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WO2025235971A1 - Procédés de détection de variation de séquence d'adn dans des sections de tissu épais - Google Patents

Procédés de détection de variation de séquence d'adn dans des sections de tissu épais

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Publication number
WO2025235971A1
WO2025235971A1 PCT/US2025/028781 US2025028781W WO2025235971A1 WO 2025235971 A1 WO2025235971 A1 WO 2025235971A1 US 2025028781 W US2025028781 W US 2025028781W WO 2025235971 A1 WO2025235971 A1 WO 2025235971A1
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tissue sample
tissue
probe
aspects
rna
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Reza Kalhor
Soichiro ASAMI
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Johns Hopkins University
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • SUMMARY 25 Disclosed herein are methods for detecting a target of interest in a tissue sample, the methods comprising: a) obtaining or having obtained the tissue sample, wherein the tissue sample comprises DNA, non-RNA molecules, and RNA, wherein the RNA comprises a target of interest; b) permeabilizing the tissue sample; c) substantially degrading the DNA in the tissue sample; and d) introducing to the tissue sample a primer or a probe specific to the 30 target of interest, wherein the primer or probe binds to the target of interest; and e) detecting the primer or probe bound to the target of interest, thereby detecting the target of interest in the tissue sample.
  • FIG. 1 is a schematic of a tissue clearing method and subsequent amplification of an RNA target of interest present in the tissue using a padlock probe and rolling circle amplification.
  • FIGS.2A-C show ACTB mRNA signal detection across a 400 micron thick mouse 15 liver sample. The 400-micron tissue slice from the mouse liver was processed using methods described herein. ACTB mRNA was targeted and the RCA amplicons were detected with a probe modified with a Cy5 dye. The sample was measured with a spinning desk confocal microscope.
  • FIG.2A shows a 3D view (10x) of the sample with RCA amplicons from Actb mRNA.
  • FIG. 2B shows the lateral view of FIG. 2A.
  • FIG. 2C shows the number of RCA 20 amplicons counted from the sample shown in FIG.2A.
  • the number of RCA amplicons were quantified with Fiji.
  • FIGS.3A-D show that the method described herein has a high RNA detection efficiency compared to another RNA detection method for thick tissues.
  • the 50-micron tissue slice from the mouse liver was processed as described herein.
  • ACTB mRNA was targeted 25 and the RCA amplicons were detected with a probe modified with a Cy5 dye using the methods described herein (FIG.3A) compared to the Melpha-X based method (FIG. 3C).
  • the sample was measured with a spinning desk confocal microscope.
  • FIGS.3C and 3D the Melpha-X RNA detection method was performed for thick slices (MelphaX, Wang, Y. et al., 2021, Cell, in which a hybridization chain reaction was used) as a side-by-side comparison. 30 The same padlock oligo and enzymes were used for FIG.3A and FIG.3C for comparison.
  • FIG. 3A shows a 3D view (20x) of the sample with RCA amplicons from Actb mRNA amplified using the methods described herein.
  • FIG.3B shows the RCA amplicons counted in FIG. 3A.
  • FIG. 3C shows a 3D view (20x) of the sample with RCA amplicons from Actb 2 Attorney Docket No.36406.0040P1 mRNA detected with the MelphaX-based method.
  • FIG. 3D shows the number of RCA amplicons counted in FIG.3C.
  • FIGS.4A-C show genomic DNA inhibited RNA detection in situ for thick tissue slices. The 400-micron tissue slice from the mouse liver was processed without DNase I 5 treatment. ACTB mRNA was targeted and the RCA amplicons were detected with a probe modified with a Cy5 dye. The sample was measured with a spinning desk confocal microscope.
  • FIG.4A shows a 3D view (10x) of the 400 micron sample stained with DAPI. The distinct genomic DNA shape was observed.
  • FIG.4B shows a lateral view (10x) of the 400 micron sample with RCA amplicons; Red: RCA amplicons, Blue: DAPI.
  • the RCA 10 amplicons were limited on the surface.
  • FIG.4C shows a 3D view (10x) of the 400 micron sample after DNase I treatment. The shape of genomic DNA was completely lost. After DNase I treatment, the uniformity of the reactions dramatically improved, which is shown in FIG 2.
  • FIG. 5 shows a schematic description of a tissue clearing method and amplification of 15 a target of interest with a mutation present in RNA.
  • FIG. 6 shows single nucleotide variants (SNVs) were selectively distinguished between C57BL/6J and BALB/cJ using the methods disclosed herein.
  • SNVs single nucleotide variants
  • Three sets of two iLock padlock oligos for C57BL/6J and BALB/cJ corresponding to each gene were separately added to the 50-micron liver slices of C57BL/6J and BALB/cJ.
  • the liver slices 20 were processed as described for FIG.5.
  • the RCA amplicons from the iLock padlock oligos targeting C57BL/6J SNVs were detected with a Cy5 probe (Red).
  • the RCA amplicons from the iLock padlock oligos targeting BALB/cJ SNVs were detected with a Cy3 probe (Green).
  • the selectivity was evaluated based on the ratio of the number of amplicons from C57BL/6J and BALB/cJ padlock oligos.
  • FIGS. 7A-B show that DNase I treatment improved the detection efficiency in the brain slices.
  • Excitatory neuron marker, Slc17a7 was detected in the 30-micron brain slices of C57BL/6J with two iLock padlock oligos. The RCA amplicons from the iLock padlock oligos were detected with a Cy5 probe (Red). Genomic DNA was visualized with DAPI 30 (Blue). The samples were measured with a spinning desk confocal microscope.
  • FIG. 8 shows selective single nucleotide variant detection with iLock oligo for C57BL/6 and Balb/c liver. A single nucleotide variant on Apoa2 gene was targeted.
  • iLock probes Two iLock probes have one nucleotide difference in the binding sites which make them complementary to C57BL/6 3 Attorney Docket No.36406.0040P1 and Balb/c transcripts, respectively. Following the protocol, iLock probes were cleaved with Taq polymerase and amplified after circularization. The amplicons were detected with Cy5 and Cy3 probes for C57BL/6 and Balb/c probes with a confocal microscope. The amplification was observed with the appropriate combinations of the iLock probes and 5 strains. The selectivity was above 98%. FIGS.
  • FIG.9A-C show selective evaluation of 254 iLock probes targeting 127 single nucleotide variants between C57BL/6J and PWK/PhJ strains on 38 genes with the B6 brain slice.
  • FIG.9A show amplifications of 254 iLock probes from the B6 hippocampus detected with Cy5 and AF750 probes with a confocal microscope. Significant number of amplicons 10 were found in Cy5 channel.
  • FIG.9B shows allele fraction of detected B6 and PWK amplicons per gene per field of view (FOV). Highly expressed genes showed low allele fraction of PWK.
  • FIG.9C shows b, ox plot of allele fractions for genes per FOV.
  • FIGS. 10A-D show scenarios for detecting allelic burst synchronization using various methodologies.
  • FIG.10A shows a schematic representation of a two-state model of stochastic transcriptional bursts (Kim, J. K. and Marioni, J. C. Genome Biol. 14, R7 (2013)).
  • FIG.10B shows a schematic representation of two potential scenarios: independent and dependent bursting, along with their expected observations. The left panel illustrates the underlying 20 promoter states and corresponding nascent RNA signals, resulting in mRNA bursts over time with a constant degradation rate.
  • FIG. 10C depicts a histogram showing an example of transcript distribution, comparing a two-state model of stochastic transcriptional bursts with a constant expression model. The two-state model was fitted using a Beta-Poisson distribution, while 25 the constant expression model was fitted with a Poisson distribution.
  • FIG.10D shows a scatter plot illustrating simulation outcomes of detectable allelic burst synchronization, measured as Pearson's correlation between alleles. Poisson sampling was applied to computationally generated, perfectly synchronized model alleles. Sampling rates were based on previously reported mRNA capture efficiencies and eSNV detection rates for both 30 scRNA-seq and direct in situ detection.
  • FIGS.11A-U show spatial transcriptomics in thick tissue with single-nucleotide resolution.
  • FIG.11A shows a schematic representation of iLock probes designed to 4 Attorney Docket No.36406.0040P1 distinguish eSNVs on RNA templates.
  • FIG.11B shows a schematic overview of two experimental workflows: one assessing eSNV detection selectivity in inbred mouse strains and the other analyzing allele-specific expression in an F1 mouse strain.
  • FIG.11C shows images of liver slices from C57BL and BALB mouse strains, showing iLock oligo reactions 5 targeting the Apoa2 eSNV on mRNA.
  • FIG. 11D shows images of liver slices from C57BL and BALB mouse strains, showing iLock oligo reactions targeting the Apoa2 eSNV on mRNA using the 3DEEP (DNase I) protocol.
  • 3DEEP DNase I
  • FIG. 11E depicts a histogram showing the number of amplicons for C57BL iLock in C57BL liver slices and BALB iLock in BALB liver slices.
  • FIG. 11F shows a heatmap displaying the expression of curated marker genes, imprinting genes, and genes with partial parent-of-origin effects in scRNA-seq data across annotated major cell types in the brain.
  • FIG.11G shows a schematic 15 representation of HybISS in situ sequencing decoding.
  • FIG.11H depicts an image showing the first round of HybISS in situ sequencing for C57BL and PWK brain slices with 254 iLock probes, including both C57BL and PWK iLock probes.
  • FIG. 11I depicts a histogram showing decoded transcript counts for bona fide genes and ghost IDs without matching iLock probes.
  • FIG.11J depicts a point plot showing the decoded 25 transcript counts and the fraction of false alleles detected in non-matched strain slices, calculated as the ratio of iLock probes targeting one strain to the total amplicons in the opposing strain's tissue.
  • FIG.11K depicts a scatter plot showing the correlation between on- target transcript counts for the genes in C57BL and PWK slices, calculated as the number of iLock amplicons targeting one strain detected in the corresponding strain’s tissue.
  • FIG.11L 30 depicts a scatter plot showing the correlation between off-target transcript rates for the genes in C57BL and PWK slices, calculated as the number of iLock amplicons targeting one strain detected in the opposing strain’s tissue.
  • FIG.11M depicts images showing the raw in situ sequencing from the first round, along with transcripts assigned to each cell using the Baysor 5 Attorney Docket No.36406.0040P1 computational pipeline.
  • FIG.11N depicts box plots showing the numbers of detected transcripts per cell between 3Diva-seq and scRNA-seq (GSE108097) for the shared gene set. P-values were calculated using the Wilcoxon signed-rank test.
  • FIG.11O depicts images showing the first round of HybISS in situ sequencing for B6PWKF1 mice, 5 using 254 iLock probes, including both C57BL and PWK iLock probes.
  • (Red) Cy5 signals from C57BL iLock amplicons and
  • FIG. 11P depicts a scatter plot showing the correlation between detected transcript counts for the genes in B6PWKF1 mice and CPM in bulk RNA-seq (GSE179711) for the shared gene set.
  • FIG. 11Q shows point plot displaying the ratios of allele-specific expression.
  • (Blue) Well- 10 characterized genomic imprinting genes.
  • FIG.11R depicts a scatter plot showing the correlation of ratios of allele-specific expression observed in 3Diva-seq and bulk RNA-seq (GECCO; Gene Expression in the Collaborative Cross).
  • FIG. 11S depicts a scatter plot showing the UMAP coordinates of single cells identified in 3Diva-seq. Annotations of the major brain cell types were provided using curated marker genes.
  • FIG.11T shows a heatmap 15 displaying the expression of curated marker genes in 3Diva-seq data across annotated major cell types in the brain.
  • FIG.11U shows point plots showing the genomic imprinting patterns of five genomic imprinting genes across the five major brain cell types.
  • FIGS.12A-I show a strong concordance in burst size and frequency between alleles across neocortex layers.
  • FIG. 12A shows histograms displaying transcript count distributions 20 and model fit results for five representative genes, comparing Beta-Poisson and Poisson model fits. Transcript distributions are fitted within the cell type where each gene is most highly expressed.
  • FIG.12B depicts a histogram showing the ratios of negative log- likelihoods (Beta-Poisson / Poisson model fitting). A ratio lower than one indicates support for the Beta-Poisson model, while a ratio higher than one indicates support for the Poisson 25 model.
  • FIG. 12A shows histograms displaying transcript count distributions 20 and model fit results for five representative genes, comparing Beta-Poisson and Poisson model fits. Transcript distributions are fitted within the cell type where each gene is most highly expressed.
  • FIG.12B depicts a histogram showing the ratios of negative log- likelihoods (Beta-Poisson / Po
  • FIG. 12C depicts scatter plots showing the correlations in burst frequency and burst size between C57BL and PWK alleles, derived from Beta-Poisson model fits of transcript count distributions for each gene in 3Diva-seq data from the cortex of B6PWKF1 mice. Transcript distributions are fitted within the cell type where each gene is most highly expressed.
  • FIG.12D depicts images showing reference annotations from the Allen Brain 30 Atlas and density-based separations of cortical layers.
  • FIG.12E depicts images showing the coordinates of detected transcripts, colored by annotated cell types and glutamatergic neurons.
  • FIG.12F shows an image displaying the density of glutamatergic neurons in the cortex, with kernel density estimation applied to glutamatergic neurons.
  • FIG. 12G depicts point plots showing burst kinetics parameters for each gene in each layer, derived from Beta-Poisson model fits of transcript count distributions.
  • FIG.12H depicts box plots showing the coefficient of variation of the eight genes for burst frequency and burst size across different 5 layers. P-values were calculated using the Wilcoxon rank-sum test. p ⁇ 0.01. FIG.
  • FIG. 12I depicts scatter plots showing the correlations in burst frequency and burst size between C57BL and PWK alleles after layer separation, derived from Beta-Poisson model fits of transcript count distributions for each gene from the cortex of B6PWKF1 mice.
  • FIGS.13A-M show widespread synchronization of transcriptional bursts between 10 alleles.
  • FIG.13A depicts scatter plots showing the correlation of transcript counts between C57BL and PWK alleles in single cells. Pearson’s r is calculated within the cell type where each gene is most highly expressed.
  • FIG.13B depicts pie charts showing the fractions of genes with allele correlations (>0.2), categorized by their estimated CPM.
  • FIG.13C depicts a scatter plot showing the correlation between allelic correlations measured in FIG.13A and 15 the mean transcript count per cell. Pearson’s r is calculated within the cell type where each gene is most highly expressed. The color represents the cell type used for calculating Pearson's r for each gene.
  • FIG.13D depicts a scatter plot showing the effect of Poisson sampling on allelic correlation and mean transcript count per cell for simulated virtual genes under perfect allelic synchronization. Colors indicate the cell type used to calculate Pearson's 20 r for each gene, as shown in FIG.13C. (Black line) Expected correlation coefficient derived from LOWESS fitting of the simulated data points.
  • FIG.13E depicts line plots showing the effect of tissue slice thickness on allelic correlations based on simulations.
  • FIG. 13F depicts box plots showing the ratios of simulated allelic correlations (after virtual slicing at the specified thickness) to the observed correlations.
  • FIG.13G depicts point and box plots showing the extent of artificial allelic correlations induced by allele misidentification, simulated using Binomial sampling with measured allele misidentification rates for each gene. (Red) Observed allelic correlations. Box plots display the outcomes of 30 1,000 repeated simulations. The colors of gene names indicate the cell type used to calculate Pearson's r for each gene, as shown in FIG.13C.
  • FIG. 13H depicts line plots showing simulated allelic correlations induced by varying allele misidentification rates, with points representing the observed allelic correlations.
  • FIG.13I depicts a scatter plot showing the 7 Attorney Docket No.36406.0040P1 minimum allele misidentification rate required to induce the observed allelic correlations.
  • FIG. 13J depicts point plots showing the extent of artificial allelic correlations induced by hidden subtypes with distinct burst kinetics parameters. The simulation generated 1, 2, 4, 8, 16, and 32 subtypes with randomly selected parameters. For each number of subtypes, the 5 simulation was repeated 1,000 times, with the average correlation coefficient between alleles visualized as blue points. (Red) Observed allelic correlations.
  • FIG. 13K depicts scatter plots showing the observed and simulated correlations between alleles. For the simulated correlations, results assuming 32 subtypes are displayed. Three 10 representative genes are shown.
  • FIG.13L depicts point plots showing the correlation coefficients between alleles after layer separation of glutamatergic neurons.
  • FIG.13M depicts a scatter plot showing the correlation between allelic correlations from FIG. 13L and mean transcript count per cell. The line represents the expected correlation coefficients based on simulation from FIG. 13D.
  • FIGS.14A-E show interchromosomal co-bursting of genes occurs as frequently as intrachromosomal co-bursting.
  • FIG.14A-E show interchromosomal co-bursting of genes occurs as frequently as intrachromosomal co-bursting.
  • FIG. 14A depicts scatter plots showing the correlation of transcript counts between paternal Slc17a7 and other alleles. Slc17a7 exhibits the highest expression level in glutamatergic neurons.
  • FIG. 14B depicts a heatmap showing correlations of transcript counts between alleles of the same or different genes. “M” represents maternal 20 alleles, and “P” denotes paternal alleles. To minimize false negatives due to low expression, genes with transcript counts exceeding 0.9 per cell were included. The maximum expression level for genes without synchronization, as shown in FIG.13C, is 0.899.
  • FIG. 14C shows a paired dot plot comparing transcript count correlations between alleles from different genes.
  • FIG.14D shows a scatter plot comparing transcript count correlations for gene pairs measured using 3Diva-seq and scRNA-seq, grouped by intra- and inter-chromosomal pairs.
  • FIG. 14E depicts histograms displaying the distributions of transcript count correlations 30 between different genes in scRNA-seq data from the mouse somatosensory cortex (Allen Brain Atlas), with genes categorized as intra- or inter-chromosomal pairs.
  • FIGS. 15A-E show the fitting of transcript count distributions using the Beta-Poisson and Poisson models.
  • FIGS.15A-E depict histograms showing 8 Attorney Docket No.36406.0040P1 transcript count distributions for each gene.
  • (Red line) Beta-Poisson model fitting.
  • FIGS. 15A-E correspond to five major cell types: glutamatergic neuron, GABAergic neuron, oligodendrocyte, astrocyte, and microglia.
  • FIGS. 16A-E (related to FIG. 13) show the synchronization of alleles of bi-allelically 5 expressed genes.
  • FIGS. 16A–E depict scatter plots showing transcript counts of alleles in single cells.
  • FIGS. 16A–E correspond to the five major cell types: glutamatergic neurons, GABAergic neurons, oligodendrocytes, astrocytes, and microglia.
  • FIGS. 17A-C (related to FIG.13) show the simulation workflows.
  • FIG.17A shows a 10 schematic representation of the simulation workflow to assess the effect of detection sampling on allelic correlations.
  • FIG.17B shows a schematic representation of the simulation workflow to evaluate the impact of allele misidentification rates on allelic correlations using Binomial sampling.
  • FIG.17C shows a schematic representation of the simulation workflow to investigate the effect of varying numbers of hidden subtypes (1, 2, 4, 8, 16, and 32) on 15 allelic correlations.
  • FIG.18A shows maximum intensity projection image of the 1 st round of 3Diva-seq from FOV8, corresponding to 10-40 ⁇ m along the Z-axis. Red points represent B6 amplicons, while blue points represent PWK amplicons.
  • FIG.18B shows images 20 illustrating the decoding process, from raw image to Baysor single-cell assignment, maternal- paternal transcript identification, and the distribution of maternal and paternal genes. For representative illustrations, four glutamatergic neurons are shown with maternal and paternal Slc17a7 transcripts, and two GABAergic neurons with maternal and paternal Gad1 transcripts. 25 FIG.
  • FIG. 19 shows the iLock probes that were designed to target 38 genes to assess allelic correlations in the brain.
  • FIG. 20 shows the bridge probes and fluorescent probes used to generate a unique color sequence for each gene-allele combination.
  • 30 DETAILED DESCRIPTION OF THE INVENTION The present disclosure can be understood more readily by reference to the following detailed description of the invention, the figures and the examples included herein. 9 Attorney Docket No.36406.0040P1 Before the present methods and compositions are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary.
  • tissue sample is meant a tissue or organ from a subject; or a solution containing one or more molecules derived from tissue material (e.g., nucleic acid), which is assayed as described herein.
  • tissue sample can be obtained via biopsy such as needle biopsy, surgical biopsy, etc.
  • the “tissue sample” can include for 15 example, a specimen from a diseased tissue (e.g., cancers, parts of a cancer, and also the cancer mass as a whole and/or tissue derived from a subject that is suspected of having a disease).
  • a diseased tissue e.g., cancers, parts of a cancer, and also the cancer mass as a whole and/or tissue derived from a subject that is suspected of having a disease.
  • the term “comprising” can include the aspects “consisting of” and “consisting essentially of.” “Comprising” can also mean “including but not limited to.” 20
  • the phrase “at least” preceding a series of elements is to be understood to refer to every element in the series. For example, “at least one” includes one, two, three, four or more.
  • the term “target of interest” is nucleic acids.
  • a target of interest can be a nucleic acid molecule which can be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others.
  • the target of 25 interest can be a target nucleic acid molecule from a tissue sample, or a secondary target such as a product of an amplification reaction, etc. It may be any length.
  • the target of interest can be RNA.
  • the term “nucleic acids” or “oligonucleotide” or grammatical equivalents herein refers to at least two nucleotides covalently linked together. Nucleic acids containing one or 30 more carbocyclic sugars are also included within the definition of nucleic acids.
  • nucleic acid will generally contain phosphodiester bonds, although in some cases (for example, in the construction of primers and probes, such as label probes), nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide 11 Attorney Docket No.36406.0040P1 (Beaucage et al., Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sblul et al., Eur. J. Biochem.81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem.
  • Nucleic acids may be single-stranded or double-stranded, as specified, or contain portions of both double-stranded or single-stranded sequences.
  • the nucleic acids may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of 12 Attorney Docket No.36406.0040P1 deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • concatemers refer to a form of target polynucleotides, containing multiple copies (e.g., monomers) of a target polynucleotide or a fragment of a target 5 nucleotide.
  • the concatamer contains a sequence of interest.
  • a concatemer can be partially double-stranded.
  • the plurality of concatemers can serve as a target nucleic acid molecule for sequencing.
  • the concatemers comprise a single-stranded RNA portion and a double-stranded RNA portion.
  • nucleotide bases are abbreviated as follows: adenine (A), cytosine (C), guanine (G), 10 thymine (T), and uracil.
  • substantially degrading the DNA in the tissue sample refers to degrading greater than 95%, 96%, 97%, 98% or 99% of the DNA that would normally be found in the tissue sample.
  • substantially removing lipids from the tissue sample refers to 15 removing greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% of the DNA that would normally be found in the tissue sample.
  • Spatial transcriptomic is a cutting edge technology, which realizes molecular pathology at scale.
  • Pathological diagnosis is common practice particularly in fields such as oncology, nephrology, and dermatology.
  • genetic diagnosis has proven exceptionally beneficial, offering insights into drug responses and 30 enhancing overall survival rates for patients.
  • Described herein are methods to detecting numerous single nucleotide variants simultaneously in tissue slices. By combining pathology analysis with the identification of these variants, the methods described herein can enhance the accuracy and predictive capabilities of disease classifications.
  • the methods integrate hydrogel and enzymatic techniques to accurately detect single nucleotide variants in RNAs within tissue sections, ensuring both high accuracy and high throughput capability.
  • the methods disclosed herein can detect a target of interest, including single 5 nucleotide variants, and said detection can be useful in diagnosing cancer and providing personalized therapeutic treatment strategies.
  • the methods disclosed herein can be used in clinical pathology with emphasis of tumor evolution, drug response, and prognosis prediction.
  • the methods described herein can simultaneous characterize >100 variants, a stark 10 contrast to the currently available products that are limited to assessing one variant at a time.
  • METHODS 15 Disclosed herein are methods for detecting a target of interest in a tissue sample. Also disclosed herein are methods for detecting RNA in a tissue sample. In some aspects, the methods comprise obtaining or having obtained a tissue sample. In some aspects, the methods can comprise permeabilizing the tissue sample. In some aspects, the methods can comprise substantially degrading the DNA in the tissue sample. In some aspects, the methods can 20 comprise introducing to the tissue sample a primer or a probe specific to the target of interest. In some aspects, the primer or probe binds to the target of interest.
  • the methods can comprise detecting the primer or probe bound to the target of interest, thereby detecting the target of interest in the tissue sample.
  • the target of interest can be RNA. 25
  • methods for detecting a single nucleotide variant in a tissue sample are also disclosed herein.
  • methods for detecting RNA in a tissue sample comprise obtaining or having obtained a tissue sample.
  • the methods can comprise permeabilizing the tissue sample.
  • the methods can comprise substantially degrading the DNA in the tissue sample.
  • the methods 30 can comprise introducing to the tissue sample a primer or a probe specific to the single nucleotide variant.
  • the primer or probe binds to the single nucleotide variant.
  • the methods can comprise detecting the primer or probe bound to the single nucleotide variant, thereby detecting the single nucleotide variant in the tissue sample.
  • the single nucleotide variant can be RNA.
  • the single nucleotide variant can be DNA.
  • the methods further comprise amplifying a probe bound to the target of interest using a polymerase. In such aspects by degrading or removing DNA from the 5 tissue sample, there is no DNA that may have been present initially in the tissue sample to serve as a template for the polymerase.
  • the methods further comprise amplifying a probe bound to the single nucleotide variant using a polymerase. In such aspects by degrading or removing DNA from the tissue sample, there is no DNA that may have been present initially in the tissue sample to 15 serve as a template for the polymerase.
  • methods of hybridizing a padlock probe to a single nucleotide variant circularizing the padlock probe and subsequently amplifying the circularized padlock probe with a DNA polymerase such as phi29 polymerase, and, by degrading or removing DNA from the tissue sample prior to hybridizing a padlock probe to a single nucleotide variant, there is no DNA that may have 20 been present initially in the tissue sample to serve as a template for the polymerase.
  • methods of making a hydrogel within a tissue sample can comprise obtaining or having obtained a fixed tissue sample; permeabilizing the tissue sample; and substantially degrading the DNA in the tissue sample; wherein a hydrogel is formed within the tissue sample.
  • the tissue sample can 25 comprise DNA, non-RNA molecules, and RNA.
  • the RNA can comprise a target of interest.
  • the non-RNA molecules can be proteins.
  • the non-RNA molecules can be lipids.
  • the proteins present in the tissue sample can be cross-linked.
  • the tissue sample can be preserved prior to the step of obtaining or having obtained the tissue sample.
  • the method can 30 comprise preserving the tissue sample using a chemical crosslinking agent.
  • the crosslinking agent can be formaldehyde, glutaraldehyde.
  • SM(PEG)12 PEGylated, long-chain SMCC crosslinker
  • SM(PEG)6 PEGylated, long-chain SMCC crosslinker
  • SM(PEG)2 PEGylated SMCC crosslinker
  • SIAB succinimidyl (4- 15 Attorney Docket No.36406.0040P1 iodoacetyl)aminobenzoate
  • BMH bismaleimidohexane
  • SBAP succinimidyl 3- (bromoacetamido)propionate
  • SMPT 4-succinimidyloxycarbonyl-alpha-methyl- ⁇ (2- pyridyldithio)toluene
  • DTSSP 3,3'-dithiobis(sulfosuccinimidyl propionate)
  • EMCH N- ⁇ - maleimidocaproic acid hydrazide
  • SM(PEG)12 PEGylated,
  • the tissue sample can have a thickness of about 20 ⁇ m to 800 ⁇ m.
  • Tissue sample can be any 3D cell structure.
  • the tissue sample can comprise DNA, non-RNA molecules, and RNA.
  • the tissue can comprise lipids.
  • the non-RNA molecules can be lipids.
  • the non-RNA molecules can be proteins.
  • the RNA can comprise a target of interest.
  • the tissue sample can be from a mammal (e.g. mammalian tissue samples).
  • the tissue sample can be from eukaryote. 20
  • the tissue sample can be a human tissue samples.
  • the tissue sample can be a liver tissue sample. In some aspects, the tissue sample can be a brain tissue sample. In some aspects, the tissue sample can be a skin sample. In some aspects, the tissue sample can be an organoid. In some aspects, the tissue sample can be fixed prior to or after the permeabilizing of the tissue sample step. In some aspects, the tissue sample can be fixed 25 by contacting the tissue sample with a chemical crosslinking agent.
  • the chemical crosslinking agent can be formaldehyde, glutaraldehyde, dimethyl suberimidate, SM(PEG)12 (PEGylated, long-chain SMCC crosslinker), SM(PEG)6 (PEGylated, long-chain SMCC crosslinker), SM(PEG)2 (PEGylated SMCC crosslinker), SIAB (succinimidyl (4- iodoacetyl)aminobenzoate), BMH (bismaleimidohexane), SBAP (succinimidyl 3-30 (bromoacetamido)propionate), SMPT (4-succinimidyloxycarbonyl-alpha-methyl- ⁇ (2- pyridyldithio)toluene), DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), EMCH (N- ⁇ - maleimidocaproic), SM
  • crosslinking reagents that contain reactive end groups that respond to the presence of specific functional groups by forming bonds between polymer chains can be used as crosslinkers in the methods disclosed 10 herein.
  • the tissue sample can have a thickness of about 15 ⁇ m to 400 ⁇ m. In some aspects, the tissue sample can have a thickness of about 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ⁇ m or any thickness in between.
  • the tissue sample can have a thickness of about 15 15 ⁇ m to 20 ⁇ m, 20 ⁇ m to 30 ⁇ m, 30 ⁇ m to 40 ⁇ m, 40 ⁇ m to 50 ⁇ m, 50 ⁇ m to 60 ⁇ m, 60 ⁇ m to 70 ⁇ m, 70 ⁇ m to 80 ⁇ m, 80 ⁇ m to 90 ⁇ m, 90 ⁇ m to 100 ⁇ m, 100 ⁇ m to 125 ⁇ m, 125 ⁇ m to 150 ⁇ m, 150 ⁇ m to 175 ⁇ m, 175 ⁇ m to 200 ⁇ m, 200 ⁇ m to 225 ⁇ m, 225 ⁇ m to 250 ⁇ m, 250 ⁇ m to 275 ⁇ m, 275 ⁇ m to 300 ⁇ m, 300 ⁇ m to 325 ⁇ m, 325 ⁇ m to 350 ⁇ m, 350 ⁇ m to 375 ⁇ m, 375 ⁇ m to 400 ⁇ m, 400 ⁇ m to 425 ⁇ m, 425 ⁇ m to 400 ⁇ m
  • the tissue sample after the step of substantially degrading the DNA in the tissue sample, can have enhanced molecular diffusion compared to the tissue sample 25 after the step of permeabilizing the tissue sample. In some aspects, the molecular diffusion can be confirmed by affirming the uniformity of RCA amplicons across the tissue samples.
  • Target of interest can be a nucleic acid molecule. In some aspects, the target of interest can be present in a tissue sample. In some aspects, the target of interest can be in a single region of the nucleic acid molecule. In some aspects, the 30 target nucleic acid molecule can be in any nucleic acid sample of interest. The source, identity, and preparation of many such nucleic acid samples are known.
  • nucleic acid samples known or identified for use in amplification or detection methods can be used for the method described herein.
  • the disclosed methods can involve utilizing RNA or RNA fragments comprising one or more targets of interest in a tissue sample.
  • the target of interest can be a RNA nucleic acid sequence.
  • the target of interest can be single-stranded.
  • the hybridization region and the amplification region within the 5 target nucleic acid molecule can be defined in terms of the relationship of the target nucleic acid molecule to the primers in a set of primers.
  • the primers can be designed to match (e.g., be complementary to) the chosen target of interest.
  • the nucleic acid sequence to be amplified and the sites of hybridization of the primers can be separate since sequences in and around the sites where the primers hybridize can be amplified.
  • the target of interest can comprise a mutation.
  • the mutation can be a single nucleotide variant (SNV).
  • SNV single nucleotide variant
  • a single nucleotide variant occurs when a single nucleotide (adenine, thymine, cytosine, or guanine) in the genome sequence (DNA or RNA) is altered.
  • Single nucleotide variants can be rare or common in a population.
  • single nucleotide variants are referred to as single nucleotide polymorphisms if 15 they are present in at least 1% of the population.
  • a SNV can occur in a protein coding region, which can result in either 1) a nucleotide substitution that does not result in a change in amino acid (synonymous change). This is possible because multiple codons (sets of three nucleotides) code for the same amino acid; 2) a nucleotide substitution leads to an amino acid substitution. This may or may not result in a pathogenic variant 20 depending on the effect of the amino acid substitution on protein function and structure.
  • the nucleic acid sample can be, for example, a nucleic acid sample from one or more cells, tissues, or other biological samples, such as tissue culture cells, tissues slices, and archeological samples such as bone or mummified tissue.
  • Tissue samples can be derived from any source including, but not limited to, eukaryotes, plants, animals, vertebrates, fish, mammals, humans, non-humans, needle aspiration biopsies, cancers, 30 tumors, tissues, cells, stool, mummified tissue, forensic sources, autopsies, archeological sources, infections, nosocomial infections, production sources, drug preparations, biological molecule productions, or protein preparations.
  • Types of useful tissue samples include eukaryotic samples, plant samples, animal samples, vertebrate samples, fish samples, 18 Attorney Docket No.36406.0040P1 mammalian samples, human samples, non-human samples, biological samples, biopsy samples, needle aspiration biopsy samples, cancer samples, tumor samples, tissue samples, cell samples, cell lysate samples, crude cell lysate samples, tissue lysate samples, stool samples, mummified tissue samples, forensic samples, autopsy samples, archeological 5 samples, infection samples, nosocomial infection samples, or protein preparation samples.
  • the target nucleic acid molecule can be damaged RNA from a damaged RNA tissue sample.
  • RNA and damaged RNA in a tissue sample are thus useful for the disclosed methods.
  • Any degraded, fragmented or otherwise damaged RNA or tissue sample containing such RNA can be used in the disclosed methods.
  • the disclosed methods can involve the sequencing of nucleic acids (e.g., a target of 15 interest, including target nucleic acid molecules).
  • the target nucleic acid molecules are RNA.
  • the target nucleic acid molecules can comprise a single nucleotide variant.
  • the target nucleic acid molecule comprises an amplification region and a hybridization region.
  • the hybridization region can include a sequences that can be 20 complementary to a primer in a set of primers.
  • the amplification region can be the portion of the amplification region that can be amplified. In some aspect, the amplification region can be downstream of or flanked by the hybridization region(s).
  • the method disclosed herein comprise permeabilizing a tissue sample.
  • the step of permeabilizing the 25 tissue sample can take place after the tissue sample is obtained.
  • lipids can be removed or partially removing lipids from a tissue sample. In some aspects, the step of removing or partially removing lipids can be optional.
  • lipids can be partially, substantially, or completely removed from the tissue sample.
  • the “permeabilizing” step of the disclosed methods can encompass lipid removal or partial lipid 30 removal and DNA degradation.
  • the step of reducing or removing lipids from the tissue sample can be carried out after the step of permeabilizing the tissue sample.
  • the tissue sample can be permeabilized by contacting the tissue sample with a tissue-permeabilizing agent.
  • the tissue-permeabilizing agent can be a 19 Attorney Docket No.36406.0040P1 detergent or sodium dodecyl sulfate (SDS).
  • the detergent can be an ionic detergent.
  • the tissue-permeabilizing agent can be any alcohol.
  • the alcohol can be methanol or ethanol. 5
  • the method disclosed herein comprises degrading DNA in a tissue sample.
  • degrading or removing DNA in the tissue sample can allow for better hydrogel formation.
  • degrading or removing DNA in the tissue sample can allow for better primer or probe hybridization to the target of interest or single nucleotide variant in the tissue sample because 10 DNA is not present as a target.
  • degrading or removing DNA in the tissue sample can allow for better diffusion of molecules into the tissue.
  • DNA can be degraded or removed or substantially degraded or removed by contacting the tissue sample with DNase (e.g., DNase I). In some aspects, DNA can be degraded or removed or substantially degraded or removed by contacting the tissue sample with exonuclease 15 enzymes.
  • the exonuclease enzyme can be DNase I. In some aspects, the exonuclease enzyme can be any dsDNA degrading enzymes (e.g., Lambda Exonuclease, Exonuclease V, and T5 Exonuclease).
  • the step of substantially degrading the DNA in the tissue sample can be carried out or performed prior to embedding the tissue sample in a hydrogel. In some 20 aspects, the step of substantially degrading the DNA in the tissue sample can be carried out or performed after embedding the tissue sample in a hydrogel.
  • Degrading non-RNA molecules in the tissue sample. In some aspects, the methods disclosed herein can further comprise substantially degrading non-RNA molecules in the tissue sample. In some aspects, non-RNA molecules can be proteins or protein molecules. In 25 some aspects, non-RNA molecules can be lipids. In some aspects, the methods disclosed herein can further comprise degrading proteins or protein molecules in the tissue sample.
  • the proteins or protein molecules can be substantially degraded by contacting the tissue sample with proteinase K.
  • the step of substantially degrading non- RNA molecules in the tissue sample can be carried out at least before the step of detecting the 30 primer or probe bound to the target of interest or single nucleotide variant.
  • the proteins or protein molecules diffuse out of the hydrogel without actively removing the proteins or protein molecules from a polymerized tissue. 20 Attorney Docket No.36406.0040P1 Hydrogels and matrices.
  • a hydrogel is a three-dimensional (3D) network of hydrophilic polymers that maintain the structure due to chemical or physical cross-linking of individual polymer chains. Hydrogels made up of hydrophilic polymers that can be crosslinked.
  • the hydrogels described herein can be chemical hydrogels or physical hydrogels.
  • chemical hydrogels can be formed by covalent cross- linking bonds.
  • physical hydrogels can have non-covalent bonds.
  • the methods disclosed herein can further comprise forming a matrix 10 within the tissue sample.
  • a matrix can be formed within the tissue sample at any time during the method.
  • the time at which the matrix is formed can depend on the type of detection of the target of interest or single nucleotide variant that is to be carried out. For example, a hydrogel might not be necessary if a hybridization chain reaction is to be carried out.
  • a protein matrix can be formed by chemical fixation. 15
  • a hydrogel matrix can be helpful.
  • the matrix can be formed or introduced after obtaining the tissue sample.
  • a matrix can be formed or introduced after the step of permeabilizing the tissue sample.
  • a matrix can be formed or introduced after the step of substantially degrading the DNA in the tissue sample.
  • a matrix can be formed or introduced after the step of 20 introducing to the tissue sample a primer or a probe that is specific to the target of interest or single nucleotide variant.
  • a protein lattice formed by chemical crosslinking during fixation can be a matrix which maintains the molecular spaces.
  • a matrix can be substituted by chemical hydrogels.
  • a chemical hydrogel matrix can be produced by introducing acrylamide monomers into tissue slices and activating 25 polymer reactions.
  • a tissue sample can be embedded in a hydrogel prior to or after the step of introducing to the tissue sample a primer or a probe specific to the target of interest or the single nucleotide variant, wherein the primer or probe binds to the target of interest or the single nucleotide variant.
  • the hydrogel 30 comprises acrylamide and bisacrylamide monomers. Other monomers that can be used include, but not limited to, acrylic acid, HEMA, and NVP.
  • the tissue sample can be embedded in the hydrogel by diffusing acrylamide monomers into the tissue sample and crosslinking them to form a polyacrylamide gel. 21 Attorney Docket No.36406.0040P1
  • the tissue sample can be embedded in the hydrogel after the step of permeabilizing the tissue sample.
  • the tissue sample can be embedded in the hydrogel after the step of substantially degrading the DNA in the tissue sample.
  • the matrix formed within the tissue sample can be cross-linking of proteins present in the tissue sample.
  • a matrix or hydrogel can be formed any time after obtaining or having obtained the tissue sample. Primers and probes.
  • the method comprises introducing to the tissue sample a primer or a probe specific to the target of interest or the 10 single nucleotide variant, wherein the primer or probe binds to the target of interest or the single nucleotide variant.
  • the probe can be an oligonucleotide probe.
  • the oligonucleotide probe can be attached to the hydrogel.
  • the oligonucleotide probe cam be covalently attached to the hydrogel.
  • the oligonucleotide probe can be modified with an acrydite moiety.
  • the target of 15 interest can be a single nucleotide variant.
  • the probe specific to the target of interest or the single nucleotide variant can be a padlock probe.
  • the padlock probe can be circularized after the step of introducing to the tissue sample the probe specific to the target of interest or the single nucleotide variant, wherein the probe binds to the target of interest or the single 20 nucleotide variant.
  • the padlock probe can be circularized after hybridizing to the target of interest or the single nucleotide variant by contacting the polymerized tissue with a ligase.
  • the disclosed methods further comprises contacting a circularized padlock probe with a primer complementary to the padlock probe.
  • the 25 primer complementary to the padlock probe can be covalently attached to a hydrogel.
  • the methods can further comprise subjecting the circularized padlock probe to rolling circle amplification (RCA) to generate an amplicon using the circularized padlock probe as a template and an oligonucleotide primer as the primer.
  • the amplicon comprises a concatemerized repeat sequence corresponding to the target of interest 30 or the single nucleotide variant.
  • the methods further comprise detecting the concatemerized repeat sequence corresponding to the target of interest or the single nucleotide variant. 22 Attorney Docket No.36406.0040P1
  • a padlock probe used in the disclosed methods can comprise terminal regions complementary to the target of interest or the single nucleotide variant.
  • an oligonucleotide primer can be used.
  • the oligonucleotide primer can be complementary to target of interest or the single nucleotide 5 variant or to a part of a padlock oligo that is included by contacting the primer with the tissue sample.
  • the methods can further comprise subjecting the circularized padlock probe to rolling circle amplification (RCA) to generate an amplicon using the circularized padlock probe as a template and an oligonucleotide primer as the primer, wherein the amplicon comprises a concatemerized repeat sequence corresponding to the RNA of 10 interest.
  • the oligonucleotide primer can be covalently attached to a hydrogel.
  • the oligonucleotide primer can be modified with an acrydite moiety and incorporated into the hydrogel during polymerization or the permeabilization step.
  • the padlock probes and oligonucleotide primers can be covalently incorporated into a polymerized or permeabilized tissue.
  • the oligonucleotide primer can be 15 modified.
  • the oligonucleotide can be modified with an acrydite moiety.
  • the amplification of the padlock probe can be rolling circle amplification. Any sequence present in a target of interest or a single nucleotide variant can serve as a primer binding site, to which a primer or probe can hybridize to.
  • a primer binding site can be from about 3 to about 30 nucleotides in length, about 15 to about 25 in length. 20 Primer oligonucleotides can be usually 6 to 25 bases in length.
  • the sequence in a primer can hybridize to another nucleic acid molecule and can be referred to as the complementary portion of the primer.
  • the complementary portion of a primer can be any length that supports specific and stable hybridization between the primer and the nucleic acid molecules (e.g., target of interest) under the reaction conditions.
  • Primers can have, for example, a length of 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucle
  • primers can have, for example, a length of less than 4 nucleotides, less than 5 nucleotides, less than 6 nucleotides, less than 7 nucleotides, less than 8 nucleotides, less 23 Attorney Docket No.36406.0040P1 than 9 nucleotides, less than 10 nucleotides, less than 11 nucleotides, less than 12 nucleotides, less than 13 nucleotides, less than 14 nucleotides, less than 15 nucleotides, less than 16 nucleotides, less than 17 nucleotides, less than 18 nucleotides, less than 19 nucleotides, less than 20 nucleotides, less than 21 nucleotides, less than 22 nucleotides, less than 23 5 nucleotides, less than 24 nucleotides, less than 25 nucleotides, less than 26 nucleotides, less than 27 nucleotides, less than 28 nucleotides
  • a “probe” can mean an oligonucleotide used in hybridization.
  • the probes can be labeled oligonucleotides having sequence complementary to detection tags or another sequence on amplified nucleic acids.
  • the complementary portion of a probe can be any length that supports specific and stable hybridization between the probe and its complementary sequence on the amplified RNA.
  • the probe can be a 15 padlock probe.
  • the length of the probe can vary.
  • the probe can have a few specific bases and many degenerate bases.
  • the length of the probe can be between 10 to 35 nucleotides, with a complementary portion of the probe being about 16 to 20 nucleotides long.
  • Probes as described herein can be labeled in a variety of ways including but not limited to direct or indirect attachment of radioactive moieties, fluorescent moieties, calorimetric moieties, and chemiluminescent moieties.
  • Probes can contain any of the detection labels described herein. Examples of detection labels include but are not limited to biotin, fluorescent molecules, and a molecular beacon.
  • Molecular beacons are probes labeled 25 with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized (Tyagi and Kramer, Nature Biotechnol. 14:303-309 (1995)).
  • the methods disclosed herein can comprise a detection step.
  • the disclosed methods can comprise a step of detecting a primer or probe bound to a target of interest.
  • the methods described herein can be used to prepare tissue samples for detecting targets of interest.
  • the detection method can be RCA detection.
  • the detection method can be hybridization chain reaction (HCR).
  • reverse transcription of target RNAs can be carried out followed by other modes of detection.
  • the amplification of the padlock probe can form a RNA concatamer.
  • the RNA concatamer can be detected.
  • the RNA concatamer can be 5 detected by fluorescent probes or in situ sequencing.
  • the detecting step can be carried out or is performed using a confocal microscope. Amplicons.
  • a target or interest can be amplified or a probe that binds to the target of interest can be amplified to form an amplicon.
  • An amplicon can be a fragment of RNA comprising the target or sequence of interest, a reverse transcribed DNA from an RNA target of interest or a DNA sequence of a probe that is specifically bound to a target of interest.
  • the amplicon can be double- stranded. In some aspects, the amplicon comprises the sequence of interest. In some aspects, the amplicon comprises the target of interest. In some aspects, the amplicon can comprise a 15 first and a second strand. In some aspects, the amplicon can be amplified and contacted with primers or probes. In some aspects, a plurality of amplicons can be immobilized on a surface. In some aspects, amplicons can be generated for disposal onto an array. In some aspects, the amplicons generated herein can comprise two or more 20 concatemers. Rolling Circle Amplification. The methods disclosed herein can further comprise rolling circle amplification (RCA).
  • RCA rolling circle amplification
  • amplification occurs with each rolling circle amplification primer, thereby forming a concatemer of tandem repeats (i.e., a TS-DNA) of segments complementary to the first-stage amplification target circle (ATC) being replicated 25 by each primer.
  • Bipolar primers can be used as second-stage primers. Since the bipolar primers have a 3'-OH at each end, they are automatically in the proper orientation for use as a primer for additional stages of amplification. In addition, because the bipolar primers have a 3'-OH at each end, they serve to curtail any strand displacement that might otherwise occur.
  • the TS-DNA 30 and second-stage, or higher order, ATCs (second-stage ATC, third-stage ATC, forth-stage ATC, and so on) complementary sequences can be arranged in any configuration within the primer sequence.
  • ATCs second-stage ATC, third-stage ATC, forth-stage ATC, and so on
  • detection labels can be directly incorporated into the primers or probes described herein or can be directly incorporated into amplified nucleic acids or can be coupled to detection molecules such as probes.
  • a detection label is any 5 molecule that can be associated with amplified nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly.
  • Many such labels for incorporation into nucleic acids or coupling to nucleic acid or antibody probes are known to those of skill in the art.
  • Examples of detection labels suitable for use in RCA are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands.
  • fluorescent labels include fluorescein (FITC), 5,6- carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • fluorescent labels are fluorescein (5- carboxyfluorescein-N-hydroxysuccinimide ester) and rhodamine (5,6-tetramethyl 15 rhodamine).
  • Preferred fluorescent labels for combinatorial multicolor coding are FITC and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection.
  • the fluorescent labels can be obtained from 20 a variety of commercial sources, including Molecular Probes, Eugene, OR and Research Organics, Cleveland, Ohio.
  • Labeled nucleotides can be used as a form of detection label since they can be directly incorporated into the products of RCA during synthesis.
  • detection labels that can be incorporated into amplified DNA or RNA include nucleotide analogs such as BrdUrd 25 (Hoy and Schimke, Mutation Research 290:217-230 (1993)), BrUTP (Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal.
  • Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic 30 Acids Res., 22:3226-3232 (1994)).
  • a preferred nucleotide analog detection label for DNA is BrdUrd (BUDR triphosphate, Sigma), and a preferred nucleotide analog detection label for RNA is Biotin-16-uridine-5'-triphosphate (Biotin-16-dUTP, Boehringher Mannheim). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling.
  • Cy3.5 and Cy7 are 26 Attorney Docket No.36406.0040P1 available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labeled probes. Detection labels that are incorporated into amplified nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art.
  • biotin 5 can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3 (4-methoxyspiro-[1,2,- dioxetane-3-2'-(5'-chloro)tricyclo [3.3.1.13,7]decane]-4-yl) phenyl phosphate; CDP- Star.RTM.
  • suitable substrates for example, chemiluminescent substrate CSPD: disodium, 3 (4-methoxyspiro-[1,2,- dioxetane-3-2'-(5'-chloro)tricyclo [3.3.1.13,7]decane]-4-yl
  • a preferred detection label for use in detection of amplified RNA is acridinium-ester- labeled DNA probe (GenProbe, Inc., as described by Arnold et al., Clinical Chemistry 15 35:1588-1594 (1989)).
  • An acridinium-ester-labeled detection probe permits the detection of amplified RNA without washing because unhybridized probe can be destroyed with alkali (Arnold et al. (1989)). Molecules that combine two or more of these detection labels are also considered detection labels. Any of the known detection labels can be used with the disclosed probes, 20 tags, and method to label and detect nucleic acid amplified using the disclosed method.
  • radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a scanner or 25 spectrophotometer, or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection label coupled to the antibody.
  • detection molecules are molecules that interact with amplified nucleic acid and to which one 30 or more detection labels are coupled.
  • Example 1 Methods for clearing and molecular analysis in thick tissue sections
  • In situ transcriptomics (also known as spatial transcriptomic) technologies, 5 which allow molecular characterization of tissues with subcellular spatial resolution, are currently employed for molecular diagnostic approaches. These approaches are 2D in practice because they are generally limited to thin tissue slices (e.g., 15 microns) due to the low efficiencies of molecular diffusion and/or tissue opacity. Attempts to combine spatial transcriptomics with tissue clearing approaches to improve molecular diffusion and optical 10 penetration have been to permit a more 3D characterization of tissues.
  • tissue clearing and molecular amplification procedures that can be used to detect RNA in tissues as thick as 400 microns.
  • the disclosed 15 methods outperform existing in situ RNA detection methods by more than ten-fold.
  • the methods disclosed herein can be further used as a molecular diagnosis approach.
  • methods that combine a tissue clearing approach and a molecular detection approach that complement each other for use in thick tissue sections are disclosed herein.
  • the methods comprise replacing the entire tissue lattice with a 20 polyacrylamide hydrogel, removing the non-RNA molecules (e.g., proteins, DNA, lipids), and creating detectable amplicons from RNA molecules that are covalently bound to the hydrogel.
  • SDS and DNase I can be used to permeabilize the tissue and remove lipids and genomic DNA from thick fixed tissue sections, leading to increased optical clarity and greatly enhancing 25 molecular diffusion.
  • tissue clarity can be measured visually. For example, molecular diffusion can be confirmed as a consequence of uniform reactions across the tissue sample.
  • Padlock probes can be applied that target the RNAs of interest together with a Rolling Circle Amplification (RCA) primer that hybridizes to the padlock 30 probes.
  • the RCA primer can be modified with an acrydite moiety.
  • the hydrogel can be introduced by diffusing acrylamide monomers into the tissue section and inducing their polymerization.
  • the RCA primers can get 28 Attorney Docket No.36406.0040P1 covalently incorporated into the polyacrylamide hydrogel, preserving their spatial position as well as that of the Padlock oligos and RNA molecule to which they are hybridized.
  • proteinase K can be used to remove proteins.
  • hydrogel polymerization and proteinase treatment can 5 effectively be carried out to replace the protein lattice of the fixed tissue with a hydrogel lattice.
  • a protein lattice is non-uniform with its physical and chemical properties (e.g., hydrophobicity, density, charge, etc.) changing in different positions within the tissue.
  • the hydrogel lattice is uniform and porous with a molecular composition, which is known to be highly permissible to molecular and enzymatic reactions.
  • a ligase enzyme can be used to circularize padlock probes and a polymerase to carry out RCA leading to local amplification of the padlock as a DNA concatemer that can be later identified using fluorescent probes or in situ sequencing chemistries.
  • the presence of a Padlock-derived RCA amplicon in each position marks the position of a specific RNA molecule within the tissue lattice.
  • the methods described herein realizes uniform and sensitive RNA detection in thick tissues by improving oligonucleotide hybridization and enzymatic reaction efficiencies.
  • the Actb transcript for example, was targeted using a Padlock probe in 400- micron thick mouse liver tissue sections (FIG.2).
  • genomic DNA inhibits hybridization and later enzymatic reactions in situ (FIGS. 4A, B).
  • DNase treatment of the thick tissue before hybridization 30 completely degrades genomic DNA (FIG.4C), which uniformly improves RNA detection across the thick tissue.
  • Tissue clearing diffusion of oligonucleotides and other molecules is the rate-limiting step in in situ applications. For example, an 8% SDS solution during hybridization that removes lipids, thus clearing the tissue and improving diffusion.
  • the sample can be 10 embedded in polyacrylamide gel that chemically captures the oligo–RNA complexes in their original cellular positions. As the hybridization rate directly determines the RNA retention rate, this permits RNAs to be retained with high efficacy.
  • Other in situ transcriptomic methods for thick tissues require multi-step chemical and hybridization reactions to reduce RNA retention rates.
  • the tissue clearing and molecular amplification strategies described herein can each be used in thin-tissue settings. Moreover, the tissue clearing aspects can be adapted to other molecular amplification strategies, including de novo and targeted in situ sequencing. Further, the methods disclosed herein are also compatible with tissue expansion.
  • the methods disclosed herein can also be used to gain a better understanding of biological 20 systems and in methods for diagnosing disorders. For example, in a clinical setting, these methods are applicable in molecular histology for detailed diagnosis of disorders such as cancer. An example of a disclosed method is described herein. Summary Procedures: 25 The tissue is fixed (4% FA), 1 day at 4°C, then sliced (400 ⁇ m) using a vibrotome. These steps are followed by 0.5% SDS pre-treatment, O/N, DNase digestion in 2% triton- x100, and 8% SDS treatment to quench DNase (for FIG.4B-C, these steps were removed).
  • Hybridization (padlock and RCA primer (+acrydite), 8% SDS) for 3 days, followed by hybridization (padlock and RCA primer (+acrydite), 0.3% SDS) for one day, then gelation to 30 capture primer-Padlock-RNA complex.
  • Tissue cleansing (1.5 ml tube, 100 ⁇ l PK+0.9 ml digestion buffer) was carried out twice following by washing.
  • padlock ligation on RNA was performed, following by rolling circle amplification, and image analysis with Fiji.
  • the fixed slices are washed with 2x saline-sodium citrate (SSC) four times on ice (1x SSC also worked; and other buffered aqueous solutions also worked).
  • the samples were next washed with 1x DNA 10 digestion buffer + 0.5% SDS at 37 °C, overnight (shaken at 1000 rpm); this step removes lipid and permeabilizes the tissue. Higher SDS concentration might cause DNase inactivation in the later steps.
  • the sample was washed with 1x DNA digestion buffer + 2% Triton- X100 + 0.4 U/ ⁇ l RNase inhibitor at 37 °C, for 2 hours, and shaken at 1000 rpm; this was carried out to remove SDS micelles, otherwise the DNase will be inactivated (this 15 concentration was used for DNase Hi-C).
  • the DNase solution was prepared as described in Table 1 and the samples were incubated in the DNase solution, at 37 °C and shaken at 1000 rpm. Table 1. DNase solution. 20 To stop the DNase reaction, the samples were placed in 2x SSC, 8% SDS solution at 37 °C for 1 hour and shaken at 1000 rpm. In some aspects, the SDS concentration can be lower.
  • FIGs.4B-C the samples the DNA digestion steps were omitted.
  • FIGs.4A and C the tissue slices were stained with DAPI and measured with a confocal microscope. 25 Next the samples were incubated in a hybridization buffer in a 1.5 ml tube for 1 hour at 37°C and shaken at 1000 rpm. 31 Attorney Docket No.36406.0040P1 Table 2. Hybridization buffer. Followinged by incubating the samples in hybridization buffer + oligos in a 1.5 ml tube (shaken at 1000 rpm). For these experiments, three oligos were used and incubated with the 5 samples overnight using an 8% SDS solution with 20% formamide (but this concentration of formamide is arbitrary). Table 3.
  • Hybridization buffer plus oligos. 10 The SDS concentration was lowered to 0.3% and the samples were incubated overnight at 37 °C and shaken at 1000 rpm. Table 4. Hybridization buffer with oligos (2% SDS). 32 Attorney Docket No.36406.0040P1 Next the samples were washed with 0.5x SSC/0.3% SDS solution, 1 ml, two times at 37 °C and shaken at 1000 rpm for1 hour each. This step is carried out to remove non-specific bindings. A SDS solution of 0.2X SSC also worked. 5 Table 5 shows the process for making a gel solution. Table 5. Gel solution.
  • tissue slices were washed with 100 ⁇ l of the gel solution without 10 ammonium persulfate (APS) or tetramethylethylenediamine (TEMED).
  • APS ammonium persulfate
  • TEMED tetramethylethylenediamine
  • the gel solution was degassed by argon bubbling.
  • the tissue slides were washed with 400 ⁇ l of the degassed gel solution without APS or TEMED, followed by being incubated for 15 mins at RT at 1000 rpm.
  • Frame-Seal Slide Chambers (Frame-SealTM in situ PCR and Hybridization Slide Chambers, 17 x 28 mm, 125 ⁇ l #SLF1201) were attached on a slide.40 U/ ⁇ l RNase inhibitor, 15 2 ⁇ l, and 5 TEMED and 5% APS, 4 ⁇ l were added to 200 ⁇ l gel solution. The tissue slices were placed on a glass slide and the activated gel solution was added. A plastic cover was used to seal the Frame-Seal Slide Chambers (Frame-SealTM in situ PCR and Hybridization Slide Chambers, 17 x 28 mm, 125 ⁇ l #SLF1201), which were then incubated at 37 °C for 5 hours.
  • the digestion buffer 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 6x SSC, 0.3% SDS
  • the digestion buffer 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 6x SSC, 0.3% SDS
  • 2x phenylmethysulfonyl fluoride was added. If incubation was short, SDS cannot be removed.
  • tissue samples were washed with 1x SSC one time, followed by a wash with 1X SplintR Ligase Buffer two times at 4 °C.
  • the tissue samples were incubate with 1.25 U/ ⁇ l 10 SplintR ligase (NEB, cat. no. M0375L) in 1X SplintR ligase buffer at 4 °C for 1 hour.
  • Table 6 SplintR ligase buffer.
  • the tissue samples were incubated at 37 °C overnight. Then, the tissue samples were 15 washed with 1x RCA buffer (New England Biolab) twice.
  • RCA solution 34 Attorney Docket No.36406.0040P1 200 ⁇ l RCA solution was added to the sample in 1.5 ml tube, and incubated at 4 °C for 1 hour and shaken at 1000 rpm, following by another incubation at 30 °C for 6 hours and shaken at 1000 rpm. The solution was replaced with a fresh solution and the tissue samples were incubated again at 30 °C overnight and shaken at 1000 rpm. Next, the tissue samples were 5 washed with 2x SSC, 1 ml, and then washed again with 2x SSC/10% formamide at RT, 1 ml.
  • a padlock oligo targeting ACTB gcagcgatatcgtcatCATAACAACAAAACAACCTCATTATCTCTCCACACACACTCCTCT CACTgttgtcgacgaccagc (SEQ ID NO: 1) was used along with an RCA-primer: 15 AGTGAGAGGAGTGTGTGTG + 5’ Acrydite (SEQ ID NO: 2), and detection probe: CATAACAACAAAACAACCTCATTATCTCTC + 5’ Cy5 (SEQ ID NO: 3).
  • Example 2 Methods for clearing and molecular analysis in thick tissue sections and in situ mutation detection Described herein are methods that can be used for in situ mutation detection. 20 Currently available commercialized in situ mutation detection techniques rely on thermodynamical differences to distinguish mutations.
  • Described herein 30 are methods that use a novel oligo design, hydrogel embedding strategy, and the Invader assay (also known as iLock padlock oligo), which in combination allows selective mutation/SNV detection in situ.
  • 35 Attorney Docket No.36406.0040P1 A summary of this procedure is shown in FIG. 5.
  • iLock padlock oligos that have two strands that compete with each other on the target mutation/SNV RNA base shown as yellow strands in FIG. 5 were designed.
  • the tissue slice is immersed with the acrydite monomer solution, which is later activated to form the hydrogel 5 lattice in the tissue.
  • the acrydite modified primer bridges the hydrogel and the iLock padlock oligos to maintain spatial information of RNAs.
  • Proteinase K was used to digest proteins, which increases optical clarity and greatly enhances molecular diffusion for enzymatic reactions.
  • Taq polymerase cleaves the flap DNA strand when the invasive structure is formed on the RNA templates, leading to the clear 10 distinguishment of mutation/SNVs from wild-type genotypes.
  • a ligase enzyme was used to circularize the cleaved iLock padlock oligos and a polymerase to carry out RCA for local amplification of the padlock that can be later identified using fluorescent probes or in situ sequencing chemistries.
  • iLock padlock oligos (2 mouse strains x 3 SNVs) were designed and these iLock padlock oligos distinguished the SNVs selectively in the 50-micron liver slices (FIG. 6). Reportedly, iLock padlock oligos showed limited efficiencies in situ [Krzywkowski, T. et al., (2019), RNA, 25(82-89)], which could be explained by the low diffusion and/or activities of enzymes in the cells/tissues.
  • FIG. 7 shows that DNase I treatment improved the detection efficiency in the brain slices.
  • Excitatory neuron marker, Slc17a7 was detected in the 30-micron brain slices of C57BL/6J with two iLock padlock oligos.
  • the brain slices were processed as described in 30 FIG. 5 except that the slice shown in FIG.7A received DNase I treatment before iLock padlock hybridization.
  • the RCA amplicons from the iLock padlock oligos were detected with a Cy5 probe (red).
  • Genomic DNA was visualized with DAPI (blue).
  • the samples were measured with a spinning desk confocal microscope.
  • 36 Attorney Docket No.36406.0040P1 The steps of the procedure are as follows.
  • the tissue is fixed in 4% PFA solution overnight at 4°C.
  • the crosslinked tissue is sectioned to a thickness of 50 ⁇ m.
  • the slices are stored in 100% methanol at -20°C before analysis to remove lipids and keep RNA intact.
  • the slice is washed with 1x SSC solution four times to remove methanol.
  • the slice is incubated at 5 37°C with the iLock padlock oligos targeting the SNVs of interest.
  • a primer that carries an acrydite modification is hybridized to the Padlock backbone simultaneously.
  • the slice is stringently washed to remove non-specific bindings of the Padlock probes.
  • acrylamide monomers are diffused into the slice and polymerized at 37°C for 2-8 hours, thus, introducing a polyacrylamide hydrogel into the tissue section.
  • the acrydite 10 moiety on the primer covalently binds the hydrogel and maintains the target RNA in place in the process.
  • proteins and lipids are degraded and removed using proteinase K treatment at 37°C.
  • 2x PMSF (2 mM) is added to the slice to quench proteinase K.
  • the slice is incubated with Taq polymerase to cleave the flap structure on the target SNVs.
  • the cleaved iLock padlock oligo is circularized by a ligase and 15 an RCA reaction is performed on these circularized templates using phi29 DNA polymerase.
  • the methods described herein can be used to provide an accurate molecular diagnosis in oncology (e.g., drug response, prognosis, and metastasis prediction).
  • this method can distinguish one base difference between RNAs, this method can be integrated into any in situ transcriptome techniques to improve their accuracy.
  • Example 3 Simultaneous detection of 16 genes in whole dorsal murine skin Sixteen genes (Table 8) were simultaneously detected from the whole P0 murine dorsal skin. Within the observed field of view measuring 749 ⁇ m x 749 ⁇ m x 250 ⁇ m, encompassing the epidermis, dermis, subcutaneous fat, and hair follicles, a total of 121,797 transcripts were detected. Mask images were generated to cover each hair follicle from the 25 raw images to extract them computationally. Histologically, the stages of hair follicles are determined by the lengths of them.
  • the premature hair follicles have shorter length while the mature ones have substantially longer shapes.
  • P0 murine dorsal skin comprises stages 1-6 hair follicles with different molecular compartments.
  • the lengths of the computationally extracted hair follicles are between 50 to 350 ⁇ m, which indicates that the 30 single hair follicles from different stages are successfully isolated.
  • the tissue sample size can be 4 to 400 ⁇ m.
  • the presence of heterogeneous cells in the developing skin hindered in-depth analysis.
  • the results demonstrated successful computational distinction and isolation of single hair follicles.
  • Hybridization buffer Next, the samples were incubated in hybridization buffer + oligos at 37 °C, for 6 days. 10 The solution was changed every day. Table 11. Hybridization buffer plus oligos. 39 Attorney Docket No.36406.0040P1 The samples were then washed with 1x SSC/0.5% SDS solution two times at 37 °C for 1 hour each. Next, a gel solution was prepared. 5 Table 12. Gel solution. The samples (e.g., slices) were then washed with 500 ⁇ l gel solution without APS or TEMED, and incubated for 30 minutes at RT. The gel solution was degassed by Argon b ubbling.
  • tissue samples were washed twice with 400 ⁇ l degassed monomer solution without APS or TEMED, and incubated for 10 m inutes at RT. Next, 5 TEMED, 4 ⁇ l, and 5% APS, 4 ⁇ l, was added to 200 ⁇ l of the monomers solution.
  • Each tissue sample e.g., slice
  • slip solution e.g., Gel Slick Solution from Lonza which helps to take off the coverslip from the sample.
  • the coverslips were applied to the glass slides that were pretreated with Bind-Silane. The tissue samples were then incubate at 37 °C for 30 minutes.
  • tissue samples were washed with PBS and the coverslips were removed, following by a wash with 2xSSC/20% 15 formamide.
  • a solution with bridge probes and dye probes were added: 2x SSC, 20% Formamide 50 nM/bridge probe (in total, 800 nM), 200 nM/dye probes (AF488, AF546, Cy5, AF750, in total 800 nM); and incubated at RT for 2 hours.
  • the tissue samples were washed with 2xSSC/20% formamide for 10 minutes at RT, followed by a wash with PBS, 6 times at 37 °C, 5 minutes each.
  • the Illumina scanning solution was added. Images were 20 taken with Nikon spinning disk confocal microscope.
  • the whole dorsal murine skin of C57BL/6J was fixed, processed with the padlock oligos, and scanned from the dorsal to ventral direction with a confocal microscope.
  • the corresponding transcripts were identified with HybISS.
  • the 5 results showed a spatial distribution of the 16 genes across the sample.
  • the developing skin sample consists of numerous hair follicles from various stages ranging from stage 2 to 6. Hair follicles were computationally isolated with manually curated mask images. The results also demonstrate that the extracted hair follicles exhibit varying lengths which reflect hair follicle stages. 10 Table 16. Probes and sequences.
  • the method utilizes SDS and DNase I to permeabilize fixed tissue sections, eliminating lipids and genomic DNA. Subsequently, iLock probes targeting expressed single nucleotide 10 variants are employed alongside a modified Rolling Circle Amplification (RCA) primer with an acrydite modification.
  • the polyacrylamide hydrogel is introduced to the tissue slice with activated monomer solutions, where the RCA primers are covalently incorporated into the polyacrylamide hydrogel, preserving their locations.
  • proteinase K is applied to remove proteins, after which the original tissue shape is strictly maintained by 15 the hydrogel.
  • Taq polymerase is used to cleave the flap structure of the iLock oligos, based on which single nucleotide differences are distinguished, followed by SplintR ligase to ligate the cleaved iLock oligos.
  • Phi29 polymerase amplifies the circularized oligos into a DNA concatemer. This concatemer, also called amplicons, can be later identified using fluorescent probes or in situ sequencing technologies. 20
  • 254 iLock probes were designed targeting 127 single nucleotide variants between C57BL/6J and PWK/PhJ strains on 38 genes (Table 20).
  • the 127 probes are bound to the RNAs from B6 genome while the other 127 probes have one nucleotide difference in their binding sites which makes them complementary to PWK transcripts.
  • B6 iLock probes were specifically 25 amplified from the B6 brain slice albeit PWK probes did not amplify with just one nucleotide differences (FIGS. 8, 9).
  • the accuracy of B6 single nucleotide detection was in total at 97%.
  • the results demonstrate that most of genes had less than 1-10% false positive ratios, with a few genes expressing lower levels demonstrating approximately 10-20% false positive rates.
  • the method demonstrates simultaneous characterization of more than 100 single nucleotide 30 variants.
  • the accurate high-throughput performance of the method was accomplished using: 1) DNase treatment: genomic DNA inhibits enzymatic reactions in situ and increases non- 48 Attorney Docket No.36406.0040P1 specific amplification of iLock oligos; and 2) Hydrogel embedding: Taq polymerase did not work in situ in tissue slices. Hydrogel embedding realized efficient Taq polymerase reactions.
  • the methods described herein have significant implications in both medical and academic settings. Genetic and histological diagnosis is a basis of ontology. The methods can 5 provide a detailed view of cancer evolution, and be used for clinical applications. The method can be used to characterize single nucleotide variants on a large scale with spatial information making it a distinctive and versatile tool in oncology, systems genomics and personalized medicine.
  • the tissue is chemically crosslinked with 4% formaldehyde 10 solution overnight at 4°C.
  • the tissue is embedded in 4% agarose gel and sectioned to 100 micron slices with vibratome.
  • the slices are stored in 100% methanol at -70 °C.
  • the slice was washed with 2x SSC/8% SDS solution and permeabilized with the fresh 2x SSC/8% SDS at 37°C overnight.
  • SDS was quenched with 2% Triton-X100.
  • the slice was washed with 2% Triton-X100/1x DNase I buffer solution.
  • the slice is incubated with DNase I solution at 37°C 15 overnight to digest genomic DNA.
  • DNase I is inactivated by 8% SDS solution followed by 5 days hybridization of iLock probes targeting the single nucleotide variants.
  • iLock probes are initially hybridized with the acrydite modified primer so that they are covalently incorporated into hydrogel in the later step. The probes are washed stringently.
  • the activated monomer solution with APS 20 and TEMED is added to the slice to form polyacrylamide hydrogel in the tissue slice, which anchors the acrydite modified primer at the original locations.
  • tissue proteins are digested with Proteinase K at 37°C overnight, replacing the tissue lattice with the hydrogel lattice.
  • Proteinase K is quenched with 2x PMSF (2 mM) at RT.
  • Single nucleotide variants are distinguished by adding Taq polymerase to the sample at 37°C for 4 hours.
  • Taq 25 polymerase cleaves the flap structure of iLock oligos when iLock oligos are hybridized on the appropriated complementary sequences without single nucleotide differences.
  • the cleaved oligos are ligated with SplintR at 37°C for 2 hours, followed by Phi29 rolling circle amplification.
  • the amplified concatemer oligos are detected with fluorescent dye modified oligos for in situ sequencing.
  • 30 49 Attorney Docket No.36406.0040P1 Table 20. Probes and sequences. 50 Attorney Docket No.36406.0040P1 51 Attorney Docket No.36406.0040P1 52 Attorney Docket No.36406.0040P1 53 Attorney Docket No.36406.0040P1 54 Attorney Docket No.36406.0040P1 55 Attorney Docket No.36406.0040P1 56 Attorney Docket No.36406.0040P1 57 Attorney Docket No.36406.0040P1 58 Attorney Docket No.36406.0040P1 59 Attorney Docket No.36406.0040P1 60 Attorney Docket No.36406.0040P1 61 Attorney Docket No.36406.0040P1 62 Attorney Docket No.36406.0040P1 63 Attorney Docket No.36406.0040P1
  • Burst timing is thought to be stochastic and independent for homologous 5 alleles, largely dictated by the local environment of each copy. However, burst dynamics in native tissues remain unknown. Described herein is a high-throughput, allele-specific spatial transcriptomics method that allows the characterization of transcriptional dynamics in thick native tissues. Applying this method to the somatosensory neocortex from the F1 hybrid of 94 Attorney Docket No.36406.0040P1 B6 and PWK mice, allele-specific mRNA levels for 36 genes across 44,256 neurons and glia were measured. Transcript distributions revealed changes in burst size and frequency in neocortical layers that were remarkably concordant for homologous alleles.
  • minute-scale transcriptional bursts are integrated into hour-scale 25 bursts in cellular mRNA abundances2 (FIG.10B).
  • Single-molecule RNA FISH (smFISH) and single-cell RNA sequencing (scRNA-seq) capture these bursts as an overdispersion of transcript counts across cell populations (FIG. 10C): most cells between bursts contain fewer transcripts than expected from constant expression, while cells during bursts contain substantially more (Bahar Halpern, K. et al. Mol. Cell 58, 147–156 (2015)).
  • Transcript counts were simulated for a bursty gene across 2,000 different expression levels in a population of 10,000 cells, with identical transcript numbers from both homologous alleles per cell (FIG. 10D, Methods). Then, a realistic scRNA-seq 25 sampling model was applied where 10,000 UMIs, representing 10% of the cellular mRNA transcriptome (100,000 mRNA transcripts per cell) (Islam, S. et al. Nat. Methods 11, 163– 166 (2014)), are sequenced, in which 10% of these UMIs carry eSNV information. These parameters reflect typical mRNA (Klein, A. M. et al. Cell 161, 1187–1201 (2015); and Tang, W., et al.
  • thick slices that capture whole cells are more accurate than single-cell transcript ratio estimates compared to thin sections in standard smFISH that 30 capture cell slices.
  • a high-throughput method allows reliable cell type identification, overcoming another limitation of smFISH.
  • a 3D in situ RNA variant sequencing (3Diva-seq) a high-throughput probe-based spatial transcriptomics method was developed that distinguishes single nucleotide variants in thick tissues with high specificity 97 Attorney Docket No.36406.0040P1 and sensitivity.
  • This method (also referred to as “3Diva-seq”) was applied to brain tissue from F1 hybrids of B6 and PWK strains (B6PWKF1) and the composition of 72 alleles was characterized from 36 genes across 44,256 cells spanning major mouse neocortical cell types.
  • the results show that a remarkably high correlation exists between maternal and paternal 5 alleles of most genes across cell types and neocortical layers, indicating that homologous promoter copies are switched ON and OFF in synchrony in temporal resolutions relevant to transcriptome composition.
  • genes on separate chromosomes co-burst at rates comparable to genes on the same chromosome, further supporting trans regulation of burst timing.
  • the 98 Attorney Docket No.36406.0040P1 iLock probe can be circularized with a ligase and amplified by rolling-circle amplification (RCA) if nucleotides in the overlap position match the SNV.
  • RCA rolling-circle amplification
  • iLocks have shown an impressive specificity of about 98–100% on purified targets (Krzywkowski, T., et al. RNA 25, 82–89 (2019); and Krzywkowski, T. and Nilsson, M. Nucleic Acids Res. 45, e161–e161 5 (2017)).
  • the tissues were first hybridized with a mixture of two iLock probes, each matching one allele of a T/C eSNV in the r.722 position of the Apoa1 transcript (NM_001305585.1). These probes were pre-hybridized with an RCA 15 primer on their backbone, featuring a 5’ acrydite modification. After hybridization, the sections were embedded in a 10% polyacrylamide gel to preserve the spatial position of the target transcripts by anchoring them to the gel matrix through their 5’ acrydite-modified RCA primers. Then, proteins and lipids were removed using Proteinase K and SDS treatment.
  • the 3DEEP clearing also enhanced specificity from 97.2% to 98.4% on average: in C57BL sections, 5 97.9% of the Apoa2 amplicons were from the C57BL iLock probe, while 98.9% of amplicons in BALB sections were from the BALB iLock probes.
  • a spatial transcriptomic method capable of identifying single nucleotide variants with exceptional specificity and sensitivity was developed and described herein. In some aspects, this method can be referred to as 3D in situ RNA variant sequencing, or 3Diva-seq for short. 10 High-throughput allele-specific spatial transcriptomics in thick tissue sections.
  • the 3Diva-seq was expanded to 38 genes to assess allelic correlations in the brain (FIG.11F, FIG. 20).
  • the gene set comprises 27 genes with differential expression in brain cell types and 11 genes with established parent-of-origin expression bias.
  • the 27 genes include one gene expressed in the non-microglial cells, four pan-neuronal genes, five glutamatergic neuron- 15 specific genes, five GABAergic neuron-specific genes, four oligodendrocyte genes, three astrocyte genes, and six microglial genes (FIG.11F). These genes can also aid cell-type annotation.
  • the genes with parent-of-origin bias include seven well-established imprinted genes (Crowley, J. J. et al. Nat.
  • FIG. 19 shows 254 iLock probes that were designed, one for each allele of each eSNV.
  • the probe backbones incorporate two 18 nt identifier sequences: a gene ID shared by the probes targeting the same gene, and an allele ID that distinguishes between C57BL and PWK variants.
  • the first hybridization round targets allele IDs, marking C57BL alleles with Cy5 and PWK alleles with AF750 (FIG.11G).
  • the subsequent four rounds target gene IDs, marking each gene with a unique sequence of AF488, AF546, Cy5, and AF750 fluorescence that can be decoded 5 using a lookup table (FIG. 11G, FIG. 19, FIG.20). While this four-color, four-round system enables decoding 256 possible color codes, 59 codes with a Hamming distance of at least two between the pairs was selected. 38 of these codes were assigned for gene identification and the remaining 21 as ‘ghost IDs’ to evaluate decoding error rates.
  • This mouse 25 has a paternal PWK allele and a maternal C57BL allele for each gene.
  • Sixteen 599 by 599 micron FOVs in the somatosensory area of the cortex were imaged (FIG. 11O).
  • Cy5 and AF750 respectively label C57BL/6J and PWK/PhJ probes
  • similar densities of the paternal and maternal amplicons were observed, pointing to consistent probe performance when both alleles are present (FIG. 11O).
  • the total number of amplicons detected for each gene were quantified and agreement with the CPM values of these genes from bulk brain cortex RNA sequencing (Yoon, G., et al. Front.
  • PWK and C57BL alleles may have differences in their cis-regulatory sequences that alter their expression to deviate from 50%.
  • Five genes in the dataset are imprinted in all or some brain cell types (Snrpn, Mest, Inpp5f, Grb10, and Ube3a) (Crowley, 10 J. J. et al. Nat. Genet.47, 353–360 (2015); Gregg, C. et al. Science 329, 643–648 (2010); and Laukoter, S. et al.
  • Beta-Poisson fit to the allele count distributions of each allele in the cell type in which it is most highly expressed (FIG. 12A and FIG. 15, Methods). 15 The negative log likelihoods of the Beta-Poisson fits compared to those of Poisson for 72 alleles (2 alleles for 36 genes) indicate Beta-Poisson as a far better fit across the board (FIG. 12B).
  • the captured cortical area was demarcated into six layers based on cell densities76 using the Allen Brain 10 Atlas as the reference (Lein, E. S. et al. Nature 445, 168–176 (2007); and Belgard, T. G. et al. Neuron 71, 605–616 (2011)) (FIGS. 12D,E,F; methods).
  • the 100-micron sample was divided in pseudosections of 1 to 50 microns and measured average detected 5 allelic correlations in these smaller slices (methods). As expected, the detected allelic correlations drop in thinner sections (FIG.13E). With 5 micron slices, the average gene shows half the allelic correlation observed in 100 micron sections (FIG. 13F). However, 50 micron sections recover the vast majority of correlation observed at 100 microns. These results signify the importance of analyzing thick tissue blocks to capture whole cells for 10 accurate quantification of transcription dynamics. Given the surprising extent of allelic correlation, the extent to which allele misidentification error—the binding and amplification of one allele’s probes on the other’s transcript—can account for the correlation was assessed.
  • Allele misidentification error was then added by swapping a fraction of the transcript labels between maternal and paternal alleles using binomial sampling (FIG.17B) based on measured allele misidentification rates (FIG. 11J) and the Pearson correlation was measured between the two alleles.
  • allelic correlation can be most sensitively measured, namely Slc17a7 and Gad1
  • allele misidentification explains less than 3% of observed correlations.
  • GABAergic neurons are considered here in one category, these neurons have more than twenty subtypes based on transcriptional profiling (Huang, Z. J. and Paul, A. Nat. Rev. Neurosci.20, 563–572 (2019)).
  • each subtype expresses a slightly different level of the target transcripts overall, potentially biasing the estimates of allelic correlation.
  • 2, 4, 8, 16, or 32 hidden subtypes 10 for each gene in the panel was simulated with exaggerated burst kinetic differences (FIG. 17C, methods). Burst kinetic parameters for each subtype were randomly sampled around each gene’s measured parameters, but ranging from one-third to three times the original value, spanning a 10-fold range.
  • Transcript counts per cell were independently generated for each allele (i.e., no 15 correlation).
  • the resulting transcript count distributions were normalized so that the mean transcript count per cell matched the measured average transcript count for the gene.
  • allelic correlations for P2ry12, Laptm5, and Tmem119 close to those expected from a synchronized model in microglia (FIGS. 13C,D), which are a relatively homogeneous cell type in the mouse 25 cortex80,81. While hidden stratification does not explain the allelic synchronization observed in the data, cortical layers enable realistic analysis of cell type stratification effects. Thus, it was assessed whether allelic correlations in each layer for genes show allelic correlations in all glutamatergic neurons. Allelic correlation was high in all layers for all genes (FIG. 13L). For 30 two of eight genes, the allelic correlation of all layers was within 0.1 Pearson correlation units of all five layers combined; for five it was within 0.3 (FIG.13L).
  • Gabra5 showed a wide range of correlations, from 0.3 to 0.7 between layers (FIG.13L). However, when simulation outcomes (FIG.13D, FIG. 17A) were used to assess the observable correlation for 108 Attorney Docket No.36406.0040P1 perfectly synchronized alleles given each gene’s expression level in each layer, it was found that the observed allelic correlation for the seven genes in all layers is near the maximum detectable (FIG.13M). These results show that the contribution of stratification to observed correlations is small. The results also demonstrate that co-bursting of the homologous copies 5 of a gene is the rule, rather than an exception, that spans cell type and cell states. Trans-acting factors orchestrate burst timing.
  • each interchromosomal gene pair represents four allele pairs on different DNA molecules whereas each intrachromosomal gene pair represents two allele pairs on the 10 same DNA molecule and another two on different molecules. It was observed that a similar fraction of the interchromosomal (83.5%) and intrachromosomal (83.1%) gene pairs exhibited some level of correlation (i.e., Pearson’s r > 0.2). Moreover, the correlation ranges for both groups were nearly identical (FIG.14E; 25th percentile: 0.22, median: 0.28, and 75 th percentile: 0.34, identical values for both groups).
  • co-bursting has important implications for cell state. Unlike independent bursting, co-bursting would ensure that the relative ratio of the protein products from a gene’s alleles remain balanced across time. It would also set limits to the variation of ratios between transcripts from different co-bursting genes whose protein products may have 15 opposing biochemical functions. Therefore, co-bursting can result in more biochemical and genetic stability than independent bursting.
  • the mechanism behind orchestrated trans regulation likely extends from the inherent bursting nature of transcription. Transcription factors and other proteins involved in gene expression also express through bursts, causing their protein levels to fluctuate. These 20 fluctuations can be integrated at target gene cis-regulatory sequences to coordinate promoter ON/OFF states, explaining the synchronized expression patterns observed here.
  • the temporal resolution of bursting and biological context are important for connecting transcriptome-level measurements showing clear synchronization to stochastic mRNA synthesis observed in live-cell measurements (Wan, Y. et al. Cell 184, 2878– 25 2895.e20 (2021); and Rodriguez, J. et al. Cell 176, 213–226.e18 (2019)).
  • 3Diva-seq measures bursts on the hour scale, as RNA quantification methods detect bursts in units of transcript half-lives (Tani, H. et al. Genome Res.22, 947–956 (2012)). Consequently, microbursts of transcription occurring minutes apart could still give rise to synchronized mRNA-level bursts (FIG.10B).
  • mRNA quantification reflects the contribution of the factors that determine mRNA levels (i.e., production, processing, 111 Attorney Docket No.36406.0040P1 transport, and degradation) whereas direct imaging isolates production alone.
  • the observed differences in dynamics may be a consequence of more complex regulation at the whole- cell level combined with temporal resolution. Third, technical considerations may explain these differences.
  • Brain tissue which experiences more constraints and cell-extrinsic 5 regulatory cues than cultured cells, may exhibit higher synchronicity. Future studies directly addressing these possibilities will be essential for creating a unified understanding. These observations also help contextualize the classic concepts of gene expression stochasticity in native mammalian tissues. Transcription bursts are the major driver of gene expression stochasticity—that is, fluctuations in a cell’s transcriptome—in mammalian 10 cells (Suter, D. M. et al. Science 332, 472–474 (2011); and Raj, A., et al. PLoS Biol. 4, e309 (2006)). The sources of gene expression stochasticity have been canonically divided into two distinct categories (Swain, P.
  • Intrinsic stochasticity arises from the inherent randomness in 15 movements, positions, and conformations of the molecules involved in transcription and affects homologous copies independently.
  • Extrinsic stochasticity stems from fluctuations in the concentrations of the non-DNA molecules involved (DNA concentration can be assumed constant) and affects homologous copies jointly.
  • the oligonucleotides were purchased from Integrated DNA Technologies. Beta- 5 Poisson model to describe two state stochastic gene expression, related to FIG. 10. Transcription burst kinetics are inferred from the steady-state transcription of single cells.
  • the two-state model of gene expression describes promoter dynamics as transitions between discrete ON and OFF states, governed by the rate constants kon and koff constants. In the ON state, transcription occurs at a rate ksyn.
  • k on and k off values were randomly sampled within the ranges observed experimentally, while k syn was randomly generated to span a wide range of expression levels.
  • values of l from a Beta distribution using the k on and k off values for 10,000 cells were sampled. This was achieved using the numpy.random.beta function in Python.
  • single- 30 cell transcript counts were generated using a Poisson distribution, where l was multiplied by ksyn to define ⁇ with which transcript counts were simulated. This resulting distribution was considered the “pre-sampling” distribution.
  • ⁇ was calculated by multiplying the transcript count by 0.005 (representing a 10% capture rate and 10% Esnv detection rate) and dividing by 2 (to account for two alleles).
  • the “sampled” distributions for alleles A and B were then generated using a Poisson distribution. Pearson’s correlation coefficients were 10 calculated between the sampled distributions of allele A and allele B, and by repeating this process, the detectable allelic correlations across varying CPM levels were determined.
  • in situ eSNV detection a similar approach was followed, with one difference in the calculation of ⁇ for the “sampled” distribution.
  • ⁇ was calculated by multiplying the transcript count by 0.1 (representing a 10% Padlock probe detection rate) and dividing by 2 15 (for two alleles).
  • cells were assumed to have a radius of 10 ⁇ m, with their volume approximated as spherical.
  • the “sampled” distribution was further sampled to obtain the “sampled” distribution for thin slices.
  • Liver sections were washed three times with PBS and then incubated in hybridization 30 buffer (2x SSC, 20% formamide, 1% Triton X-100, 0.2% SDS, 0.1 mg/ml salmon sperm DNA, 0.4 U/ ⁇ l murine RNase inhibitor) at 4 degrees for 30 minutes.
  • hybridization 30 buffer (2x SSC, 20% formamide, 1% Triton X-100, 0.2% SDS, 0.1 mg/ml salmon sperm DNA, 0.4 U/ ⁇ l murine RNase inhibitor
  • Hybridization was performed in a solution containing 2x SSC, 20% formamide, 1% Triton X-100, 0.2% SDS, 0.1 mg/ml salmon sperm DNA, 0.4 U/ ⁇ l murine RNase inhibitor, and probes specific for 115 Attorney Docket No.36406.0040P1 Apoa2 of C57BL/6J and BALB/cJ (100 Nm each, C57BL/6J: TATATCCCTATATAAGGTTCATTAAACTGCCATAACAACAAAACAACCTCATTAT CTCTCCACACACACTCCTCTCACTCCGGTTTCTCCTCA (SEQ ID NO: 327), BALB/cJ: 5 TATATCCCTATATGAGGTTCATTAAACTGCACTCTATAACATCCAATACTACACC AAATCCACACACACTCCTCTCACTCCGGTTTCTCCTCG; (SEQ ID NO: 328)), along with a 400 nM acydite-modified RCA primer (/5Acryd/AGTGAGAGGAGTGTGTGTGTG
  • reaction was quenched with 2x PMSF in 2x SSC/0.1% Triton X-100 at RT for 30 minutes, followed by washes in 2x SSC/0.1% Triton X-100, twice, and 2x SSC five times, 5 minutes each. Flap cleavage, ligation, and RCA steps were then performed.
  • Samples were washed with 1x ThermoPol buffer (supplemented with MgSO 4 at 8 mM) and incubated in flap cleavage 25 solution (1x ThermoPol buffer with 8 mM MgSO4, 0.2% BSA, 0.4 U/ ⁇ l murine RNase inhibitor, and 0.2 631 U/ ⁇ l Taq polymerase) at 4 degrees for 30 minutes, then at 37 degrees for 6 hours. Samples were washed three times with 1x SplintR buffer at 4 degrees, followed by incubation in SplintR solution (1x SplintR buffer, 0.4 U/ ⁇ l murine RNase inhibitor, and 1.25 U/ ⁇ l SplintR) at 4 degrees for 1 hour and at 30 degrees overnight.
  • 1x ThermoPol buffer supplied with MgSO 4 at 8 mM
  • flap cleavage 25 solution (1x ThermoPol buffer with 8 mM MgSO4, 0.2% BSA, 0.4 U/ ⁇ l murine RNase inhibitor, and 0.2 631 U/ ⁇ l
  • RCA was performed by incubating samples in RCA solution (1x Phi29 buffer, 0.2% BSA, 6360.25 mM dNTP, 0.8 U/ ⁇ l Phi29 polymerase) at 37 degrees for 6 hours. Samples were then washed sequentially in 2x SSC, followed by 2x SSC/20% formamide.
  • liver sections were incubated in fluorescent probe solution (5 ng/ml DAPI, 500 nM 639 Cy5-conjugated probe for C57BL/6J iLock 640 (/5Cy5/CATAACAACAAAACAACCTCATTATCTCTC; (SEQ ID NO: 330)), 500 nM Cy3-conjugated probe for 641 BALB/cJ iLock 5 (/5Cy3/ACTCTATAACATCCAATACTACACCAAATC; (SEQ ID NO: 331) in 2x SSC/20% formamide at RT for 1 hour.
  • fluorescent probe solution 5 ng/ml DAPI, 500 nM 639 Cy5-conjugated probe for C57BL/6J iLock 640 (/5Cy5/CATAACAACAAAACAACCTCATTATCTCTC; (SEQ ID NO: 330)
  • 500 nM Cy3-conjugated probe for 641 BALB/cJ iLock 5 /5Cy3/ACTCTATAACATCCAATACTACA
  • the samples After being rinsed in PBS, the samples were 15 placed in ice-cold 4% formaldehyde in 1x PBS (pH 7.4) and left to incubate overnight at 4 degrees. Following fixation, they were rinsed in PBS (pH 7.4), and submerged sequentially in 15% and 30% sucrose solutions at 4 degrees until they sank. The samples were embedded in O.C.T. compound and frozen using liquid nitrogen. Cryosectioning was performed at 20 ⁇ m using a LEICA CM1860 Cryostat, and the sections were transferred into 100% methanol. The 20 methanol-fixed samples were subsequently stored at -70 degrees until further use.
  • Liver sections were washed three times with PBS, then incubated at RT for 1 hour in DNase I buffer, which contained 1x DNA digestion buffer and 2% Triton X-100. Genomic DNA digestion was performed by placing the samples in DNase I solution (1x DNA digestion buffer, 2% Triton X-100, 0.4 U/ ⁇ l murine RNase inhibitor, and 0.2 U/ ⁇ l DNase I) and 25 incubating overnight at 37 degrees. To neutralize the DNase I activity, the samples were treated twice with a solution of 2x SSC, 125 mM EDTA, and 1% beta-mercaptoethanol at RT for 30 minutes.
  • Proteinase K was applied to degrade tissue structure. The samples were incubated overnight at 37 degrees with gentle rotation in a Proteinase K solution containing 100 ⁇ l Proteinase K and 900 ⁇ l digestion buffer (50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 6x SSC, and 2% SDS). The reaction was terminated by treating the samples with 2x PMSF in 2x SSC/0.1% Triton X-100 at RT for 30 minutes, 20 followed by brief washes in 2x SSC/0.1% Triton X-100. Flap cleavage, ligation, and RCA steps were then performed.
  • Samples were washed with 1x ThermoPol buffer (supplemented with MgSO 4 at 8 mM), twice, and incubated in flap cleavage solution (1x ThermoPol buffer with 8 mM MgSO4, 0.2% BSA, 0.4 U/ ⁇ l murine RNase inhibitor, and 0.5 U/ ⁇ l Taq polymerase) at 37 degrees for 4 hours.
  • Samples were 25 washed five times with 2x SSC solution, briefly, followed by two-times washes in 1x SplintR buffer. Then, samples were incubated in SplintR solution (1x SplintR buffer, 0.4 U/ ⁇ l murine RNase inhibitor, and 1.25 U/ ⁇ l SplintR) at 37 degrees for 2 hours.
  • RCA was performed by incubating samples in RCA solution (1x Phi29 buffer, 0.2% BSA, 0.25 mM dNTP, 0.8 U/ ⁇ l Phi29 30 polymerase) at 30 degrees, overnight. Samples were then washed sequentially in 2x SSC, followed by 2x SSC/40% formamide.
  • liver sections were incubated in fluorescent probe solution (500 nM Cy5-conjugated 700 probe for C57BL/6J iLock (/5Cy5/CATAACAACAAAACAACCTCATTATCTCTC; (SEQ ID NO: 330)), 500 nM 118 Attorney Docket No.36406.0040P1 Cy3-conjugated probe for BALB/cJ iLock (/5Cy3/ACTCTATAACATCCAATACTACACCAAATC; (SEQ ID NO: 331))) in 2x SSC/40% formamide at RT for 1 hour. After a PBS wash, samples were imaged using a Nikon Eclipse Ti2 confocal microscope with a 40x water immersion objective. 5 Image analysis.
  • the iLock probe consists of several important elements arranged from its 5' to 3' ends: a 13-nt flap sequence (TATATCCCTATAT (SEQ ID NO: 332) or ATATACCCATATA (SEQ ID NO: 333), chosen based on the target RNA to prevent unintended binding), followed by a 20-nt complementary sequence that hybridizes to the 25 target RNA. This is linked to a 19-nt RCA primer binding sequence (CACACACACTCCTCTCACT (SEQ ID NO: 334)) and an 18-nt HybISS bridge probe binding sequence, separated by a 2-nt gap. Another 20-nt complementary sequence follows, hybridizing to the target RNA.
  • RNA sequences centered on the SNV at the 21st position were generated using the GRCm38 reference mouse genome.
  • RefSeq sequences for each gene were used as a filter.
  • the RNA sequences found within the corresponding RefSeq annotations were retained for further 5 analysis. These sequences were then subjected to BLAST search filtering to remove potential non-specific targets. After refining the potential target sequences, the presence of indels surrounding the eSNVs was confirmed.
  • RNA sequences for PWK/PhJ were modified to incorporate indels, and iLock oligos were designed accordingly.
  • iLock probes were prioritized based on GC content (ideally 50%) and the absence of repetitive sequences (e.g., 10 GGGG or CCCC). Each gene was assigned four iLock probes whenever possible; for genes with fewer available eSNVs, the maximum number of probes was generated. The iLock probes were then synthesized from IDT using oPools service.
  • HybISS bridge probe design HybISS bridge probes, which had been successfully detected were used.
  • Permeabilization was performed overnight at 37°C in 2 ⁇ SSC/8% SDS/1% ⁇ -mercaptoethanol. In the following day, SDS was neutralized with 2 ⁇ SSC/2% Triton X-100 at 37°C for 1 hour. The solution was then replaced with 1 ⁇ DNase I buffer containing DNA digestion buffer and 2% Triton X-100. Genomic DNA digestion was 120 Attorney Docket No.36406.0040P1 carried out by incubating the samples overnight at 37°C in DNase I solution (1 ⁇ DNA digestion buffer, 2% Triton X-100, 0.4 766 U/ ⁇ l murine RNase inhibitor, and 0.2 U/ ⁇ l DNase I).
  • DNase I activity was quenched by incubating the samples in 2 ⁇ SSC/8% SDS at 37 degrees for 2 hours.
  • the samples were washed with a hybridization buffer (2 ⁇ SSC, 20% 5 formamide, 8% SDS) followed by hybridization with a solution containing 2 ⁇ SSC, 20% formamide, 8% SDS, and iLock probes (100 nM each, 254 probes) for C57BL/6J and PWK/PhJ eSNVs, along with a 40 ⁇ M acrydite-modified RCA primer (/5Acryd/AGTGAGAGGAGTGTGTGTG; SEQ ID NO: 329). Hybridization was performed at 37°C with shaking for five days.
  • Post-hybridization washes were carried out in 1 ⁇ 10 SSC/0.5% SDS at 37°C for 30 773 minutes, twice, followed by two washes in 1 ⁇ SSC/0.1% Triton X-100 and two washes in 1 ⁇ SSC, 774 each at 37°C for 30 minutes.
  • tissue sections were treated with a monomer solution (10% acrylamide, 7760.02% bis-acrylamide, and 2 ⁇ SSC) at room temperature for 30 minutes.
  • the monomer solution was deoxygenated by argon bubbling. Polymerization was initiated by 15 adding 2 ⁇ l murine RNase inhibitor, 4 ⁇ l 5% TEMED, and 4 ⁇ l 5% APS to 200 ⁇ l of the monomer solution.
  • Tissue slices were placed on a glass plate with the activated monomer solution, covered with a coverslip, and gently pressed to remove excess solution for flattening. Polymerization was completed by incubating the samples at 37°C for 1 hour. After polymerization was confirmed, the gelled samples underwent Proteinase K digestion to break 20 down tissue structure. Samples were incubated overnight at 37°C with gentle rotation in Proteinase K solution (100 ⁇ l Proteinase K, 900 ⁇ l digestion buffer: 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 2 ⁇ SSC, 2% SDS).
  • the reaction was quenched by incubating the samples in 2 ⁇ PMSF in 2 ⁇ SSC/0.1% Triton X-100 at room temperature for 30 minutes. Finally, the samples were washed five times in 2 ⁇ SSC/0.1% Triton X-100, with each wash lasting 10 25 minutes. Flap cleavage, ligation, and RCA steps were then performed. Samples were washed with 1x ThermoPol buffer (supplemented with MgSO 4 at 8 mM) and incubated in flap cleavage solution (1x ThermoPol buffer with 8 mM MgSO4, 0.2% BSA, 0.4 U/ ⁇ l murine RNase inhibitor, and 0.2 791 U/ ⁇ l Taq polymerase) at 37 degrees for 4 hours.
  • Samples were 30 washed two times with 1x SplintR buffer, followed by incubation in SplintR solution (1x SplintR buffer, 0.4 U/ ⁇ l murine RNase inhibitor, and 1.25 U/ ⁇ l SplintR) at 37 degrees for 2 hours.
  • RCA was performed by incubating samples in RCA solution (1x Phi29 buffer, 0.2% BSA, 0.5 mM dNTP, 0.04 mM 121 Attorney Docket No.36406.0040P1 1 mM 5-(3-aminoallyl)-dUTP, 0.02 U/ ⁇ l Thermostable Inorganic Pyrophosphatase, 0.8 U/ ⁇ l Phi29 polymerase) at 30 degrees, overnight. Samples were then washed sequentially in 2x SSC. After amplification, samples were anchored to a glass-bottom plate to minimize 5 deformation during image-based sequencing.
  • a second gelation step was performed to anchor the amplicons within a secondary gel and secure the first gel structure onto the glass plate.
  • samples were treated with 10 mM Acryloyl-X in 2 ⁇ SSC (10% DMSO) at room temperature for 2 hours, modifying the 5- AA-2'-dUTP within the amplicons with acrylamide.
  • a monomer solution (2.5% acrylamide, 10 0.125% bis-acrylamide, 2 ⁇ SSC, 0.1% Triton X-100) was prepared, with Triton X-100 added after deoxygenation via argon bubbling. Samples were washed with 200 ⁇ l of this monomer solution.
  • the glass-bottom plate was activated by sequential washes with water and ethanol, followed by treatment with a bind silane solution (1 ml ethanol, 5 ⁇ l bind silane, 50 ⁇ l acetic acid) at room temperature for 30 minutes. After removing the bind silane 15 solution, the plate was dried, washed again with ethanol, and dried completely.
  • the monomer solution was activated by adding 4 ⁇ l of 5% TEMED and 4 ⁇ l of 5% APS to 200 ⁇ l of the monomer solution. The sample was then transferred to the activated plate with the activated monomer solution, covered with a coverslip to flatten the sample, and incubated at 37°C for 1 hour to solidify the secondary gel.
  • bridge probe solutions were prepared by combining 2 ⁇ SSC, 20% formamide, and 10 nM of each bridge probe (86 probes in total, 860 nM), along with 250 nM of each fluorescent probe modified with the dye at 5’ end (AF488: 30 TTCTATGTGACCGGGTACAG (SEQ ID NO: 335), AF546: GTACTCGGCTTTCCAACTGT (SEQ ID NO: 336, Cy5: CATATCTGTGACTGCCTGTC (SEQ ID NO: 337, and AF750: GGAGATAACGATCCCTCACA (SEQ ID NO: 338). These solutions were prepared in advance and stored at -70°C until use.
  • In situ sequencing was performed using a Nikon Eclipse Ti2 confocal microscope equipped with a 25 ⁇ silicon objective (CFI Plan Apochromat Lambda S 25XC Sil, NA: 1.05, FOV: 599 ⁇ m ⁇ 599 ⁇ m ⁇ 1 ⁇ m) and four lasers corresponding to the AF488, AF546, Cy5, and AF750 channels.
  • the bridge probes were stripped using a stripping solution (0.1 ⁇ SSC, 70% formamide) at 60°C for 10 10 minutes, repeated six times. The hybridization and imaging steps were repeated for each sequencing round. Decoding sequencing images. To decode transcripts from the images, nd2 files were first preprocessed.
  • transcripts were decoded, and a confidence score was computed for each amplicon.
  • the confidence score was calculated based on signal intensity using the following equation: 25 where is the sequencing round (ranging from 2 to 5), is the channel (AF488, AF546, Cy5, and AF750), and represents the expected code at round for a given gene.
  • the scores were computed for the genes for each amplicon, and the gene with the highest score was assigned to each. Decoding accuracy was estimated by calculating the fraction of detected 30 negative control barcodes.
  • the score threshold was determined to improve decoding accuracy while maintaining detection sensitivity. To ensure consistency, the same score threshold was applied throughout the study for gene decoding. 123 Attorney Docket No.36406.0040P1 Selectivity evaluation of 3Diva-seq.
  • transcript coordinates Prior to running Baysor, allele-level transcript information was converted to gene level, and XY transcript coordinates (in pixels) were scaled to micrometers.
  • the average parent-of-origin biases for the tested gene set in 3Diva-seq were calculated by using GECCO bulk RNA-seq data.
  • the resulting parental fractions were then 124 Attorney Docket No.36406.0040P1 compared to those observed in 3Diva-seq.
  • a scatter plot was used for visualization, and similarity between datasets was assessed using Pearson’s correlation.
  • Seurat cell type annotation related to FIG.11. Cell type annotation in cortex slices was conducted using a defined gene set. Cells with fewer than ten transcripts were excluded from 5 the analysis. Transcript counts for each cell were SC transformed (Hafemeister, C. and Satija, R.
  • Beta-Poisson fitting a pipeline was used (Larsson, A. J. M. et al. Nature 565, 251–254 (2019)), available at //github.com/sandberg-lab/txburst. After confirming the likelihood of a two-state transcription model in the brain, preprocessing methods were compared for Beta-Poisson fitting.
  • Beta-Poisson fitting was performed using the pipeline developed by Larsson et al. (Larsson, A. J. M. et al. Nature 565, 251–254 (2019)), available at GitHub (//github.com/sandberg-lab/txburst). Low-quality 30 fittings, such as values near parameter boundaries or burst sizes below 1, were excluded based on previously established filtering criteria (Larsson, A. J. M. et al. Nature 565, 251– 254 (2019)).
  • burst frequency and burst size were 125 Attorney Docket No.36406.0040P1 compared. Pearson’s correlation was used to assess the similarity of these burst parameters between alleles.
  • Cortex layer annotation related to FIG.12. Glutamatergic neurons in the cortex were stratified into six layers based on their spatial coordinates. As a reference for 5 layer annotation, histological images with annotated neural densities were used. The spatial coordinates of glutamatergic neurons in the dataset were visualized, and a density map was generated using kernel density estimation. Based on density variations, layer annotations were applied. Layers were defined as follows: Layer 1 included the outermost cortical surface with sparse glutamatergic neurons.
  • Layer 4 was defined as the densest layer, while Layer 5 had the lowest density.
  • the region between L5 and the corpus callosum was designated as Layer 6.
  • Burst frequency and burst size were visualized separately for each allele and layer. The variation in these parameters was quantified using the coefficient of variation, and a box plot was used to illustrate variability across tested genes. 20 Correlations in burst kinetics parameters between alleles, after layer separation, were visualized using a scatter plot. Evaluation of allelic synchronizations, related to FIG. 13. Among the 28 bi-allelically expressed genes, each gene was assigned to the cell type showing the highest expression level.
  • Beta-Poisson distribution 15 parameterized by k on , k off , and k syn , values of l from a Beta distribution for 23,179 cells was generated, matching the number of glutamatergic neurons in the study, based on the randomly-derived kon and koff values. This process was executed using the numpy.random.beta function in Python. Next, using the sampled l values, single-cell transcript counts were simulated with a Poisson distribution. The value of l was multiplied by 20 k syn to define ⁇ , the rate parameter, which was used to simulate the transcript counts. Assuming perfect synchronization between the maternal and paternal alleles, identical transcript counts were assigned to both alleles.
  • Poisson sampling was then independently applied to the transcript counts of both alleles to assess how Poisson sampling during in situ detection influences allelic correlation observations.
  • ⁇ was calculated by 25 multiplying the transcript count by 0.05 (representing a 10% capture rate and 50% matching iLock probe binding to the corresponding allele).
  • Post-sampling transcript counts for both alleles were generated using a Poisson distribution.
  • Pearson’s correlation coefficients were computed between the sampled distributions of both alleles.
  • a scatter plot was used. Expected correlations across expression levels were estimated by applying locally weighted scatterplot smoothing (LOWESS). Simulation of slice thickness effect on allelic correlations, related to FIG.13.
  • transcript counts were generated based on burst kinetics parameters derived from the measured transcript counts. 10 Using a Beta-Poisson distribution and these kinetics parameters, transcript counts were simulated for the corresponding number of cells. Independent simulations for the two alleles resulted in uncorrelated allele distributions. To incorporate allele misidentification, simulated transcript counts were swapped between alleles according to gene-specific allele misidentification rates, using Binomial sampling. Pearson’s correlation coefficients were then 15 computed between alleles after this swapping. The simulation was repeated 1,000 times, and a box plot was used to visualize the expected artificial allelic correlations.
  • scRNA-seq data of glutamatergic neurons was obtained from the somatosensory cortex via the Allen Brain Atlas portal (//portal.brain-map.org/atlases-and-data/rnaseq/mouse-whole-cortex-and- hippocampus-10x).
  • Mouse gene annotation data, including chromosomal locations, was obtained from Larsson et al.’s pipeline (github.com/sandberg-lab/txburst). Genes with an 25 average of >1 transcript per cell were selected for further analysis. Based on chromosomal information, gene pairs were categorized as being on the same chromosome or different chromosomes.
  • Pearson’s correlation coefficients were then computed for these genes, and their distribution was visualized using a box plot. For the shared gene set, a scatter plot was used to compare the consistency of Pearson’s correlations between 3Diva-seq and scRNA- 30 seq data. 129

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Abstract

Sont divulgués des procédés de détection d'un variant nucléotidique unique dans un échantillon de tissu. Les procédés comprennent : a) l'obtention, présente ou par le passé, de l'échantillon de tissu, l'échantillon de tissu comprenant de l'ADN, des molécules autres que l'ARN et de l'ARN, l'ARN comprenant un variant mononucléotidique ; b) la perméabilisation de l'échantillon de tissu ; c) la dégradation quasi-totale de l'ADN dans l'échantillon de tissu ; et d) l'introduction dans l'échantillon de tissu d'une amorce ou d'une sonde spécifique du variant mononucléotidique, l'amorce ou la sonde se liant au variant mononucléotidique ; et e) la détection de l'amorce ou de la sonde liée au variant mononucléotidique, ce qui permet de détecter le variant mononucléotidique dans l'échantillon de tissu.
PCT/US2025/028781 2024-05-09 2025-05-09 Procédés de détection de variation de séquence d'adn dans des sections de tissu épais Pending WO2025235971A1 (fr)

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