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WO2025029627A1 - Procédés, compositions et kits de détection spatiale de variants génétiques - Google Patents

Procédés, compositions et kits de détection spatiale de variants génétiques Download PDF

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Publication number
WO2025029627A1
WO2025029627A1 PCT/US2024/039737 US2024039737W WO2025029627A1 WO 2025029627 A1 WO2025029627 A1 WO 2025029627A1 US 2024039737 W US2024039737 W US 2024039737W WO 2025029627 A1 WO2025029627 A1 WO 2025029627A1
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WIPO (PCT)
Prior art keywords
probe
capture
nucleic acid
biological sample
target nucleic
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PCT/US2024/039737
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English (en)
Inventor
Paulius Mielinis
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10X Genomics, Inc.
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Application filed by 10X Genomics, Inc. filed Critical 10X Genomics, Inc.
Publication of WO2025029627A1 publication Critical patent/WO2025029627A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells.
  • the specific position of a cell within a tissue e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment
  • High-throughput methods are available for spatial analysis to determine the identity, abundance, and distribution of nucleic acid genetic variants within cells of a biological sample, for example, a tissue sample or tissue section.
  • Such methods include array-based spatial transcriptomics assays.
  • the biological sample can be placed on an array or on a substrate and aligned with an array 7 to improve specificity and efficiency for the identification and/or characterization of a nucleic acid (e.g., DNA or RNA) within the biological sample.
  • Increased sensitivity 7 for differentiating between wild-ty pe nucleic acids and genetic variant nucleic acids is important for accurately identifying genetic variants.
  • probes specific to either wild-type nucleic acids or nucleic acids with genetic variants can compete against each other resulting in inaccurate determination of the presence, location, and/or frequency of a genetic variant. Therefore, there exists a need for improved compositions, methods, and kits with increased accuracy and sensitivity 7 for identifying genetic variants (e.g., single nucleotide variants or polymorphisms, insertions, deletions, etc.) in spatial analysis assays.
  • genetic variants e.g., single nucleotide variants or polymorphisms, insertions, deletions, etc.
  • the present disclosure features methods, compositions, and kits that can be used to analyze the presence, location, and/or frequency of genetic variants in a target nucleic acid in a biological sample using a target specific nucleic acid probe (e.g., a first probe), a blocking oligonucleotide, and a second probe in combination with capture probes in an array format.
  • a probe e.g., a first probe
  • the extended probe When the first probe is extended, for example, with a DNA polymerase or a reverse transcnptase (RT), the extended probe will incorporate a complementary sequence of a genetic variant or w ild-type sequence.
  • accuracy of the extended probe incorporating the complementary sequence of the genetic variant depends on several factors. Mistakes in extension by DNA polymerase or RT or competition between probes in traditional templated ligation reactions can produce inaccurate readouts of the presence, location, and/or frequency of genetic variants in the biological sample.
  • the methods, compositions, and kits disclosed herein include a blocking oligonucleotide including: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3' blocking moiety.
  • the blocking oligonucleotide prevents the first probe from being extended beyond the blocking oligonucleotide such that the extended probe can be ligated to a second probe via branch ligation.
  • the resulting ligation products can be about the same length and more conducive for subsequent analysis (e.g., sequencing), leading to increased sensitivity and accuracy.
  • a location of a genetic variant in a target nucleic acid in a biological sample including: (a) providing an arrayincluding a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) contacting a plurality of first probes and a plurality of blocking oligonucleotides w ith the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary 7 to the target nucleic acid at a region that is downstream of the genetic variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary' to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety ; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic
  • the 3' blocking moiety is selected from the group consisting of: a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.
  • the genetic variant includes a single nucleotide variant or polymorphism.
  • the target nucleic acid includes at least two, at least three, at least four, at least five or more single nucleotide variants or polymorphisms.
  • second probe includes a 5' phosphate group.
  • the capture probe capture domain is substantially complementary 7 to the capture probe capture domain on the array.
  • the ligating includes chemical ligation or enzy matic ligation.
  • the enzymatic ligation includes use of a ligase.
  • the ligase includes a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.
  • the ligase includes a pre-activated T4 DNA ligase.
  • the extending the first probe using the target nucleic acid as a template in (d) includes the use of a polymerase or a reverse transcriptase.
  • the reverse transcriptase is a non-displacing reverse transcriptase.
  • the non-displacing reverse transcriptase includes a Thermococcus gorgonarius mutant reverse transcription xenopolymerase.
  • the method includes releasing the ligation product from the target nucleic acid.
  • the releasing includes the use of an endoribonuclease.
  • the endoribonuclease includes RNase H, RNase A, RNase C, and/or RNase I, and optionally, where the RNase H includes RNase Hl, RNase H2, or both.
  • the capture probe includes one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.
  • the method includes contacting the biological sample with a permeabilization reagent.
  • the permeabilization reagent includes pepsin, proteinase K. or collagenase.
  • the method includes extending the capture probe using the ligation product as a template, thereby generating an extended capture probe. In some embodiments, the method includes extending the ligation product using the capture probe as a template, thereby generating an extended ligation product. In some embodiments, the method includes amplifying the extended capture probe or the extended ligation product.
  • the determining in (h) includes sequencing.
  • the biological sample is disposed on the array. In some embodiments, the biological sample is disposed on a substrate. In some embodiments, the method includes aligning the substrate including the biological sample with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array.
  • the biological sample is a tissue sample. In some embodiments, the biological sample is a tissue section. In some embodiments, the tissue section is a fixed tissue section. In some embodiments, the fixed tissue section is a formalin- fixed, paraffin-embedded tissue section, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone-fixed tissue section. In some embodiments, the tissue section is a fresh frozen tissue section. In some embodiments, the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.
  • the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, optionally, mRNA.
  • the first probe includes a functional domain, where the functional domain is a primer binding site or a sequence specific site.
  • a 3' end of the first probe includes one or more mismatched nucleotides. In some embodiments, a 3' end of the first probe includes a dideoxynucleotide. In some embodiments, the dideoxynucleotide includes ddATP, ddCTP, ddGTP, or ddTTP.
  • Also provided herein are methods for determining a presence or absence of a single nucleotide variant in a biological sample including: (a) providing an array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is downstream of the single nucleotide variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic at a region that is upstream of the single nucleotide variant and (ii) a 3' blocking moiety; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic
  • the determining in (h) includes correlating a location of the single nucleotide variant on the array to a location of the single nucleotide variant in the biological sample.
  • the 3' blocking moiety is selected from the group consisting of: a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.
  • the target nucleic acid includes at least one single nucleotide variant. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more single nucleotide variants.
  • the second probe includes a 5' phosphate group. In some embodiments, the second probe includes a capture probe capture domain that is substantially complementary to the capture probe capture domain on the array.
  • the ligating includes chemical ligation or enzymatic ligation.
  • the enzymatic ligation includes use of a ligase.
  • the ligase includes a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.
  • the ligase includes a pre-activated T4 DNA ligase.
  • the extending the first probe using the target nucleic acid as a template in (d) includes the use of a polymerase or a reverse transcriptase.
  • the reverse transcriptase enzyme is a non-displacing reverse transcriptase.
  • the method includes releasing the ligation product from the target nucleic acid.
  • the releasing includes the use of an endoribonuclease.
  • the endoribonuclease includes RNase H, RNase A, RNase C, and/or RNase I, and optionally, where the RNase H includes RNase Hl, RNase H2, or both.
  • the capture probe includes one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.
  • the method includes contacting the biological sample with a permeabilization reagent.
  • the permeabilization reagent includes pepsin, proteinase K, or collagenase.
  • the method includes extending the capture probe using the ligation product as a template thereby generating an extended capture probe. In some embodiments, the method includes extending the ligation product using the capture probe as a template, thereby generating an extended ligation product.
  • the method includes amplifying the extended capture probe and/or the extended ligation product.
  • the determining in (h) includes sequencing.
  • the biological sample is disposed on the array. In some embodiments, the biological sample is disposed on a substrate. In some embodiments, the method includes aligning the substrate including the biological sample with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array.
  • the biological sample is a tissue sample. In some embodiments, the biological sample is a tissue section. In some embodiments, the tissue section is a fixed tissue section. In some embodiments, the fixed tissue section is a formalin- fixed, paraffin-embedded tissue section, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone-fixed tissue section. In some embodiments, the tissue section is a fresh frozen tissue section. In some embodiments, the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.
  • the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, optionally, mRNA. In some embodiments, the first probe includes a functional domain, where the functional domain is a primer binding site or a sequence specific site.
  • a 3' end of the first probe includes one or more mismatched nucleotides. In some embodiments, a 3' end of the first probe includes a dideoxynucleotide. In some embodiments, the dideoxynucleotide includes ddATP, ddCTP, ddGTP, or ddTTP.
  • compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a first probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the first probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe including a partially double-stranded nucleic acid molecule, where the second probe includes a capture probe capture domain.
  • the 3' blocking moiety is selected from the group consisting of a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.
  • the target nucleic acid includes a genetic variant. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more genetic variants. In some embodiments, the genetic variant includes an insertion or a deletion. In some embodiments, the genetic variant includes a single nucleotide variant or polymorphism.
  • the second probe includes a 5' phosphate group. In some embodiments, the second probe includes a capture probe capture domain that is substantially complementary to the capture probe capture domain on the array.
  • the composition includes a polymerase or a reverse transcriptase.
  • the reverse transcriptase is a non-displacing reverse transcriptase.
  • the composition includes a biological sample.
  • the biological sample is a tissue sample.
  • the tissue sample is a tissue section.
  • the tissue section is a fixed tissue section.
  • the fixed tissue section is a formalin-fixed, paraffin-embedded tissue section, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone- fixed tissue section.
  • the tissue section is a fresh frozen tissue section.
  • the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.
  • the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, optionally, mRNA.
  • a 3' end of the first probe includes one or more mismatched nucleotides. In some embodiments, a 3' end of the first probe includes a dideoxynucleotide. In some embodiments, the dideoxynucleotide includes ddATP, ddCTP. ddGTP, or ddTTP.
  • kits including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode, and (ii) a capture domain; (b) (i) a plurality of first probes, where a first probe of the plurality of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is dow nstream of a genetic variant, and (ii) a plurality of second probes, where a second probe of the plurality of second probes includes a partially double-stranded nucleic acid molecule and where the second probe includes a capture probe capture domain; (c) a plurality of blocking oligonucleotides, where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3'
  • the capture probe includes one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.
  • the ligase includes a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.
  • the ligase is a pre-activated T4 DNA ligase.
  • the kit includes a polymerase. In some embodiments, the kit includes a reverse transcriptase. In some embodiments, the reverse transcriptase enzyme is a non-displacing reverse transcriptase.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
  • FIG. 1A shows an exemplary sandwiching process where a first substrate (e.g., a slide), including a biological sample, and a second substrate (e g., array slide) are brought into proximity with one another.
  • a first substrate e.g., a slide
  • a second substrate e.g., array slide
  • FIG. IB shows a fully formed sandwich configuration creating a chamber formed from the one or more spacers, the first substrate, and the second substrate.
  • FIG. 2A shows a perspective view' of an exemplary sample handling apparatus in a closed position.
  • FIG. 2B shows a perspective view of an exemplary sample handling apparatus in an open position.
  • FIG. 3A shows the first substrate angled over (superior to) the second substrate.
  • FIG. 3B shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium.
  • FIG. 3C shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate.
  • FIG. 4A shows a side view of the angled closure workflow.
  • FIG. 4B shows a top view of the angled closure workflow.
  • FIG. 5 is a schematic diagram showing an example of a barcoded capture probe, as described herein.
  • FIG. 6 shows a schematic illustrating a cleavable capture probe.
  • FIG. 7 shows exemplary capture domains on capture probes.
  • FIG. 8 shows an exemplary arrangement of barcoded features within an array.
  • FIG. 9A shows an exemplary workflow for performing templated capture and producing a ligation product
  • FIG. 9B shows an exemplary' workflow for capturing a ligation product from FIG. 9A on a substrate.
  • FIG. 10 is a schematic diagram of an exemplary analyte capture agent.
  • FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 1124 and an analyte capture agent 1126.
  • FIG. 12 from top to bottom is a schematic diagram depicting a method of detecting a genetic variant (e.g., SNPs) using a first probe and a blocking oligonucleotide hybridized to a target nucleic acid.
  • the first probe can be extended to incorporate complementary sequences of genetic variants until the extended probe reaches or nearly reaches the blocking oligonucleotide.
  • partially double-stranded second probe including a capture probe capture domain can then be ligated to the extended probe.
  • FIG. 13 is a schematic diagram depicting specificity improvements of the method shown in FIG. 12 by incorporation of either a mismatched base (top) or a dideoxynucleotide (bottom) to limit extension of the first probe and non-specific ligation. Unextended first probes are shown on the far right.
  • Spatial analysis methodologies described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context.
  • Spatial analysis methods can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell.
  • a spatial barcode e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample
  • a capture domain that is capable of binding to an analyte (e.g.,
  • Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte.
  • the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.
  • a '‘barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe).
  • a barcode can be part of an analyte, or independent of an analyte.
  • a barcode can be attached to an analyte.
  • a particular barcode can be unique relative to other barcodes.
  • an “analyte” can include any biological substance, structure, moiety, or component to be analyzed.
  • target can similarly refer to an analyte of interest.
  • Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
  • non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory', viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments.
  • viral proteins e.g., viral capsid, viral envelope, viral coat, viral accessory', viral glycoproteins, viral spike, etc.
  • the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria. Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference.
  • an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • a “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject.
  • the biological sample is a tissue sample.
  • the biological sample e.g.. tissue sample
  • TMA tissue microarray
  • a tissue microarray contains multiple representative tissue samples - which can be from different tissues or organisms - assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time.
  • Tissue microarrays can be paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these tissue cores into a single recipient (microarray) block at defined array coordinates.
  • the biological sample as used herein can be any suitable biological sample described herein or known in the art.
  • the biological sample is a tissue sample.
  • the tissue sample is a solid tissue sample.
  • the biological sample is a tissue section (e.g., a fixed tissue section).
  • the tissue is flash-frozen and sectioned. Any suitable method described herein or known in the art can be used to flash-freeze and section the tissue sample.
  • the biological sample, e.g., the tissue is flash-frozen using liquid nitrogen before sectioning.
  • the biological sample, e.g., a tissue sample is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane.
  • the biological sample e.g., the tissue
  • a matrix e.g., optimal cutting temperature (OCT) compound to facilitate sectioning.
  • OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens.
  • the sectioning is performed by cryosectioning, for example, using a microtome.
  • the methods further comprise a thawing step, after the cryosectioning.
  • the biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat. In addition to the subjects described above, the biological sample can be obtained from non-mammalian organisms (e.g.. a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungus, an amphibian, or a fish (e.g., zebrafish)). A biological sample can be obtained from a prok ar ote such as a bacterium, e.g., Escherichia coll.
  • a prok ar ote such as a bacterium, e.g., Escherichia coll.
  • a biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX).
  • the biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy.
  • Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities.
  • an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid.
  • Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy.
  • a disease e.g., cancer
  • pre-disposition to a disease e.g., cancer
  • Bio samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
  • Biological samples can include one or more diseased cells.
  • a diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.
  • the biological sample e.g., the tissue sample
  • a fixative including alcohol for example methanol.
  • acetone or an acetone-methanol mixture can be used instead of methanol.
  • the fixation is performed after sectioning.
  • the biological sample is fixed using a fixative including an alcohol (e.g.. methanol or acetone-methanol mixture) after freezing and/or sectioning.
  • the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone-methanol mixture). In some instances when methanol, acetone, or an acetone- methanol mixture is used to fix the biological sample, the sample is not decrosslinked at a later step. In instances when the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone- methanol) fixation or acetone fixation, the biological sample is referred to as “fresh frozen 7 ’.
  • fresh frozen 7 e.g., methanol, acetone- methanol
  • fixation of the biological sample e.g., using acetone and/or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g., glass slide, such as a positively charged glass slide).
  • acetone and/or alcohol e.g., methanol, acetone-methanol
  • the biological sample e.g.. the tissue sample
  • the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PF A) or formalin.
  • the fixative induces crosslinks within the biological sample.
  • the biological sample is dehydrated via sucrose gradient.
  • the fixed biological sample is treated with a sucrose gradient and then embedded in a matrix e.g., OCT compound.
  • the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix e.g., OCT compound after fixation.
  • the sample when a fixed frozen tissue sample is treated with a sucrose gradient, the sample can be rehydrated with an ethanol gradient.
  • the PFA or formalin fixed biological sample which can be optionally dehydrated via sucrose gradient and/or embedded in OCT compound, is then frozen e.g., for storage or shipment.
  • the biological sample is referred to as “fixed frozen”.
  • a fixed frozen biological sample is not treated with methanol.
  • a fixed frozen biological sample is not paraffin embedded.
  • a fixed frozen biological sample is not deparaffinized.
  • a fixed frozen biological sample is rehydrated using an ethanol gradient.
  • the biological sample e.g., a fixed frozen tissue sample
  • a citrate buffer can be used for antigen retrieval to decrosslink antigens and fixation medium in the biological sample for antigen retrieval.
  • any suitable decrosslinking agent can be used in addition to or alternatively to citrate buffer.
  • the biological sample e.g., a fixed frozen tissue sample
  • the biological sample can further be stained, imaged, and/or destained.
  • a fresh frozen tissue sample or fixed frozen tissue sample is stained (e.g., via eosin and/or hematoxylin), imaged, destained (e.g.. via HC1), or a combination thereof.
  • the sample is treated with isopropanol prior to being stained (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HC1), or a combination thereof.
  • the sample when a fixed frozen tissue sample is treated with a sucrose gradient, the sample can be rehydrated using an ethanol gradient before being stained, (e.g.. via eosin and/or hematoxylin), imaged, destained (e.g., via HC1), decrosslinked (e.g., via TE buffer or citrate buffer), or a combination thereof.
  • the biological sample can undergo further fixation (e.g., while mounted on a substrate), stained, imaged, and/or destained.
  • a fixed frozen biological sample may be subject to an additional fixing step (e.g., using PF A) before optional ethanol rehydration, staining, imaging, and/or destaining.
  • the biological sample can be fixed using PAXgene.
  • the biological sample can be fixed using PAXgene in addition, or alternatively to, a fixative disclosed herein or known in the art (e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde).
  • PAXgene is a non-cross-linking mixture of different alcohols, an acid, and a soluble organic compound that preserves morphology and bio-molecules.
  • PAXgene provides a two-reagent fixative system in which tissue is firstly fixed in a solution containing methanol and acetic acid then stabilized in a solution containing ethanol. See. Ergin B.
  • the fixative is PAXgene.
  • a fresh frozen tissue sample is fixed with PAXgene.
  • a fixed frozen tissue sample is fixed with PAXgene.
  • the biological sample e.g., the tissue sample is fixed, for example in methanol, acetone, acetone-methanol, PF A, PAXgene or is formalin-fixed and paraffin-embedded (FFPE).
  • the biological sample comprises intact cells.
  • the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet.
  • FFPE samples are used in some instances in the RNA-templated ligation (RTL) methods disclosed herein.
  • RNA integrity of fixed (e.g., FFPE) samples can be lower than a fresh sample, thereby capturing RNA directly from fixed samples, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule, can be more difficult.
  • RTL probes that hybridize to RNA target sequences in the trans criptome, RNA analytes can be captured without requiring that both a poly(A) tail and target sequences remain intact. Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples.
  • the biological sample e.g., tissue sample
  • the biological sample can be stained, and imaged prior, during, and/or after each step of the methods described herein. Any of the methods described herein or known in the art can be used to stain and/or image the biological sample.
  • the imaging occurs prior to destaining the sample.
  • the biological sample is stained using an H&E staining method.
  • the tissue sample is stained and imaged for about 10 minutes to about 2 hours (or any of the subranges of this range described herein). Additional time may be needed for staining and imaging of different types of biological samples.
  • the tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject.
  • the sample is a mouse sample.
  • the sample is a human sample.
  • the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen.
  • the sample is a human or mouse breast tissue sample.
  • the sample is a human or mouse brain tissue sample.
  • the sample is a human or mouse lung tissue sample.
  • the sample is a human or mouse tonsil tissue sample.
  • the sample is a human or mouse liver tissue sample. In some instances, the sample is a human or mouse bone, skin, kidney, thymus, testes, or prostate tissue sample. In some embodiments, the tissue sample is derived from normal or diseased tissue. In some embodiments, the sample is an embryo sample.
  • the embry o sample can be a non-human embry o sample. In some instances, the sample is a mouse embryo sample.
  • the biological sample (e.g.. a fixed and/or stained biological sample) is imaged.
  • the biological sample is visualized or imaged using bright field microscopy.
  • the biological sample is visualized or imaged using fluorescence microscopy.
  • the biological sample can be visualized or imaged using additional methods of visualization and imaging known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy.
  • the sample is stained and imaged prior to adding reagents for analyzing captured analytes as disclosed herein to the biological sample.
  • the methods include staining the biological sample.
  • the staining includes the use of hematoxylin and/or eosin.
  • stains include histological stains (e g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains).
  • a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI (4',6-diamidino-2- phenylindole), eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red. Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin.
  • biological stains including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI (4',6-diamidino-2- phenylindole), eosin, ethidium bromide, acid fuchsine, hematoxylin, Ho
  • the biological sample can be stained using known staining techniques, including Can-Grunw ald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky 7 , silver, Sudan, Wright’s, and/or Periodic Acid Schiff (PAS) staining techniques.
  • PAS staining is typically performed after formalin or acetone fixation.
  • the staining includes the use of a detectable label, such as a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
  • a detectable label such as a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
  • a biological sample is permeabilized with one or more permeabilization reagents.
  • permeabilization of a biological sample can facilitate analyte capture.
  • Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)( 13) or the Exemplary Embodiments Section of PCT Publication No.
  • the method includes permeabilizing the biological sample.
  • the biological sample can be permeabilized to facilitate transfer of the extension products to the capture probes on the array.
  • the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, and methanol), a detergent (e g., saponin, Triton X-100TM, Tween-20TM, or sodium dodecyl sulfate (SDS)), an enzyme (an endopeptidase, an exopeptidase, a protease), or a combination thereof.
  • an organic solvent e.g., acetone, ethanol, and methanol
  • a detergent e g., saponin, Triton X-100TM, Tween-20TM, or sodium dodecyl sulfate (SDS)
  • an enzyme an endopeptidase, an exopeptidase, a protease
  • the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X-100TM, Tween-20TM, or a combination thereof.
  • the endopeptidase is pepsin.
  • the endopeptidase is Proteinase K. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, which is herein incorporated by reference.
  • Array-based spatial analysis methods can involve the transfer of one or more analytes or derivatives thereof from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array.
  • a 'capture probe refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample.
  • the capture probe is a nucleic acid or a polypeptide.
  • the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI) and a capture domain).
  • UMI unique molecular identifier
  • the capture probe includes a homopolymer sequence, such as a poly(T) sequence.
  • a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for nextgeneration sequencing (NGS)).
  • NGS nextgeneration sequencing
  • a cleavage domain and/or a functional domain e.g., a primer-binding site, such as for nextgeneration sequencing (NGS)
  • NGS nextgeneration sequencing
  • Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. which is herein incorporated by reference.
  • a capture probe and a nucleic acid analyte interaction occurs because the sequences of the two nucleic acids are substantially complementary to one another.
  • two nucleic acid sequences can be complementary when at least 60% of the nucleotide residues of one nucleic acid sequence are complementary to nucleotide residues of the other nucleic acid sequence.
  • the complementary residues within a particular complementary nucleic acid sequence need not always be contiguous with each other, but can be interrupted by one or more non-complementary residues within the complementary nucleic acid sequence.
  • At least 60%, but less than 100%, of the residues of one of the two complementary nucleic acid sequences are complementary to residues of the other nucleic acid sequence.
  • at least 70%, 80%, 90%, 95% or 99% of the residues of one nucleic acid sequence are complementary to residues of the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence.
  • the biological sample is mounted on a first substrate and the substrate comprising the array of capture probes is a second substrate.
  • one or more analytes or analyte derivatives can then be released from the biological sample and migrate to the second substrate comprising an array of capture probes.
  • the release and migration of the analytes or analyte derivatives to the second substrate comprising the array of capture probes occurs in a manner that preserves the original spatial context of the analytes in the biological sample.
  • This method can be referred to as a sandwiching process, which is described e.g., in U.S. Patent Application Pub. No. 2021/0189475 and PCT Pub. Nos. WO 2021/252747 Al, WO 2022/061152 A2, and WO 2022/140028 Al, each of which is herein incorporated by reference.
  • FIG. 1A shows an exemplary sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102. and a second substrate (e.g., array slide 104 including an array having spatially barcoded capture probes 106) are brought into proximity with one another.
  • a liquid reagent drop e.g., permeabilization solution 105
  • the permeabilization solution 105 may release analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) that can be captured by the capture probes of the array 106.
  • the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the capture probes (e.g., aligned in a sandwich configuration).
  • the second substrate e.g., array slide 104 is in an inferior position to the first substrate (e.g., slide 103).
  • the first substrate e.g., slide 103
  • the second substrate e.g., slide 104
  • a reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates.
  • the reagent medium may be a permeabilization solution, which permeabilizes and/or digests the biological sample 102. In some embodiments, where the biological sample 102 has been pre-permeabilized, the reagent medium is not a permeabilization solution.
  • the reagent medium may also comprise one or more of a monovalent salt, a divalent salt, ethylene carbonate, and/or glycerol. In some embodiments, analytes (e.g...
  • mRNA transcripts and/or analyte derivatives (e.g., intermediate agents; e.g., ligation products) of the biological sample 102 may release from the biological sample, and actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106.
  • migration of the analyte or analyte derivative (e.g., intermediate agent; e.g., ligation product) from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration).
  • electrophoretic migration are described in WO 2020/176788, and U.S. Patent Application Pub. No. 2021/0189475, each of which is hereby incorporated by reference in its entirety.
  • one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including spatially barcoded capture probes 106).
  • the one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.
  • the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and 500 microns, between about 2 microns and 400 microns, between about 2 microns and 300 microns, between about 2 microns and 200 microns, between about 2 microns and 100 microns, between about 2 microns and 25 microns, or between about 2 microns and 10 microns), measured in a direction orthogonal to the surface of first substrate that supports the biological sample.
  • a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and
  • the separation distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In some embodiments, the separation distance is less than 50 microns. In some embodiments, the separation distance is less than 25 microns. In some embodiments, the separation distance is less than 20 microns.
  • the separation distance may include a distance of at least 2 pm.
  • FIG. IB shows a fully formed sandwich configuration 125 creating a chamber 150 formed from the one or more spacers 110, the first substrate (e.g.. the slide 103), and the second substrate (e.g., the slide 104 including an array 106 having spatially barcoded capture probes) in accordance with some example implementations.
  • the first substrate e.g. the slide 103
  • the second substrate e.g., the slide 104 including an array 106 having spatially barcoded capture probes
  • the liquid reagent e.g., the permeabilization solution 105 fills the volume of the chamber 150 and may create a permeabilization buffer that allows analytes (e.g., mRNA transcripts and/or other molecules) or analyte derivatives (e.g., intermediate agents; e.g., ligation products) to diffuse from the biological sample 102 toward the capture probes of the second substrate (e.g., slide 104).
  • flow of the permeabilization buffer may deflect transcripts and/or molecules from the biological sample 102 and may affect diffusive transfer of analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) for spatial analysis.
  • the first substrate (e.g., slide 103), and the second substrate (e.g., slide 104) may reduce or prevent flow from undesirable movement (e.g., convective movement) of transcripts and/or molecules during the diffusive transfer from the biological sample 102 to the capture probes.
  • the sandwiching process methods described above can be implemented using a variety of hardware components.
  • the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sample handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in. e.g., US. Patent Application Pub. No. 2021/0189475, and PCT Publ. No. WO 2022/061152 A2, each of which is incorporated by reference in its entirety.
  • the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a biological sample.
  • the first retaining mechanism can be configured to retain the first substrate disposed in a first plane.
  • the sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane.
  • the sample holder can further include an alignment mechanism connected to one or both of the first member and the second member.
  • the alignment mechanism can be configured to align the first and second members along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane.
  • the adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and/or move the first member along an axis orthogonal to the first plane.
  • the adjustment mechanism includes a linear actuator.
  • the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and/or the second member.
  • the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and/or the second member.
  • the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0. 1 mm/sec.
  • the linear actuator is configured to move the first member, the second member, or both the first member and the second member with an amount of force of at least 0. 1 lbs.
  • FIG. 2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations.
  • the sample handling apparatus 200 includes a first member 204, a second member 210, optionally an image capture device 220, a first substrate 206, optionally a hinge 215, and optionally a mirror 216.
  • the hinge 215 may be configured to allow the first member 204 to be positioned in an open or closed configuration by opening and/or closing the first member 204 in a clamshell manner along the hinge 215.
  • FIG. 2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations.
  • the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206.
  • the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206.
  • the first substrate 206 and/or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200, such as within the first member 204 and the second member 210, respectively.
  • the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration.
  • an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and/or the second member 210 to form the sandwich configuration for permeabilization of the sample (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration).
  • the adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration.
  • the biological sample (e.g., sample 102 from FIG. 1A) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample is aligned with the barcoded array of the second substrate (e.g., the slide 104 from FIG. 1A), e.g., when the first and second substrates are aligned in the sandwich configuration.
  • Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g.. via an automated alignment mechanism).
  • spacers may be applied to the first substrate 206 and/or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching.
  • the permeabilization solution e.g., permeabilization solution 305
  • the first member 204 may then close over the second member 210 and form the sandwich configuration.
  • Analytes or analyte derivatives e g., intermediate agents, e.g., ligation products
  • the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and/or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas.
  • FIGs. 3A-3C depict a side view' and a top view' of an exemplary angled closure w orkflow' 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary implementations.
  • FIG. 3A depicts the first substrate (e.g., slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304).
  • reagent medium e.g., permeabilization solution
  • FIG. 3A depicts the reagent medium on the right-hand side of side view, it should be understood that such depiction is not meant to be limiting as to the location of the reagent medium on the spacer.
  • FIG. 3B shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate (e.g., a side of the slide 303 angled toward the slide 304) may contact the reagent medium 305.
  • the dropped side of the slide 303 may urge the reagent medium 305 toward the opposite direction (e.g., towards an opposite side of the spacer 310. towards an opposite side of the slide 303 relative to the dropped side).
  • the reagent medium 305 may be urged from right to left as the sandwich is formed.
  • the first substrate and/or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate.
  • FIG. 3C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the second substrate and maintaining a separation distance and optionally the approximately parallel arrangement between the two substrates.
  • the spacer 310 fully encloses and surrounds the biological sample 302 and the capture probes 306, and the spacer 310 form the sides of chamber 350 which holds a volume of the reagent medium 305.
  • FIG. 3C depicts the first substrate (e.g., the slide 303 including biological sample 302) angled over (superior to) the second substrate (e.g., slide 304) and the second substrate comprising the spacer 310
  • an exemplary 7 angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate comprising the spacer 310.
  • the reagent medium be free from air bubbles between the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present between the substrates may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and/or elimination of air bubbles between the two substrates (e.g., slide 303 and slide 304) during permeabilization (e.g., step 104). In some aspects, bubble formation between the substrates may be reduced or eliminated using a variety' of filling methods and/or closing methods. In some instances, the first substrate and the second substrate are arranged in an angled sandwich assembly as described herein. For example, during the sandwiching of the two substrates (e.g., the slide 303 and the slide 304), an angled closure workflow may be used to suppress or eliminate bubble formation.
  • FIG. 4A is a side view of the angled closure workflow 400 in accordance with some exemplary implementations.
  • FIG. 4B is a top view of the angled closure workflow 400 in accordance with some exemplary implementations.
  • reagent medium 401 is positioned to the side of the substrate 402 contacting the spring.
  • the dropped side of the angled substrate 406 contacts the reagent medium 401 first.
  • the contact of the substrate 406 with the reagent medium 401 may form a linear or low curvature flow front that fills the gap between the two substrates 406 and 402 uniformly with the slides closed.
  • the substrate 406 is further lowered tow ard the substrate 402 (or the substrate 402 is raised up toward the substrate 406) and the dropped side of the substrate 406 may contact and may urge the reagent medium toward the side opposite the dropped side, thereby creating a linear or low curvature flow front that may prevent or reduce bubble trapping betw een the substrates.
  • the reagent medium 401 fills the gap between the substrate 406 and the substrate 402.
  • the linear flow front of the liquid reagent may be formed by squeezing the reagent medium 401 volume along the contact side of the substrate 402 and/or the substrate 406. Additionally, capillary' flow' may also contribute to filling the gap area.
  • the reagent medium (e.g., 105 in FIG 1A) comprises a permeabilization agent.
  • the permeabilization agent can be removed from contact with the biological sample (e.g., by opening the sample holder).
  • Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, or methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin. Triton X- 100TM, Tween-20TM, or sodium dodecyl sulfate (SDS)). and enzymes (e.g.. trypsin or other proteases (e g., proteinase K).
  • the detergent is an anionic detergent (e.g., SDS orN-lauroylsarcosine sodium salt solution).
  • the reagent medium comprises a lysis reagent.
  • Lysis solutions can include ionic surfactants, such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents.
  • the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, elastase, and proteinase K.
  • the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises an RNase.
  • the RNase is selected from RNase A, RNase C, RNase H, and RNase I.
  • the reagent medium comprises one or more of sodium dodecyl sulfate (SDS) or a sodium salt thereof, proteinase K, pepsin, N-lauroylsarcosine. and RNase.
  • the reagent medium comprises polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG is from about 2K to about 16K.
  • the PEG is about 2K, about 3K, about 4K, about 5K, about 6K, about 7K, about 8K, about 9K, about 10K, about UK, about 12K. about 13K. about 14K. about 15K. or about 16K.
  • the PEG is present at a concentration from about 2% to about 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v/v).
  • a dried permeabilization reagent is applied or formed as a layer on the first substrate, the second substrate, or both prior to contacting the biological sample with the array.
  • a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.
  • the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1 minute, about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45 minutes, or about an hour. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1-60 minutes.
  • the device is configured to control a temperature of the first and second substrates. In some embodiments, the temperature of the first and second members is lowered to a first temperature that is below room temperature.
  • a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location.
  • One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes).
  • Another method is to release or cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.
  • capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g.. a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes, which is herein incorporated by reference).
  • a template e.g. a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of PCT Publication No. WO2020/176788 and/or U.S
  • capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligation products that sen e as proxies for the template.
  • a template e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof
  • an “extended capture probe’ 7 refers to a capture probe having additional nucleotides added to a terminus (e.g., 3' or 5' end) of the capture probe thereby extending the overall length of the capture probe.
  • an “extended 3' end” indicates additional nucleotides were added to the most 3' nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e g., a DNA polymerase or a reverse transcriptase).
  • a polymerase e g., a DNA polymerase or a reverse transcriptase
  • extending the capture probe includes adding to a 3’ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture probe capture domain.
  • the capture probe is extended using a reverse transcriptase.
  • the capture probe is extended using one or more DNA polymerases.
  • the extended capture probes include the sequence of the capture domain, the sequence of the spatial barcode of the capture probe, and the complementary sequence of the template used for extension of the capture probe.
  • extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for subsequent analysis, e.g., sequencing.
  • extended capture probes e.g., DNA molecules
  • can act as templates for an amplification reaction e.g., a polymerase chain reaction.
  • an “extended probe” refers to a first probe hybridized to a target nucleic acid and is extended by one or more nucleotides using the target nucleic acid (e.g., RNA or DNA) as a template.
  • First probes are substantially complementary to a target nucleic acid at a region of the target nucleic acid that is downstream (i.e.. to the 3' direction) of one or more genetic variant(s).
  • These hybridized first probes can be extended with a polymerase or a reverse transcriptase, and the resulting extended probes comprising the complementary sequence of one or more genetic variant(s) can be ligated to a second probe (e.g.. second probes described herein) via branch ligation.
  • blocking oligonucleotide refers to an oligonucleotide hybridized to a target nucleic acid at a region of the target nucleic acid that is upstream (i.e., to the 5' direction) of one or more genetic variant(s).
  • the blocking oligonucleotide can be substantially complementary (as defined herein) to the target nucleic acid.
  • the blocking oligonucleotide includes a 3' blocking moiety.
  • Various blocking moieties are known in the art (e.g., moieties sufficient to block extension). Non-limiting examples of blocking moieties include a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.
  • Spatial information can provide information of medical importance.
  • the methods described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g..).
  • biomarkers e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment
  • a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.
  • Exemplary methods for identifying spatial information of biological and/or medical importance can be found in U.S. Patent Application Publication Nos. 2021/0140982, 2021/0198741, and 2021/0199660, each of which is herein incorporated by reference.
  • Spatial information can provide information of biological importance.
  • the methods described herein can allow for: identification of transcriptome and/or proteome expression profiles (e g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor or proximity based analysis); determination of up-regulated and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in healthy and diseased tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).
  • a substrate can function as a support for direct or indirect attachment of capture probes to features of the array.
  • a "feature” is an entity 7 that acts as a support or repository for various molecular entities used in spatial analysis.
  • some or all of the features in an array are functionalized for analyte capture.
  • Exemplary substrates are described in Section (II)(c) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
  • Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (IT)(d)(iii), and (II)(d)(iv) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference.
  • analytes and/or intermediate agents can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes).
  • capture probes e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes.
  • contact contacted
  • contacting a biological sample with a substrate refers to any contact (e.g.. direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e g., hybridize)) with analytes from the biological sample.
  • FIG. 5 is a schematic diagram showing an exemplary capture probe, as described herein. As shown, the capture probe 502 is optionally coupled to a feature 501 by a cleavage domain 503, such as a disulfide linker. The capture probe can include a functional sequence 504 that is useful for subsequent processing.
  • the functional sequence 504 can include all or a part of sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence), all or a part of a sequencing primer sequence, (e.g., a R1 primer binding site, a R2 primer binding site), or a combination thereof.
  • the capture probe can also include a spatial barcode 505.
  • the capture probe can also include a unique molecular identifier (UMI) sequence 506. While FIG. 5 shows the spatial barcode 505 as being located upstream (5') of UMI sequence 506, it is to be understood that capture probes wherein UMI sequence 506 is located upstream (5') of the spatial barcode 505 is also suitable for use in any of the methods described herein.
  • the capture probe can also include a capture domain 507 to facilitate capture of a target analyte.
  • the capture domain can have a sequence complementary to a sequence of a nucleic acid analyte.
  • the capture domain can have a sequence complementary to a connected probe described herein.
  • the capture domain can have a sequence complementary to an analyte capture sequence present in an analyte capture agent.
  • the capture domain can have a sequence complementary to a splint oligonucleotide.
  • a splint oligonucleotide in addition to having a sequence complementary' to a capture domain of a capture probe, can have a sequence complementary to a sequence of a nucleic acid analyte, a portion of a connected probe described herein, a capture handle sequence described herein, and/or a methylated adaptor described herein.
  • FIG. 6 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to analytes within the sample.
  • the capture probe 601 can contain a cleavage domain 602, a cell penetrating peptide 603, a reporter molecule 604, and a disulfide bond (-S-S-).
  • 605 represents all other parts of a capture probe, for example a spatial barcode and a capture domain.
  • FIG. 7 is a schematic diagram of an exemplary 7 multiplexed spatially-barcoded feature.
  • the feature 701 can be coupled to spatially -barcoded capture probes, wherein the spatially-barcoded probes of a particular feature can possess the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with more than one target analyte.
  • a feature may include four different ty pes of spatially -barcoded capture probes, each ty pe of spatially-barcoded capture probe possessing the spatial barcode 702.
  • One type of capture probe associated with the feature can include the spatial barcode 702 in combination with a poly(T) capture domain 703, designed to capture mRNA target analytes.
  • a second type of capture probe associated with the feature can include the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis.
  • a third type of capture probe associated with the feature can include the spatial barcode 702 in combination with a capture domain complementary to the analyte capture agent of interest 705.
  • a fourth type of capture probe associated with the feature can include the spatial barcode 702 in combination with a capture probe that can specifically bind a nucleic acid molecule 706 that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only four different capture probe-barcoded constructs are shown in FIG.
  • capture-probe barcoded constructs can be tailored for analyses of any given analyte associated with a nucleic acid and capable of binding with such a construct.
  • the schemes shown in FIG. 7 can also be used for concurrent analysis of other analytes disclosed herein, including, but not limited to: (a) mRNA, a lineage tracing construct, cell surface or intracellular proteins and/or metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq), cell surface or intracellular proteins and metabolites, and a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense oligonucleotide as described herein); (c) mRNA, cell surface or intracellular proteins and/or metabolites, a barcoded labelling agent (e.g., the MHC multimers
  • the functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio. Oxford Nanopore, etc., and the requirements thereof.
  • functional sequences can be selected for compatibility with noncommercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.
  • functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.
  • the spatial barcode 505 and functional sequence 504 are common to all of the probes attached to a given feature.
  • the UMI sequence 506 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.
  • FIG. 8 depicts an exemplary arrangement of barcoded features within an array. From left to right, FIG. 8 shows (left) a slide including six spatially-barcoded arrays, (center) an enlarged schematic of one of the six spatially -barcoded arrays, showing a grid of barcoded features in relation to a biological sample, and (right) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (e.g., labelled as ID578, ID579, ID580, etc ).
  • more than one analyte type e.g., nucleic acids and proteins
  • a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference.
  • spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample).
  • a plurality of molecules e.g., a plurality of nucleic acid molecules
  • a plurality of barcodes e.g., a plurality of spatial barcodes
  • a biological sample e.g., to a plurality of cells in a biological sample for use in spatial analysis.
  • the biological sample after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis.
  • Some such methods of spatial analysis are described in Section (III) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. which is herein incorporated by reference.
  • spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte.
  • spatial analysis can be performed using RNA-templated ligation (RTL).
  • RTL RNA-templated ligation
  • Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug 21; 45(14):el28, which is herein incorporated by reference.
  • RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule).
  • the oligonucleotides are DNA molecules.
  • one of the oligonucleotides includes at least two ribonucleic acid bases at the 3' end and/or the other oligonucleotide includes a phosphorylated nucleotide at the 5' end.
  • one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence or a non-homopolymeric sequence).
  • a ligase e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase
  • a ligase e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase
  • the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides.
  • a polymerase e.g..
  • a DNA polymerase can extend one of the oligonucleotides prior to ligation.
  • the ligation product is released from the analyte.
  • the ligation product is released using an endonuclease (e.g., RNase H).
  • the ligation product is removed using heat.
  • the ligation product is removed using potassium hydroxide (KOH).
  • the released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.
  • the disclosed methods can include contacting the biological sample with a deoxyribonuclease (DNase).
  • DNase can be an endonuclease or exonuclease.
  • the DNase digests single-stranded and/or doublestranded DNA.
  • Suitable DNases include, without limitation, a DNase I and a DNase II. Use of a DNase as described can mitigate false positive sequencing data from off target gDNA ligation events.
  • FIG. 9A A non-limiting example of templated ligation methods disclosed herein is depicted in FIG. 9A.
  • a biological sample is contacted with a substrate including a plurality of capture probes and contacted with (a) a first probe 901 having a target-hybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a targethybridization sequence 905 and a capture domain (e.g., a poly(A) sequence) 906, the first probe 901 and the second probe 904 hybridize 910 to an analyte 907.
  • a ligase 921 ligates 920 the first probe to the second probe 904, thereby generating a ligation product 922.
  • the ligation product 922 is then released 930 from the analyte 931 by digesting the analyte 907 using an endoribonuclease 932.
  • the sample is permeabilized 940 and the ligation product 941 can hybridize to a capture probe on the substrate.
  • the ligation product 9001 includes a capture probe capture domain 9002, which can bind to a capture probe 9003 (e.g., a capture probe immobilized, directly or indirectly, on a substrate 9004).
  • methods provided herein include contacting 9005 a biological sample with a substrate 9004, wherein the capture probe 9003 is affixed to the substrate (e.g.. immobilized to the substrate, directly or indirectly).
  • the capture probe capture domain 9002 of the ligated product 9001 specifically binds to the capture domain 9006.
  • the capture probe can also include a unique molecular identifier (UMI) 9007, a spatial barcode 9008, a functional sequence 9009, and a cleavage domain 9010.
  • UMI unique molecular identifier
  • methods provided herein include permeabilization of the biological sample such that the capture probe can more easily capture the ligation products (i.e., compared to no permeabilization).
  • reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can be used to extend the capture probes 9011 to produce spatially-barcoded full-length cDNA 9012 and 9013 from the captured ligation products.
  • the extended ligation products can be denatured 9014, released from the capture probe and transferred (e.g.. to a clean tube) for amplification, and/or library construction.
  • the spatially-barcoded ligation products can be amplified 9015 via PCR prior to library construction.
  • P5 9016, and P7 9019 sequences can be used as flow cell capture probes and i5 9017 and i7 9018 can be used as sample indexes.
  • the amplicons can then be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites.
  • an analyte capture agent refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte.
  • the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence.
  • an analyte binding moiety barcode refers to a barcode that is associated with or otherwise identifies the analyte binding moiety.
  • analyte capture sequence refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe.
  • an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g.. cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of PCT Publication No. WO2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference.
  • FIG. 10 is a schematic diagram of an exemplary analyte capture agent 1002 comprised of an analyte-binding moiety 1004 and an analyte-binding moiety barcode domain 1008.
  • the exemplary analyte-binding moiety 1004 is a molecule capable of binding to an analyte 1006 and the analyte capture agent 1002 is capable of interacting with a spatially- barcoded capture probe.
  • the analyte binding moiety 1004 can bind to the analyte 1006 with high affinity and/or with high specificity.
  • the analyte capture agent 1002 can include: (i) an analyte binding moiety barcode domain 1008, which serves to identify the analyte binding moiety, and (ii) an analyte capture sequence, which can hybridize to at least a portion or an entirety of a capture domain of a capture probe.
  • the analyte-binding moiety 1004 can include a polypeptide and/or an aptamer.
  • the analyte-binding moiety’ 1004 can include an antibody or antibody fragment (e.g.. an antigen-binding fragment).
  • FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1124 and an analyte capture agent 1126.
  • the feature- immobilized capture probe 1124 can include a spatial barcode 1108 as well as functional sequence 1106 and a UMI 1110. as described elsewhere herein.
  • the capture probe can be affixed 1104 to a feature, such as a bead 1102.
  • the capture probe 1124 can also include a capture domain 1112 that is capable of binding to an analyte capture agent 1126.
  • the analyte binding moiety barcode domain of the analyte capture agent 1126 can include a functional sequence 1118, analyte binding moiety barcode 1116.
  • the analyte capture agent 1126 can also include a linker 1120 that allows the analytebinding moiety barcode domain (e.g., including the functional sequence 1118, analyte binding moiety barcode 1116, and analyte capture sequence 1114) to couple to the analyte binding moiety 1122.
  • the linker 1120 is a cleavable linker.
  • the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, chemical-cleavable linker, thermal-cleavable linker, or an enzyme cleavable linker.
  • the cleavable linker is a disulfide linker.
  • a disulfide linker can be cleaved by use of a reducing agent, such as dithiothreitol (DTT), beta-mercaptoethanol (BME). or tris(2- carboxyethyl)phosphine (TCEP).
  • sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample.
  • Various methods can be used to obtain the spatial information.
  • specific capture probes and their captured analytes are associated with specific locations in an array of features on a substrate.
  • specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.
  • specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array.
  • the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.
  • each array feature location represents a position relative to a coordinate reference point (e g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.
  • Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of PCT Publication No. W02020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. See, for example, the Exemplary 7 embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed. . . ” of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. which is herein incorporated by reference.
  • spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.
  • Suitable systems for performing spatial analysis can include components, such as a chamber (e.g., a flow cell or a sealable, fluid-tight chamber) for containing a biological sample.
  • the biological sample can be mounted, for example, in a biological sample holder.
  • One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow.
  • One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.
  • the systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid-state storage medium such as, but not limited to. a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium).
  • the control unit can optionally be connected to one or more remote devices via a network.
  • the control unit (and components thereof) can generally perform any of the methods and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the methods or features described herein.
  • the systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images.
  • the systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.
  • one or more light sources e.g., LED-based, diode-based, lasers
  • the systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components, such as application specific integrated circuits.
  • the software instructions when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the methods or functions described herein.
  • the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Publication No. W0202I/102003 and/or U.S. Patent Application Publication No. 2021/0150707, each of which is incorporated herein by reference in its entirety.
  • the biological sample Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two-dimensional and/or three- dimensional map of the analyte presence and/or level are described in PCT Publication No. W02020/053655 and spatial analysis methods are generally described in PCT Publication No. W02021/102039 and/or U.S. Patent Application Publication No. 2021/0155982, each of which is incorporated herein by reference in its entirety.
  • a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. W02020/123320, WO 2021/102005, and/or U.S. Patent Application Publication No. 2021/0158522, each of which is incorporated herein by reference in its entirety.
  • fiducial markers e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. W02020/123320, WO 2021/102005, and/or U.S. Patent Application Publication No. 2021/0158522, each of which is incorporated herein by reference in its entirety.
  • Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, or to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances.
  • the present disclosure features methods, compositions, and kits that can be used to analyze the presence, location, and/or frequency of genetic variants in a target nucleic acid in a biological sample using a target specific nucleic acid probe (e.g., a first probe), a blocking oligonucleotide, and a second probe in combination with capture probes in an arrayed format.
  • a probe e.g., a first probe
  • the first probe is extended, for example, with a DNA polymerase or a reverse transcriptase, the extended probe will incorporate a complementary sequence of a genetic variant or wild-tj pe sequence.
  • the methods, compositions, and kits disclosed herein include a blocking oligonucleotide including: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3' blocking moiety.
  • the blocking oligonucleotide prevents the first probe from being extended beyond the blocking oligonucleotide such that the extended probe can be ligated to a second probe via branch ligation.
  • the second probe is a partially double-stranded molecule that can be ligated to the extended probe.
  • the second probe also includes a capture probe capture domain that is substantially complementary to a capture domain of a capture probe on a spatial array.
  • Branch ligation (e g., 3' branch ligation) is described in Wang, L., 3' Branch ligation: a novel method to ligate non-compl ementary DNA to recessed or internal 3' OH ends in DNA or RNA, DNA Res., 26(1): 45-53 (2019), which is incorporated herein by reference in its entirety.
  • 3' branch ligation can be performed using T4 DNA ligase at non- conventional DNA ends formed by nicks, gaps, and overhangs.
  • blunt-end DNA donors e.g., second probes
  • the partially double-stranded DNA molecule includes a dideoxy 3' terminus which inhibits self-ligation.
  • the longer strand of the partially double-stranded DNA molecule e.g., the second probe
  • DNA acceptor oligonucleotide complexes can be assembled with a first probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the first probe is extended to or near the blocking oligonucleotide.
  • the partially doublestranded DNA molecule e.g.. second probe
  • Branch ligation can be effective for ligation of nucleic acid molecules having nicks (e.g., lacking phosphates), gaps (e.g., 1 nucleotide to 8 nucleotides), 3' recessive ends, or blunt ends.
  • nicks e.g., lacking phosphates
  • gaps e.g., 1 nucleotide to 8 nucleotides
  • 3' recessive ends e.g., blunt ends.
  • a location of a genetic variant in a target nucleic acid in a biological sample including: (a) providing an array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of the genetic variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid; (d)
  • Also provided herein are methods for determining a presence or absence of a single nucleotide variant in a biological sample including: (a) providing an array including a plurality of capture probes, where a capture probe of the plurality' of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is downstream of the single nucleotide variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic at a region that is upstream of the single nucleotide variant and (ii) a 3' blocking moiety; (c) hybridizing the first probe and the blocking oligonucleotide to the target
  • array-based spatial transcriptomics methods can measure analyte and/or gene expression data for a variety of analytes (e.g.. target nucleic acids), or proxies thereof (e.g., ligation products), within a biological sample at high spatial resolution, while retaining native spatial context.
  • analytes e.g.. target nucleic acids
  • proxies thereof e.g., ligation products
  • a genetic variant includes any genetic variation or modification compared to a canonical nucleic acid.
  • non-limiting examples of genetic variants include one or more single nucleotide variants or polymorphisms, one or more nucleotide deletions, one or more nucleotide insertions, one or more nucleic acid rearrangements, and one or more nucleotide duplications.
  • a genetic variant is at least one single nucleotide variant (SNV) or single nucleotide polymorphism (SNP).
  • a genetic variant includes multiple SNVs or SNPs (e.g., 1. 2. 3, 4. 5 or more SNVs or SNPs ).
  • the target nucleic acid comprises numerous genetic variants at a particular location, for example a “hot spot” of variants clustered in a particular location on the target nucleic acid.
  • the target nucleic acid is RNA.
  • RNA include various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA.
  • the RNA can be a transcript (e.g., present in a tissue section).
  • the RNA can be small (e.g.. less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length).
  • Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA).
  • the RNA can be double-stranded RNA or single-stranded RNA.
  • the RNA can be circular RNA.
  • the RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).
  • the RNA can be from an RNA virus, for example RNA viruses from Group III, IV or V of the Baltimore classification system.
  • the RNA can be from a retrovirus, such as a virus from Group VI of the Baltimore classification system.
  • the target nucleic acid is DNA.
  • Non-limiting examples of DNA analytes include genomic DNA. methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and viral DNA.
  • the first probe is extended after hybridizing to the target nucleic acid (e.g., hybridized at a region that is upstream of a genetic variant), thereby generating an extended probe.
  • the first probe can be extended by a polymerase or a reverse transcriptase. Extension is performed with an enzyme (e.g., a polymerase, a reverse transcriptase) that is non-strand displacing.
  • an enzyme e.g., a polymerase, a reverse transcriptase
  • Non-limiting examples of non-strand displacing polymerases are described herein. For example, a Thermococcus gorgonarius mutant reverse transcription xenopolymerase as found in U.S. Provisional Patent Application No. 63/467,541 lacks strand displacement activity.
  • the first probe includes a functional domain (e.g., a primer binding site or a sequencing specific site).
  • the blocking oligonucleotide includes a 3' blocking moiety.
  • Non-limiting examples of 3’ blocking moieties include a carbon spacer (e.g., one, two, three, or more carbon molecules), an inverted nucleotide, dideoxynucleotides, and nucleotides with a substituted 3' OH group.
  • the extended probe and the second probe e.g., a partially double-stranded DNA molecule
  • the second probe includes a capture probe capture domain (e.g., a sequence capable of hybridizing to the capture domain of a capture probe).
  • the first probe includes one or more mismatched nucleotides at its 3' end. In some embodiments, the first probe includes a dideoxynucleotide at its 3' end. In some embodiments, the dideoxynucleotide includes one of ddATP, ddCTP, ddGTP. or ddTTP. Modifications at the 3' end of the first probe as described above can increase specificity of the extension reaction and reduce the likelihood of ligation of first probes (i.e., unextended first probes or first probes that have not undergone nucleic acid extension) to second probes. In some embodiments, the second probe includes a 5' phosphate group. The 5' phosphate group permits ligation to a 3' hydroxyl group (i.e., a 3' hydroxyl group of an extended first probe).
  • the extended probe and the second probe are ligated via chemical ligation. In some embodiments, the extended probe and the second probe are ligated via enzymatic ligation. In some embodiments, enzymatic ligation includes the use of a ligase.
  • the ligase is one or more of a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single-stranded DNA ligase, a T4 DNA ligase, and a combination thereof. In some embodiments, the ligase is a pre-activated T4 DNA ligase.
  • the ligation product is released or removed from the target nucleic acid.
  • the ligation product is removed from the target nucleic acid using an endoribonuclease.
  • the endoribonuclease is RNase H, RNase A, RNase C, and/or RNase I.
  • the endoribonuclease is RNase H.
  • RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA, when hybridized to DNA. RNase H is part of a conserved family of ribonucleases which are present in many different organisms.
  • RNase H There are two primary classes of RNase H: RNase Hl and RNase H2. Retroviral RNase H enzymes are similar to the prokaryotic RNase Hl. All of these enzymes share the characteristic that they are able to cleave the RNA component of an RNA: DNA heteroduplex.
  • the RNase H is RNase Hl , RNase H2, or RNase Hl , or RNase H2.
  • the RNase H includes but is not limited to RNase HII from Pyrococcus furiosus, RNase HII from Pyrococcus horikoshi, RNase HI from Thermococcus litoralis. RNase HI from Thermus thermophilus, RNAse HI from E. coll, or RNase HII from E. coll.
  • the target nucleic acid is released from the ligation product via denaturation.
  • denaturation includes the use of heat.
  • the denaturation includes the use of potassium hydroxide.
  • denaturation includes the use of both heat and potassium hydroxide.
  • the ligation product can hybridize to a capture domain of a capture probe via the capture probe capture domain of the second probe (e.g., incorporated into the ligation product).
  • the capture probe is extended using the hybridized ligation product as a template, thereby generating an extended capture probe (as defined herein).
  • the ligation product is extended (e.g., extended towards the array) using the capture probe as a template, thereby generating an extended ligation product.
  • both the capture probe and the ligation product are extended using each other, respectively, as a template.
  • the extended capture probe and/or the extended ligation product are removed (e.g., cleaved, denatured, etc.) from the array. In some embodiments, the extended capture probe and/or the extended ligation product are amplified, thereby producing amplicons of the extended capture probe and/or the extended ligation product.
  • the determining step includes sequencing. In some embodiments, the sequencing includes high-throughput sequencing. In some embodiments, the determining includes correlating a location of the genetic variant(s) on the array to a location of the genetic variant(s) in the biological sample.
  • the method includes washing the array to remove unhybridized first probes and/or unhybridized blocking probes.
  • the array includes one or more features.
  • features are directly or indirectly attached or fixed to a substrate.
  • the features are not directly or indirectly attached or fixed to a substrate, but instead, for example, are disposed within an enclosed or partially enclosed three-dimensional space (e.g., wells or divots).
  • the plurality 7 of capture probes can be located on features on a substrate.
  • features include, but are not limited to, a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead (e.g., a hydrogel bead).
  • the biological sample can be stained. In some embodiments, the biological sample is stained after fixation. In some embodiments, the biological sample is stained before fixation.
  • the staining includes optical labels as described herein, including, but not limited to, fluorescent (e.g., fluorophore), radioactive (e.g., radioisotope), chemiluminescent (e.g., a chemiluminescent compound), a bioluminescent compound, calorimetric, or colorimetric detectable labels. In some embodiments, the staining includes a fluorescent antibody directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample.
  • a target analyte e.g., cell surface or intracellular proteins
  • the staining includes an immunohistochemistry stain directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample.
  • a target analyte e.g., cell surface or intracellular proteins
  • the staining includes a chemical stain, such as hematoxylin and eosin (H&E) or periodic acid-schiff (PAS).
  • staining the biological sample includes the use of a biological stain including, but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue.
  • significant time e.g., days, months, or years
  • significant time can elapse between staining and/or imaging the biological sample.
  • the biological sample is imaged. In some embodiments, the biological sample is imaged after fixation. In some embodiments, the biological sample is imaged before fixation. In some embodiments, imaging includes one or more of expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy.
  • the biological sample is permeabilized. Permeabilization of a biological sample can occur on a substrate where the substrate is aligned with the array such that at least a portion of the biological sample is aligned with at least a portion of the array or directly on an array including a plurality of capture probes.
  • the biological sample is permeabilized with a protease.
  • the protease is one or more of pepsin, Proteinase K, and collagenase.
  • the biological sample can be applied to any of the variety' of arrays described herein.
  • the plurality of capture probes includes, e.g., in a 5' to a 3' direction, a spatial barcode and a capture domain.
  • the capture domain hybridizes to a capture sequence.
  • the capture domain is a poly(T) capture domain.
  • the capture domain is not a poly(T) sequence.
  • the capture domain is a fixed sequence.
  • a “fixed sequence” is anon-random sequence.
  • the capture domain and the capture probe capture domain of the second probe each can be any sequence as long as the respective sequences are substantially complementary' to one another to facilitate hybridization.
  • a capture probe can include one or more functional domains, and/or a cleavage domain.
  • a functional domain typically includes a functional nucleotide sequence for a subsequent analytical step in the overall analysis procedure.
  • the functional domain can include a sequencing specific site or a primer binding site.
  • the functional domain can include an amplification (e.g., PCR) sequence.
  • a capture probe includes a unique molecular identifier as described herein. In some embodiments, the unique molecular identifier is located 5' to the capture domain in the capture probe. The methods disclosed herein can be performed on any type of biological sample.
  • the biological sample is a fresh tissue sample.
  • the biological sample is a frozen tissue sample. In some embodiments, the biological sample was previously frozen. In some embodiments, the biological sample is a fixed tissue sample. In some embodiments, the fixed tissue sample is a formalin-fixed, paraffin embedded (FFPE) sample.
  • FFPE formalin-fixed, paraffin embedded
  • the biological sample is a tissue section. In some embodiments, the sample is a fresh tissue section. In some embodiments, the biological sample is a frozen tissue section. In some embodiments, the biological sample was previously frozen. In some embodiments, the biological sample is a fixed tissue section. In some embodiments, the fixed tissue section is a formalin-fixed, paraffin embedded (FFPE) section, an acetone-fixed tissue section, a methanol-fixed tissue section, or a paraformaldehyde-fixed tissue section.
  • FFPE formalin-fixed, paraffin embedded
  • Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy.
  • the biological sample can include one or more diseased cells.
  • a diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, cancer, and/or diseases associated with a genetic variant (e.g., any of the genetic variants described herein).
  • the biological sample comprises nucleic acids with one or more genetic variants.
  • the one or more genetic variants is associated with a disease or disease state.
  • the biological sample includes cancer or tumor cells.
  • Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.
  • the biological sample is a heterogeneous sample.
  • the biological sample is a heterogeneous sample that includes tumor or cancer cells and/or stromal cells.
  • FFPE samples generally are heavily cross-linked and fragmented, and therefore this ty pe of sample allows for limited RNA recovery' using conventional detection techniques.
  • methods of targeted RNA capture provided herein are less affected by RNA degradation associated with FFPE fixation than other methods (e.g., methods that take advantage of oligo-dT capture and reverse transcription of mRNA).
  • methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.
  • the FFPE sample or section is deparaffmized, permeabilized, equilibrated, and blocked before target probe oligonucleotides are added.
  • deparaffmization includes using xylenes.
  • deparaffinization includes multiple washes with xylenes.
  • deparaffmization includes multiple washes with xylenes followed by removal of xylenes using multiple rounds of graded alcohol followed by washing the sample with water.
  • the water is deionized water.
  • the target nucleic acids or complements thereof and other proxies of target nucleic acids can be prepared for subsequent applications, such as generation of a sequencing library and next-generation sequencing.
  • Generating sequencing libraries are known in the art.
  • the target nucleic acids, proxies of target nucleic acids and/or their complements thereof can be purified and collected for subsequent amplification steps.
  • the amplification products can be amplified using PCR, where primer binding sites flank the spatial barcode and target nucleic acid, or a complement thereof, generating a library associated with a particular spatial barcode.
  • the library preparation can be quantitated and/or quality controlled to verify the success of the library preparation steps.
  • the library amplicons are sequenced and analyzed to decode spatial information of the target nucleic acid or proxy thereof (e.g., a ligation product).
  • the amplicons can then be enzymatically fragmented and/or size-selected in order to provide for desired amplicon size.
  • sequences can be added to the amplicons thereby allowing for capture of the library preparation on a sequencing flow cell (e.g., on Illumina sequencing instruments).
  • i7 and i5 can index sequences be added as sample indexes if multiple libraries are to be pooled and sequenced together.
  • Read 1 and Read 2 sequences can be added to the library preparation for sequencing purposes.
  • the aforementioned sequences can be added to a library preparation sample, for example, via End Repair, A-tailing, Adaptor Ligation, and/or PCR.
  • the cDNA fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites, although other methods are known in the art.
  • compositions for the spatial detection of genetic variants via branched ligation.
  • compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a first probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the first probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe including a partially double-stranded nucleic acid molecule, where the second probe includes a capture probe capture domain.
  • compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) an extended probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the extended probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe including a partially double-stranded nucleic acid molecule, and where the second probe includes a capture probe capture domain.
  • compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) an extended probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the extended probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe ligated to the extended probe, wherein the second probe includes a partially double-stranded nucleic acid molecule, and where the second probe includes a capture probe capture domain.
  • compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; and (b) a partially double-stranded ligation product including a capture probe capture domain.
  • compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; and (b) a partially double-stranded ligation product hybridized to the capture probe capture domain.
  • the 3' blocking moiety is selected from the group consisting of a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.
  • the target nucleic acid includes at least one genetic variant. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more genetic variants. In some embodiments, at least one genetic variant includes one or more indels. In some embodiments, at least one genetic variant includes one or more single nucleotide variants or polymorphisms.
  • the second probe includes a 5' phosphate group.
  • the extended probe is immediately adjacent to the blocking oligonucleotide. In some embodiments, the extended probe is at least one nucleotide away from the blocking oligonucleotide. In some embodiments, the extended probe is at least 2, at least 3, at least 4. at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least, 16, at least 17, at least 18, at least 19. at least 20 or more nucleotides away from the blocking oligonucleotide.
  • the second probe includes a capture probe capture domain that is capable of hybridizing to the capture probe capture domain on the array.
  • the capture probe capture domain sequence is substantially complementary to a sequence of the capture probe capture domain on the array.
  • the composition includes a polymerase enzyme or a reverse transcriptase enzyme.
  • the reverse transcriptase enzyme is a nondisplacing reverse transcriptase enzyme.
  • the composition includes a biological sample.
  • the biological sample is a tissue sample.
  • the tissue sample is a tissue section.
  • the tissue section is a fixed tissue section.
  • the fixed tissue section is a formalin-fixed, paraffin-embedded tissue section, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone- fixed tissue section.
  • the tissue section is a fresh frozen tissue section.
  • the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.
  • the target nucleic acid is DNA. In some embodiments, the target nucleic acid is mRNA.
  • the first probe includes one or more mismatched nucleotides at its 3' end.
  • the one or more mismatched nucleotides are not complementary to the target nucleic acid to which the first probe hybridizes.
  • the first probe includes a dideoxynucleotide at its 3' end.
  • the dideoxy nucleotide includes one of ddATP, ddCTP, ddGTP, or ddTTP.
  • kits for the spatial detection of genetic variants via branched ligation including: (a) a spatial array including a plurality' of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode, and (ii) a capture domain; (b) (i) a plurality of first probes, where a first probe of the plurality’ of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is dow nstream of a genetic variant, and (ii) a plurality of second probes, where a second probe of the plurality of second probes includes a partially double-stranded nucleic acid molecule and where the second probe includes a capture probe capture domain; (c) a plurality of blocking oligonucleotides, where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes:
  • the capture probes can further include capture probe include one or more functional domains (e.g., a sequencing specific site, a primer binding site, etc.), a unique molecular identifier, a cleavage domain, and any combination thereof.
  • functional domains e.g., a sequencing specific site, a primer binding site, etc.
  • unique molecular identifier e.g., a unique molecular identifier, a cleavage domain, and any combination thereof.
  • the first probe includes a functional domain.
  • the second probe includes a 5' phosphate group.
  • the ligase is one or more of a T4 RNA ligase (Rnl2), a PBCV- 1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.
  • the ligase is a pre-activated T4 DNA ligase.
  • the kit includes a polymerase enzyme. Any suitable polymerase enzyme can be used with the methods described herein, where the polymerase includes non-displacing activity.
  • the kit includes a reverse transcriptase enzyme. In some embodiments, the reverse transcriptase enzyme is a nondisplacing reverse transcriptase enzyme.
  • FIG. 12 shows an exemplary method for spatially detecting genetic variant(s). More specifically, beginning at the top, a first probe and a blocking oligonucleotide can be contacted with a biological sample.
  • the first probe includes a sequence that is substantially complementary to a target nucleic acid in the biological sample and hybridizes to the target nucleic acid at a region that is downstream (i.e. , 3') of the genetic variant(s).
  • the first probe further includes a functional domain (e.g., any of the functional domains described herein).
  • the blocking oligonucleotide is also substantially complementary to the target nucleic acid and hybridizes to the target nucleic acid at a region that is upstream (i.e., 5') of the genetic variant(s).
  • the blocking oligonucleotide includes a 3' blocking moiety. Any suitable blocking moiety can be used.
  • 3' blocking moieties include a carbon spacer (e g., one, two, three, four or more carbon molecules), an inverted nucleotide, one or more dideoxynucleotides, and a nucleotide with a substituted 3' OH group.
  • the first probe can be extended to generate an extended first probe that incorporates complementary sequence(s) of the genetic variant(s) in the target nucleic acid if present.
  • the extended first probe is extended with a polymerase (e.g., a DNA polymerase).
  • the extended first probe is extended with a reverse transcriptase (e.g., any suitable reverse transcriptase).
  • neither the polymerase nor the reverse transcriptase possesses strand displacing activity, i.e., the extended first probe is extended using a non-displacing extension enzyme.
  • the blocking oligonucleotide stops extension of the non-displacing extension enzy me (e.g., the polymerase or the reverse transcriptase).
  • the extended probe is extended completely to the blocking oligonucleotide.
  • the extended probe is extended one or more nucleotides short of the blocking oligonucleotide (i.e., leaving a “gap’ ? ) between the extended first probe and the blocking oligonucleotide. In some examples, this gap can be the result of steric hindrance based on the selected polymerase or reverse transcriptase.
  • a second probe can be contacted with the biological sample where the second probe is a partially double-stranded molecule that includes a capture probe capture domain.
  • a branch-like structure can be formed between the extended first probe, the second probe, and the blocking oligonucleotide.
  • the extended first probe and the second probe can be ligated to one another, thereby generating a ligation product.
  • the second probe comprises a partially double-stranded DNA molecule, which is phosphorylated on the 5' end of the double-stranded DNA molecule.
  • the double-stranded DNA molecule can also include a capture probe capture domain sequence that can hybridize to a capture domain of a capture probe on a spatial array, i.e., the capture probe capture domain sequence is substantially complementary to a sequence of the capture probe capture domain.
  • the phosphorylated, partially double-stranded DNA molecule, or the second probe can be added to the biological sample and ligated to the first probe, for example, using a T4 DNA ligase, etc.
  • this method of branch ligation allows non-complementary ends to be ligated together, such that the extended first probe can be ligated to a double-stranded DNA molecule that includes a capture probe capture domain that, in turn, can be captured by a capture probe on a spatial array.
  • the 3' branch ligation described herein is further described in Wang, L. et al., as discussed above.
  • FIG. 13 is a schematic diagram depicting specificity improvements by incorporating either a mismatched base (top) or a dideoxynucleotide (bottom) to limit unintended extension of the first probe.
  • Reverse transcriptases having exonucleolytic activity can be used to remove the one or more mismatched bases at the 3' end of the first probe.
  • the reverse transcriptase can be used to digest the mismatched base(s) prior to extension (i.e., reverse transcription) of the hybridized first probe.
  • a dideoxynucleotide can be incorporated at the 3' end of the first probe to increase specificity of the extension reaction and reduce non-specific ligation.
  • a modification at the 3' end of the first probe using one or more ddNTPs, a 3' flap, etc. can be removed in a 3 '-5' direction by the reverse transcriptase, while the reverse transcriptase facilitates polymerization in the 5 '-3' direction of the first probe.
  • FIG. 13 also shows unextended probes on the far right with either a mismatched base or a dideoxynucleotide. Ligation cannot occur between the first probe and the second probe if the first probe has not been extended.
  • the target nucleic acid can be released from the ligation product.
  • Any suitable method of releasing the target nucleic acid can be used, including, but not limited to RNase treatment (e.g., any of the RNases described herein) and/or denaturation. Denaturation can be performed with potassium hydroxide and/or heat.
  • the released ligation product can include the capture probe capture domain from the second probe which interacts with (e.g., hybridizes to) a capture domain of a capture probe on a spatial array (e.g., any of the arrays described herein).
  • the captured ligation product and/or the capture probe can be extended using each other, respectively, as a template.
  • the resulting products e.g., extended capture probes, extended ligation products

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Abstract

La présente divulgation concerne des procédés, des compositions et des kits pour la détection spatiale de variants génétiques dans un échantillon biologique à l'aide d'une ligature ramifiée.
PCT/US2024/039737 2023-07-31 2024-07-26 Procédés, compositions et kits de détection spatiale de variants génétiques WO2025029627A1 (fr)

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