probe complexes (900) is located inside the cellular sample. In some embodiments, at least 2-10,000, or any range therebetween target probe complexes (900) are located inside the cellular sample. In some embodiments, the at least 2-10,000 target probe complexes (900) that are located inside the cellular sample can bind at least 2-10,000 different target polynucleotides, or any range therebetween.

[00255] The present disclosure provides compositions comprising a plurality of target probe complexes (900) and a cellular sample, and comprising any one or any combination of two or more components which include a plurality of target polynucleotides, rolling circle reagents and / or a plurality of compaction oligonucleotides. In some embodiments, the plurality of target probe complexes (900), the plurality of target polynucleotides, the rolling circle reagents and / or the plurality of compaction oligonucleotides are located inside the cellular sample.

[00256] In some embodiments, individual target probe complexes (900) inside the cellular sample comprise a target probe (600) attached at their terminal 3' ends with a nascent polymerase-catalyzed extension product. In some embodiments, the nascent extension product comprises a sequence that is complementary to at least a portion of the circularized barcoded oligonucleotide (500).

[00257] In some embodiments, individual target probe complexes (900) inside the cellular sample comprise a target probe (600) attached at their terminal 3' ends with a nucleic acid concatemer molecule. In some embodiments, the concatemer molecule comprises a plurality of tandem repeat polynucleotide units wherein each unit comprises a sequence that is complementary to the circularized barcoded oligonucleotide (500).

[00258] In some embodiments, the cellular sample harbors a plurality of target polynucleotides including DNA (e.g., genomic DNA), cDNA, RNA, or a mixture thereof. In some embodiments, the plurality of target polynucleotides are located inside the cellular sample.

[00259] In some embodiments, the cellular sample harbors a plurality of target polynucleotides. In some embodiments, the plurality of target polynucleotides have different sequences. In some embodiments, the cellular sample harbors 1-25 different target polynucleotides, or harbors 25-50 different target polynucleotides, or harbors 50-75 different target polynucleotides, or harbors 75-100 different target polynucleotides, or harbors any range therebetween of different target polynucleotides. In some embodiments, the cellular sample harbors more than 100 different target polynucleotides, or more than 250 different target polynucleotides, or more than 500 different target molecules, or more than 1000 different target polynucleotides, or more. In some embodiments, the cellular sample harbors more than 10,000 different target polynucleotides.

[00260] In some embodiments, the cellular sample comprises a whole cell (e.g., a single cell), a plurality of whole cells, an intact tissue or sectioned cellular sample. In some embodiments, the cellular sample comprises a fresh cellular sample, a freshly-frozen cellular sample, a sectioned cellular sample, or a formalin-fixed, paraffin-embedded (FFPE) cellular sample. In some embodiments, the cellular sample can be fixed and / or permeabilized. In some embodiments, the cellular sample comprises an expanded cellular sample, e.g., a patient-derived cell line or patient-derived organoid, that has been cultured in a simple or complex cell culture media.

[00261] In some embodiments, the cellular sample can be deposited (e.g., seeded) onto a support. In some embodiments, the support comprises a planar or non-planar support. In some embodiments, the support comprises a solid or semi-solid support. In some embodiments, the support comprises a porous, semi-porous or non-porous support. The support can be made of any material such as glass, plastic or a polymer material. In some embodiments, the surface of the support can be coated with one or more compounds to produce a passivated layer on the support. In some embodiments, the passivated layer forms a porous or semi-porous layer.

[00262] In some embodiments, the cellular sample can be deposited (e.g., seeded) onto a support which is passivated with a coating that promotes proliferation, migration, differentiation and / or adhesion of cultured cell or living ex vivo cells or tissue samples. In some embodiments, the cellular sample can be deposited on a support that lacks immobilized capture primers which can bind target polynucleotides from the cellular sample. In some embodiments, the support comprises a coated support. In some embodiments, the support can be coated with a solubilized basement membrane matrix. Suitable methods for coating a support with solubilized basement membrane matrix will be known to persons of ordinary skill in the art. In some embodiments, the solubilized basement membrane matrix is secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, for example MATRIGEL®. In some embodiments, the support can be coated with a gel matrix, including for example collagen gel, alginate gel or lactate gel. In some embodiments, the support can be coated with one or more animal-derived protein including for example aggrecan, brevican, collagen (e.g., collagen type I, II, III or IV), fibronectin, elastin, laminin, laminin / fibronectin, laminin / poly-D-lysine, laminin / poly-D-ornithine, vitronectin, osteopontin, gelatin (e.g., porcine), fibrillin, fibrinogen, plasminogen, plasmin, tenascin, hyaluronic acid proteoglycan, keratan sulfate proteoglycan, heparan sulfate proteoglycan, chondroitin sulfate proteoglycan, syndecan-1 (e.g., proteoglycan), and IGF binding protein. In some embodiments, the support can be coated with one or more compounds that generate a charged coated surface. In some embodiments, the support is coated with a poly-amino acid including and without limitation, a poly-lysine compound (e.g., poly-L-lysine (PLL) or poly-D-lysine (PDL)), an arginine compound, a poly-arginine compound, a poly-ornithine compound, or an amino-terminated compound (e.g., including amino-terminated PEG). The support can be coated with an unbranched compound, a branched compound, or a mixture of unbranched and branched compounds. In some embodiments, the support can be coated with modified peptides, including, for example and without limitation, cationic anti-microbial peptides or dual surface anti-microbial peptides. In some embodiments, the support can be coated with polycyclic peptide antibiotics comprising thioether amino acids lanthionine or methyllanthionine and / or unsaturated amino acids dehydroalaine and 2-aminoisobutryic acid. In some embodiments, the support can be coated with at least one small peptide such as melittin. In some embodiments, the support can be coated with a compound that promotes integrin-mediated cell adhesion. For example, and without limitation, the support can be coated with tripeptide arginyl-glycyl-aspartic acid (Arg-Gly-Asp; also known as RGD). In some embodiments, the support can be coated with short peptides of an extracellular matrix protein, including and without limitation, Arg-Gly-Asp (RGD), RGD-coupled alginate, Ile-Lys-Val-Ala-Val (IKVAV), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV), Val-Ala-Pro-Gly (VAPG), Phe-Gly-Leu (FGL), fibronectin domains, and laminin. In some embodiments, the support can be coated with amines or polymers having -NH2 groups which promote cell adhesion, including for example polyethyleneimine (PEI) or polydopamine (PDA). In some embodiments, the support is coated with any compound that is presently known in the art to be useful for promoting proliferation, migration, differentiation and / or adhesion of cultured cells or living ex vivo cells or tissue samples. In some embodiments, the cellular sample can be deposited (e.g., seeded) onto an uncoated support. Methods for Generating Concatemers Inside a Cellular Sample Using Target Probe Complexes

[00263] The present disclosure provides methods for generating a plurality of concatemer molecules inside a cellular sample using a plurality of target probe complexes (900). In some embodiments, the plurality of target probe complexes (900) can be used to generate a plurality of concatemer molecules in situ. In some embodiments, a plurality of target polynucleotides can be detected by generating a plurality of concatemer molecules inside a cellular sample using a plurality of target probe complexes (900) and sequencing the plurality of concatemer molecules. In some embodiments, a plurality of the target probe complexes (900) inside the cellular sample are subjected to rolling circle amplification to generate the plurality of concatemer molecules. In some embodiments, the plurality of concatemer molecules inside the cellular sample can be sequenced in separate batches. In some embodiments, the plurality of concatemer molecules inside the cellular sample can be sequenced simultaneously.

[00264] In some embodiments, the present disclosure provides a method for generating a plurality of nucleic acid concatemer molecules in situ, comprising step (a): providing a cellular sample deposited (e.g., seeded) on a support wherein the cellular sample harbors a plurality of polynucleotides including at least one target polynucleotide.

[00265] In some embodiments, in step (a), the cellular sample harbors a plurality of target polynucleotide including DNA (e.g., genomic DNA), cDNA, RNA, or a mixture thereof. In some embodiments, the plurality of target polynucleotides are located inside the cellular sample. In some embodiments, the cellular sample harbors a plurality of target polynucleotides having different sequences, as described above. In some embodiments, the cellular sample harbors a plurality of target polynucleotides comprising recombinant DNA, cDNA and / or RNA. In some embodiments, the cellular sample harbors a plurality of RNA which comprises coding RNA or non-coding RNA (ncRNA). In some embodiments, the coding RNA comprises mRNA. In some embodiments, the non-coding RNA comprises tRNA, rRNA, microRNA (miRNA), mature microRNA, immature microRNA or a combination thereof. In some embodiments, the cellular sample harbors a plurality of RNA comprising un-spliced RNA, partially spliced RNA, fully spliced RNA and / or RNA splice variants.

[00266] In some embodiments, in step (a), the cellular sample comprises a whole cell (e.g., a single cell), a plurality of whole cells, an intact tissue or sectioned cellular sample. In some embodiments, the cellular sample comprises a fresh cellular sample, a freshly-frozen cellular sample, a sectioned cellular sample, or an FFPE cellular sample. In some embodiments, the cellular sample can be fixed and / or permeabilized. In some embodiments, the cellular sample comprises an expanded cellular sample that has been cultured in a simple or complex cell culture media.

[00267] In some embodiments, in step (a), the cellular sample comprises an intracellular matrix and / or a cross-linked matrix comprising a hydrogel, swellable hydrogel, or crosslinked matrix.

[00268] In some embodiments, in step (a), the cellular sample comprises an intracellular matrix wherein the cellular sample is infused with a swellable poly electrolyte hydrogel (e.g., see U.S. patent No. 10,309,879 and Chen 2015 Science 347:543, the contents of these documents are herein incorporated by reference in their entireties). In some embodiments, a fixed and permeabilized cellular sample can be infused with sodium acrylate, acrylamide and a cross-linker, e.g., N-N’-methylenebisacrylamide. In some embodiments, the cellular sample can be infused with an ammonium persulfate (APS) initiator and tetramethylethylenediamine (TEMED) accelerator to achieve polymerization inside the cellular sample. In some embodiments, the cellular sample can be infused with a protease, e.g., proteinase K, for proteolysis and incubated in a digestion buffer. In some embodiments, the gel inside the cellular sample can be swelled by addition of water. In some embodiments, the cellular sample lacks an intracellular matrix.

[00269] In some embodiments, in step (a), the cellular sample can be deposited (seeded) onto a support as described herein.

[00270] In some embodiments, in step (a), the cellular sample can be deposited (e.g., seeded) onto a support as described herein. In some embodiments, the support is coated with any of the coatings describe herein. In some embodiments, the cellular sample can be deposited (e.g., seeded) onto an uncoated support.

[00271] In some embodiments, in step (a), the cellular sample can be deposited (e.g., seeded) on a support that lacks immobilized capture primers which can bind target polynucleotides from the cellular sample. Alternatively, the support includes a plurality of immobilized capture primers which can bind target polynucleotides from the cellular sample.

[00272] In some embodiments, the method for generating a plurality of nucleic acid concatemer molecules in situ comprises step (b): providing a plurality of the nucleic acid target probe complexes (900), wherein individual target probe complexes (900) comprise a target probe (600) and a circularized barcoded oligonucleotide (500) which are bound together (e.g., FIGS. 15, 16 and 17). In some embodiments, the circularized barcoded oligonucleotide (500) comprises (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) target a barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; and (iii) a universal circularized region (400) that binds a universal probe region of the target probe (600). In some embodiments, the target probe (600) comprises an oligonucleotide having (i) a target binding moiety (700) which selectively binds a target polynucleotide; and (ii) a universal probe region (800) that binds the universal circularized region (400).

[00273] In some embodiments, at step (b), the universal pre-circularized region (400) of the circularized barcoded oligonucleotide is hybridized to the universal probe region (800) of the target probe (600). In some embodiments, the duplex region formed by hybridization between the universal circularized region (400) and the universal probe region (800) is fully or partially complementary.

[00274] In some embodiments, at step (b), a target barcode sequence (300) can be linked to a given universal circularized region (400), and the given universal circularized region (400) hybridizes to the universal probe region (800) which is directly linked to the target binding moiety (700). Thus, in an assembled target probe complex, the target barcode sequence (300) corresponds to a given target binding moiety (700). In some embodiments, step (b) comprises providing at least 2-10,000 target probe complexes (900), between 50 and 5,000 target probe complexes (900), between 100 and 5,000 target probe complexes (900), between 500 and 10,000 target probe complexes (900), between 100 and 1,000 target probe complexes (900), or any range therebetween. In some embodiments, the at least 2-10,000 target probe complexes (900) can bind at least 2-10,000 different target polynucleotides.

[00275] In some embodiments, at step (b), individual circularized barcoded oligonucleotides (500) comprise single-stranded covalently closed oligonucleotides, which comprise: (i) a sequencing primer binding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300) that corresponds to a target binding moiety (700) of the target probe; and (iii) a universal circularized region (400) that binds a universal probe region of the target probe (600) (e.g. FIG. 15).

[00276] In some embodiments, at step (b), individual circularized barcoded oligonucleotides (500) comprise a single-stranded covalently closed oligonucleotides which comprise DNA, RNA or chimeric DNA / RNA. In some embodiments, individual circularized barcoded oligonucleotides (500) comprise canonical nucleotides or nucleotide analogs or a combination thereof.

[00277] In some embodiments, at step (b), the sequencing primer binding site sequence (200) can be 10-50 nucleotides in length, or any range therebetween. In some embodiments, the target barcode sequence (300) can be 3-20 nucleotides in length, or any range therebetween. In some embodiments, the universal circularized region (400) can be 10-200 nucleotides in length, or any range therebetween. In some embodiments, the universal circularized region (400) can be 20-75 nucleotides in length, or any range therebetween.

[00278] In some embodiments, at step (b), the circularized barcoded oligonucleotide (500) comprises a universal circularized region (400) which includes a first sub-region (410) and a second sub-region (420). In some embodiments, the first sub-region (410) binds a portion of the universal probe region (800), and the second sub-region (420) binds a portion of the universal probe region (800). In some embodiments, the universal probe region (800) includes a first sub-region (810) and a second sub-region (820), which binds the second subregion and first sub-region of the circularized barcoded oligonucleotide (e.g., FIG. 16).

[00279] In some embodiments, at step (b), the circularized barcoded oligonucleotide (500) comprises any combination of: (iv) a sample index sequence (310); (v) a batch barcode sequence (320); and / or (vi) a compaction oligonucleotide binding site sequence (415) (or a complementary sequence thereof). In some embodiments, the circularized barcoded oligonucleotide (500) comprises any combination, and arranged in any order: (iv) a sample index sequence (310); (v) a batch barcode sequence (320), and / or (vii) a compaction oligonucleotide binding site sequence (415) (e.g., FIG. 17). In some embodiments, the sample index sequence (310) can be used to distinguish cellular samples from different sources, e.g., in a multiplex assay. In some embodiments, a batch barcode sequence (320) can be used for batch sequencing.

[00280] In some embodiments, at step (b), the target barcode sequence (300) of the circularized barcoded oligonucleotide (500) comprises a short random sequence comprising 3-20 nucleotides in length, or any range therebetween (e.g., NNN or NNNN). In some embodiments, the short random sequence is designed to provide nucleotide diversity and color balance, e.g., of detectable reporter moieties, when sequencing concatemers generated by amplifying the circularized barcoded oligonucleotides (500). In some embodiments, the circularized barcoded oligonucleotide (500) lacks a short random sequence.

[00281] In some embodiments, in the short random sequence each base “N” at a given position is independently selected from A, G, C, T or U. In some embodiments, the random sequence lacks consecutive repeat sequences having 2 or 3 of the same nucleo-base, for example AA, TT, CC, GG, UU, AAA, TTT, CCC, GGG or UUU.

[00282] In some embodiments, in a population of target probe complexes (900), the short random sequence comprises a high diversity sequence which includes approximately equal proportions of all four nucleotides (e.g., A, G, C, T and / or U) that will be represented in each cycle of a sequencing run.

[00283] In some embodiments, the short random sequence (e.g., NNN) includes, but is not limited to, AGC, AGT, GAC, GAT, CAT, CAG, TAG, TAC. The skilled artisan will recognize that many more random sequences can be prepared (e.g., 64 possible combinations) where each base “N” at a given position in the random sequence is independently selected from A, G, C, T or U.

[00284] In some embodiments, at step (b), individual target probes (600) comprise singlestranded oligonucleotides. In some embodiments, individual target probes (600) comprise DNA, RNA or chimeric DNA / RNA. In some embodiments, individual target probes (600) comprise canonical nucleotides or nucleotide analogs or a combination of both.

[00285] In some embodiments, at step (b), individual target probes (600) comprise a target binding moiety (700) and a universal probe region (800) (e.g., FIGS. 15 and 22). In some embodiments, individual target probes (600) comprise a target binding region which selectively binds a target polynucleotide. In some embodiments, the target binding moiety (700) is joined directly or non-directly to a universal probe region (800) which is designed to bind to the universal circularized region (400) of a circularized barcoded oligonucleotide (500). In some embodiments, the target binding moiety (700) can be 5-100 nucleotides in length, 15-75 nucleotides in length, 20-50 nucleotides in length, or any range therebetween. In some embodiments, the target binding moiety (700) can be 15-75 nucleotides in length, or any range therebetween. In some embodiments, the target binding moiety (700) comprises a poly-T sequence having 3-50 consecutive thymine bases, or any range therebetween. In some embodiments, the universal probe region (800) can be 5-100 nucleotides in length, or any range therebetween. In some embodiments, the universal probe region (800) can be 15-75 nucleotides in length, or any range therebetween.

[00286] In some embodiments, at step (b), the universal probe region comprises a first sub-region (810) and a second sub-region (820). In some embodiments, the first sub-region (810) of the target probe (600) binds the second sub-region of the universal circularized region (420). In some embodiments, the second sub-region (820) of the target probe (600) binds the first sub-region of the universal circularized region (410). See for example and without limitation, FIGS. 16, 17 and 22.

[00287] In some embodiments, at step (b), the target probes (600) comprise a 3' OH extendible end, or a 3' non-extendible end that can be converted into a 3' OH extendible end. In some embodiments, the target probes (600) comprise a 5' end that inhibits ligation.

[00288] In some embodiments, at step (b), the target probes (600) comprise one or more phosphorothioate linkage at their 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the target probes (600) comprise one or more phosphorothioate linkages at an internal position to confer endonuclease resistance. In some embodiments, the target probes (600) comprise one or more 2’-O-methylcytosine bases at their 5' and / or 3' ends, or at an internal position. In some embodiments, the 5' end of the target probes (600) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the target probes (600) comprises a terminal 3' OH group or a terminal 3' blocking group.

[00289] In some embodiments, at step (b), the target probe (600) comprises an optional linker region (710) which is located proximal to the target binding moiety (700) (e.g., FIGS. 17 and 22). In some embodiments, the linker region (710) comprises a polynucleotide having a sequence that does not hybridize to the target polynucleotide. In some embodiments, the linker region (710) comprises a polynucleotide having a sequence that does not hybridize to any portion of the circularized barcoded oligonucleotide (500). In some embodiments, the linker region (710) comprises a spacer, e.g., an 18-carbon spacer (e.g., a hexa-ethyleneglycol spacer), multiple C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the linker region (710) comprises a polyethylene glycol moiety, for example and without limitation, a PEG2, a PEG3 or a PEG4 spacer. In some embodiments, a PEG2 spacer comprises two polyethylene glycol units, a PEG3 spacer comprises three polyethylene glycol units, and a PEG4 spacer comprises four polyethylene glycol units.

[00290] In some embodiments, at step (b), the target probe (600) further comprises a compaction oligonucleotide binding region (815) (or a complementary sequence thereof) which is located between the first sub-region (810) and the second sub-region (820). Exemplary embodiments of various target probes (600) are shown in FIG. 22.

[00291] In some embodiments, the method for generating a plurality of nucleic acid concatemer molecules in situ comprises step (c): contacting the cellular sample with the plurality of the nucleic acid target probe complexes (900), wherein the contacting is conducted under a condition suitable for moving the plurality of the nucleic acid target probe complexes (900) into the cellular sample and selectively binding individual target probe complexes (900) to at least a portion of their cognate target polynucleotides inside the cellular sample.

[00292] In some embodiments, the contacting of step (c) comprises contacting at least a first sub-population of target probe complexes (first target probe complexes, 900-1) and a second sub-population of target probe complexes (second target probe complexes, 900-2) with the cellular sample, wherein the contacting is conducted under conditions suitable for moving the first and second sub-populations of nucleic acid target probe complexes (900) into the cellular sample and selectively binding individual target probe complexes (e.g., (9001) and (900-2), respectively) to at least a portion of their cognate target polynucleotides inside the cellular sample.

[00293] In some embodiments, the contacting of step (c) can be conducted in a nucleic acid hybridization solution. In some embodiments, the target binding moiety (700) of a given target probe complex (900) selectively binds at least a portion of a target polynucleotide. In some embodiments, the target binding moiety (700-1) of a first target probe complex (900-1) selectively binds a first portion of a first target polynucleotide, and the target binding moiety (700-2) of a second target probe complex (900-2) selectively binds a second portion of the same first target polynucleotide. In some embodiments, the target binding moiety (700-1) of a first target probe complex (900-1) selectively binds at least a portion of a first target polynucleotide, and the target binding moiety (700-2) of a second target probe complex (9002) selectively binds at least a portion of a second target polynucleotide, where the first and second target polynucleotides are different target polynucleotides. In some embodiments, the target binding moi eties of the first and second target probe complexes ((900-1) and (900-2), respectively) have different sequences. In some embodiments, the concentration of target probe complexes in the plurality ((900-1) and / or (900-2)) can be about 1-5 uM. In some embodiments, the concentration of target probe complexes in the plurality ((900-1) and / or (900-2)) can be about 1-500 nM, or about 500-1000 nM, or about 0.1 nM - 1 nM, or about 15 nM, or about 5-25 nM, or about 25-50 nM, or about 50-100 nM.

[00294] In some embodiments, at step (c), the cellular sample is contacted with the plurality of the nucleic acid target probe complexes (900) in the presence of a hybridization solution. In some embodiments, the hybridization solution comprises a saline-sodium citrate (SSC) solution. In some embodiments, the hybridization solution comprises a 2X, 3X, 4X, 5X, 7X, 8X, 9X or 10X SSC solution. In some embodiments, the hybridization solution comprises a 2X, 3X or 5X SSC solution. In some embodiments, the hybridization solution comprises acetonitrile. In some embodiments, the hybridization solution comprises DMSO. In some embodiments, the hybridization solution comprises Denhardt’s solution which includes Ficoll (e.g., Ficoll 400), polyvinylpyrrolidone and BSA. In some embodiments, the hybridization solution comprises betaine. In some embodiments, the hybridization solution comprises trehalose. In some embodiments, the hybridization solution comprises guanidinium isothiocyanate (GITC). In some embodiments, the hybridization solution comprises any one or any combination of two or more of a pH buffering agent, a chelator, a detergent, a denaturing agent, a crowding agent and / or a blocking agent. In some embodiments, the hybridization solution comprises a sodium salt (e.g., NaCl). In some embodiments, the pH buffering agent comprises HEPES (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or MES (e.g., 2-(A-morpholino)ethanesulfonic acid). In some embodiments, the chelator comprises EDTA (ethylenediaminetetraacetic acid) and / or EGTA (ethylene glycol tetraacetic acid). In some embodiments, the detergent comprises Tween-20, Triton-X 100 and / or SDS. In some embodiments, the denaturing agent comprises formamide, 2-pyrollidone, urea and / or ethylene carbonate. In some embodiments, the crowding agent comprises dextran sulfate and / or PEG, for example and without limitation, 1KPEG, 2KPEG, 4KPEG, 5KPEG and / or PEG200. In some embodiments, the blocking agent comprises BSA.

[00295] In some embodiments, the method for generating a plurality of nucleic acid concatemer molecules in situ comprises step (d): contacting the plurality of target probe complexes (900) (e.g., which are bound to their cognate target polynucleotides) with a rolling circle amplification reaction mixture, and conducting a rolling circle amplification reaction inside the cellular sample, thereby generating a plurality of concatemer molecules inside the cellular sample, wherein individual concatemer molecules have a sequence that is complementary to their cognate circularized barcoded oligonucleotide (500) or their cognate nucleic acid target probe complexes (900). In some embodiments, the concatemer molecules comprise a plurality of tandem repeat polynucleotide units wherein each unit comprises a sequence that is complementary to the circularized barcoded oligonucleotide (500). In some embodiments, a given concatemer molecule corresponds to a given target polynucleotide. In some embodiments, the polynucleotide unit of a concatemer molecule comprises: a target barcode sequence (300), a sequencing primer binding site sequence (200) (or a complementary sequence thereof), and a universal circularized region (400). In some embodiments, the polynucleotide unit of a concatemer molecule comprises: a target barcode sequence (300), a sequencing primer binding site sequence (200) (or a complementary sequence thereof), a universal circularized region second sub-region (420), and universal circularized first sub-region (410). In some embodiments, the polynucleotide unit of a concatemer molecule comprises: a batch barcode sequence (320), a sample index sequence (310), a target barcode sequence (300), a sequencing primer binding site sequence (200) (or a complementary sequence thereof), a universal circularized region second sub-region (420), a compaction oligonucleotide binding site sequence (415) (or a complementary sequence thereof), and universal circularized first sub-region (410).

[00296] In some embodiments, at step (d), the rolling circle amplification reagents comprise a plurality of strand-displacing DNA polymerases and a plurality of nucleotides. In some embodiments, in the assembled target probe complex (900) the 3' end of the target probe (600) provides an initiation site for the rolling circle amplification. In some embodiments, a soluble amplification primer is not needed to conduct rolling circle amplification. In some embodiments, the plurality of strand-displacing DNA polymerases comprises polymerases that exhibit the ability to displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include any of the strand displacing polymerases described herein.

[00297] In some embodiments, at step (d), the plurality of nucleotides comprises any combination of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, at least one of the nucleotides comprises a scissile moiety which can be converted to an abasic site as described herein. In some embodiments, nucleotides comprising a scissile moiety include uridine, 8-oxo-7,8-dihydrogunine and deoxyinosine. In some embodiments, the plurality of nucleotides comprises at least one nucleotide labeled with a detectable reporter moiety. For example, a detectable reporter moiety can comprise a fluorophore.

[00298] In some embodiments, at step (d), the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides. In some embodiments, the compaction oligonucleotides bind portions of the concatemer molecules generated by conducting a rolling circle amplification region. In some embodiments, individual compaction oligonucleotides comprise a 5' region that binds a first portion of the concatemer molecule and a 3' region that binds a second portion of the same concatemer molecule. In some embodiments, the 5' and 3' regions of a compaction oligonucleotide can hybridize to binding sites in a concatemer molecule to pull together distal portions of the concatemer molecule causing compaction of the concatemer molecule, e.g., to form a DNA nanoball. In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a universal probe region (800) sequence. In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a universal circularized region (400). In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a first sub-region (410) of a universal circularized region. In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a second sub-region (420) of a universal circularized region. In some embodiments, a compaction oligonucleotide can bind a portion of the concatemer molecule having a compaction oligonucleotide binding site sequence (415).

[00299] In some embodiments, at step (d), when the rolling circle amplification reaction includes a plurality of nucleotides which includes dUTP, the resulting concatemer molecule can be cross-linked to a cross-linking reactive group by treating the cellular sample with a succinimide ester (NHS), maleimide (Sulfo-SMCC), imidoester (DMP), carbodiimide (DCC, EDC) or phenyl azide. In some embodiments, polymerization of the cross-linking reactive group can be initiated with light or ultraviolet (UV) light. In some embodiments, the resulting concatemer molecule can be cross-linked to an intracellular matrix, e.g., by treating the cellular sample with a cross-linked agarose, cross-linked dextran or cross-linked polyethylene glycol (PEG), polyacrylamide, cellulose alginate or polyamide. In some embodiments, the PEG comprises a sulfo-NHS ester moiety at one or both ends, for example a PEGylated bis(sulfosuccinimidyl)suberate) (e.g., BS(PEG)9 from Thermo Fisher Scientific®, catalog No. 21582). In some embodiments, the rolling circle amplification reaction lacks a crosslinking reactive group. In some embodiments, the concatemer molecule is not cross-linked to an intracellular matrix.

[00300] In some embodiments, the method for generating a plurality of nucleic acid concatemer molecules in situ comprises step (e): sequencing the plurality of concatemer molecules inside the cellular sample. In some embodiments, the sequencing of step (e) can be performed using any one of the methods described for steps (el), (e2) or (e3) described below.

[00301] In some embodiments, the sequencing of step (el) comprises use of first batch sequencing primers comprising terminal 3’ extendible ends, second batch sequencing primers comprising terminal 3’ blocked ends, a plurality of sequencing polymerases, and nucleotide reagents comprising nucleotides and / or multivalent molecules comprising a plurality of nucleotide moieties. The sequencing of step (el) is described below.

[00302] In some embodiments, the sequencing of step (e2) comprises use of first and second batch sequencing primers having terminal 3’ extendible ends, a plurality of sequencing polymerases, and nucleotide reagents comprising nucleotides and / or multivalent molecules comprising a plurality of nucleotide moieties. The sequencing of step (e2) is described below.

[00303] In some embodiments, the sequencing of step (e3) comprises use of multivalent probes comprising a plurality of target-specific oligonucleotide probe arms. The sequencing-by-hybridization of step (e3) is described below. Sequencing Step (el)

[00304] In some embodiments, the method of the disclosure comprises step (el): sequencing the concatemer molecules inside the cellular sample. In some embodiments, the concatemer molecules serve as the template molecules to be sequenced. In some embodiments, the plurality of concatemer molecules comprise at least first and second concatemer molecules. In some embodiments, the sequencing of step (el) comprises contacting the cellular sample with a plurality of soluble sequencing primers, a plurality of sequencing polymerases, and a plurality of nucleotide reagents (e.g., nucleotides and / or multivalent molecules comprising a plurality of nucleotide moieties), wherein the contacting is conducted under a condition suitable for moving the plurality of soluble sequencing primers, the plurality of sequencing polymerases and the plurality of nucleotide reagents into the cellular sample and suitable for hybridizing the plurality of sequencing primers with their cognate concatemer molecules. In some embodiments, the entire cellular sample is contacted with a plurality of the multivalent molecules. In some embodiments, a portion of the cellular sample is contacted with the plurality of multivalent molecules. In some embodiments, the sequencing of step (el) comprises contacting the plurality of concatemer molecules inside the cellular sample with the plurality of soluble sequencing primers, the plurality of sequencing polymerases, and the plurality of nucleotide reagents (e.g., nucleotides and / or multivalent molecules comprising a plurality of nucleotide moieties), and conducting at least one polymerase-catalyzed sequencing cycle thereby generating a plurality of sequencing read products inside the cellular sample.

[00305] In some embodiments, in the sequencing of step (el), the plurality of soluble sequencing primers comprises the same sequence. In some embodiments, the plurality of soluble sequencing primers comprises at least a first and second sub-population of sequencing primers having different sequences.

[00306] In some embodiments, in the sequencing of step (el), the sequencing comprises batch sequencing the plurality of concatemer molecules inside the cellular sample. In some embodiments, the plurality of concatemer molecules of step (d) comprises at least a first and a second sub-population of concatemer molecules (e.g., first and second batches of concatemer molecules), wherein the sequencing further comprises: (1) sequencing the first sub-population of concatemer molecules while inhibiting sequencing the second subpopulation of concatemer molecules, wherein sequencing the first sub-population of concatemer molecules comprises contacting the first sub-population of the concatemer molecules with a first plurality of soluble sequencing primers (e.g., first batch sequencing primers) and conducting a first plurality of sequencing cycles thereby generating a first plurality of sequencing read products; and (2) sequencing the second sub-population of concatemer molecules while inhibiting sequencing the first sub-population of concatemers, wherein sequencing the second sub-population of concatemer molecules comprises contacting the second sub-population of the concatemer molecules with a second plurality of soluble sequencing primers (e.g., second batch sequencing primers) and conducting a second plurality of sequencing cycle thereby generating a second plurality of sequencing read products. In some embodiments, the first batch sequencing conducted in step (1) comprises conducting no more than 50 polymerase-catalyzed sequencing cycles, thereby generating a plurality of first batch sequencing read products that are no longer than 50 bases. In some embodiments, the first batch sequencing conducted in step (1) comprises conducting 50-200 polymerase-catalyzed sequencing cycles, thereby generating a plurality of first batch sequencing read products that are 50-200 bases in length. In some embodiments, the second batch sequencing conducted in step (2) comprises conducting no more than 50 polymerase-catalyzed sequencing cycles, thereby generating a plurality of second batch sequencing read products that are no longer than 50 bases. In some embodiments, the second batch sequencing conducted in step (2) comprises conducting 50-200 polymerase-catalyzed sequencing cycles, thereby generating a plurality of second batch sequencing read products that are 50-200 bases in length.

[00307] In some embodiments, the sequencing of step (el) comprises contacting the first and second sub-population of concatemer molecules essentially simultaneously with a plurality of soluble first and second batch sequencing primers.

[00308] In some embodiments, the sequencing of step (el) comprises: step (1) sequencing the first sub-population of concatemer molecules while inhibiting sequencing the second subpopulation of concatemer molecules by (i) contacting the first sub-population of concatemer molecules with a plurality of soluble first batch sequencing primers under conditions suitable to hybridize individual first batch sequencing primers to the first sub-population of concatemer molecules wherein individual soluble first batch sequencing primers in the plurality comprise an extendible terminal 3’ end which permits polymerase-catalyzed extension of the first batch sequencing primers that are hybridized to the first sub-population of concatemer molecules, and conducting a first plurality of sequencing cycles thereby generating a first plurality of sequencing read products; and (ii) contacting the second subpopulation of concatemer molecules with a plurality of soluble second batch sequencing primers under conditions suitable to hybridize individual second batch sequencing primers to the second sub-population of concatemer molecules, wherein individual soluble second batch sequencing primers in the plurality comprise a terminal 3’ reversible blocking moiety which blocks polymerase-catalyzed extension of the second batch sequencing primers that are hybridized to the second sub-population of concatemer molecules.

[00309] In some embodiments, in the sequencing of step (el) (1), the sequencing comprises conducting no more than 50 polymerase-catalyzed sequencing cycles of the first concatemer molecules, thereby generating a plurality of first batch sequencing read products that are no longer than 50 bases. In some embodiments, the sequencing comprises conducting 50 - 200 polymerase-catalyzed sequencing cycles of the first concatemer molecules, thereby generating a plurality of first batch sequencing read products that are 50 - 200 bases in length. In some embodiments, the sequencing comprises sequencing only the target barcode sequence (300) of the first concatemer molecules. In some embodiments, the sequencing comprises sequencing the sample index sequence (310) of the first concatemer molecules. In some embodiments, the sequencing comprises sequencing the batch barcode sequence (320) of the first concatemer molecules. In some embodiments, the sequencing comprises sequencing any combination of (i) the target barcode sequence (300) of the concatemer molecules, (ii) the sample index sequence (310) of the concatemers, and / or (iii) the batch barcode sequence (320) of the first concatemer molecules.

[00310] In some embodiments, in the sequencing of step (el) (1), the plurality of first sequencing read products inside the cellular sample can be detected by imaging. In some embodiments, at least a sub-set of the plurality of the first sequencing read products inside the cellular sample are detected essentially simultaneously by imaging. In some embodiments, a first sub-population of concatemer molecules are sequenced using first-batch sequencing primers, thereby generating a first batch of sequencing read products inside the cellular sample which are detected by imaging the first batch of sequencing read products.

[00311] In some embodiments, the sequencing of step (el) (1) comprises sequencing at least a portion of the first sub-population of concatemer molecules inside the cellular sample using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, individual cycle times can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm2 and the cycle time for scanning large area (> 10 mm2) can be less than 5 minutes.

[00312] In some embodiments, the sequencing of step (el) comprises: step (2) blocking the terminal 3 ’ end of the first plurality of sequencing read products to inhibit polymerase-catalyzed extension of the first batch sequencing primers that are hybridized to the first subpopulation of concatemer molecules (e.g., by incorporating a dideoxynucleotide nucleotide triphosphate at the terminal 3’ end of the first plurality of sequencing read products).

[00313] In some embodiments, the sequencing of step (el) comprises: step (3) converting the terminal 3’ reversible blocking moi eties of the second batch sequencing primers to terminal 3’ extendible moi eties wherein the converting permits polymerase-catalyzed extension of the second batch sequencing primers that are hybridized to the second subpopulation of concatemer molecules.

[00314] In some embodiments, the sequencing of step (el) comprises: step (4) conducting a second plurality of sequencing cycles thereby generating a second plurality of sequencing read products.

[00315] In some embodiments of step (el) (4), the sequencing comprises conducting no more than 50 polymerase-catalyzed sequencing cycles of the second concatemer molecules, thereby generating a plurality of second batch sequencing read products that are no longer than 50 bases. In some embodiments, in the sequencing of step (el) (4), the sequencing comprises conducting 50 - 200 polymerase-catalyzed sequencing cycles of the second concatemer molecules, thereby generating a plurality of second batch sequencing read products that are 50 - 200 bases in length. In some embodiments, the sequencing comprises sequencing only the target barcode sequence (300) of the second concatemer molecules. In some embodiments, the sequencing comprises sequencing the sample index sequence (310) of the second concatemer molecules. In some embodiments, the sequencing comprises sequencing the batch barcode sequence (320) of the second concatemer molecules. In some embodiments, the sequencing comprises sequencing any combination of (i) the target barcode sequence (300) of the concatemer molecules, (ii) the sample index sequence (310) of the concatemers, and / or (iii) the batch barcode sequence (320) of the second concatemer molecules.

[00316] In some embodiments of step (el) (4), the plurality of second sequencing read products inside the cellular sample can be detected by imaging. In some embodiments, at least a sub-set of the plurality of the second sequencing read products inside the cellular sample are detected essentially simultaneously by imaging. In some embodiments, a second sub-population of concatemer molecules are sequenced using second-batch sequencing primers, thereby generating a second batch of sequencing read products inside the cellular sample which are detected by imaging the second batch of sequencing read products.

[00317] In some embodiments, the sequencing of step (el) (1) comprises sequencing at least a portion of the first sub-population of concatemer molecules inside the cellular sample using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, the sequencing of step (el) (4) comprises sequencing at least a portion of the second sub-population of concatemer molecules inside the cellular sample using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, individual sequencing cycle times for steps (el) (1) and / or (el) (4) can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) for steps (el) (1) and / or (el) (4) can exceed 1 mm2 and the sequencing cycle time for scanning a large area (> 10 mm2) can be less than 5 minutes. For in situ sequencing, the limit of optical resolution may impede the ability to perform highly multiplex sequencing. The batch sequencing workflow can enable sequencing a desired subset (e.g., a batch) of the concatemer molecules using selected batch-specific sequencing primers to reduce over-crowding signals and images. The use of batch-specific sequencing primers can produce optical images that provide intense signal and are highly resolvable. Conducting multiple rounds of sequencing on the same cellular sample, e.g., using different batch-specific sequencing primers, enables multiplex sequencing.

[00318] In some embodiments, in the sequencing of step (el), the sequencing comprises re-iterative sequencing of the same plurality of concatemer molecules inside the cellular sample, which comprises: (1) contacting the plurality of concatemer molecules with a plurality of soluble first batch sequencing primers and a plurality of nucleotide reagents, and conducting a plurality of polymerase-catalyzed sequencing cycles, thereby generating a plurality of first batch sequencing read products inside the cellular sample; (2) removing the plurality of first batch sequencing read products from the plurality of concatemer molecules while retaining the plurality of concatemer molecules inside the cellular sample; and (3) reiteratively sequencing the same plurality of concatemer molecules inside the cellular sample by repeating steps (1) - (2) at least once. In some embodiments, steps (1)-(2) can be repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, steps (1) - (2) can be repeated up to 10 times, up to 20 times, up to 30 time, up to 40 times, or up to 50 times. In some embodiments, the re-iterative sequencing conducted in step (1) comprises conducting no more than 50 polymerase-catalyzed sequencing cycles using the plurality of first batch sequencing primers thereby generating a plurality of first batch sequencing read products that are no longer than 50 bases. In some embodiments, steps (1) -(3) of the re-iterative sequencing of step (el) can be conducted using a plurality of soluble second batch sequencing primers instead of first batch sequencing primers. Sequencing Step (e2)

[00319] In some embodiments, the method of the disclosure comprises step (e2): sequencing the concatemer molecules inside the cellular sample. In some embodiments, the concatemer molecules serve as the template molecules to be sequenced. In some embodiments, the plurality of concatemer molecules of step (d) comprise at least first and second concatemer molecules. In some embodiments, the sequencing of step (e2) comprises separately contacting a first sub-population of concatemer molecules with the plurality of the first batch sequencing primers, and contacting a second sub-population of concatemer molecules with the plurality of the second batch sequencing primers.

[00320] In some embodiments, the sequencing of step (e2) comprises contacting the cellular sample with a plurality of soluble first batch sequencing primers, a plurality of sequencing polymerases, and a plurality of nucleotide reagents (e.g., nucleotides and / or multivalent molecules comprising a plurality of nucleotide moieties), wherein the contacting is conducted under a condition suitable for moving the plurality of soluble first batch sequencing primers, the plurality of sequencing polymerases and the plurality of nucleotide reagents into the cellular sample and hybridizing the plurality of first batch sequencing primers with their cognate first sub-population of concatemer molecules. In some embodiments, the entire cellular sample is contacted with a plurality of the multivalent molecules. In some embodiments, a portion of the cellular sample is contacted with the plurality of multivalent molecules.

[00321] In some embodiments, the sequencing of step (e2) comprises contacting the cellular sample with a plurality of soluble second batch sequencing primers, a plurality of sequencing polymerases, and a plurality of nucleotide reagents, wherein the contacting is conducted under a condition suitable for moving the plurality of soluble second batch sequencing primers, the plurality of sequencing polymerases and the plurality of nucleotide reagents into the cellular sample and hybridizing the plurality of second batch sequencing primers with their cognate second sub-population of concatemer molecules. In some embodiments, the entire cellular sample is contacted with the plurality of the multivalent molecules. In some embodiments, a portion of the cellular sample is contacted with the plurality of multivalent molecules.

[00322] In some embodiments, the sequencing of step (e2) comprises: step (1) contacting the plurality of first concatemer molecules inside the cellular sample with a plurality of first batch sequencing primers having terminal 3’ extendible ends and a plurality of nucleotide reagents, and conducting at least one polymerase-catalyzed sequencing cycle thereby generating a plurality of first batch sequencing read products inside the cellular sample. In some embodiments of step (e2) (1), the plurality of first batch sequencing primers comprises the same sequence. In some embodiments, the first batch sequencing primers hybridize selectively to the sequencing primer binding site sequences (200-1) of the first concatemer molecules. In some embodiments, the first batch sequencing primers do not hybridize to (or hybridize very little to) the sequencing primer binding site sequences (200-2) of the second concatemer molecules.

[00323] In some embodiments, in the sequencing of step (e2) (1), the plurality of first batch sequencing read products inside the cellular sample can be detected by imaging. In some embodiments, the plurality of first batch sequencing read products inside the cellular sample are detected essentially simultaneously by imaging.

[00324] In some embodiments, in the sequencing of step (e2) (1), the sequencing comprises conducting no more than 50 polymerase-catalyzed sequencing cycles of the first concatemer molecules, thereby generating a plurality of first batch sequencing read products that are no longer than 50 bases. In some embodiments, in the sequencing of step (e2) (1), the sequencing comprises conducting 50 - 200 polymerase-catalyzed sequencing cycles of the first concatemer molecules, thereby generating a plurality of first batch sequencing read products that are 50 - 200 bases in length. In some embodiments, the sequencing of step (e2) (1) comprises sequencing only the target barcode sequence (300) of the first concatemer molecules. In some embodiments, the sequencing of step (e2) (1) comprises sequencing the sample index sequence (310) of the first concatemer molecules. In some embodiments, the sequencing of step (e2) (1) comprises sequencing the batch barcode sequence (320) of the first concatemer molecules. In some embodiments, the sequencing of step (e2) (1) comprises sequencing any combination of (i) the target barcode sequence (300) of the concatemer molecules, (ii) the sample index sequence (310) of the concatemers, and / or (iii) the batch barcode sequence (320) of the first concatemer molecules.

[00325] In some embodiments, the sequencing of step (e2) (1) comprises sequencing at least a portion of the plurality of the first concatemer molecules inside the cellular sample using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, individual cycle times can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm2 and the cycle time for scanning large area (> 10 mm2) can be less than 5 minutes.

[00326] In some embodiments, the sequencing of step (e2) comprises: step (2) removing the plurality of first batch sequencing read products from the plurality of first concatemer molecules while retaining the plurality of concatemer molecules inside the cellular sample, wherein the plurality of first batch sequencing read products are removed by enzymatic degradation or a chemical de-hybridization reagent.

[00327] In some embodiments, the sequencing of step (e2) comprises: step (3) contacting the plurality of second concatemer molecules inside the cellular sample with a plurality of second batch sequencing primers and a plurality of nucleotide reagents, and conducting at least one polymerase-catalyzed sequencing cycle thereby generating a plurality of second batch sequencing read products inside the cellular sample. In some embodiments of step (e2) (3), the plurality of second batch sequencing primers comprises the same sequence. In some embodiments, the first batch sequencing primers and the second batch sequencing primers comprise different sequences. In some embodiments, the second batch sequencing primers hybridize selectively to the sequencing primer binding site sequence (200-2) of the second concatemer molecules. In some embodiments, the second batch sequencing primers do not hybridize to (or hybridize very little to) the sequencing primer binding site sequence (200-1) of the first concatemer molecules.

[00328] In some embodiments, in the sequencing of step (e2) (3), the plurality of second batch sequencing read products inside the cellular sample can be detected by imaging. In some embodiments, the plurality of second batch sequencing read products inside the cellular sample are detected essentially simultaneously by imaging.

[00329] In some embodiments, in the sequencing of step (e2) (3), the sequencing comprises conducting no more than 50 polymerase-catalyzed sequencing cycles of the second concatemer molecules, thereby generating a plurality of second batch sequencing read products that are no longer than 50 bases. In some embodiments, in the sequencing of step (e2) (3), the sequencing comprises conducting 50 - 200 polymerase-catalyzed sequencing cycles of the second concatemer molecules, thereby generating a plurality of second batch sequencing read products that are 50 - 200 bases in length. In some embodiments, the sequencing of step (e2) (3) comprises sequencing only the target barcode sequence (300) of the second concatemer molecules. In some embodiments, the sequencing of step (e2) (3) comprises sequencing the sample index sequence (310) of the second concatemer molecules. In some embodiments, the sequencing of step (e2) (3) comprises sequencing the batch barcode sequence (320) of the second concatemer molecules. In some embodiments, the sequencing of step (e2) (3) comprises sequencing any combination of (i) the target barcode sequence (300) of the concatemer molecules, (ii) the sample index sequence (310) of the concatemers, and / or (iii) the batch barcode sequence (320) of the second concatemer molecules.

[00330] In some embodiments, the sequencing of step (e2) (3) comprises sequencing at least a portion of the plurality of the second concatemer molecules inside the cellular sample using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, individual cycle times can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm2 and the cycle time for scanning large area (> 10 mm2) can be less than 5 minutes.

[00331] In some embodiments, the sequencing of step (e2) comprises: step (4) optionally removing the plurality of second batch sequencing read products from the plurality of second concatemer molecules while retaining the plurality of concatemer molecules inside the cellular sample, wherein the plurality of second batch sequencing read products are removed by enzymatic degradation or de-hybridization reagent.

[00332] In some embodiments, additional batch sequencing primers can be used to conduct subsequent batch sequencing according to steps (e2) (1) - (4). For example, additional batch sequencing primers include a third, fourth, fifth, sixth, seventh, eighth, ninth and tenth batch sequencing primers. In some embodiments, steps (e2) (1)-(4) can be conducted using up to 100 different batch sequencing primers.

[00333] For in situ sequencing, the limit of optical resolution may impede the ability to perform highly multiplex sequencing. The batch sequencing workflow can enable sequencing a desired subset (e.g., a batch) of the concatemers using selected batch-specific sequencing primers to reduce over-crowding signals and images. The use of batch-specific sequencing primers can produce optical images that provide intense signal and are highly resolvable. Conducting multiple rounds of sequencing on the same cellular sample, e.g., using different batch-specific sequencing primers, enables multiplex sequencing.

[00334] In some embodiments of step (e2), the sequencing comprises re-iterative sequencing of the same plurality of concatemer molecules inside the cellular sample, which comprises: (1) contacting the plurality of first concatemer molecules with a plurality of first batch sequencing primers and a plurality of nucleotide reagents (e.g., nucleotides and / or multivalent molecules comprising a plurality of nucleotide moieties), and conducting a plurality of polymerase-catalyzed sequencing cycles, thereby generating a plurality of first batch sequencing read products inside the cellular sample; (2) removing the plurality of first batch sequencing read products from the plurality of concatemer molecules while retaining the plurality of concatemer molecules inside the cellular sample; and (3) reiteratively sequencing the same plurality of first concatemer molecules inside the cellular sample by repeating steps (1) - (2) at least once. In some embodiments, steps (1) - (2) can be repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, steps (1) - (2) can be repeated up to 10 times, up to 20 times, up to 30 time, up to 40 times, or up to 50 times. In some embodiments, the re-iterative sequencing conducted in step (1) comprises conducting no more than 50 polymerase-catalyzed sequencing cycles using the plurality of first batch sequencing primers thereby generating a plurality of first batch sequencing read products that are no longer than 50 bases. In some embodiments, steps (1) - (3) of the re-iterative sequencing of step (e2) can be conducted using a plurality of second batch sequencing primers instead of first batch sequencing primers in order to re-iteratively sequence the second concatemer molecules. Sequencing Step (e3)

[00335] In some embodiments, the method of the disclosure comprises step (e3): conducting a sequencing-by-hybridization method comprising contacting the plurality of concatemer molecules inside the cellular sample with a plurality of detectably labeled multivalent probes and conducting at least one probe hybridization cycle thereby generating a plurality of concatemer-probe complexes inside the cellular sample. In some embodiments, the plurality of concatemer-probe complexes inside the cellular sample can be detected by imaging. In some embodiments, the concatemer molecules serve as the template molecules to be detected and sequenced. In some embodiments, the plurality of concatemer molecules comprise at least first and second concatemer molecules. In some embodiments, the detection of the concatemer-probe complexes confirms the presence of a concatemer molecule comprising a target sequence. In some embodiments, sequencing inside a cellular sample can be conducted by hybridizing a plurality of multivalent probes to concatemer molecules inside the cellular sample. In some embodiments, individual multivalent probes comprise a core attached to a plurality of probe arms wherein individual probe arms comprises a polymer arm linked to a target-specific oligonucleotide probe (e.g., FIGS. 5A and 5B).

[00336] In some embodiments, detecting the presence of a first and second target sequences inside a cellular sample comprises using at least two different multivalent probes which selectively hybridize to their cognate target sequences on the first or second concatemer molecules. In some embodiments, a method for detecting the presence of a first and second target sequences inside a cellular sample comprises: step (1) contacting the cellular sample with a first sub-population of detectably labeled multivalent probes under a condition suitable for moving the first sub-population of detectably labeled multivalent probes into the cellular sample and suitable for selectively hybridizing the first subpopulation of detectably labeled multivalent probes to at least a first portion of individual concatemer molecules in the first sub-population thereby generating a first sub-population of detectably labeled concatemer-probe complexes, wherein individual detectably labeled multivalent probes in the first sub-population comprise a (i) core, (ii) a plurality of probe arms wherein individual probe arms comprise a first target-specific probe sequence, and (iii) a detectable reporter moiety that uniquely identifies the first target-specific probe sequence, wherein individual detectably labeled concatemer-probe complexes of the first subpopulation generate a signal inside the cellular sample; step (2) detecting the signal generated by the first sub-population of detectably labeled concatemer-probe complexes inside the cellular sample thereby confirming the presence of a first sub-population of concatemer molecules comprising a first target sequence; step (3) removing the detectably labeled multivalent probes of the first sub-population from the first sub-population of detectably labeled concatemer-probe complexes while retaining the first sub-population of concatemer molecules; step (4) contacting the cellular sample with a second sub-population of detectably labeled multivalent probes under a condition suitable for moving the second sub-population of detectably labeled multivalent probes into the cellular sample and selectively hybridizing the second sub-population of detectably labeled multivalent probes to at least a second portion of individual concatemer molecules in the second sub-population thereby generating a second sub-population of detectably labeled concatemer-probe complexes, wherein individual detectably labeled multivalent probes in the second sub-population comprise a (i) core, (ii) a plurality of probe arms wherein individual probe arms comprise a second targetspecific probe sequence, and (iii) a detectable reporter moiety that uniquely identifies the second target-specific probe sequence, wherein individual detectably labeled concatemer-probe complexes of the second sub-population generate a signal inside the cellular sample; step (5) detecting the signal generated by the second sub-population of detectably labeled concatemer-probe complexes inside the cellular sample thereby confirming the presence of a second sub-population of concatemer molecules comprising a second target sequence; and step (6) optionally removing the detectably labeled multivalent probes of the second subpopulation from the second sub-population of detectably labeled concatemer-probe complexes while retaining the second sub-population of concatemer molecules.

[00337] In some embodiments, when step (e3) comprises using at least two different multivalent probes, the contacting of steps (1) and (4) can be conducted essentially simultaneously or separately. In some embodiments, the cellular sample can be contacted with the first and second sub-population of detectably labeled multivalent probes essentially simultaneously or separately. In some embodiments the detecting of steps (2) and (5) can be conducted essentially simultaneously or separately. In some embodiments, the first and second sub-population of detectably labeled concatemer-probe complexes inside the cellular sample can be detected essentially simultaneously or separately. In some embodiments, the detecting of steps (2) and (5) can be performed by imaging the signals generated by the first and second sub-populations of detectably labeled concatemer-probe complexes.

[00338] In some embodiments, when step (e3) comprises using at least two different multivalent probes, the contacting of step (1) comprises selectively hybridizing the first subpopulation of detectably labeled multivalent probes to the first sub-population of concatemer molecules while hybridizing non-labeled blocking oligonucleotides to the second subpopulation of concatemer molecules.

[00339] In some embodiments, when step (e3) comprises using at least two different multivalent probes, the contacting of step (4) comprises selectively hybridizing the second sub-population of detectably labeled multivalent probes to the second sub-population of concatemer molecules while hybridizing non-labeled blocking oligonucleotides to the first sub-population of concatemer molecules.

[00340] In some embodiments, when step (e3) comprises using at least two different multivalent probes, the selectively hybridizing of steps (1) and / or (4) comprises selectively hybridizing the detectably labeled multivalent probe to the target barcode sequence (300) of the concatemer molecules. In some embodiments, the selectively hybridizing of steps (1) and / or (4) comprises selectively hybridizing the detectably labeled multivalent probe to the sample index sequence (310) of the concatemer molecules. In some embodiments, the selectively hybridizing of steps (1) and / or (4) comprises selectively hybridizing the detectably labeled multivalent probe to the batch barcode sequence (320) of the concatemer molecules. In some embodiments, the selectively hybridizing of steps (1) and / or (4) comprises selectively hybridizing the detectably labeled multivalent probe to any combination of (i) the target barcode sequence (300) of the concatemer molecules, (ii) the sample index sequence (310) of the concatemer molecules, and / or (iii) the batch barcode sequence (320) of the concatemer molecules.

[00341] In some embodiments, when step (e3) comprises using at least two different multivalent probes, the detecting of steps (2) and / or (5) comprises using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, the selectively hybridizing of steps (1) and / or (4) can be achieved in less than about 2 minutes, or can be achieved in about 2-5 minutes, or about 5-10 minutes, or about 10-20 minutes, or about 20-60 minutes, or any range therebetween. In some embodiments, the field of view (FOV) can exceed 1 mm2 and the cycle time for scanning large area (> 10 mm2) can be about 2 minutes, or can be achieved in about 2-5 minutes, or about 5-10 minutes, or about 10-20 minutes, or about 20-60 minutes, or any range therebetween. For in situ detecting, the limit of optical resolution may impede the ability to perform highly multiplex detection. The batch detection workflow can enable detecting a desired subset (e.g., a batch) of the concatemer molecules using selected batch-specific multivalent probes to reduce over-crowding signals and images. The use of batch-specific multivalent probes can produce optical images that provide intense signal and are highly resolvable. Conducting multiple rounds of sequencing-by-hybridization on the same cellular sample, e.g., using different batch-specific multivalent probes, enables multiplex sequencing-by-hybridization.

[00342] In some embodiments, at step (e3), detecting the presence of four target sequences inside a cellular sample comprises using at least four different multivalent probes which selectively hybridize to their cognate target sequences on the first or second concatemer molecules. In some embodiments, the method comprises detecting the presence of a first, second, third and fourth target sequences inside a cellular sample. In such embodiments, the method comprises: step (1) contacting the cellular sample with a first sub-population of detectably labeled multivalent probes under a condition suitable for moving the first subpopulation of detectably labeled multivalent probes into the cellular sample and selectively hybridizing the first sub-population of detectably labeled multivalent probes to a first target sequence of individual concatemer molecules in the first sub-population thereby generating a first sub-population of detectably labeled concatemer-probe complexes, wherein individual detectably labeled multivalent probes in the first sub-population comprise a (i) core, (ii) a plurality of probe arms wherein individual probe arms comprise a first target-specific probe sequence, and (iii) a detectable reporter moiety that uniquely identifies the first target-specific probe sequence, wherein individual detectably labeled concatemer-probe complexes of the first sub-population generate a signal inside the cellular sample; step (2) detecting the signal generated by the first sub-population of detectably labeled concatemer-probe complexes inside the cellular sample thereby confirming the presence of the first sub-population of concatemer molecules comprising the first target sequence; step (3) removing the detectably labeled multivalent probes of the first sub-population from the first sub-population of detectably labeled concatemer-probe complexes while retaining the first sub-population of concatemer molecules; step (4) contacting the cellular sample with a second sub-population of detectably labeled multivalent probes under a condition suitable for moving the second sub-population of detectably labeled multivalent probes into the cellular sample and selectively hybridizing the second sub-population of detectably labeled multivalent probes to a second target sequence of individual concatemer molecules in the first sub-population thereby generating a second sub-population of detectably labeled concatemer-probe complexes, wherein individual detectably labeled multivalent probes in the second subpopulation comprise a (i) core, (ii) a plurality of probe arms wherein individual probe arms comprise a second target-specific probe sequence, and (iii) a detectable reporter moiety that uniquely identifies the second target-specific probe sequence, wherein individual detectably labeled concatemer-probe complexes of the second sub-population generate a signal inside the cellular sample; step (5) detecting the signal generated by the second sub-population of detectably labeled concatemer-probe complexes inside the cellular sample thereby confirming the presence of the first sub-population of concatemer molecules comprising the second target sequence; step (6) removing the detectably labeled multivalent probes of the second sub-population from the second sub-population of detectably labeled concatemer-probe complexes while retaining the first sub-population of concatemer molecules; step (7) contacting the cellular sample with a third sub-population of detectably labeled multivalent probes under a condition suitable for moving the third sub-population of detectably labeled multivalent probes into the cellular sample and selectively hybridizing the third subpopulation of detectably labeled multivalent probes to a third target sequence of individual concatemer molecules in the second sub-population of concatemer molecules thereby generating a third sub-population of detectably labeled concatemer-probe complexes, wherein individual detectably labeled multivalent probes in the third sub-population comprise a (i) core, (ii) a plurality of probe arms wherein individual probe arms comprise a third targetspecific probe sequence, and (iii) a detectable reporter moiety that uniquely identifies the third target-specific probe sequence, wherein individual detectably labeled concatemer-probe complexes of the third sub-population generate a signal inside the cellular sample; step (8) detecting the signal generated by the third sub-population of detectably labeled concatemer-probe complexes inside the cellular sample thereby confirming the presence of a second subpopulation of concatemer molecules comprising the third target sequence; step (9) removing the detectably labeled multivalent probes of the third sub-population from the third subpopulation of detectably labeled concatemer-probe complexes while retaining the second subpopulation of concatemer molecules; step (10) contacting the cellular sample with a fourth sub-population of detectably labeled multivalent probes under a condition suitable for moving the fourth sub-population of detectably labeled multivalent probes into the cellular sample and selectively hybridizing the fourth sub-population of detectably labeled multivalent probes to a fourth target sequence of individual concatemer molecules in the second sub-population of concatemer molecules thereby generating a fourth sub-population of detectably labeled concatemer-probe complexes, wherein individual detectably labeled multivalent probes in the fourth sub-population comprise a (i) core, (ii) a plurality of probe arms wherein individual probe arms comprise a fourth target-specific probe sequence, and (iii) a detectable reporter moiety that uniquely identifies the fourth target-specific probe sequence, wherein individual detectably labeled concatemer-probe complexes of the fourth sub-population generate a signal inside the cellular sample; step (11) detecting the signal generated by the fourth sub-population of detectably labeled concatemer-probe complexes inside the cellular sample thereby confirming the presence of the second sub-population of concatemer molecules comprising the fourth target sequence; and step (12) optionally removing the detectably labeled multivalent probes of the fourth sub-population from the fourth sub-population of detectably labeled concatemer-probe complexes while retaining the second sub-population of concatemer molecules.

[00343] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the contacting of steps (1) and (4) can be conducted essentially simultaneously or separately. In some embodiments, the cellular sample can be contacted with the first and second sub-population of detectably labeled multivalent probes essentially simultaneously or separately. In some embodiments, the detectably labeled multivalent probes of the first sub-population can hybridize to the first target sequence on the first subpopulation of concatemer molecules, and the detectably labeled multivalent probes of the second sub-population can hybridize to the second target sequence on the first sub-population of concatemer molecules, wherein the first target sequence overlaps or does not overlap with the second target sequence.

[00344] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the detecting of steps (2) and (5) can be conducted essentially simultaneously or separately. In some embodiments, the first and second sub-population of detectably labeled concatemer-probe complexes inside the cellular sample can be detected essentially simultaneously or separately. In some embodiments, the detecting of steps (2) and (5) can be performed by imaging the signals generated by the first and second subpopulations of detectably labeled concatemer-probe complexes.

[00345] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the contacting of steps (7) and (10) can be conducted essentially simultaneously or separately. In some embodiments, the cellular sample can be contacted with the third and fourth sub-population of detectably labeled multivalent probes essentially simultaneously or separately. In some embodiments, the detectably labeled multivalent probes of the third sub-population can hybridize to the third target sequence on the second sub-population of concatemer molecules, and the detectably labeled multivalent probes of the fourth sub-population can hybridize to the fourth target sequence on the second subpopulation of concatemer molecules, wherein the third target sequence overlaps or does not overlap with the fourth target sequence.

[00346] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the detecting of steps (8) and (11) can be conducted essentially simultaneously or separately. In some embodiments, the third and fourth sub-population of detectably labeled concatemer-probe complexes inside the cellular sample can be detected essentially simultaneously or separately.

[00347] In some embodiments, the detecting of steps (8) and (11) can be performed by imaging the signals generated by the third and fourth sub-populations of detectably labeled concatemer-probe complexes.

[00348] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the contacting of step (1) comprises selectively hybridizing the first subpopulation of detectably labeled multivalent probes to the first target sequence on the first sub-population of concatemer molecules while hybridizing non-labeled blocking oligonucleotides to the second target sequence on the first sub-population of concatemer molecules and / or while hybridizing non-labeled blocking oligonucleotides to the third target sequences on the second sub-population of concatemer molecules and / or while hybridizing non-labeled blocking oligonucleotides to the fourth target sequences on the second subpopulation of concatemer molecules.

[00349] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the contacting of step (4) comprises selectively hybridizing the second sub-population of detectably labeled multivalent probes to the second target sequence on the first sub-population of concatemer molecules while hybridizing non-labeled blocking oligonucleotides to the first target sequence on the first sub-population of concatemer molecules, the third target sequences on the second sub-population of concatemer molecules, and / or the fourth target sequences on the second sub-population of concatemer molecules.

[00350] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the contacting of step (7) comprises selectively hybridizing the third subpopulation of detectably labeled multivalent probes to the third target sequence on the second sub-population of concatemer molecules while hybridizing non-labeled blocking oligonucleotides to the first target sequence on the first sub-population of concatemer molecules, the second target sequences on the first sub-population of concatemer molecules, and / or the fourth target sequences on the second sub-population of concatemer molecules.

[00351] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the contacting of step (10) comprises selectively hybridizing the fourth sub-population of detectably labeled multivalent probes to the fourth target sequence on the second sub-population of concatemer molecules while hybridizing non-labeled blocking oligonucleotides to the first target sequence on the first sub-population of concatemer molecules, the second target sequences on the first sub-population of concatemer molecules, and / or the third target sequences on the second sub-population of concatemer molecules.

[00352] In some embodiments, when step (e3) comprises using at least four different multivalent probes, the selectively hybridizing of steps (1), (4), (7) and / or (10) comprises selectively hybridizing the detectably labeled multivalent probe to the target barcode sequence (300) of the concatemer molecules. In some embodiments, the selectively hybridizing of steps (1), (4), (7) and / or (10) comprises selectively hybridizing the detectably labeled multivalent probe to the sample index sequence (310) of the concatemer molecules. In some embodiments, the selectively hybridizing of steps (1), (4), (7) and / or (10) comprises selectively hybridizing the detectably labeled multivalent probe to the batch barcode sequence (320) of the concatemer molecules. In some embodiments, the selectively hybridizing of steps (1), (4), (7) and / or (10) comprises selectively hybridizing the detectably labeled multivalent probe to any combination of (i) the target barcode sequence (300) of the concatemer molecules, (ii) the sample index sequence (310) of the concatemer molecules, and / or (iii) the batch barcode sequence (320) of the concatemer molecules.

[00353] In some embodiments, in the sequencing of step (e3) using at least four different multivalent probes, the detecting of steps (2), (5), (8) and / or (11) comprises using an optical imaging system comprising a field-of-view (FOV) greater than 1.0 mm2. In some embodiments, the selectively hybridizing of steps (1), (4), (7) and / or (10) can be achieved in less than about 2 minutes, or can be achieved in about 2-5 minutes, or about 5-10 minutes, or about 10-20 minutes, or about 20-60 minutes, or any range therebetween. In some embodiments, the field of view (FOV) can exceed 1 mm2 and the cycle time for scanning large area (> 10 mm2) can be about 2 minutes, or can be achieved in about 2-5 minutes, or about 5-10 minutes, or about 10-20 minutes, or about 20-60 minutes, or any range therebetween. First and Second Sub-Populations of Target Probe Complexes

[00354] In some embodiments, in the method for generating a plurality of nucleic acid concatemer molecules in situ of step (b) above, the plurality of the nucleic acid target probe complexes (900) includes at least a first and second sub-population of target probe complexes (900) (e.g., a first and second set of target probe complexes).

[00355] In some embodiments, at step (b) above, individual target probe complexes in the first sub-population of target probe complexes (900-1) comprise a first circularized barcoded oligonucleotide (500-1) and a first target probe (600-1). In some embodiments, the first circularized barcoded oligonucleotide (500-1) comprises: (i) a first sequencing primer binding site sequence (200-1) (or a complementary sequence thereof), (ii) a first target barcode sequence (300-1) that corresponds to a target binding moiety of the first target probe (600-1), and (iii) a first universal circularized region (400-1) that binds a universal probe region of the first target probe (600-1). In some embodiments, the first target probe (600-1) comprises: an oligonucleotide having (i) a first target binding moiety (700-1) which selectively binds at least a portion of a first target polynucleotide, and (ii) a first universal probe region (800-1) that binds the first universal circularized region (400-1). In some embodiments, individual target probe complexes in the first sub-population comprise a first universal circularized region (400-1) hybridized to a first universal probe region (800-1), thereby forming a first target probe complex (900-1).

[00356] In some embodiments, at step (b) above, individual target probe complexes in the second sub-population of target probe complexes (900-2) comprise a second circularized barcoded oligonucleotide (500-2) and a second target probe (600-2). In some embodiments, the second circularized barcoded oligonucleotide (500-2) comprises: (i) a second sequencing primer binding site sequence (200-2) (or a complementary sequence thereof); (ii) a second target barcode sequence (300-2) that corresponds to a target binding moiety of the second target probe (600-2), and (iii) a second universal circularized region (400-2) that binds a universal probe region of the second target probe (600-2). In some embodiments, the second target probe (600-2) comprises: an oligonucleotide having (i) a second target binding moiety (700-2) which selectively binds at least a portion of a second target polynucleotide, and (ii) a second universal probe region (800-2) that binds the second universal circularized region (400-2). In some embodiments, individual target probe complexes in the second subpopulation comprise a second universal circularized region (400-2) hybridized to a second universal probe region (800-2), thereby forming a second target probe complex (900-2).

[00357] In some embodiments, at step (b) above, the first target probe complex (900-1) comprises a first sequencing primer binding site sequence (200-1) which is the same or different from the second sequencing primer binding site sequences (200-2) of the second target probe complex (900-2). In some embodiments, the first target probe complex (900-1) comprises a first target barcode sequence (300-1) having the same or a different sequence from the second target barcode sequence (300-2) of the second target probe complex (900-2). In some embodiments, the first target probe complex (900-1) comprises a first universal circularized region (400-1) having the same or a different sequence from the second universal circularized region (400-2) of the second target probe complex (900-2). In some embodiments, the first target probe complex (900-1) comprises a first target binding moiety (700-1) having the same or a different sequence from the second target binding moiety (7002) of the second target probe complex (900-2). In some embodiments, the first target probe complex (900-1) comprises a first universal probe region (800-1) having the same or a different sequence from the second universal probe region (800-2) of the second target probe complex (900-2). First and Second Sub-Populations of Target Probe Complexes

[00358] In some embodiments, in the method for generating a plurality of nucleic acid concatemer molecules in situ of step (b) above, the first sub-population of target probe complexes (900-1) and the second sub-population of target probe complexes (900-2) comprise: (i) a first target binding moiety (700-1) and a second target binding moiety (700-2) having different sequences; (ii) a first universal probe region (800-1) and a second universal probe region (800-2) having different sequences; (iii) a first target barcode sequence (300-1) and a second target barcode sequence (300-2) having different sequences; and (iv) a first sequencing primer binding site sequence (200-1) and a second sequencing primer binding site sequence (200-2) having the same sequence. In some embodiments, the first and second target binding moieties ((700-1) and (700-2)) can bind to different target polynucleotides, for example a first and second target polynucleotide. In some embodiments, the first and second target binding regions ((700-1) and (700-2)) can bind to different portions of the same target polynucleotides, for example a first and second region of the same target polynucleotide wherein the first and second regions overlap or do not overlap with each other. First and Second Sub-Populations of Target Probe Complexes

[00359] In some embodiments, in the method for generating a plurality of nucleic acid concatemer molecules in situ of step (b) above, the first sub-population of target probe complexes (900-1) and the second sub-population of target probe complexes (900-2) comprise: (i) a first target binding moiety (700-1) and a second target binding moiety (700-2) having different sequences; (ii) a first universal probe region (800-1) and a second universal probe region (800-2) having different sequences; (iii) a first target barcode sequence (300-1) and a second target barcode sequence (300-2) having different sequences; and (iv) a first sequencing primer binding site sequence (200-1) and a second sequencing primer binding site sequence (200-2) having different sequences. In some embodiments, the first and second target binding moieties ((700-1) and (700-2)) can bind to different target polynucleotides, for example first and second target polynucleotides. In some embodiments, the first and second target binding moieties ((700-1) and (700-2)) can bind to different portions of the same target polynucleotides, for example a first and second region of the same target polynucleotide, wherein the first and second regions overlap or do not overlap with each other. First and Second Sub-Populations of Target Probe Complexes

[00360] In some embodiments, in the method for generating a plurality of nucleic acid concatemer molecules in situ of step (b) above, the first sub-population of target probe complexes (900-1) and the second sub-population of target probe complexes (900-2) comprise: (i) a first target binding moiety (700-1) and a second target binding moiety (700-2) have the same sequence or have a sequence that binds the same target polynucleotide; (ii) a first universal probe region (800-1) and a second universal probe region (800-2) having different sequences; (iii) a first target barcode sequence (300-1) and a second target barcode sequence (300-2) having different sequences; and (iv) a first sequencing primer binding site sequence (200-1) and a second sequencing primer binding site sequence (200-2) having different sequences. In some embodiments, the first and second target binding moieties ((7001) and (700-2)) can bind to different portions of the same target polynucleotides, for example a first and second region of the same target polynucleotide wherein the first and second regions overlap or do not overlap with each other. Nucleotide Reagents: Steps (el) and (e2)

[00361] In some embodiments of the method of sequencing the plurality of nucleic acid concatemer molecules in situ at steps (el) and (e2) above, the plurality of nucleotide reagents comprises a plurality of nucleotides that are detectably labeled, non-labeled, or a combination thereof. In some embodiments, individual nucleotides are linked to a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the plurality of nucleotide reagents comprises a plurality of detectably labeled nucleotide analogs comprising a plurality of chain terminating nucleotides, wherein the chain terminating moiety is linked to the 3' nucleotide sugar position to form a 3' blocked nucleotide analog. In some embodiments, the chain terminating moiety can be removed to convert the 3' blocked nucleotide analog to an extendible nucleotide having a 3' OH group on the sugar. In some embodiments, the labeled nucleotide analogs are linked to different fluorophores that correspond to the nucleo-bases adenine, cytosine, guanine, thymine or uracil, wherein the different fluorophores emit a fluorescent signal (“color signal”) during sequencing. In some embodiments, a sequencing cycle comprises (1) contacting a plurality of concatemer molecule / sequencing primer duplexes with a plurality of sequencing polymerases and a plurality of detectably labeled chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual detectably labeled chain terminating nucleotides into the terminal ends of the sequencing primers, (2) detecting and imaging the color signal (e.g. a fluorescent signal with a particular emission spectrum) emitted by the incorporated chain terminating nucleotides, and (3) removing the chain terminating moieties from the incorporated nucleotides (e.g., unblocking) and retaining the concatemer molecule / sequencing primer duplexes. In some embodiments, one sequencing cycle comprises steps (1) - (3). In some embodiments, steps (1) - (3) can be repeated at least once. In some embodiments, steps (1) - (3) can be repeated at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 times. In some embodiments, steps (1) - (3) can be repeated 50-100 times, or can be repeated 100-150 times, or can be repeated 150-200 times, or can be repeated more than 200 times. In some embodiments, a washing step can be conducted prior to step (2) to remove detectably labeled chain terminating nucleotides that are not bound to the sequencing polymerases and the concatemer molecule / sequencing primer duplexes. In some embodiments, the plurality of detectably labeled chain terminating nucleotides comprises at least one non-labeled chain terminating nucleotide. In some embodiments, the sequencing cycles are conducted on the plurality of concatemer molecules inside the cellular sample to generate a plurality of sequencing read products. In some embodiments, the sequence of the sequencing read product can be determined and aligned with a reference sequence to confirm the presence of target polynucleotides inside the cellular sample. In some embodiments, the sequences of the sequencing read products can be aligned after each round of generating the sequencing read products, or after generating a set of reiterative sequencing read products. In some embodiments, the sequencing reactions are conducted on a sequencing apparatus having a detector, e.g., that captures fluorescent signals from the sequencing reactions inside the cellular sample. The sequencing apparatus can be configured to relay the fluorescent signal data captured by the detector to a computer or other suitable system that is programmed to display images of one or more fluorescent signals, e.g., which are colocalized in the cellular sample, wherein individual fluorescent signals correspond to different target polynucleotides or target polypeptides. In some embodiments, when the sequencing is conducted using different fluorescently-labeled nucleotide reagents that correspond to different nucleo-bases (e.g., A, G, C, T / U), the images can have different colored fluorescent signals co-localized in the same cellular sample at different sequencing cycles.

[00362] In some embodiments of the method of sequencing the plurality of nucleic acid concatemer molecules in situ at steps (el) and (e2) above, the plurality of nucleotide reagents comprises (i) a plurality of nucleotides and (ii) a plurality of multivalent molecules. In some embodiments, the sequencing of steps (el) and (e2) comprise a two-stage sequencing method. In some embodiments, the first stage employs a plurality of multivalent molecules. In some embodiments, individual multivalent molecules comprise a core attached to a plurality of nucleotide-arms, wherein the nucleotide-arms are attached to a nucleotide moiety (e.g., FIGS. 1-4A). In some embodiments, individual multivalent molecules are labeled with a detectably reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the core of the multivalent molecule is labeled with a fluorophore, and wherein the fluorophore which is attached to a given core of the multivalent molecule corresponds to the nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil) of the nucleotide arm. In some embodiments, at least one of the nucleotide arms of the multivalent molecule comprises a linker and / or nucleotide base that is attached to a fluorophore. In some embodiments, the fluorophore which is attached to a given nucleotide base corresponds to the nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil) of the nucleotide arm. In some embodiments, the second stage employs a plurality of nucleotides. In some embodiments, individual nucleotides comprise chain terminating nucleotides comprising a chain terminating moiety linked to the 3' nucleotide sugar position to form a 3' blocked nucleotide analog. In some embodiments, the chain terminating moiety can be removed to convert the 3' chain terminating nucleotide analog to an extendible nucleotide having a 3' OH group on the sugar group. In some embodiments, a sequencing cycle comprises: (1) contacting a plurality of concatemer molecule / sequencing primer duplexes with a first plurality of sequencing polymerases to form a plurality of complexed polymerases wherein individual complexed polymerases comprise a concatemer molecule / sequencing primer duplex bound with a first sequencing polymerase; (2) contacting the complexed polymerases with a plurality of detectably labeled multivalent molecules under conditions suitable for binding a complementary nucleotide moiety of one of the multivalent molecules to individual complexed polymerases, thereby forming individual multivalent-binding complexes, and the conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer; (3) detecting and imaging the fluorescent signal and color (color signal) emitted by the detectably labeled multivalent molecules that are bound to the complexed polymerases (e.g., multivalent-binding complexes); (4) removing the first sequencing polymerases and the bound detectably labeled multivalent molecules, and retaining the concatemer / sequencing primer duplexes; (5) contacting the retained concatemer / sequencing primer duplexes with a second plurality of sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers; and (6) removing the chain terminating moieties from the incorporated chain terminating nucleotides (e.g., unblocking) and retaining the concatemer molecule / sequencing primer duplexes. In some embodiments, the chain terminating nucleotides of step (5) comprises non-labeled or detectably labeled chain terminating nucleotides or a mixture of both. In some embodiments, one sequencing cycle comprises steps (1) - (6). In some embodiments, steps (1) - (6) can be repeated at least once. In some embodiments, steps (1) - (6) can be repeated at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 times. In some embodiments, steps (1) - (6) can be repeated 50100 times, or can be repeated 100-150 times, or can be repeated 150-200 times, or can be repeated more than 200 times. In some embodiments, a washing step can be conducted prior to step (3) to remove detectably labeled multivalent molecules that are not bound to the first sequencing polymerases and the concatemer molecule / sequencing primer duplexes. In some embodiments, a washing step can be conducted prior to step (6) to remove chain terminating nucleotides that are not bound to the second sequencing polymerases and the concatemer molecule / sequencing primer duplexes. In some embodiments, the sequencing cycles are conducted on the plurality of concatemer molecules inside the cellular sample to generate a plurality of sequencing read products. In some embodiments, the sequence of the sequencing read product can be determined and aligned with a reference sequence to confirm the presence of target polypeptides inside the cellular sample. In some embodiments, the sequences of the sequencing read products can be aligned after each round of generating the sequencing read products, or after generating a set of reiterative sequencing read products. In some embodiments, the sequencing reactions are conducted on a sequencing apparatus having a detector, e.g., that captures fluorescent signals from the sequencing reactions inside the cellular sample. The sequencing apparatus can be configured to relay the fluorescent signal data captured by the detector to a computer or other suitable system that is programmed to display images of one or more fluorescent signals, e.g., which are colocalized in the cellular sample, wherein individual fluorescent signals correspond to different target polynucleotides or target polypeptides. In some embodiments, when the sequencing is conducted using different fluorescently-labeled multivalent molecules that correspond to different nucleo-bases (e.g., A, G, C, T / U), the images can have different colored fluorescent signals co-localized in the same cellular sample at different sequencing cycles.

[00363] In some embodiments, when the sequencing of steps (el) and (e2) comprise sequencing with detectably labeled multivalent molecules, and when step (2) in which multivalent-binding complexes are formed and step (3) in which the bound detectably labeled multivalent molecules are imaged and detected, the conditions are gentle compared to sequencing workflows that employ detectably labeled chain terminating nucleotides. For example, steps (2) and (3) can be conducted at a gentle temperature of about 35 -45 °C, or about 39 - 42 °C, or any range therebetween. Steps (2) and (3) can be conducted at a gentle temperature which can help retain the compact size and shape of a DNA nanoball during multiple sequencing cycles (e.g., up to 200 or more cycles) which can improve FWHM (full width half maximum) of a spot image of the DNA nanoball inside a cellular sample. In some embodiments, the DNA nanoball does not unravel during multiple sequencing cycles. In some embodiments, the spot image of the DNA nanoball does not enlarge during multiple sequencing cycles. In some embodiments, the spot image of the DNA nanoball remains a discrete spot during multiple sequencing cycles. The spot image can be represented as a Gaussian spot, and / or the size can be measured as a FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 pm or smaller.

[00364] In some embodiments, in the sequencing of steps (el) and (e2) above, in the sequencing of steps (1) and (2), out-of-sync phasing and / or pre-phasing events can occur during synchronized polymerase-catalyzed sequencing reactions employing detectably labeled multivalent molecules. During sequencing, a fluorescent signal can be detected which corresponds to binding of complementary nucleotide moiety of a multivalent molecule to the complexed polymerase thereby forming a multivalent-binding complex. Thus, phasing and pre-phasing events can be detected and monitored using binding of labeled multivalent molecules. In some embodiments, when conducting up to 50 sequencing cycles with detectably labeled multivalent molecules, the phasing and / or pre-phasing rate can be less than about 5%, or less than about 1%, or less than about 0.01%, or less than about 0.001%. By contrast, the phasing and / or pre-phasing rates for conducting up to 50 sequencing cycles using labeled chain terminator nucleotides can be about 5% or higher. Introduction - Analyte Detection Complexes

[00365] The present disclosure provides analyte detection complexes and methods that employ analyte detection complexes for in situ detection and identification of target analytes. In some embodiments, individual analyte detection complexes comprise antibody bridge circle complexes (1700) comprising an antibody which can bind a target analyte wherein the antibody is attached to a bridge circle complex (1600). In some embodiments, the bridge circle complex (1600) comprises a circularized barcoded oligonucleotide (1400) and a bridge oligonucleotide (1500) (e.g., FIGS. 23-27, 33-35).

[00366] In some embodiments, individual analyte detection complexes comprise bipartite complexes comprising a primary antibody which can bind a target analyte and the primary antibody is bound to a secondary antibody. In some embodiments, the secondary antibody is attached to a bridge circle complex (1600). In some embodiments, the bridge circle complex (1600) comprises a circularized barcoded oligonucleotide (1400) and a bridge oligonucleotide (1500). In some embodiments, the secondary antibody can be attached to any of the bridge circle complexes (1600) shown in FIGS. 23A, 24A, 25A, 26A and 27A. In some embodiments, the primary antibody is not directly attached to a bridge circle complex.

[00367] In some embodiments, target analytes comprise polypeptides, lipids, nucleic acids, polysaccharides or a combination thereof. In some embodiments, target analytes can be located inside a cellular sample, including, for example and without limitation, a single cell, multiple cells, a tissue, an organ, a tumor, or portions thereof.

[00368] In some embodiments, the antibody bridge circle complex (1700) and the bipartite complex comprise a circularized barcoded oligonucleotide (1400). In some embodiments, the circularized barcoded oligonucleotide (1400) comprises a covalently closed circular oligonucleotide harboring a sequencing primer binding site sequence (1100), a target barcode sequence (1200) that corresponds to the target analyte, and a universal sequence (1300) that binds at least a portion of the bridge oligonucleotide (1500) (e.g., FIGS. 23A-23B). The sequencing primer binding site sequence (1100) can be a universal sequence or a batchspecific sequencing primer binding site sequence.

[00369] In some embodiments, the antibody bridge circle complex (1700) and the bipartite complex comprise a bridge oligonucleotide (1500). In some embodiments, the bridge oligonucleotide (1500) comprises a linear oligonucleotide comprising a region that binds to a universal sequence in a circularized barcoded oligonucleotide (1400), and one end of the bridge oligonucleotide (1500) is attached to a primary or secondary antibody. In some embodiments, the 3’ end of the bridge oligonucleotide (1500) can serve as an initiation site for a rolling circle amplification reaction and the circularized barcoded oligonucleotide (1400) can serve as a template molecule for generating a concatemer molecule carrying a plurality of tandem copies of the sequencing primer binding sites (1100) and the barcode sequence (1200). The barcode sequences on the concatemer molecule can be detected and / or sequenced. Thus the barcode sequences on the concatemer molecule serve as a surrogate for detecting the presence of the target analytes and for identifying the target analyte.

[00370] The modular designs of the circularized barcoded oligonucleotides (1400) and the bridge oligonucleotides (1500) can offer flexibility for preparing different sets of bridge circle complexes (1600) wherein each set of bridge circle complexes (1600) can be attached to a different primary antibody which can selectively bind a different target analyte. In some embodiments, detection and identification of different target analytes inside a cellular sample relies on different bridge circle complexes (1600) carrying different sequence primer binding site sequences (1100) and different target barcode sequences (1200).

[00371] The modular designs of the circularized barcoded oligonucleotides (1400) and the bridge oligonucleotides (1500) can offer flexibility for preparing different sets of bridge circle complexes (1600) wherein each set of bridge circle complexes (1600) can be attached to a secondary antibody which binds a different primary antibody that can selectively bind a different target analyte. In some embodiments, detection and identification of different target analytes inside a cellular sample relies on different bridge circle complexes (1600) carrying different sequence primer binding site sequences (1100) and different target barcode sequences (1200).

[00372] Different sets (e.g., sub-populations) of circularized barcoded oligonucleotides (1400) can be designed to include different target barcode sequences (1200) and different universal sequences (1300), or to include different target barcode sequences (1200) and the same universal sequence (1300). Different sets (e.g., sub-populations) of antibody bridge circle complexes (1700) can be designed so that in a given sub-population of antibody bridge circle complexes (1700) the bridge oligonucleotides (1500) can hybridize to a circularized barcoded oligonucleotides (1400) comprising a particular barcode sequence (1200), and one end of the bridge oligonucleotide can be attached to an antibody, wherein the particular barcode sequence (1200) corresponds to the target analyte. Different sets (e.g., subpopulations) of bipartite complexes can be designed so that in a given sub-population of bipartite complexes the bridge oligonucleotides (1500) can hybridize to a circularized barcoded oligonucleotides (1400) comprising a particular barcode sequence (1200), and one end of the bridge oligonucleotide can be attached to a secondary antibody, wherein the particular barcode sequence (1200) corresponds to the target analyte.

[00373] The bridge circle complexes (1600) of the instant disclosure can offer the advantage of employing circularized barcoded oligonucleotides, rather than existing padlock probe based technologies that require enzymatic ligation or polymerase-catalyzed gap fill-in reaction. The circularized barcoded oligonucleotides (1400) are already covalently closed circular oligonucleotides, which obviates the requirement for enzymatic closure of a nick or gap. The circularized barcoded oligonucleotide (1400) thereby reduces the number of enzymatic steps in the reaction, reducing cost of the workflow, and simplifying the workflow.

[00374] The antibody bridge circle complexes (1700) and bipartite complexes can bind to target analytes to enable high target specificity and sensitivity. The antibody bridge circle complexes (1700) and bipartite complexes can be used to detect target analytes inside a cellular sample, and the barcoded concatemers can be sequenced inside the cellular sample to indicate the presence and identify the target analytes.

[00375] The antibody bridge circle complexes (1700) and bipartite complexes can have many uses, including but not limited to detection and identification of a particular target analyte or at least two different target analytes (e.g., co-localization inside a cellular sample), batch sequencing, reiterative sequencing and multiplex workflows. Bridge Circle Complexes

[00376] The present disclosure provides a plurality of bridge circle complexes (1600) wherein individual bridge circle complexes comprise two oligonucleotides including a circularized barcoded oligonucleotide (1400) and a linear bridge oligonucleotide (1500). In some embodiments, a circularized barcoded oligonucleotide (1400) and a bridge oligonucleotide (1500) are bound together. In some embodiments, a bridge circle complex (1600) is attached to a primary antibody which forms an antibody bridge circle complex (1700) (e.g., FIGS. 23B, 24B, 25B, 26B, 27B, 33B, 34A, 34B and 35). In some embodiments, a bridge circle complex (1600) is attached to a secondary antibody which forms a portion of a bipartite complex . In a bipartite complex, the bridge circle complex is attached to the secondary antibody and is not attached to the primary antibody.

[00377] In some embodiments, the circularized barcoded oligonucleotide (1400) comprises (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte; and (iii) a universal circularized region (1300) that binds a universal...

Claims

1. A multivalent analyte detection reagent, comprising: a multivalent affinity reagent (1900) comprising a core attached to a plurality of polymer arms,• wherein individual polymer arms are attached to affinity moieties that can bind a target analyte,• wherein at least one of the polymer arms attached to the affinity moieties comprises a linear polymer arm, a forked polymer arm or a branched polymer arm, and• optionally wherein the multivalent affinity reagent (1900) comprises at least one fluorophore.

2. A multivalent analyte detection reagent, comprising: a multivalent oligo reagent (2000) comprising a core attached to a plurality of polymer arms, wherein individual polymer arms are attached to affinity moieties that can bind a target analyte and at least one polymer arm is attached to an oligonucleotide,• wherein at least one of the polymer arms attached to the affinity moieties comprises a linear polymer arm, a forked polymer arm or a branched polymer arm,• wherein the at least one polymer arm attached to the oligonucleotide comprises a linear polymer arm, a forked polymer arm or a branched polymer arm,• wherein the oligonucleotide comprises a sequencing primer binding site and a target barcode sequence corresponding to the affinity moieties attached to individual polymer arms, and• optionally wherein the multivalent oligo reagent (2000) comprises at least one fluor ophore.

3. A multivalent analyte detection reagent, comprising: a multivalent heterofunctional reagent (2500) comprising a multi-arm polymer attached to at least one oligonucleotide and a plurality of affinity moieties that can bind a target analyte,• wherein the multi-arm polymer comprises a forked multi-arm polymer or a branched multi-arm polymer,• wherein the oligonucleotide comprises a sequencing primer binding site and a target barcode sequence corresponding to the plurality of affinity moieties attached to the multi-arm polymer, and• optionally wherein the multivalent heterofunctional reagent (2500) comprises at least one fluorophore.

4. A plurality of multivalent analyte detection reagents, comprising: a plurality of multivalent oligo reagents (2000) of claim 2, wherein the plurality of multivalent oligo reagents comprises:a) a first sub-population of multivalent oligo reagents (2000-1), wherein individual multivalent oligo reagents in the first sub-population comprise a core attached to a plurality of polymer arms, and wherein individual polymer arms are attached to first affinity moieties that can bind a first target analyte, and at least one of the polymer arms is attached to an oligonucleotide comprising a first sequencing primer binding site (2010-1) and a first target barcode sequence (2020-1) corresponding to the first affinity moieties that is at least 2 nucleotides in length;b) a second sub-population of multivalent oligo reagents (2000-2), wherein individual multivalent oligo reagents in the second sub-population comprise a core attached to a plurality of polymer arms, and wherein individual polymer arms are attached to second affinity moieties that can bind a second target analyte, and at least one of the polymer arms is attached to an oligonucleotide comprising a second sequencing primer binding site (2010-2) and a second target barcode sequence (2020-2) corresponding to the second affinity moieties that is at least 2 nucleotides in length,• wherein the first and second target barcode sequences are different,• wherein the first and second affinity moieties are different, and• wherein the first and second sequencing primer binding sites are the same sequence or different sequences;c) wherein one nucleo-base in a first position in the first target barcode sequence (2020-1) generates a first color signal in a first sequencing cycle, and wherein one nucleo-base in a first position in the second target barcode sequence (2020-2) generates a second color signal in the same first sequencing cycle, and wherein the first and second color signals are different;d) wherein one nucleo-base in a second position in the first target barcode sequence (2020-1) generates a third color signal in a second sequencing cycle, and wherein one nucleo-base in a second position in the second target barcode sequence (2020-2) generates a fourth color signal in the same second sequencing cycle, and wherein the third and fourth color signals are different; ande) wherein the first color signal identifies the first target analyte, and wherein the fourth color signal identifies the second target analyte.

5. The plurality of multivalent analyte detection reagents of claim 4, wherein the first and third color signal can be the same color signal or the different color signals.

6. The plurality of multivalent analyte detection reagents of claim 4 or 5, wherein the second and fourth color signals can be the same color signal or different color signals.

7. A plurality of multivalent analyte detection reagents, comprising: a plurality of multivalent heterofunctional reagents (2500) of claim 3, wherein the plurality of multivalent heterofunctional reagents comprisesa) a first sub-population of multivalent heterofunctional reagents (2500-1), wherein individual multivalent heterofunctional reagents in the first subpopulation comprise a multi-arm polymer attached to (1) at least one oligonucleotide comprising a first sequencing primer binding site (2510-1) and a first target barcode sequence (2520-1) at least 2 nucleotides in length and (2) a plurality of affinity moieties that can bind a first target analyte, wherein the first target barcode sequence (2520-1) corresponds to the first affinity moieties;b) a second sub-population of multivalent heterofunctional reagents (2500-2), wherein individual multivalent heterofunctional reagents in the second subpopulation comprise a multi-arm polymer attached to (1) at least one oligonucleotide comprising a second sequencing primer binding site (2510-2) and a second target barcode sequence (2520-2) at least 2 nucleotides in length and (2) a plurality of affinity moieties that can bind a second target analyte, wherein the second target barcode sequence (2520-2) corresponds to the second affinity moieties;• wherein the first and second target barcode sequences are different,• wherein the first and second affinity moieties are different, and• wherein the first and second sequencing primer binding sites are the same sequence or different sequences;c) wherein one nucleo-base in a first position in the first target barcode sequence (2520-1) generates a first color signal in a first sequencing cycle, and wherein one nucleo-base in a first position in the second target barcode sequence (2520-2) generates a second color signal in the same first sequencing cycle, and wherein the first and second color signals are different;d) wherein one nucleo-base in a second position in the first target barcode sequence (2520-1) generates a third color signal in a second sequencing cycle, and wherein one nucleo-base in a second position in the second target barcode sequence (2520-2) generates a fourth color signal in the same second sequencing cycle, and wherein the third and fourth color signals are different; ande) wherein the first color signal identifies the first target analyte, and wherein the fourth color signal identifies the second target analyte.

8. The plurality of multivalent analyte detection reagents of claim 7, wherein the first and third color signal can be the same color signal or the different color signals.

9. The plurality of multivalent analyte detection reagents of claim 7 or 8, wherein the second and fourth color signals can be the same color signal or different color signals.

10. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the targetanalyte is located on the exterior of a cell, is embedded in a cell membrane, is located in the cytoplasm, is located on an cell organelle, is located inside a cell, or a combination thereof.

11. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte is located on a cellular organelle or structure or inside a cellular organelle or structure, optionally wherein the cellular organelle or structure comprises a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a microtubule, a centriole, a spindle, an actin filament, a flagellum, a cilium, a peroxisome, a lysosome, a chloroplast or a combination thereof.

12. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises a polypeptide, a protein, a protein fragment, an enzyme or an antibody.

13. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, a homopolysaccharide or a heteropolysaccharide.

14. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises a lipid, a triglyceride, a phospholipid, a steroid, a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a saccharolipid, a polyketide, a sterol lipid or a prenol lipid.

15. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises an oligonucleotide, a polynucleotide, DNA, cDNA or RNA.

16. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the targetanalyte comprises a cell surface receptor comprising an ion channel receptor, a G-protein coupled receptor or an enzyme-linked receptor.

17. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises a cluster of differentiation (CD) comprising a cell receptor CD, a cell ligand CD, a cell signaling CD, or a cell adhesion CD.

18. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises a cytokine, an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor, a chemokine, a vascular growth factor, a platelet derived growth factor, a lymphokine, a monokine or a colony stimulating factor.

19. The multivalent analyte detection reagent of any one of claims 1-3 or the plurality of multivalent analyte detection reagents of any one of claims 4-9, wherein the target analyte comprises a peptide hormone.

20. A method for detecting a plurality of target analytes inside a cellular sample, comprising:a) providing a cellular sample deposited on a support, wherein the cellular sample comprises a plurality of analytes including at least one target analyte;b) providing a multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of any one of claims 1-19, wherein individual multivalent analyte detection reagents comprise a fluorophore;c) contacting the cellular sample with the plurality of multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for moving the plurality of multivalent analyte detection reagents into the cellular sample and suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes inside the cellular sample to form a plurality of multivalent reagent-analyte complexes inside the cellular sample;d) washing the cellular sample to remove multivalent analyte detection reagents that are not bound to a cognate target analyte, under a condition suitable toretain the plurality of f multivalent reagent-analyte complexes inside the cellular sample; ande) detecting the plurality of multivalent reagent-analyte complexes inside the cellular sample by imaging fluorescent signals emitted from the plurality of multivalent reagent-analyte complexes inside the cellular sample, thereby detecting the plurality of target analytes inside a cellular sample.

21. The method of claim 20, wherein the multivalent analyte detection reagents comprise fluorophore-labeled multivalent affinity reagents comprising at least a first and second sub-population of fluorophore-labeled multivalent affinity reagents,• wherein individual fluorophore-labeled multivalent affinity reagents in the first sub-population (1900-1) bind a first target analyte and comprise a first fluorophore,• wherein individual fluorophore-labeled multivalent affinity reagents in the second sub-population (1900-2) bind a second target analyte and comprise a second fluorophore,• wherein the first and second target analytes are different target analytes, and• wherein the first and second fluorophores emit fluorescent signals that are distinguishable from each other.

22. The method of claim 20, wherein the multivalent analyte detection reagents comprise fluorophore-labeled multivalent oligo reagents comprising at least a first and second sub-population of fluorophore-labeled multivalent oligo reagents,• wherein individual fluorophore-labeled multivalent oligo reagents in the first sub-population (2000-1) bind a first target analyte and comprise a first fluorophore,• wherein individual fluorophore-labeled multivalent oligo reagents in the second sub-population (2000-2) bind a second target analyte and comprise a second fluorophore,• wherein the first and second target analytes are different target analytes, and• wherein the first and second fluorophore emit fluorescent signals that are distinguishable from each other.

23. The method of claim 20, wherein the multivalent analyte detection reagents comprise fluorophore-labeled multivalent heterofunctional reagents comprising at least a first and second sub-population of fluorophore-labeled multivalent heterofunctional reagents,• wherein individual fluorophore-labeled multivalent heterofunctional reagents in the first sub-population (2500-1) bind a first target analyte and comprise a first fluorophore,• wherein individual fluorophore-labeled multivalent heterofunctional reagents in the second sub-population (2500-2) bind a second target analyte and comprise a second fluorophore,• wherein the first and second target analytes are different target analytes, and• wherein the first and second fluorophore emit fluorescent signals that are distinguishable from each other.

24. A method for conducting a sequencing-based workflow for detecting a plurality of target analytes inside a cellular sample, comprising:a) providing a cellular sample deposited on a support, wherein the cellular sample harbors a plurality of analytes including at least one target analyte;b) providing the multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of any one of claims 2-19;c) contacting the cellular sample with a plurality of the multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for moving the plurality of multivalent analyte detection reagents into the cellular sample and suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes inside the cellular sample to form a plurality of multivalent reagent-analyte complexes, wherein individual multivalent reagent-analyte complexes comprise affinity moieties of an individual multivalent analyte detection reagent bound to a plurality of cognate target analytes, and wherein individual multivalent reagent-analyte complexes comprise a sequencing primer binding site and a target barcode sequence;d) washing the cellular sample to remove multivalent analyte detection reagents inside the cellular sample that are not bound to a cognate target analyte, undera condition suitable to retain the plurality of multivalent reagent-analyte complexes inside the cellular sample; ande) sequencing the target barcodes of the plurality of multivalent reagent-analyte complexes, wherein the sequencing is conducted inside the cellular sample.

25. The method of claim 24, wherein the sequencing of step e) comprises:a) hybridizing the sequencing primer binding sites with a plurality of sequencing primers, thereby forming a plurality of nucleic acid duplexes, and contacting the plurality of nucleic acid duplexes with a first plurality of sequencing polymerases to form a first plurality of complexed polymerases, wherein individual first complexed polymerases comprise a duplex bound with a first sequencing polymerase;b) contacting the first complexed polymerases with a plurality of first fluorophore-labeled multivalent molecules, wherein individual first fluorophore-labeled multivalent molecules comprise a core attached to a plurality of nucleotide-arms attached to nucleotide moieties, and wherein the contacting is conducted under a condition suitable for binding a complementary nucleotide moiety of one of the first fluorophore-labeled multivalent molecules to individual first complexed polymerases at a position that is opposite of a nucleotide in the target barcode region thereby forming a plurality of first multivalent-binding complexes, wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer;c) detecting a color signal emitted by the first fluorophore-labeled multivalent molecules that are bound to the first complexed polymerases;d) removing the first sequencing polymerases and the first fluorophore-labeled multivalent molecules and retaining the nucleic acid duplexes;e) contacting the nucleic acid duplexes retained at step (d) with a second plurality of sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers thereby extending the sequencing primers by one nucleotide;f) removing the chain terminating moieties from the chain terminating nucleotides incorporated at step (e) and retaining the nucleic acid duplexes; andg) contacting the nucleic acid duplexes of step (f) with a plurality of second fluorophore-labeled multivalent molecules and a third plurality of sequencing polymerases to form a plurality of second multivalent-binding complexes wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer, and repeating steps (c) - (f) at least once.

26. The method of claim 24 or 25, wherein the plurality of multivalent analyte detection reagents comprise at least a first and second sub-population of multivalent oligo reagents,• wherein individual multivalent oligo reagents in the first sub-population (2000-1) bind a first target analyte and comprise a first sequencing primer binding site and a first target barcode,• wherein individual multivalent oligo reagents in the second sub-population (2000-2) bind a second target analyte and comprise a second sequencing primer binding site and a second target barcode,• wherein the first and second target analytes are different target analytes,• wherein the first and second sequencing primer binding sites have different sequences, and• wherein the first and second target barcodes have different sequences.

27. The method of claim 24 or 25, wherein the plurality of multivalent analyte detection reagents comprise multivalent heterofunctional reagents comprising at least a first and second sub-population of multivalent oligo reagents,• wherein individual multivalent heterofunctional reagents in the first subpopulation (2500-1) bind a first target analyte and comprise a first sequencing primer binding site and a first target barcode,• wherein individual multivalent heterofunctional reagents in the second subpopulation (2500-2) bind a second target analyte and comprise a second sequencing primer binding site and a second target barcode,• wherein the first and second target analytes are different target analytes,• wherein the first and second sequencing primer binding sites have different sequences, and• wherein the first and second target barcodes have different sequences.

28. The method of claim 26 or 27, wherein sequencing the target barcodes comprises sequencing the first sub-population of target barcodes while inhibiting sequencing the second sub-population of target barcodes by:a) contacting the first sub-population of sequencing primer binding sites inside the cellular sample with a plurality of first batch sequencing primers under conditions suitable to hybridize individual first batch sequencing primers to the first sub-population of sequencing primer binding sites, wherein individual soluble first batch sequencing primers comprise an extendible terminal 3’ end which permits polymerase-catalyzed extension of the first batch sequencing primers that are hybridized to the first sub-population of sequencing primer binding sites;b) contacting the second sub-population of sequencing primer binding sites inside the same cellular sample with a plurality of second batch sequencing primers under conditions suitable to hybridize individual second batch sequencing primers to the second sub-population of sequencing primer binding sites, wherein individual soluble second batch sequencing primers comprise a terminal 3’ reversible blocking moiety which blocks polymerase-catalyzed extension of the second batch sequencing primers that are hybridized to the second sub-population of sequencing primer binding sites; andc) conducting a first plurality of sequencing cycles inside the cellular sample using a sequencing polymerase thereby generating a first plurality of sequencing read products of the first target barcodes, wherein the first and second batch sequencing primers are soluble.

29. The method of claim 26 or 27, wherein sequencing the target barcodes comprises conducting batch sequencing of the first and second target barcodes by:a) contacting the first sub-population of sequencing primer binding sites inside the cellular sample with a plurality of first batch sequencing primers having terminal 3’ extendible ends and a plurality of nucleotide reagents, and conducting a first plurality of polymerase-catalyzed sequencing cycles thereby generating a plurality of first batch sequencing read products inside the cellular sample;b) removing the plurality of first batch sequencing read products from the first sub-population of sequencing primer binding sites while retaining the first sub-population of multivalent reagent-analyte complexes inside the cellular sample, wherein the plurality of first batch sequencing read products are removed by enzymatic degradation or a chemical de-hybridization reagent;c) contacting the second sub-population of sequencing primer binding sites inside the same cellular sample with a plurality of second batch sequencing primers and a plurality of nucleotide reagents, and conducting a second plurality of polymerase-catalyzed sequencing cycles thereby generating a plurality of second batch sequencing read products inside the cellular sample; andd) optionally removing the plurality of second batch sequencing read products from the second sub-population of sequencing primer binding sites while retaining the second sub-population of multivalent reagent-analyte complexes inside the cellular sample, wherein the plurality of second batch sequencing read products are removed by enzymatic degradation or de-hybridization reagent.

30. A method for conducting cell painting, comprising:a) providing a plurality of barcoded multivalent reagent-analyte complexes inside a cellular sample, wherein the plurality of barcoded multivalent reagentanalyte complexes comprises the plurality of multivalent analyte detection reagents of claim 4, wherein individual barcoded multivalent reagent-analyte complexes comprise individual multivalent oligo reagents from either the first sub-population of multivalent oligo reagents (2000-1) or the second subpopulation of multivalent oligo reagents (2000-2), thereby generating a first and second sub-population of barcoded multivalent reagent-analyte complexes;b) conducting a first sequencing cycle inside the cellular sample, wherein the first sequencing cycle comprises sequencing essentially simultaneously a first nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagent-analyte complexes using a plurality of sequencing primers having the same sequence or different sequences, wherein the first nucleo-base position of the first target barcode generates a first color signal and the first nucleo-base position of the second barcode generates a second color signal, wherein the first and second color signals are distinguishable from each other in the first sequencing cycle, and wherein the first color signal identifies the first target analyte;c) conducting a second sequencing cycle inside the cellular sample, wherein the second sequencing cycle comprises sequencing essentially simultaneously the second nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagent-analyte complexes, wherein the second nucleo-base position of the first target barcode generates the second color signal, wherein the second nucleo-base position of the second barcode generates the first color signal, wherein the first and second color signals are distinguishable from each other in the second sequencing cycle, and wherein the first color signal identifies the second target analyte;d) imaging the first and second color signals generated inside the cellular sample in the first sequencing cycle at step (b) and identifying the first target analyte wherein the imaging of step (d) can be conducted essentially simultaneously with the sequencing of step (b); ande) imaging the first and second color signals generated inside the cellular sample in the second sequencing cycle at step (c) and identifying the second target analyte, wherein the imaging of step (e) can be conducted essentially simultaneously with the sequencing of step (c).

31. A method for conducting cell painting, comprising:a) providing a plurality of barcoded multivalent reagent-analyte complexes inside a cellular sample, wherein the plurality of barcoded multivalent reagentanalyte complexes comprises the plurality of multivalent analyte detection reagents of claim 7, wherein individual barcoded multivalent reagent-analytecomplexes comprise individual multivalent heterofunctional reagents from either the first sub-population of multivalent heterofunctional reagents (25001) or the second sub-population of multivalent heterofunctional reagents (2500-2), thereby generating a first and second sub-population of barcoded multivalent reagent-analyte complexes;b) conducting a first sequencing cycle inside the cellular sample, wherein the first sequencing cycle comprises sequencing essentially simultaneously a first nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagent-analyte complexes using a plurality of sequencing primers having the same sequence or different sequences, wherein the first nucleo-base position of the first target barcode generates a first color signal and the first nucleo-base position of the second barcode generates a second color signal, wherein the first and second color signals are distinguishable from each other in the first sequencing cycle, and wherein the first color signal identifies the first target analyte;c) conducting a second sequencing cycle inside the cellular sample, wherein the second sequencing cycle comprises sequencing essentially simultaneously the second nucleo-base position of the first and second target barcodes in the first and second sub-populations of barcoded multivalent reagent-analyte complexes, wherein the second nucleo-base position of the first target barcode generates the second color signal, wherein the second nucleo-base position of the second barcode generates the first color signal, wherein the first and second color signals are distinguishable from each other in the second sequencing cycle, and wherein the first color signal identifies the second target analyte;d) imaging the first and second color signals generated inside the cellular sample in the first sequencing cycle at step (b) and identifying the first target analyte wherein the imaging of step (d) can be conducted essentially simultaneously with the sequencing of step (b); ande) imaging the first and second color signals generated inside the cellular sample in the second sequencing cycle at step (c) and identifying the second target analyte, wherein the imaging of step (e) can be conducted essentially simultaneously with the sequencing of step (c).

32. The method of claim 30 or 31, wherein the first sequencing cycle comprises:a) hybridizing the first sequencing primer binding sites with a plurality of first sequencing primers thereby forming a plurality of nucleic acid duplexes, or hybridizing the second sequencing primer binding sites with a plurality of second sequencing primers, thereby forming a plurality of nucleic acid duplexes, and contacting the plurality of nucleic acid duplexes with a first plurality of sequencing polymerases to form a first plurality of complexed polymerases, wherein individual complexed polymerases comprise a duplex bound with a first sequencing polymerase;b) contacting the first complexed polymerases with a plurality of first fluorophore-labeled multivalent molecules, wherein individual fluorophore-labeled multivalent molecules comprise a core attached to a plurality of nucleotide-arms attached to a nucleotide moieties, and wherein the contacting is conducted under a condition suitable for binding a complementary nucleotide moiety of one of the first fluorophore-labeled multivalent molecules to individual first complexed polymerases at a position that is opposite of a nucleotide in the target barcode region thereby forming a plurality of first multivalent-binding complexes, wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer;c) detecting a color signal emitted by the first fluorophore-labeled multivalent molecules that are bound to the first complexed polymerases;d) removing the first sequencing polymerases and the first fluorophore-labeled multivalent molecules and retaining the nucleic acid duplexes;e) contacting the nucleic acid duplexes retained at step (d) with a second plurality of sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers thereby extending the sequencing primers by one nucleotide; andf) removing the chain terminating moieties from the incorporated chain terminating nucleotides and retaining the nucleic acid duplexes.

33. The method of claim 32, wherein the second sequencing cycle comprises:a) contacting the plurality of nucleic acid duplexes with a plurality of first sequencing polymerases to form a first plurality of complexed polymerases, wherein individual complexed polymerases comprise a duplex bound with a first sequencing polymerase;b) contacting the first complexed polymerases with a plurality of fluorophore-labeled multivalent molecules, wherein individual fluorophore-labeled multivalent molecules comprise a core attached to a plurality of nucleotide-arms attached to a nucleotide moieties, and wherein the contacting is conducted under a condition suitable for binding a complementary nucleotide moiety of one of the fluorophore-labeled multivalent molecules to individual first complexed polymerases at a position that is opposite of a nucleotide in the target barcode region thereby forming individual multivalent-binding complexes, wherein the contacting conditions are suitable for inhibiting incorporation of the complementary nucleotide moiety into the terminal end of the sequencing primer;c) detecting a color signal emitted by the fluorophore-labeled multivalent molecules that are bound to the first complexed polymerases;d) removing the first sequencing polymerases and the bound fluorophore-labeled multivalent molecules and retaining the nucleic acid duplexes;e) contacting the nucleic acid duplexes retained at step (d) with a plurality of second sequencing polymerases and a plurality of chain terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of individual chain terminating nucleotides into the terminal end of individual sequencing primers thereby extending the sequencing primers by one nucleotide; andf) removing the chain terminating moieties from the chain terminating nucleotides incorporated at step (e) and retaining the nucleic acid duplexes.

34. The method of claim 33, comprising repeating steps (a)-(f) at least once.

35. A method for detecting a plurality of target analytes in a sample, comprising:a) providing a sample comprising a plurality of analytes including at least one target analyte;b) providing a multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of any one of claims 1-19, wherein individual multivalent analyte detection reagents comprise a fluorophore;c) contacting the sample with the plurality of multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes in the sample to form a plurality of multivalent reagent-analyte complexes, wherein the multivalent reagent-analyte complex exhibits physical, chemical and / or optical characteristics that are distinguishable from the multivalent analyte detection reagent that is not bound to its cognate target analyte;d) removing the multivalent analyte detection reagents that are not bound to a cognate target analyte, under a condition suitable to retain the plurality of multivalent reagent-analyte complexes; ande) detecting the plurality of the retained multivalent reagent-analyte complexes.

36. The method of claim 35, wherein in step (a) the target analyte in the sample is in solution, embedded in a gel or immobilized to a support.

37. The method of claim 35 or 36, wherein the detecting of step (e) comprises:• measuring a change in optical properties of the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents;• measuring a color change of the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; or• measuring an increased fluorescence intensity of the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents.

38. The method of any one of claims 35-37, wherein the detecting of step (e) comprises:• using atomic mass spectrometry to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents;• using flow cytometry to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents;• using capillary electrophoresis to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents;• using capillary electrochromatography to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents;• using gel electrophoresis to measure a change in the plurality of multivalent reagent-analyte complexes compared to the plurality of the unbound multivalent analyte detection reagents; or• using a microarray to measure a change in the plurality of multivalent reagentanalyte complexes compared to the plurality of the unbound multivalent analyte detection reagents.

39. A method for conducting a sequencing-based workflow for detecting a plurality of target analytes in a sample, comprising:a) providing a sample comprising a plurality of analytes including at least one target analyte;b) providing a plurality of the multivalent analyte detection reagent or plurality of multivalent analyte detection reagents of any one of claims 2-19, wherein multivalent oligo reagents or the multivalent heterofunctional reagents comprise a fluorophore;c) contacting the sample with a plurality of the multivalent analyte detection reagents, wherein the contacting is conducted under a condition suitable for binding individual multivalent analyte detection reagents to at least a portion of their cognate target analytes in the sample to form a plurality of multivalent reagent-analyte complexes;d) removing the multivalent analyte detection reagents that are not bound to a cognate target analyte, under a condition suitable to retain the plurality of multivalent reagent-analyte complexes; ande) sequencing the target barcodes of the plurality of multivalent reagent-analyte complexes thereby identifying the target analyte that was bound to the multivalent analyte detection reagent.

40. The method of any one of claims 35-39, wherein the at least one target analyte comprises molecules from air, water, soil or food.

41. The method of any one of claims 35-39, wherein the at least one target analyte comprises molecules isolated from viruses, fungi, prokaryotes or eukaryotes.

42. The method of any one of claims 35-39, wherein the at least one target analyte comprises molecules isolated from human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect or bacteria.

43. The method of any one of claims 35-39, wherein the at least one target analyte comprises molecules isolated from blood, urine, serum, lymph, tumor, saliva, anal secretions, vaginal secretions, amniotic samples, perspiration, semen, environmental samples or culture samples.

44. The method of any one of claims 35-39, wherein the at least one target analyte comprises molecules isolated from head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

45. The method of any one of claims 25-30 or 32-34, wherein individual fluorophore-labeled multivalent molecules comprise: (a) a core, (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide moiety, and (c) a fluorophore, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety.

46. The method of claim 45, wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits.

47. The method of claim 45 or 46, wherein the plurality of nucleotide arms attached to a given core have the same type of nucleotide moiety, and wherein the nucleotide moiety comprises dATP, dGTP, dCTP, dTTP or dUTP.

48. The method of any one of claims 45-47, wherein the plurality of multivalent molecules comprise one type of a multivalent molecule wherein each multivalent molecule in the plurality has the same type of nucleotide moiety selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

49. The method of any one of claims 45-47, wherein the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules each type having nucleotide moieties selected from a group consisting of dATP, dGTP, dCTP, dTTP and / or dUTP.

50. The method of any one of claims 47-49, wherein the individual fluorophore-labeled multivalent molecules comprise a fluorophore that corresponds to the nucleotide moiety.

51. A method for conducting multi-omic detection and identification, comprising:a) providing a cellular sample deposited on a support, wherein the cellular sample harbors a plurality of target polynucleotides and a plurality of target analytes;b) contacting the cellular sample with a plurality of barcoded target probe complexes (900), wherein the contacting is conducted under a condition suitable for moving the plurality of target probe complexes (900) into the cellular sample and for selectively binding the plurality of target probe complexes (900) to their corresponding target polynucleotides inside the cellular sample,• wherein individual barcoded target probe complexes (900) in the plurality comprise a circularized barcoded oligonucleotide (500) hybridized to a linear target probe (600), wherein the circularized barcoded oligonucleotide (500) comprises (i) a sequencing primerbinding site sequence (200) (or a complementary sequence thereof); (ii) a target barcode sequence (300), and (iii) a universal circularized region (400), wherein the linear target probe (600) comprises an oligonucleotide having (i) a target binding moiety (700) which selectively binds a target polynucleotide and (ii) a universal probe region (800), wherein the 3’ end of the linear target probe (600) is extendible, wherein the universal circularized region (400) is hybridized to the universal probe region (800) of the target probe (600), and wherein target barcode sequence (300) corresponds to the target binding moiety (700) of the linear target probe (600);c) removing an excess of target probe complexes (900) that are not bound to their corresponding target polynucleotides and retaining the plurality of target probe complexes (900) bound to their corresponding target polynucleotides;d) contacting the cellular sample with a plurality of barcoded bipartite complexes , wherein the contacting is conducted under a condition suitable for moving the plurality of barcoded bipartite complexes into the cellular sample and for binding the plurality of barcoded bipartite complexes to their corresponding target analytes inside the cellular sample,• wherein individual barcoded bipartite complexes comprise a primary antibody bound to a secondary antibody, wherein the primary antibody selectively binds a target analyte, wherein the secondary antibody is attached to a bridge circle complex, the bridge circle complex comprising a circularized barcoded oligonucleotide (1400) hybridized to a linear bridge oligonucleotide (1500), wherein the circularized barcoded oligonucleotide (1400) comprises: (i) a sequencing primer binding site sequence (1100) (or a complementary sequence thereof); (ii) a target barcode sequence (1200) that corresponds to a target analyte and (iii) a universal circularized region (1300), wherein the linear bridge oligonucleotide (1500) comprises a universal sequence region, wherein the 5’ end of the linear bridge oligonucleotide (1500) is attached to the secondary antibody, wherein the 3’ end of the linear bridge oligonucleotide (1500) is extendible, and wherein the universal sequence region of the bridge oligonucleotide (1500) is hybridized tothe universal circularized region (1300) of the circularized barcoded oligonucleotide (1400);e) removing an excess of barcoded bipartite complexes that are not bound to their corresponding target analytes and retaining the plurality of bipartite complexes bound to their corresponding target analytes;f) contacting the cellular sample with a rolling circle amplification reagent comprising a plurality of strand-displacing DNA polymerases and a plurality of nucleotides, under a condition suitable for moving the rolling circle amplification reagent into the cellular sample;g) conducting a rolling circle amplification reaction inside the cellular sample thereby generating a plurality of barcoded concatemer molecules comprising (i) a first sub-population of barcoded concatemer molecules generated from the plurality of barcoded target probe complexes (900) wherein individual barcoded concatemer molecules of the first sub-population comprise a plurality of the target barcode sequence (300), and (ii) a second sub-population of barcoded concatemer molecules generated from the plurality of barcoded bipartite complexes wherein individual barcoded concatemer molecules of the second sub-population comprise a plurality of the target barcode sequence (1200); andh) sequencing the target barcode sequence (300) of the first sub-populations of barcoded concatemer molecules and sequencing the target barcode sequence (1200) of the second sub-populations of barcoded concatemer molecules wherein the sequencing is conducted inside the cellular sample.

52. The method of claim 51, wherein the cellular sample comprises a whole single cell, a plurality of whole cells, an intact tissue, sectioned cell or a sectioned tissue sample.

53. The method of claim 51, wherein the cellular sample comprises a fresh cellular sample, a freshly-frozen cellular sample, or a formalin-fixed and paraffin-embedded (FFPE) cellular sample.

54. The method of claim 51, wherein the cellular sample comprises a fixed and permeabilized cellular sample.

55. The method of any one of claims 51-54, wherein the plurality of target polynucleotides comprise DNA, cDNA, RNA, or a mixture thereof.

56. The method of any one of claims 51-55, wherein the plurality of target analytes comprises polypeptides, lipids, nucleic acids, polysaccharides or a combination thereof.

57. The method of any one of claims 51-56, wherein the plurality of nucleotides in the rolling circle amplification reagent comprises dATP, dGTP, dCTP, dTTP and / or dUTP.

58. The method of any one of claims 51-57, wherein the rolling circle amplification reagent further comprises a plurality of compaction oligonucleotides, wherein individual compaction oligonucleotides comprise a 5' region that binds a first portion of one of the concatemer molecules and a 3' region that binds a second portion of the same concatemer molecule to pull together distal portions of the concatemer molecule thereby causing compaction of the concatemer molecule.

59. The method of any one of claims 51-58, wherein the sequencing comprises sequencing the first and second sub-population of barcoded concatemer molecules essentially simultaneously.

60. The method of any one of claims 51-58, wherein the sequencing comprises sequencing the first and second sub-population of barcoded concatemer molecules in separate batches.

61. The method of any one of claims 51-60, wherein the sequencing comprisesa) contacting the first sub-population of barcoded concatemer molecules with a plurality of a first sequencing primer, a plurality of sequencing polymerases, and a plurality of detectably labeled multivalent molecules, andb) contacting the second sub-population of barcoded concatemer molecules with a plurality of a second sequencing primer, a plurality of sequencing polymerases, and a plurality of detectably labeled multivalent molecules, wherein individual multivalent molecules comprise a core attached to a plurality of nucleotide-arms where individual nucleotide arms comprise a core attachment moiety, a spacer, a linker, and a nucleotide moiety.

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