WO2024254316A1 - Compositions and methods for identifying blood clots in biological samples - Google Patents

Abstract

Provided herein are methods, systems, and kits to interrogate a biological sample using templated ligation. The methods disclosed herein identify blood clots in biological samples by detecting mismatched templated ligation probe pairs in areas proximal to and/or within blood clots of biological samples compared to areas without blood clots. The methods disclosed herein also include diagnosing blood clots by detecting the mismatched ligation probe pairs in the biological samples.

Description

COMPOSITIONS AND METHODS FOR IDENTIFYING BLOOD CLOTS IN BIOLOGICAL SAMPLES

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial Nos. 63/506.689, filed June 7. 2023, which is incorporated by reference herein.

BACKGROUND

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) can affect, e.g., the cell’s morphology, differentiation, fate, viability, proliferation, behavior, and signaling and crosstalk with other cells in the tissue.

Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provides substantial analyte data for dissociated tissue (e g., single cells), but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).

Blood clots in tissue samples can be indicative of injuries, strokes, or disease, and identification of these clots can provide a deeper understanding of the pathology of a tissue sample. Hematoxylin and eosin (H&E) staining can reveal the location of clots, but smaller clots may be harder to discern and their identification may be dependent on the resolution of a microscopy image of a tissue sample. There remains a need to develop methods to identify blood clots in biological samples independent of pathology and/or tissue staining.

SUMMARY

Provided herein are methods and compositions for detecting blood clots in a biological sample. The present disclosure relies on a surprising observation of an increased amount of chimeric ligation products in areas where blood clots are located in a biological sample. Typically, chimeric ligation products occur at very low levels (e.g., less than 1% of reads in a library) and are thus treated as background noise and are eliminated from analysis. However, as described in the present disclosure, it has been observed that these normally unusable sequencing reads reproducibly occur at measurably higher levels in specific locations of biological samples and co-localize with blood clots.

Thus, compositions, kits and methods of use thereof in identifying the presence of one or more blood clots in biological samples are described here. In some instances, the methods disclosed herein can be used to identify blood clots, diagnose diseases or conditions associated therewith, and inform treatments of such blood clots or diseases or conditions associated therewith.

In one embodiment, disclosed herein is a method of determining presence of a blood clot or pool of blood in a biological sample. In some instances, the method includes: (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary to different nucleic acid analytes in the biological sample or wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100, 150, 200, 250, 300, 350. 400, 450, 500 nucleotides, or more apart; (b) generating a chimeric ligation product by ligating the first probe and the second probe; and (c) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 200 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 300 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary' to a first sequence and a second sequence in the same target nucleic acid that are 400 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 500 nucleotides or more apart.

In some instances, disclosed is a method for determining a location of a chimeric nucleic acid in a biological sample, the method comprising: (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to the chimeric nucleic acid present in the biological sample, and wherein the second probe comprises a capture probe capture domain sequence; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; (d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality of capture probes; and (e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the chimeric nucleic acid in the biological sample.

In some instances, disclosed is a method of determining a location of a blood clot or pool of blood in a biological sample, the method comprising: (a) contacting a plurality⁷ of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample, and wherein the second chimeric probe comprises a capture probe capture domain sequence; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; (d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality of capture probes; and (e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the blood clot or pool of blood in the biological sample.

In some instances, disclosed is a method of determining presence of a blood clot or pool of blood in a biological sample, the method comprising: (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; and (d) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

Also disclosed herein is a method for determining a location of a chimeric nucleic acid in a biological sample, the method comprising: (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to the chimeric nucleic acid present in the biological sample, and wherein the second probe comprises a capture probe capture domain sequence; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; (d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array of capture probes; and (e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the chimeric nucleic acid in the biological sample.

In some instances, disclosed is a method of determining a location of a blood clot in a biological sample, the method comprising: (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample, and wherein the second chimeric probe comprises a capture probe capture domain sequence; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; (d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array of capture probes; and (e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the blood clot in the biological sample. In some instances, disclosed is a method of determining presence of a blood clot in a biological sample, the method comprising: (a) contacting a plurality’ of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; and (d) determining the presence of the chimeric ligation product, or a complement thereof, optionally wherein a location of the ligation product corresponds to a location of the blood clot in the biological sample.

In some instances, disclosed is a method of determining presence of a blood clot in a biological sample, the method comprising: (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary- to different nucleic acid analytes in the biological sample; (b) generating a chimeric ligation product by ligating the first probe and the second probe; and (c) determining the presence of the chimeric ligation product, or a complement thereof, optionally wherein a location of the ligation product corresponds to a location of the blood clot in the biological sample.

In some instances, the methods also include mounting the biological sample on a first substrate. In some instances, the methods also include amplifying the chimeric ligation product. In some instances, the determining step comprises hybridizing a detectable probe to the chimeric ligation product; and detecting the presence of the detectable probe. In some instances, the detectable probe comprises a fluorescent label or a chromogenic label. In some instances, the chimeric ligation products in a first region of the biological sample are greater than a second region of the biological sample; or in a test sample compared to a reference sample.

In some instances, the chimeric ligation product comprises a probe pair selected from the group consisting of: PRDM4-GTF2A1; SLC30A5-VPS33B; IZUM01R-DD0; OR7D2- UQCRH; ZNF841-IL18RAP; MYB-NBPF3; SLC25A51-SKA1; OR6C2-SMIM29; IGKC- IGKC; FAM72B-NSL1; CAMKMT-RIDA; 0LFML1-ZFYVE16; and any combination thereof.

In some instances, the contacting the plurality of probes with the biological sample comprises contacting the biological sample with 5000 or more probe pairs, wherein a probe pair of the 5000 or more probe pairs comprises the first probe and the second probe. In some instances, the contacting the plurality of probes with the biological sample comprises contacting the biological sample with 100 or more probe pairs, wherein a probe pair of the 100 or more probe pairs comprises the first probe and the second probe. In some instances, the capture probe capture domain sequence is substantially complementary to a capture domain of the capture probe. In some instances, the first probe further comprises a primer sequence. In some instances, the capture probe capture domain sequence comprises a polyadenylated sequence or a complement thereof.

In some instances, the contacting the plurality of probes with the biological sample comprises contacting the biological sample with a single pair of probes that is capable of forming chimeric ligation products in the presence of blood clots or pools of blood in the biological sample. In some instances, the contacting the plurality of probes with the biological sample comprises contacting the biological sample with two or more pairs, (e.g., two pairs, three pairs, four pairs, five pairs, six pairs, seven pairs, eight pairs, nine pairs, ten or more pairs), of probes that are capable of forming chimeric ligation products in the presence of blood clots or pools of blood in the biological sample. In some instances, the single pair or two or more pairs of probes are selected from the group consisting of: PRDM4- GTF2A1; SLC30A5-VPS33B: IZUMO1R-DDO; OR7D2-UQCRH; ZNF841-IL18RAP; MYB-NBPF3; SLC25A51-SKA1; OR6C2-SMIM29; IGKC-IGKC; FAM72B-NSL1; CAMKMT-RIDA; OLFML1-ZFYVE16; and any combination thereof.

In some instances, the first probe and/or the second probe is a DNA probe. In some instances, the first probe and the second probe hybridize to adjacent sequences on the chimeric nucleic acid. In some instances, the adjacent sequences abut one another. In some instances, the adjacent sequences are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from one another.

In some instances, the methods also include generating an extended first probe, wherein the extended first probe comprises a sequence substantially complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second chimeric probe. In some instances, the methods further include generating an extended second probe using a polymerase, wherein the extended second probe comprises a sequence substantially complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe.

In some instances, the array of capture probes comprising the plurality is on a first substrate.

In some instances, the array of capture probes comprising the plurality is on a second substrate. In some instances, 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 array of capture probes. In some instances, the biological sample is aligned with at least a portion of the array of capture probes, releasing the chimeric ligation product from the biological sample.

In some instances, the methods also include contacting the biological sample with a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents comprises an analyte binding moiety and a capture agent barcode domain, wherein the analyte binding moiety specifically binds to a protein from the biological sample, and wherein the capture agent barcode domain comprises an analyte binding moiety barcode and a capture handle sequence; and hybridizing the capture handle sequence to a second capture domain of a second capture probe on the array of capture probes, wherein the second capture probe further comprises a second spatial barcode. In some instances, the methods include determining the sequence of (i) the capture agent barcode domain; and (ii) the second spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the location of the protein in the biological sample. In some instances, the generating the chimeric ligation product comprises ligating the first probe to the second probe using enzymatic ligation or chemical ligation, wherein the enzymatic ligation utilizes a ligase.

In some instances, the generating the chimeric ligation product comprises ligating the extended first probe to the second probe using enzymatic ligation, wherein the enzymatic ligation utilizes a ligase. In some instances, the ligase is one or more of a T4 RNA ligase (Rnl2), a Chlorella virus ligase, a single-stranded DNA ligase, or a T4 DNA ligase. In some instances, the generating the chimeric ligation product comprises ligating the extended first probe to the second probe using chemical ligation. In some instances, the methods also include releasing the chimeric ligation product. In some instances, the releasing the chimeric ligation product comprises contacting the biological sample with a reagent medium comprising an agent for releasing the chimeric ligation product. In some instances, the agent for releasing the chimeric ligation product comprises a nuclease. In some instances, the nuclease comprises an RNase, optionally wherein the RNase is selected from RNase A, RNase C, RNase H, or RNase I. In some instances, the reagent medium further comprises a permeabilization agent, optionally wherein the permeabilization agent comprises a protease. In some instances, the protease is selected from try psin, pepsin, elastase, or proteinase K. In some instances, the reagent medium further comprises a detergent. In some instances, the reagent medium further comprises polyethylene glycol (PEG). In some instances, the capture probe comprises a poly(T) sequence (e.g., at the capture domain sequence). In some instances, the capture probe comprises a sequence (e.g., at the capture domain sequence) substantially complementary to a sequence in the first probe or the second probe. In some instances, the capture probe further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, and combinations thereof.

In some instances, the chimeric nucleic acid is DNA. In some instances, the DNA is genomic DNA. In some instances, the chimeric nucleic acid is RNA. In some instances, the RNA is mRNA.

In some instances, the biological sample is a tissue sample. In some instances, the tissue sample is a solid tissue sample. In some instances, the solid tissue sample is a tissue section. In some instances, the biological sample is a fixed tissue sample. In some instances, the fixed tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample. In some instances, the FFPE tissue sample is an FFPE tissue section. In some instances, the FFPE tissue sample is deparaffinized and decrosslinked prior to step (a). In some instances, the biological sample is a fresh frozen tissue sample. In some instances, the biological sample is a fresh frozen tissue section. In some instances, the biological sample is fixed and stained prior to step (a). In some instances, the biological sample is stained using immunofluorescence, immunohistochemistry, or hematoxylin and/or eosin.

In some instances, the methods also include imaging the biological sample. In some instances, the biological sample is obtained from a subject having deep vein thrombosis, a pulmonary embolism, or an arterial thrombosis. In some instances, the biological sample is obtained from a subject having antiphospholipid antibody syndrome, Factor V Leiden, a prothrombin gene mutation, a Protein C deficiency, a Protein S deficiency, hemophilia, or an ATIII deficiency. In some instances, the biological sample is derived from skin, liver, brain, lungs, heart, kidney, spleen, pancreas, and/or tonsils of a subject.

In some instances, the subject is selected from a mouse, a rat, a rabbit, a guinea pig, an ungulate, a horse, a sheep, a pig, a goat, a cow. a cat. or a dog. In some instances, the subject is a human.

In some instances, the method also includes extending the chimeric ligation product using the capture probe as a template, thereby generating an extended chimeric ligation product. In some instances, the method also includes extending the capture probe using the chimeric ligation product as a template, thereby generating an extended capture probe. In some instances, determining the sequence of (i) and (ii) comprises determining the sequence of the extended chimeric ligation product, or a complement thereof, or the extended capture probe, or the complement thereof. In some instances, the determining comprises sequencing the extended chimeric ligation product, or a complement thereof, or the extended capture probe, or the complement thereof.

Also disclosed herein are methods of diagnosing a subject with a blood clot. In some instances, the method include performing any one of the methods described herein, and diagnosing the subject as having a blood clot. In some instances, subject has had a ministroke.

Also disclosed herein are methods of diagnosing a subject with a one or more of anti phospholipid antibody syndrome, Factor V Leiden, a prothrombin gene mutation, a Protein C deficiency, a Protein S deficiency, hemophilia, or an ATIII deficiency. In some instances, the method includes performing any one of the methods described herein, and diagnosing the subject as having one or more of antiphospholipid antibody syndrome, Factor V Leiden, a prothrombin gene mutation, a Protein C deficiency, a Protein S deficiency, hemophilia, or an ATIII deficiency. In some instances, subject has had a mini-stroke.

In some instances, disclosed herein are methods of identifying a subject as having an increased likelihood of having a blood clot. In some instances, the methods include performing any one of the methods described herein, and identifying a subject as having an increased likelihood of having the blood clot.

In some instances, the methods further include administering to the subject one or more anticoagulant medications. In some instances, the one or more anticoagulant medication is selected from aspirin, warfarin, heparin, low-molecular weight heparin, fondaparinux, rivaroxaban, apixaban. dabigatran, or any combination thereof. In some instances, the blood clot is removed using minimally invasive interventional radiology.

Also provided herein is a system comprising: (a) a support device configured to retain a first substrate and a second substrate, wherein a biological sample is placed on the first substrate, and wherein the second substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (b) a delivery means to deliver a first probe and a second probe to the biological sample, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to adjacent sequences of an analyte, wherein the second probe comprises a capture probe binding domain, and wherein the first probe and the second probe are capable of being ligated together to form a chimeric ligation product; and (c) a location within the first substrate or the second substrate comprising a reagent medium comprising a permeabilization agent and optionally an agent for releasing the chimeric ligation product.

In some instances, the permeabilization agent is pepsin or proteinase K. In some instances, the agent for releasing the chimeric ligation product is an RNAse, optionally wherein the RNAse is selected from RNase A, RNase C, RNase H, or RNase I. In some instances, the system further includes an alignment mechanism on the support device to align the first substrate and the second substrate. In some instances, the alignment mechanism comprises a linear actuator, wherein the first substrate comprises a first member and the second substrate comprises a second member, and optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to a plane or the first member and/or the second member, and/or 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, and/or 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, and/or 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.

In some instances, the system includes chimeric ligation products. In some instances, the chimeric ligation product comprises a probe pair selected from the group consisting of: PRDM4-GTF2A1; SLC30A5-VPS33B; IZUMO1R-DDO; OR7D2-UQCRH: ZNF841- IL18RAP; MYB-NBPF3; SLC25A51 -SKA1 ; OR6C2-SMIM29; IGKC-IGKC; FAM72B- NSL1; CAMKMT-RIDA; OLFML1-ZFYVE16; and any combination thereof.

Finally, also provided herein is a kit comprising: (a) a support device configured to retain a first substrate and a second substrate, wherein a biological sample is placed on the first substrate, and wherein the second substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (b) a delivery means to deliver a first probe and a second probe to the biological sample, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to adjacent sequences of an analyte, wherein the second probe comprises a capture probe binding domain, and wherein the first probe and the second probe are capable of being ligated together to form a chimeric ligation product; (c) a location within the first substrate or the second substrate comprising a reagent medium comprising a permeabilization agent and optionally an agent for releasing the chimeric ligation product; and (d) instructions for performing the any one of the methods disclosed herein. In some instances, the permeabilization agent is pepsin or proteinase K. In some instances, the agent for releasing the chimeric ligation product is an RNAse, optionally wherein the RNAse is selected from RNase A, RNase C, RNase H, or RNase I. In some instances, the kit also includes an alignment mechanism on the support device to align the first substrate and the second substrate. In some instances, the alignment mechanism comprises a linear actuator, wherein the first substrate comprises a first member and the second substrate compnses a second member, and optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to a plane or the first member and/or the second member, and/or 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, and/or 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, and/or 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.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

The term “about” or “approximately” as used herein means within an acceptable error range for the particular value as determined by one of ordinary⁷ skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about"’ is implicit and in this context means within an acceptable error range for the particular value.

The term “substantially complementary” used herein means that a first sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-40, 40-60, 60-100, or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions. Substantially complementary also means that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations known to those skilled in the art.

The term “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 every⁷ item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

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.

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 and exemplary workflow for performing templated capture and producing a ligation product, and 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. 12A shows a hematoxylin and eosin (H&E) stain image of a human spleen sample. Arrow indicates location of a blood clot.

FIGs. 12B and 12C are heatmaps showing counts of unique molecular identifiers (UMIs) of chimeric/mismatched ligation products for the human spleen sample of FIG. 12A, wherein the sample was contacted with templated ligation probe pairs. Each image shows a serial sample from the human spleen sample.

FIGs. 12D and 12E are heatmaps showing UMI counts of chimeric/mismatched ligation products for the human spleen sample of FIG. 12A, wherein the sample was contacted with templated ligation probe pairs and oligonucleotide-tagged antibodies.

FIG. 12F shows detection of blood clots in three areas (labeled as Al, A2, and A3) in the H&E stained image of the human spleen sample of FIG. 12A (right), with a corresponding heatmap showing UMI counts of chimeric/mismatched ligation products at Al and A3 (left). FIG. 13A shows an H&E stained image of a human tonsil sample. Arrow and outline indicate location of a blood clot in the sample.

FIG. 13B shows a heatmap of UMIs mapped to chimeric/mismatched ligation products in the human spleen sample of FIG. 13 A.

FIG. 14A shows an H&E stained image of a human kidney sample. Representative arrows indicate exemplary locations of a blood clot.

FIGs. 14B-14G show heatmaps of UMI counts mapped to chimeric/mismatched ligation products to serial tissue sections of the human kidney sample of FIG. 14A.

DETAILED DESCRIPTION

A. Spatial Analysis Methods

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. 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. For example, 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.

Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos. 11,447,807, 1 1,352,667, 11,168,350, 11,104,936, 11,008,608, 10,995,361, 10,913,975, 10,774,374, 10,724,078, 10,640,816, 10,494,662, 10,480,022, 10,364.457, 10,317,321, 10,059,990, 10,041,949, 10,030,261, 10.002,316, 9,879,313, 9,783,841, 9.727,810, 9,593.365, 8,951,726, 8,604,182. and 7,709,198; U.S. Patent Application Publication Nos. 2020/0239946, 2020/0080136, 2020/0277663, 2019/0330617, 2020/0256867, 2020/0224244, 2019/0085383, and 2013/0171621; PCT Publication Nos. WO2018/091676, W02020/176788, WO2017/144338, and WO2016/057552; Non-patent literature references Rodriques et al., Science 363(6434): 1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458. 2015; Trejo et al., PLoS ONE 14(2) :e0212031, 2019; Chen et al.. Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36: 1197-1202. 2018; and the Visium Spatial Gene Expression Reagent Kits User Guide (e.g.. Rev F, dated January 2022) and/or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), both of which are available at the 1 Ox Genomics Support Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in its entirety. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.

Some general terminology that may be used in this disclosure can be found in Section (I)(b) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. Typically, 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. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “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. Examples of 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

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. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample (e.g.. tissue sample) is a tissue microarray (TMA). 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 may 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. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a solid tissue sample. In some embodiments, the biological sample is a tissue section (e.g., a fixed tissue section). In some embodiments, 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. In some embodiments, the biological sample, e.g., the tissue, is flash-frozen using liquid nitrogen before sectioning. In some embodiments, the biological sample, e.g., a tissue sample, is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane.

In some embodiments, the biological sample, e.g., the tissue, is embedded in 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. In some embodiments, the sectioning is performed by cryosectioning, for example using a microtome. In some embodiments, 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 some instances, the biological sample is from a human. In addition to the subjects described above, the biological sample can be obtained from non-mammalian organisms (e.g.. a plant, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans a fungus, an amphibian, or a fish (e.g., zebrafish)). Examples of subjects include, but are not limited to, a mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate (i.e. human or non-human primate); a plant such as Arabidopsis thaliana, com, sorghum, oat, wheat, rice, canola, or soybean; an algae such as Chlamydomonas reinhardtiv, a nematode such as Caenorhabditis elegans,' an insect such as Drosophila melanogaster , mosquito, fruit fly, or honey bee; an arachnid such as a spider; a fish such as zebrafish; a reptile; an amphibian such as a frog or Xenopus laevis,' a Dictyostelium discoideum, a fungi such as Pneumocystis carinii. Takifugu rubripes. yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombc. or a Plasmodium falciparum .

A biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli. Staphylococci or Mycoplasma pneumoniae,' an archaeon; a virus such as Hepatitis C virus or human immunodeficiency vims; or a viroid. 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. In some embodiments, 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.

Biological 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.

In some embodiments, the biological sample, e.g.. the tissue sample, is fixed in a fixative including alcohol, for example, methanol. In some embodiments, instead of methanol, acetone or an acetone-methanol mixture can be used. In some embodiments, the fixation is performed after sectioning. In some instances, when the biological sample is fixed using a fixative including an alcohol (e.g., methanol or acetone-methanol mixture), the biological sample is not decrosslinked afterward. In some preferred embodiments, the biological sample is fixed using a fixative including an alcohol (e.g., methanol or an acetone- methanol mixture) after freezing and/or sectioning. In some instances, 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”. In some embodiments, 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).

In some embodiments, the biological sample, e.g.. the tissue sample, is fixed e.g.. immediately after being harvested from a subject. In such embodiments, the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PF A) or formalin. In some embodiments, the fixative induces crosslinks within the biological sample. In some embodiments, after fixing, e.g., by formalin or PFA, the biological sample is dehydrated via sucrose gradient. In some instances, the fixed biological sample is treated with a sucrose gradient and then embedded in a matrix, e.g., OCT compound. In some instances, the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix, e.g., OCT compound after fixation. In some embodiments when a fixed frozen tissue sample is treated with a sucrose gradient, the sample can be rehydrated using an ethanol gradient. In some embodiments, 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. In such instances, the biological sample is referred to as “fixed frozen”. In preferred embodiments, a fixed frozen biological sample is not treated with methanol. In preferred embodiments, a fixed frozen biological sample is not paraffin embedded. Thus, in preferred embodiments, a fixed frozen biological sample is not deparaffinized. In some embodiments, a fixed frozen biological sample is rehydrated using an ethanol gradient.

In some instances, the biological sample (e.g.. a fixed frozen tissue sample) is treated with a citrate buffer. Citrate buffer can be used to decrosslink antigens and fixation medium for antigen retrieval in the biological sample. Thus, any suitable decrosslinking agent can be used in addition, or alternatively, to citrate buffer. In some embodiments, for example, the biological sample (e.g., a fixed frozen tissue sample) is decrosslinked using TE buffer.

In any of the foregoing, the biological sample can further be stained, imaged, and/or destained. For example, in some embodiments, 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. In some embodiments, when a fresh frozen tissue sample is fixed in methanol, 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. In some embodiments 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. In some embodiments, the biological sample can undergo further fixation (e.g., while mounted on a substrate), stained, imaged, and/or destained. For example, 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.

In any of the foregoing, the biological sample can be fixed using PAXgene. For example, 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 morphology⁷ and biomolecules. 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. et al., J Proteome Res. 2010 Oct 1 ;9(10):5188-96; Kap M. et al., PLoS One.; 6(1 l):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol.; 146(I):25-40 (2016), each of which is hereby incorporated by reference in its entirety, for a description and evaluation of PAXgene for tissue fixation. Thus, in some embodiments, when the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, the fixative is PAXgene. In some embodiments, a fresh frozen tissue sample is fixed with PAXgene. In some embodiments, a fixed frozen tissue sample is fixed with PAXgene.

In some embodiments, 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). In some embodiments, the biological sample comprises intact cells. In some embodiments, 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. A limitation of direct RNA capture for fixed samples is that the RNA integrity of fixed (e.g., FFPE) samples can be lower than of 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. By utilizing RTL probes that hybridize to RNA target sequences in the transcriptome. 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, 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. In some embodiments, the imaging occurs prior to destaining the sample. In some embodiments, the biological sample is stained using an H&E staining method. In some embodiments, 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. In some instances, the sample is a mouse sample. In some instances, the sample is a human sample. In some embodiments, the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. In some instances, the sample is a human or mouse breast tissue sample. In some instances, the sample is a human or mouse brain tissue sample. In some instances, the sample is a human or mouse lung tissue sample. In some instances, the sample is a human or mouse tonsil tissue sample. In some instances, 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 embryo sample can be a non-human embryo sample. In some instances, the sample is a mouse embryo sample.

Biological samples are also described in Section (I)(d) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. The following embodiments can be used with any of the methods described herein. In some embodiments, the biological sample (e.g., a fixed and/or stained biological sample) is imaged. In some embodiments, the biological sample is visualized or imaged using bright field microscopy. In some embodiments, 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. In some embodiments, the sample is stained and imaged prior to adding reagents for analyzing captured analytes, as disclosed herein, to the biological sample.

In some embodiments, the methods include staining the biological sample. In some embodiments, the staining includes the use of hematoxylin and/or eosin. Non-limiting examples of stains include histological stains (e g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, 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. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation.

In some embodiments, 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.

In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(l 3) or the Exemplary' Embodiments Section of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. Briefly, any of the methods described herein includes permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of extension products to the capture probes on the array. In some embodiments, the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, or methanol), a detergent (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), an enzyme (e.g., an endopeptidase, an exopeptidase, or a protease), or a combination thereof. In some embodiments, the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X- 100™, Tween-20™, or a combination thereof. In some embodiments, the endopeptidase is pepsin. In some embodiments, 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. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI) and a capture domain). In some instances, the capture probe includes a homopolymer sequence, such as a poly(T) sequence. In some embodiments, 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)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. which is herein incorporated by reference. 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.

In some instances, a capture probe and a nucleic acid analyte interaction (or any other nucleic acid to nucleic acid interaction) occurs because the sequences of the two nucleic acids are substantially complementary to one another. By “substantial,” “substantially,” and the like, 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. In some embodiments, 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. In some embodiments, at least 70%, 80%, 90%, 95%, or 99% of the residues of one nucleic acid sequence are complementary⁷ 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 complementary⁷ to residues of the other nucleic acid sequence. In some embodiments, the biological sample is mounted on a first substrate and the substrate comprising the array of capture probes is a second substrate. In this configuration, one or more analytes or analyte derivatives (e.g., intermediate agents; e.g.. ligation products) are then released from the biological sample and migrate to the second substrate comprising an array of capture probes. In some embodiments, 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 2022/061 152 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. As shown in FIG. 1A, a liquid reagent drop (e.g., permeabilization solution 105) is introduced on the second substrate in proximity' to the capture probes 106 and in between the biological sample 102 and the second substrate (e.g., slide 104 including an array having spatially barcoded capture probes 106). 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.

During the exemplary' sandwiching process, 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). As shown, the second substrate (e.g., array slide 104) is in an inferior position to the first substrate (e.g., slide 103). In some embodiments, the first substrate (e.g., slide 103) may be positioned superior to 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 wherein the biological sample 102 has been pre-permeabihzed. the reagent medium is not a permeabilization solution. Herein, 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. Alternatively, in certain embodiments, 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). Exemplary methods of electrophoretic migration are descnbed in WO 2020/176788 and U.S. Patent Application Pub. No. 2021/0189475, each of which is hereby incorporated by reference in its entirety.

As further shown, 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 show n as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.

In some embodiments, the one or more spacers 110 is configured to maintain a separation distance betw een first and second substrates that is betw een about 2 microns (pm) and about 1 mm (e.g., betw een about 2 pm and about 800 pm, betw een about 2 pm and about 700 pm, between about 2 pm and about 600 pm, between about 2 pm and about 500 pm, between about 2 pm and about 400 pm. between about 2 pm and about 300 pm, between about 2 pm and about 200 pm, betw een about 2 pm and about 100 pm, between about 2 pm and about 25 pm, or between about 2 pm and about 10 pm), measured in a direction orthogonal to the surface of first substrate that supports the biological sample. In some instances, 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 pm. In some embodiments, the separation distance is less than 50 pm. In some embodiments, the separation distance is less than 25 pm. In some embodiments, the separation distance is less than 20 pm. 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. In the example of FIG. IB, 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). In some aspects, 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. A partially or fully sealed chamber 150 resulting from the one or more spacers 110. the first substrate (e.g.. slide 103), and the second substrate (e.g., slide 104) may reduce or prevent 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. For example, 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., U.S. Patent Application Pub. No. 2021/0189475 and PCT Publ. No. WO 2022/061152 A2, each of which is incorporated by reference in its entirety.

In some embodiments of a sample holder, 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.

In some embodiments, the adjustment mechanism includes a linear actuator. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. As shown, 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. As shown, the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206. In the example of FIG. 2B, 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.

In some aspects, when the sample handling apparatus 200 is in an open position (e.g., in FIG. 2B), 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. As noted, the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration.

In some aspects, after the first member 204 closes over the second member 210, 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 the permeabilization step (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.

In some embodiments, 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). After or before alignment, 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. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 206 and/or the second substrate 212. 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) may be captured by the capture probes of the array and may be processed for spatial analysis.

In some embodiments, during the permeabilization step, 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.

Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. 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). As shown, reagent medium (e.g., permeabilization solution) 305 is located on the spacer 310 toward the right-hand side of the side view' in FIG. 3A. While 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). For example, in the side view of FIG. 3B the reagent medium 305 may be urged from right to left as the sandwich is formed.

In some embodiments, 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. As shown in the top view of FIG. 3C, 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

While 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. it should be understood that an exemplary angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate comprising the spacer 310.

It may be desirable that the reagent medium be free from air bubbles betw een the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present betw een 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 betw een the two substrates (e.g., slide 303 and slide 304) during a permeabilization step (e.g., step 104). In some aspects, it may be possible to reduce or eliminate bubble formation between the substrates 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 tw o 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. As shown at step 405, reagent medium 401 is positioned to the side of the substrate 402.

At step 410, 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.

At step 415, the substrate 406 is further lowered toward 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 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 between the substrates.

At step 420, the reagent medium 401 fills the gap betw een 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.

In some embodiments, the reagent medium (e.g., 105 in FIG. 1A) comprises a permeabilization agent. In some embodiments, following initial contact betw een the biological sample and 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- 100™, Tween-20™, SDS), and enzymes (e.g., trypsin or other proteases (e.g.. proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution).

In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and SDS. More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. In some embodiments, the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, elastase, and proteinase K. In some embodiments, the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises an RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, and RNase I. In some embodiments, the reagent medium comprises one or more of SDS or a sodium salt thereof, proteinase K, pepsin, N- lauroylsarcosine. and RNase.

In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG molecular weight is from about 2K to about 16K. In some embodiments, 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. In some embodiments, 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).

In certain embodiments, 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. For example, a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.

In some instances, 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.

In some instances, 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.

There are at least two methods to associate 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 cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.

In some cases, 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). In some cases, 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 serve as proxies for the template.

As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to a terminus (e.g.. a 3^? or 5’ end) of the capture probe, thereby extending the overall length of the capture probe. For example, 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). In some embodiments, 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 domain of the capture probe. In some embodiments, the capture probe is extended using a reverse transcriptase. In some embodiments, the capture probe is extended using one or more DNA polymerases. In some embodiments, 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.

In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes using the captured analyte as a template, sequencing (e g., of a cleaved extended capture probe and/or a cDNA molecule complementary' to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference. Some quality control measures are described in Section (II)(h) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference.

Spatial information can provide information of medical importance. For example, 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., diagnosis) of 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 in its entirety.

Spatial information can provide information of biological importance. For example, 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).

For spatial array-based methods, a substrate may function as a support for direct or indirect attachment of capture probes to features of the array. A '’feature’’ is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, 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. which is herein incorporated by reference. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(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.

Generally, analytes and/or intermediate agents (or portions thereof) 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 or wells) comprising capture probes). As used herein, “contact,’’ “contacted,” and/or “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. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, which is herein incorporated by reference.

In some embodiments, a substrate including an array includes a plurality of beads. For example, a substrate can include a monolayer of beads where each bead occupies a unique position on the substrate. In some instances, the beads can be immobilized on the substrate and can each contain a plurality of capture probes. In some instances, the capture probes on a particular bead have the same barcode, which is unique, and thus differs from the barcodes of capture probes on other beads. Thus, the barcode contained by the capture probes on each bead can serve as a spatial barcode that is associated with a distinct position on the substrate.

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 combinations 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 cell. 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’ multiplexed spatially-barcoded feature. In FIG. 7, 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. For example, a feature may include four different types of spatially-barcoded capture probes, each type 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 ty pe 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. 7. 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. For example, 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/or 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 described herein), and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). In some embodiments, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature) change, or any other known perturbation agents.

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. In some embodiments, 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. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.

In some embodiments, the spatial barcode 505 and functional sequence 504 are common to all of the probes attached to a given feature. In some embodiments, 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.).

In some embodiments, more than one analyte type (e.g.. nucleic acids and proteins) from 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. In some cases, 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). In some embodiments, a plurality of molecules (e.g., a plurality⁷ of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, 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.

In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). 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 in its entirety. Typically. RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, 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. In some instances, one of the tw o oligonucleotides includes a capture probe binding domain (e.g., a poly(A) sequence or anon-homopolymeric sequence). After hybridization to the analyte, 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) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap betw een the hybridized oligonucleotides. In some instances, a polymerase (e.g.. a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e g., RNase H). In some instances, the ligation product is removed using heat. In some instances, the ligation product is removed using 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.

In some instances, one or both of the oligonucleotides may hybridize to genomic DNA (gDNA), which can lead to false positive sequencing data from ligation events on gDNA (off target) in addition to the desired (on target) ligation events on target nucleic acids (e.g., mRNA). Thus, in some embodiments, the disclosed methods can include contacting the biological sample with a deoxyribonuclease (DNase). The DNase can be an endonuclease or exonuclease. In some embodiments, 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.

A non-limiting example of templated ligation methods disclosed herein is depicted in FIG. 9A. After 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 901 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 is able to hybridize to a capture probe on the substrate. Methods and compositions for spatial detection using templated ligation have been described in PCT Publication. No. WO 2021/133849 Al, U.S. Pat. Nos. 11,332.790 and 11,505,828, each of which is incorporated by reference in its entirety.

In some embodiments, as shown in FIG. 9B, 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). In some embodiments, 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). In some embodiments, 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. In some embodiments, 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). In some embodiments, polymerization (e.g., reverse transcription (RT)) reagents can be added to permeabilized biological samples. Incubation with the polymerization 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 (e.g., ligation products). The ligation products can be extended using the capture probe as a template to include a complement of the capture probe, thereby generating extended ligation products.

In some embodiments, 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, i5 9017, i7 9018, and P7 9019 sequences 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.

In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, 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. In some embodiments, 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. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherw ise interact with a capture domain of a capture probe. In some cases, 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. W02020/176788 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 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 functional sequence 1118, analyte binding moiety barcode 1116. and an analyte capture sequence 1114 that is capable of binding (e.g., hybridizing) to the capture domain 1112 of the capture probe 1124. The analyte capture agent 1126 can also include a linker 1120 that allows the analyte binding 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. In some embodiments, the linker 1120 is a cleavable linker. In some embodiments, the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, chemical-cleavable, thermal-cleavable, or an enzy me cleavable linker. In some instances, 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).

During analysis of spatial information, 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. In some embodiments, specific capture probes and the captured analytes are associated with specific locations in an array of features on a substrate. For example, 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. Alternatively, 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 each spatial barcode is uniquely associated with a single feature of the array. Where necessary, 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.

When sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location or a fiducial marker) of 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 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. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022) and/or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), each of which is herein incorporated by reference in its entirety.

In some embodiments, 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. 2020/0277663. or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for . Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of PCT Publication No. W02020/123320, which is herein incorporated by reference.

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 steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps 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.

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 method steps or functions described herein.

In some cases, 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. W02021/102003 and/or U.S. Patent Application Publication No. 2021/0150707, each of which is incorporated herein by reference in its entirety.

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.

In some cases, 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 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, 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.

B. Methods and Compositions for Identifying Chimeric Ligation Products

(a) Introduction

The presence of blood clots in tissue samples can be indicative of injuries, strokes, or disease. Efficient and accurate identification of these blood clots can therefore provide improved pathohistological analysis of a biological (e.g., tissue) sample. In some cases, H&E staining can reveal the location of blood clots, but smaller clots may be harder to discern and their identification may be dependent on the resolution of an image of the tissue sample.

As described in the working Example below, it has been discovered that analysis of gene expression using a templated ligation probe-based assay provides an additional mechanism of blood clot identification. Typically, the templated ligation probe-based assay uses whole transcriptome probe sets (containing thousands of probe pairs collectively targeting a majority of the transcriptome) to spatially resolve RNA expression levels across biological samples. The templated ligation probes come in pairs that are designed to both specifically hybridize to a portion of a target gene, followed by their coupling (e.g., ligation). Chimeras occur when probes mismatch, e.g., when two unpaired probes are ligated together. Typically, chimeras occur at low levels (e.g., less than 1% of all sequencing reads in a nucleic acid sequencing 1 i brary) and are considered a non-specific error. However, the present disclosure is based, in part, on the observation that high levels of chimeras occur near or within blood clots across multiple tissue sample replicates. It has been further observed the same mismatched probe pairs occur at high levels across replicates of the same tissue sample type, with some occurring at high levels in multiple tissue types. In some embodiments, the targets of the probes in these mismatched probe pairs (chimeras) are associated with aspects of blood flow or clotting (e.g., platelet recruitment, B cell expression).

Provided herein are methods, devices, compositions, and systems for analyzing the presence, location and/or abundance of a chimeric ligation product in a biological sample. As disclosed herein, there is a marked association between an increased amount of chimeric ligation products and the presence of blood clots in a biological sample. In addition, in some instances, the method disclosed herein can be used to diagnose blood clots in a biological sample.

As used herein, a “chimeric ligation product” or “mismatched ligation product” refers to a nucleic acid molecule that includes two probes (e g., nucleic acid probes) coupled together (e.g., by ligation) in which the probes are designed to hybridize to different target nucleic acid analytes or in which the two probes are not members of a designed probe pair (e g., the two probes are not designed to hybridize within proximity' of each other on a target nucleic acid). For example, the two probes can each contain nucleic acid sequences that are substantially complementary to sequences from different target nucleic acid analytes. For example, a PDRM4-GTF2A1 chimeric ligation product can include a first probe designed to target (e.g., hybridize to) a first analyte (e.g., PRDM4) and a second probe designed to target (e.g., hybridize to) a second analyte (e.g., GTF2A1) in a biological sample. In some embodiments, the two probes can each contain nucleic acid sequences that are substantially complementary’ to a first sequence and a second sequence that are far apart (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500 nucleotides, or more) on the same target nucleic acid analyte. In some biological samples (as will be relevant herein), the two probes hybridize to adjacent or near-adjacent sequences of a nucleic acid analyte in the biological sample.

In some instances, the first probe and the second probe hybridize to a chimeric nucleic acid in the biological sample. A “chimeric nucleic acid” is a nucleic acid (e.g., DNA or RNA) to which templated ligation probes that are designed to target two specific (and in some embodiments, different) nucleic acids, hybridize. For example, a chimeric nucleic acid can be a single, hybrid nucleic acid molecule that contains sequences from different nucleic acids juxtaposed to each other. In some instances, the chimeric nucleic acid can include a sequence that is not 100% complementary to the first probe and/or the second probe. In some instances, the chimeric nucleic acid is not the same nucleic acid target as the target of the first probe and/or the second probe.

In some instances, the chimeric nucleic acid can be the target of the first probe. In this instance, the second probe (which has been designed to target a different and second nucleic acid) also hybridizes to an adjacent or nearby sequence of the target of the first probe. In some instances, the chimeric nucleic acid can be the target of the second probe. In this instance, the first probe (which has been designed to target a first nucleic acid) also hybridizes to an adjacent or nearby sequence of the target of the second probe. In another instance, the first probe and the second probe can hybridize to adjacent or nearby sequences of a completely different target nucleic acid (compared to the target nucleic acid(s) to which each probe was designed to hybridize).

The methods used herein can include contacting a plurality of first probes and second probes to the biological sample and then detecting chimeric ligation products. In some instances, the plurality of probes can include a probe set having hundreds or even thousands (e.g., 100, 200. 300, 400, 500, 600, 700, 800, 900. 1000, 2000, 3000, 4000. 5000, 7500, 10.000. 15.000, 18,000, 20,000 or more) probe pairs. In some instances, a single chimeric ligation product (e.g., PRDM4-GTF2A1) or two or more chimeric ligation products can be used to detect blood clots. In some embodiments, the methods used herein can include contacting a single pair of probes (e.g., probes that are known to or capable of forming chimeric ligation products in the presence of blood clots, pools of blood, or red blood cells) to the biological sample and then detecting any chimeric ligation products formed therefrom. For example, individual probes known to form a PDRM4-GTF2A1 chimeric ligation product, can be contacted with the biological sample in accordance with the methods disclosed herein, and the presence of one or more PDRM4-GTF2A1 chimeric ligation products in the biological sample can be determined.

In some embodiments, the methods used herein can include contacting two pairs, three pairs, four pairs, five pairs, six pairs, seven pairs, eight pairs, nine pairs, ten pairs, or more than ten pairs of probes (e.g., that are known to or capable of forming chimeric ligation products in the presence of blood clots, pools of blood, or red blood cells) to the biological sample and then detecting any chimeric ligation products formed therefrom. For example, individual probes known to form a chimeric ligation product (e.g., a chimeric ligation product from Tables 2 or 3), can be contacted with the biological sample in accordance with the methods disclosed herein, and the presence of one or more chimeric ligation products (e.g., one or more chimeric ligation product from Tables 2 or 3) in the biological sample can be determined.

In some instances, the methods involve disposing a biological sample (e.g., a tissue section) onto a substrate comprising a plurality of capture probes (e.g., an array comprising a plurality⁷ of capture probes). In other instances, as described above and in FIGs. 1-4, the methods include aligning (e.g., sandwiching) a first substrate having the biological sample with a second substrate that includes a plurality of capture probes, thereby ‘"sandwiching⁷’ the biological sample (e.g., a tissue section) between the two substrates. Upon bringing the biological sample in contact with or proximal to the substrate having a plurality of capture probes (in either instance), the location and/or abundance of a nucleic acid, protein, chimeric ligation product, and/or other analyte in a biological sample can be determined, as provided herein. This method includes an advantage in that steps prior to analyte or analyte-derived molecule capture by the capture probe, can be performed on a substrate that does not have an array of capture probes, thereby providing a method that is cost effective.

In some embodiments, disclosed herein are methods for determining a location of a chimeric nucleic acid in a biological sample. In some instances, the methods include: (a) contacting the biological sample with a first substrate; (b) contacting a plurality of probes to the biological sample, wherein a first probe and a second probe of the plurality⁷ of probes comprise sequences that are substantially complementary to the chimeric nucleic acid, and wherein the second probe comprises a capture probe capture domain sequence; (c) hybridizing the first probe and the second probe to the chimeric nucleic acid; (d) generating a chimeric ligation product by ligating the first probe and the second probe; (e) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality of capture probes; and (f) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the chimeric nucleic acid in the biological sample.

In additional embodiments, disclosed herein are methods of determining a location of a blood clot or pool of blood in a biological sample. In some instances, the methods include: (a) contacting the biological sample with a first substrate; (b) contacting a plurality of probes to the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid, and wherein the second chimeric probe comprises a capture probe capture domain sequence; (c) hybridizing the first probe and the second probe to the chimeric nucleic acid; (d) generating a chimeric ligation product by ligating the first probe and the second probe; (e) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality of capture probes; and (f) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the blood clot or pool of blood in the biological sample.

In some embodiments, the methods of determining the presence of a blood clot or pool of blood in a biological sample can include (a) contacting a plurality of probes to the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid; (c) generating a chimeric ligation product by ligating the first probe and the second probe; and (d) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample. In some instances of this embodiment, a location where the ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

In some instances, the methods of determining presence of a blood clot in a biological sample include: (a) contacting a plurality of probes to the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary to different nucleic acid analytes in the biological sample; (b) generating a chimeric ligation product by ligating the first probe and the second probe; and (c) determining the presence of the chimeric ligation product, or a complement thereof. In some instances of this embodiment, a location of the ligation product corresponds to a location of the blood clot in the biological sample.

In some instances, the methods of determining presence of a blood clot or pool of blood in a biological sample include (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary to different nucleic acid analytes in the biological sample or wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100, 150, 200, 250, 300. 350, 400. 450, 500 nucleotides, or more apart; (b) generating a chimeric ligation product by ligating the first probe and the second probe; and (c) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 200 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 300 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary’ to a first sequence and a second sequence in the same target nucleic acid that are 400 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 500 nucleotides or more apart.

In some instances, the location where the chimeric ligation product is detected corresponds to or indicates a location of one or more red blood cells in the biological sample.

In any of the foregoing, it is to be understood that generating a chimeric ligation product can include, but is not limited to, ligating the first probe and the second probe, so as long as the method generates a single connected probe that comprises the first and second unpaired probes. In some embodiments, generating a chimeric ligation product includes coupling the first probe and the second probe via a suitable approach disclosed herein (e.g., enzy matic or chemical ligation).

In some instances, the methods, devices, compositions, and systems disclosed herein provide efficient release of an analyte derived molecule (e.g.. a chimeric ligation product) from a biological sample so that it can be captured and/or detected using methods disclosed herein. Additional embodiments are further disclosed herein.

(b) Detecting Chimeric Ligation Products In some embodiments, the methods, devices, compositions, and systems described herein utilize templated ligation (e.g., RNA-templated ligation) to detect a chimeric ligation product. As used herein, '‘RNA-templated ligation." or “RTL” or simply '‘templated ligation’’ is a process wherein individual probes (e.g., a first probe, a second probe) in a probe pair hybridize to adjacent or proximal sequences of an analyte (e.g., an RNA or DNA molecule, e.g., a chimeric nucleic acid) in a biological sample (e.g., a tissue sample). The RTL probes are then coupled (e.g., ligated) together, thereby creating a connected probe (e.g., a ligation product). RNA-templated ligation is disclosed in PCT Publ. No. WO 2021/133849 Al, U.S. Pat. No. 11,618,897, and U.S. Publ. No. US 2021/0285046 Al, each of which is incorporated by reference in its entirety.

An advantage to using templated ligation is that it allows for enhanced detection of analytes (e.g., low expressing analytes) because both probes must hybridize to the analyte in order for the coupling (e.g., ligating) reaction to occur. Thus, enhancing specificity of the analytes detected. As used herein, “coupling” refers to an interaction between two probes that results in a single connected probe that comprises the two probes. In some instances, coupling is achieved through ligation. In some instances, coupling is achieved through extension of one probe to the second probe followed by ligation. In some instances, coupling is achieved through hybridization (e.g., using a third probe that is hybridized to each of the two probes) followed by extension of one probe or gap filling of the sequence between the two probes using the third probe as a template.

The connected probe (e g., chimeric ligation product) that results from the coupling (e.g., ligation) of the two probes can serve as a proxy for the target analyte. Further, it is appreciated that probe pairs can be designed to cover any gene of interest. For example, a pair of probes can be designed so that each analyte, e.g.. a whole exome, a transcriptome, a genome, can conceivably be detected using probe pairs. Normally, probe pairs are designed to target a specific single nucleic acid. However, it has been observed that a first probe and a second probe from different probe pairs, and in some embodiments, for targeting different nucleic acid analytes, can be ligated together to form a chimeric ligation product.

In some instances, a chimeric ligation product comprises a probe pair listed in Table 1 and/or selected from the group consisting of: PRDM4-GTF2A1; SLC30A5-VPS33B; IZUMO1R-DDO; OR7D2-UQCRH; ZNF841-IL18RAP; MYB-NBPF3; SLC25A51-SKA1; OR6C2-SMIM29; IGKC-IGKC (probes for IGKC and IGKC target different locations of the IGKC analyte); FAM72B-NSL1; CAMKMT-RIDA; OLFML1-ZFYVE16; and any combination thereof. In some instances, a chimeric ligation product comprises any combination of two probes selected from PRDM4, GTF2A1, SLC30A5, VPS33B,

IZUM01R, DDO, OR7D2, UQCRH, ZNF841, IL18RAP, MYB, NBPF3, SLC25A51, SKA1, OR6C2, SMIM29, IGKC, FAM72B, NSL1, CAMKMT, RIDA, OLFML1, and ZFYVE16. In the foregoing examples, the probe name indicates the target nucleic acid analyte to which the probe is designed to hybridize.

Table 1. Probe Pair Targets.

In some embodiments, the process of transferring the connected probe (e.g., a chimeric ligation product) from the biological sample to the capture probe is performed on a single substrate, e.g.. glass slide. In some embodiments, the process of transferring the connected probe (e.g., a chimeric ligation product) from a first substrate to a second substrate is referred to as a “sandwich’’ process. The sandwich process is described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety. Described herein are methods in which an array with capture probes located on a second substrate and a biological sample located on a first substrate, are arranged such that the array is brought into proximity with the biological sample (e.g.. the first and second substrates are sandwiched together). In some embodiments, the array and the biological sample can be brought into proximity (e.g., sandwiched), without the aid of a substrate holder.

In some embodiments, the methods as disclosed herein include hybridizing one or more probe pairs (e.g.. RTL probes) to adjacent or nearby sequences of a target analyte (e.g., RNA, e.g., mRNA, DNA, a chimeric nucleic acid). In some embodiments, the probe pairs include sequences that are complementary' or substantially complementary to a chimeric nucleic acid. For example, in some embodiments, each probe in a chimeric ligation product includes a sequence that is complementary’ or substantially complementary to a chimeric nucleic acrd present in the biologrcal sample (e.g.. at, or proximal to. a blood clot, pool of blood, or red blood cells). In some embodiments, each target analyte includes a first target region and a second target region. In some embodiments, the methods include providing a plurality of first probes and a plurality of second probes, wherein a pair of probes for a target analyte comprises both a first and second probe from the plurality’. In some embodiments, a first probe is designed to hybridize to a first target region of the analyte, and the second probe is designed to hybridize to a second, adjacent or proximal target region of the analyte.

In some instances, the probes are DNA molecules. In some instances, the first probe is a DNA molecule. In some instances, the second probe is a DNA molecule. In some instances, one probe in the pair of probes comprises at least two ribonucleic acid bases at the 3’ end while the other probe in the pair of probes comprises a phosphorylated nucleotide at the 5’ end. In some instances, the first probe comprises at least two ribonucleic acid bases at the 3’ end. In some instances, the second probe comprises a phosphorylated nucleotide at the 5’ end.

RTL probes can be designed using methods known in the art. In some instances, probe pairs are designed to cover an entire transcriptome of a species (e.g., a mouse or a human). In some instances, RTL probes are designed to cover a subset of a transcriptome (e.g., a mouse or a human). In some instances, the methods disclosed herein utilize about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000. about 15.000, about 20,000, or more probe pairs.

In some embodiments, one of the probes of the pair of probes includes a capture probe capture domain sequence. The capture probe capture domain sequence can be a poly(A) sequence, or a complement thereof. Thus, in some embodiments, one of the probes of the pair of probes for RTL includes a poly(A) sequence or a complement thereof. In some instances, the poly(A) sequence or a complement thereof is on the 5^? end of one of the probes. In some instances, the poly(A) sequence or a complement thereof is on the 3’ end of one of the probes. In some embodiments, one probe of the pair of probes for RTL includes a degenerate or unique molecular identifier (UMI) sequence. In some embodiments, the UMI sequence is specific to a particular target or set of targets. In some instances, the UMI sequence or a complement thereof is on the 5’ end of one of the probes. In some instances, the UMI sequence or a complement thereof is on the 3’ end of one of the probes.

In some instances, the first and second target regions of a chimeric nucleic acid are directly adjacent to one another. In some embodiments, the complementary sequences to which the first probe and the second probe hybridize are 1, 2. 3, 4, 5. 6. 7, 8, 9. 10. about 15. about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, or about 150 nucleotides away from each other. Gaps between the first and second probes may be filled prior to coupling (e.g., ligation), using, for example, dNTPs in combination with a polymerase such as polymerase mu, DNA polymerase, RNA polymerase, reverse transcriptase, VENT polymerase, Taq polymerase, and/or any combinations, derivatives, and variants (e.g., engineered mutants) thereof. In some embodiments, when the first and second probes are separated from each other by one or more nucleotides, deoxyribonucleotides are used to extend and couple (e.g., ligate) the first and second probes.

In some instances, the first probe and the second probe hybridize to a chimeric nucleic acid on the same mRNA transcript. In some instances, the first probe and the second probe hybridize to a chimeric nucleic acid on the same exon. In some instances, the first probe and the second probe hybridize to a chimeric nucleic acid on different exons. In some instances, the first probe and the second probe hybridize to a chimeric nucleic acid that is the result of a translocation event (e.g., in the setting of cancer). The methods provided herein make it possible to identify alternative splicing events, translocation events, and mutations that change the hybridization rate of one or both probes (e.g., single nucleotide polymorphisms, insertions, deletions, point mutations). In some embodiments, the first and/or second probe as disclosed herein includes at least two ribonucleic acid bases at the 3’ end; a functional sequence; a phosphory lated nucleotide at the 5’ end; and/or a capture probe binding domain. In some embodiments, the functional sequence is a primer sequence.

The “capture probe binding domain” is a sequence that is complementary to a particular capture domain present in a capture probe. In some embodiments, the capture probe binding domain includes a poly(A) sequence. In some embodiments, the capture probe binding domain includes a poly -uridine sequence, a poly -thymidine sequence, or a combination thereof. In some embodiments, the capture probe binding domain includes a random sequence (e.g., a random hexamer or octamer). In some embodiments, the capture probe binding domain is complementary to a capture domain in a capture probe that detects a particular chimeric nucleic acid of interest. In some embodiments, a capture probe binding domain blocking moiety⁷ that interacts with the capture probe binding domain is provided. In some embodiments, a capture probe binding domain blocking moiety includes a sequence that is complementary or substantially complementary⁷ to a capture probe binding domain. In some embodiments, a capture probe binding domain blocking moiety prevents the capture probe binding domain from binding the capture probe when present. In some embodiments, a capture probe binding domain blocking moiety⁷ is removed prior to binding the capture probe binding domain (e.g., present in a connected probe (e.g., a chimeric ligation product)) to a capture probe. In some embodiments, a capture probe binding domain blocking moiety comprises a poly-uridine sequence, a poly-thymiidine sequence, or a combination thereof.

Hybridization of the probes to a chimeric nucleic acid can occur at a target having a sequence that is 100% complementary to the probe(s). In some embodiments, hybridization can occur at a chimeric nucleic acid having a sequence that is at least (e.g., at least about) 80%, at least (e.g., at least about) 85%, at least (e.g., at least about) 90%, at least (e.g., at least about) 95%, at least (e.g., at least about) 96%, at least (e.g., at least about) 97%, at least (e.g., at least about) 98%, or at least (e.g., at least about) 99% complementary⁷ to the probe(s). After hybridization, in some embodiments, the first probe is extended. After hybridization, in some embodiments, the second probe is extended. For example, in some instances a first probe hybridizes to a target sequence of a chimeric nucleic acid upstream from a second probe, whereas in other instances a first probe hybridizes to a target sequence of a chimeric nucleic acid downstream of a second probe.

In some embodiments, methods disclosed herein include a wash step after hybridizing the first and the second probes. The wash step removes any unbound first or second probes and can be performed using any technique known in the art. In some embodiments, a prehybridization buffer is used to wash the sample. In some embodiments, a phosphate buffer is used. In some embodiments, multiple wash steps are performed to remove unbound or non- specifically bound probes. For example, it is advantageous to decrease the amount of unhybridized probes present in a biological sample as they may interfere with downstream applications and methods. Decreasing the amount of unhybridized or non-specifically bound probes present can also improve the specificity and sensitivity of the assay.

In some embodiments, after hybridization of probes (e.g., first and the second probes) to a chimeric nucleic acid, the probes (e.g., the first probe and the second probe) are coupled (e.g., ligated) together, creating a single chimeric connected probe (e.g., a chimeric ligation product).

Ligation can be performed chemically, as described herein and in Maruyama et al., Nucleic Acids Res. 2017 Jul 7; 45(12): 7042-7048; Manuguerra et al., Chem Commun (Camb). 2018 May 4; 54(36): 4529-4532; and Fantoni et al., Chem. Rev. 2021, 121, 12, 7122-7154, each of which is incorporated by reference in its entirety. Chemical ligation enables covalent bond formation between two oligonucleotide strands. An example of chemical ligation includes click chemistry and includes (i) nucleophilic substitutions; (ii) additions to C-C multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziridination); (iii) nonaldol like chemistry (e.g., N-hydroxysuccinimide active ester couplings); and (iv) cycloadditions (e.g., Diels-Adler reaction, Huisgen’s cycloaddition). Chemical ligation benefits from the highly efficient click chemistry' approach templated by D A nanostructures, and produces modified DNA that is compatible with polymerase enzymes.

Ligation can be performed enzymatically. For example, the first and second probes are hybridized to the first and second target regions of the chimeric nucleic acid, and the probes are subjected to a nucleic acid reaction to ligate them together. For example, the probes may be subjected to an enzy matic ligation reaction using a ligase (e.g., T4 RNA ligase (Rnl2), a SplintR ligase, or a T4 DNA ligase). See, e.g.. Zhang L., et al.; Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation RNA Biol. 2017; 14(1): 36-44 for a description of KOD ligase. A skilled artisan wi 11 understand that various reagents, buffers, cofactors, etc. may be included in a ligation reaction depending on the ligase being used.

In some embodiments, the method further includes hybridizing the chimeric ligation product to a capture domain of a capture probe. Once hybridized, the chimeric ligation product can be extended using the capture probe as a template to generate an extended chimeric ligation product, the capture probe can be extended using the chimeric ligation product as a template to generate an extended capture probe, or a combination thereof. Extending the chimeric ligation product using the capture probe as a template generates an extended chimeric ligation product including a sequence complementary to the capture probe (e.g., including the spatial barcode, UMI, functional domains, etc.). The chimeric ligation product and/or capture probe can be extended from the 3’ end using a polymerase (e.g., a DNA polymerase).

In some embodiments, the method further includes amplifying the chimeric connected probe (e.g.. the chimeric ligation product) prior to the releasing step. In some embodiments, the entire chimeric connected probe (e.g., a chimeric ligation product) is amplified. In some embodiments, only part of the chimeric connected probe (e.g., a chimeric ligation product) is amplified. In some embodiments, amplification is isothermal. In some embodiments, amplification is not isothermal. Amplification can be performed using any of the methods described herein such as, but not limited to, a strand-displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, an isothermal amplification reaction, and/or a loop-mediated amplification reaction. In some embodiments, amplifying the chimeric connected probe (e.g., a chimeric ligation product) creates an amplified connected probe (e.g.. a ligation product) that includes (i) all or part of sequence of the chimeric connected probe (e.g.. a chimeric ligation product) specifically bound to the capture domain, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof.

In some embodiments, the method further includes determining (i) all or a part of the sequence of the chimeric connected probe (e.g., the chimeric ligation product), or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof. In some embodiments, the method further includes using the determined sequence of (i) and (ii) to determine the location of the chimeric ligation product (and therefore location of a blood clot) in the biological sample. In some embodiments, the sequences of (i) and/or (ii) are determined from an extended capture probe or a complement thereof, or an extended chimeric ligation product, or a complement thereof. In some embodiments, determining the sequences of (i) and/or (ii) includes sequencing an extended capture probe or a complement thereof, or an extended chimeric ligation product, or a complement thereof. In some embodiments, after coupling (e.g., ligation) of the first and second probes to create a chimeric ligation product, the chimeric connected probe (e.g., a chimeric ligation product) is released from the analyte. To release the chimeric connected probe (e.g., a chimeric ligation product), an endoribonuclease (e.g., RNase A, RNase C, RNase H, or RNase I) can be used. An endoribonuclease such as RNase H specifically cleaves RNA in RNA:DNA hybrids. In some embodiments, the chimeric connected probe (e.g., a chimeric ligation product) is released enzymatically. In some embodiments, an endoribonuclease is used to release the chimeric connected probe from the analyte. In some embodiments, the endoribonuclease is RNase H. In some embodiments, the RNase H is RNase Hl or RNase H2.

In some embodiments, the releasing of the chimeric connected probe (e.g., a chimeric ligation product) includes contacting the biological sample with a reagent medium comprising an agent for releasing the chimeric connected probe (e.g., a chimeric ligation product). In some embodiments, the reagent medium comprises a permeabilization agent and an agent for releasing the chimeric connected probe (e.g., a chimeric ligation product), thereby permeabilizing the biological sample and releasing the chimeric connected probe (e g., a chimeric ligation product) from the analyte. In some embodiments, the agent for releasing the chimeric connected probe (e.g., a chimeric ligation product) comprises a nuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease includes an RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, or RNase I.

In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG is from about PEG 2K to about PEG 16K. In some embodiments, the PEG is PEG 2K, 3K, 4K, 5K. 6K, 7K, 8K, 9K, 10K, 1 IK, 12K, 13K, 14K, 15K, or 16K. In some embodiments, the PEG is present at a concentration from about 2% to 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v/v).

In some embodiments, the reagent medium includes a wetting agent. Wetting agents increase the spreading and penetrating properties of a liquid by lowering its surface tension. Examples of wetting agents include surfactants, a soap, an alcohol, gum arabic, oxgall, and compositions comprising fatty acids.

In some instances, after generation of the chimeric connected probe (e.g., a chimeric ligation product), the methods disclosed herein include simultaneous treatment of the biological sample with a permeabilization agent such as proteinase K (to permeabilize the biological sample) and a releasing agent such as an endonuclease such as RNase H (to release the chimeric connected probe (e.g., a chimeric ligation product) from the analyte). In some instances, the permeabilization step and releasing step occur at the same time. In some instances, the permeabilization step occurs before the releasing step. In some embodiments, the permeabilization agent comprises a protease. In some embodiments, the protease is selected from trypsin, pepsin, elastase, or Proteinase K. In some embodiments, the protease is an endopeptidase. Endopeptidases that can be used include but are not limited to trypsin, chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamyl endopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN), endopeptidase LysC and endopeptidase LysN. In some embodiments, the endopeptidase is pepsin.

In some embodiments, the reagent medium further includes a detergent. In some embodiments, the detergent is selected from sodium dodecyl sulfate (SDS), sarkosyl, saponin, Triton X-100™, or Tween-20™. In some embodiments, the reagent medium includes less than 5 w/v% of a detergent selected from sodium dodecyl sulfate (SDS) and sarkosyl. In some embodiments, the reagent medium includes as least 5% w/v% of a detergent selected from SDS and sarkosyl. In some embodiments, the reagent medium does not include SDS or sarkosyl.

In some embodiments, the biological sample and the array are contacted with the reagent medium for about 1 to about 60 minutes. In some embodiments, the biological sample and the array are contacted with the reagent medium for about 30 minutes.

In some embodiments, the chimeric connected probe (e.g., a chimeric ligation product) includes a capture probe binding domain, which can hybridize to a capture probe (e.g., a capture probe immobilized, directly or indirectly, on a substrate). In some embodiments, the capture probe includes a spatial barcode and the capture domain. In some embodiments, the capture probe binding domain of the chimeric connected probe (e.g., a chimeric ligation product) binds (e.g., hybridizes) to the capture domain of the capture probe.

In some embodiments, methods provided herein include mounting a biological sample on a first substrate, then aligning the first substrate with a second substrate including a nucleic acid array, such that at least a portion of the biological sample is aligned with at least a portion of the nucleic acid array, wherein the nucleic acid array includes a plurality of capture probes. After hybridization of the chimeric connected probe (e.g., a chimeric ligation product) to the capture probe, downstream methods as disclosed herein can be performed.

In some embodiments, at least 50% of connected probes (e.g., a ligation products) released from the portion of the biological sample aligned with the portion of the array are captured by capture probes of the portion of the array. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of connected probe (e.g., a ligation products) are detected by the array aligned with the biological sample.

In some embodiments, the capture probe (e.g.. capture domain) includes a poly(T) sequence. In some embodiments, the capture probe (e.g., capture domain) includes a sequence specific to an analyte. In some embodiments, the capture probe includes a functional domain. In some embodiments, the capture probe further includes one or more functional domains, a UMI, a cleavage domain, and combinations thereof. In some embodiments, the capture probe binding domain of a probe comprised in a (chimeric) ligation product includes a poly(A) sequence. In some embodiments, the capture probe binding domain includes a sequence complementary to a capture domain of a capture probe that detects a target analyte of interest. In some embodiments, the analyte is RNA. In some embodiments, the analyte is mRNA.

In some embodiments, the chimeric connected probe (e.g., a chimeric ligation product) (e.g., the analyte derived molecule) includes a capture probe binding domain, which can hybridize to a capture probe (e.g., a capture probe immobilized, directly or indirectly), on a substrate. Methods provided herein also include contacting a biological sample with a substrate directly, wherein the capture probe is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). After hybridization of the chimeric connected probe (e.g., a chimeric ligation product) to the capture probe, downstream methods as disclosed herein (e.g., sequencing, in situ analysis such as RCA) can be performed.

In some embodiments, the method further includes analyzing a different analyte, such as a protein, in the biological sample (e.g., multiplexing). In some embodiments, the analysis of the different analyte includes (a) further contacting the biological sample with a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents includes an analyte binding moiety and a capture agent barcode domain, wherein the analyte binding moiety specifically binds to the different analyte, and wherein the capture agent barcode domain includes an analyte binding moiety barcode and an capture handle sequence that is complementary to a capture domain of a capture probe; and (b) hybridizing the analyte capture sequence to the capture domain.

In some embodiments, detection of chimeric ligation products can be performed without capture of the chimeric ligation product on an array. For example, a chimeric ligation product can inform the presence of a blood clot in a biological sample without the context of spatiality. In some embodiments, the chimeric ligation products can be detected in situ using fluorescent-based or chemical-based approaches.

In some instances, the methods include determining presence of a blood clot in a biological sample, the method comprising: (a) contacting a plurality of probes to the biological sample, wherein a first probe and a second probe of the plurality comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid; (c) generating a chimeric ligation product by ligating the first probe and the second probe; and (d) determining the presence of the chimeric ligation product, or a complement thereof. In some instances, the methods include determining presence of a blood clot in a biological sample, the method comprising: (a) contacting a plurality of probes to the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary' to different nucleic acid analytes in the biological sample; (b) generating a chimeric ligation product by ligating the first probe and the second probe; and (c) determining the presence of the chimeric ligation product, or a complement thereof. In some embodiments, a location of the ligation product corresponds to a location of the blood clot in the biological sample.

In some instances, the methods include determining a location of a blood clot or pool of blood in a biological sample. In some instances, these methods include (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality⁷ of probes comprise sequences that are substantially complementary⁷ to a chimeric nucleic acid in the biological sample, and wherein the second chimeric probe comprises a capture probe capture domain sequence; (b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; (d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality of capture probes; and (e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the blood clot or pool of blood in the biological sample.

In some instances, the methods include determining presence of a blood clot or pool of blood in a biological sample. In some instances, the methods include (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample: (b) hybridizing the first probe and the second probe to the chimeric nucleic acid; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; and (d) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

In some instances, the methods include determining presence of a blood clot or pool of blood in a biological sample. In some instances, the methods include (a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary to different nucleic acid analytes in the biological sample or wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100, 150, 200, 250, 300. 350, 400, 450. 500 nucleotides, or more apart; (b) generating a chimeric ligation product by ligating the first probe and the second probe; and (c) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 200 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 300 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 400 nucleotides or more apart. In some instances, the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 500 nucleotides or more apart. In some instances, the location where the chimeric ligation product is detected corresponds to or indicates a location of one or more red blood cells in the biological sample.

The methods described herein provide for in situ detection of the chimeric ligation product. In some instances, a detectable probe (fluorescent-based or chemically-based) can be used. In some instances, the detectable probe hybridizes to part or all of the chimeric ligation product. In some instances, after hybridization, the biological sample is visualized or imaged using light or fluorescence microscopy. In some instances, amplification reactions can be performed to increase the amount of chimeric ligation products (e.g., PCR amplification, in situ amplification, clonal amplification), prior to using detectable probes.

(c) Diagnostic and Treatment Methods

Provided in this disclosure are methods of diagnosing and treating blood clots, and/or conditions or diseases associated with blood clots in a subject. Diagnosis of blood clots can be based on the methods of detecting one or more chimeric ligation products, blood clots or pools of blood in a biological sample from a subject.

“Treatment” or “treating” describes administration of composition to a subject or a system with an undesired condition. The condition can include one or more symptoms of a disease, pathological state, or disorder. Treatment includes medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological state, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological state, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, that is. treatment employed to supplement another specific therapy directed tow ard the improvement of the associated disease, pathological state, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention.

“Prevention” or “preventing” means to administer a composition to a subject or a system at risk for the condition. The condition can be a predisposition to a disease. The effect of the administration of the composition to the subject can be the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or minimization of the chances that a particular event or characteristic will occur.

As used herein, the terms “effective amount'’ or “therapeutically effective amount” means a dosage sufficient to alleviate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subjectdependent variables (e.g., age, immune system health, weight, etc.), the disease or disorder being treated, as well as the route of administration, the pharmacokinetics of the agent being administered and the pharmacodynamic effects of the agent.

In some instances, the chimeric ligation product comprises a probe pair selected from the group consisting of: PRDM4-GTF2A1; SLC30A5-VPS33B; IZUM01R-DD0; OR7D2- UQCRH; ZNF841-IL18RAP; MYB-NBPF3; SLC25A51-SKA1; OR6C2-SMIM29; IGKC- IGKC; FAM72B-NSL1; CAMKMT-RIDA; 0LFML1-ZFYVE16; and any combination thereof. In some instances, a chimeric ligation product comprises any combination of two probes selected from PRDM4, GTF2A1, SLC30A5, VPS33B, IZUM01R, DDO, OR7D2, UQCRH, ZNF841, IL18RAP, MYB, NBPF3, SLC25A51, SKA1, OR6C2, SMIM29, IGKC, FAM72B, NSL1, CAMKMT, RIDA, 0LFML1, and ZFYVE16, where the probe name indicates the target nucleic acid analyte to which the probe is designed to hybridize.

In some instances, diagnosis can include detection of one or more chimeric ligation products combined with an additional method of diagnosis (e.g., eosin staining). For instance, a tissue sample can be stained using H&E, hematoxylin alone, eosin alone, or stained using immunofluorescence or immunohistochemistry.

In some instances, diagnosis of a blood clot includes identifying an increase in one or more chimeric ligation products in an area of a biological sample compared to other areas of the same biological sample. In some instances, diagnosis of a blood clot includes identifying an increase in one or more chimeric ligation products in an area of a biological sample compared to other areas of a different biological sample (e.g.. either of the same tissue type, a different tissue type, or a reference sample).

In some instances, the blood clot is in a blood vessel, such as an artery or a vein. In some instances, the blood clot is associated with a chronic disease. In some instances, the blood clot is associated with an acute disease. In some instances, the blood clot causes or is associated with deep vein thrombosis. In some instances, the blood clot is associated with a miscarriage.

In some instances, the blood clot is an intestinal blood clot, a stomach blood clot, a lingual blood clot, a lung blood clot (e.g., a pulmonary embolus), a thyroid blood clot, a thymic blood clot, a testicular blood clot, a hepatic blood clot, a pancreatic blood clot, an epithelial blood clot, a lung blood clot, a kidney blood clot, a gastruloid, a cardiac blood clot, a tonsil blood clot, a spleen blood clot, or a retinal blood clot. In some instances, the blood clot is located in the eye (e.g., in the retina).

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.

In some instances, the blood clot is indicative of a stroke. In some instances, the blood clot is indicative of a mini-stroke.

In some instances, the blood clot can be associated with a genetic disposition. For instance, the blood clot can occur in a subject with Factor V Leiden, a prothrombin gene mutation (such as G20210A), genetic defects that cause deficiencies of natural proteins that prevent clotting such as antithrombin, protein C and protein S, genetic defects that cause elevated levels of fibrinogen or dysfunctional fibrinogen, genetic defects that cause elevated levels of factor VIII and other factors including factor IX and XI. or in a subject having an abnormal fibrinolytic system.

In some instances, the blood clot is associated with an acquired disease, including but not limited to antiphospholipid syndrome (APS), disseminated intravascular coagulation (DIC), cancer and medications that treat cancer, recent trauma or surgery, central venous catheter placement, obesity, pregnancy, supplemental estrogen use, hormone replacement therapy, prolonged immobility, a sedentary lifestyle, heart attack, congestive heart failure, stroke, heparin-induced thrombocytopenia, autoimmune disorders, antiphospholipid antibody syndrome, deep vein thrombosis, pulmonary embolism, myeloproliferative disorders such as polycythemia vera or essential thrombocytosis, paroxysmal nocturnal hemoglobinuria, inflammatory bowel syndrome, vitamin B deficiency, HIV, sepsis, infections, nephrotic syndrome, and any combination thereof.

In some instances, provided herein is a method of treating or preventing blood clots in a subject in need thereof, comprising administering an effective amount of a therapeutic agent to the subject, wherein the subject has been identified as having or previously having a blood clot (e.g.. by detecting chimeric ligation products in a sample from the subject as disclosed herein). In some embodiments, the amount of therapeutic agent administered is an amount effective to reduce one or more symptoms of a blood clot. Symptoms of a blood clot include difficulty breathing, faster than normal or irregular heartbeat, low blood pressure, lightheadedness, or fainting, shortness of breath, chest pain or discomfort, reddish or bluish skin discoloration, and leg swelling or cramping. In some embodiments, the amount of therapeutic agent administered is an amount effective to dissolve or dislodge a blood clot.

In some embodiments, the methods can include confirming a suspected diagnosis of a blood clot in a subject (e.g., by detecting chimeric ligation products in a sample from the subject as disclosed herein). For example, a blood clot can block part an artery of the heart muscle which may lead to a heart attack. Non-limiting examples of ways to confirm a diagnosis of myocardial infarction include an electrocardiogram (ECG), holter monitoring, echocardiogram, stress test, cardiac catheterization, cardiac computerized tomography (CT) scan, or cardiac magnetic resonance imaging (MRI).

In some embodiments, the methods can further include selecting a treatment for the subject. In some embodiments, the methods can further include administering a treatment for reducing the risk of having a blood clot. In some embodiments, a treatment for reducing the risk of having a blood clot can be a treatment that reduces the rate of progression of the risk of developing a blood clot in a subject. In some embodiments, a treatment for reducing the risk of developing a blood clot can include lifestyle changes, such as stopping smoking, treatments to lower the blood pressure, treatments to lower the level of cholesterol, treatment of diabetes, exercise (e.g., at least 30 minutes most days of the week), change of diet (e.g., eating a low-fat and low-sodium diet, and limiting alcohol), weight control, stress management or treatment (e.g., muscle relaxation and deep breathing), depression management, and heart procedures (e.g., angioplasty), surgery (e.g., bypass surgery), or implanting a device (e.g., a pacemaker or a defibrillator).

In some embodiments, a treatment for a blood clot, treatment for reducing the risk of developing a blood clot, and/or preventing or treating a disorder/disease associated with blood clots can include administering one or more therapeutic or prophylactic medications. Such medications include, but not limited to, an anticoagulant medications. In some instances, the anticoagulant medication includes aspirin, warfarin (Coumadin® or Jantoven®), heparin, low-molecular weight heparin, fondaparinux, an injection, or an oral anticoagulants such as rivaroxaban, apixaban. or dabigatran. In some cases, blood clots can be removed using minimally invasive interventional radiology procedures. Also provided herein are methods of identifying a subject as having an increased likelihood of having a blood clot using any one of the methods provided herein to detect an increase in a chimeric ligation product in a biological sample from the subject. In some embodiments, the methods can further comprise monitoring the identified subject for the development of symptoms of blood clot. The methods provided herein also include methods of monitoring risk of having a blood clot in a subject by detecting one or more chimeric ligation products in one or more biological samples from the subject over time.

(d) Compositions. Systems, and Kits

In some embodiments, also provided herein are compositions, systems, and kits that include one or more reagents to detect one or more chimeric ligation products. In some instances, the composition, system, and/or kit includes (a) a support device configured to retain a first substrate and a second substrate, wherein a biological sample is placed on the first substrate, and wherein the second substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (b) a first probe and a second probe, wherein the first probe and the second probe each comprise a sequence that is substantially complementary' to adjacent sequences of a chimeric nucleic acid, wherein the second probe comprises a capture probe binding domain, and wherein the first probe and the second probe are capable of being ligated together to form a chimeric ligation product; (c) a reagent medium comprising a permeabilization agent and optionally an agent for releasing the chimeric ligation product from the chimeric nucleic acid; and optionally (d) instructions for performing any of the methods disclosed herein.

In some instances, the composition, system, and/or kit includes a permeabilization agent such as pepsin or proteinase K. The agent for releasing the chimeric ligation product can be an RNAse, optionally wherein the RNAse is selected from RNase A, RNase C, RNase H, or RNase I.

The composition, system, and/or kit can also include an alignment mechanism on the support device to align the first substrate and the second substrate. The alignment mechanism can comprise a linear actuator, wherein the first substrate comprises a first member and the second substrate comprises a second member, and optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to the plane or the first member and/or the second member, and/or 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, and/or 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, and/or 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.

In some instances, the chimeric ligation product comprises a probe pair selected from the group consisting of: PRDM4-GTF2A1; SLC30A5-VPS33B; IZUMO1R-DDO; OR7D2- UQCRH; ZNF841-IL18RAP; MYB-NBPF3; SLC25A51-SKA1; OR6C2-SMIM29; IGKC- IGKC; FAM72B-NSL1; CAMKMT-RIDA; OLFML1-ZFYVE16; and any combination thereof. In some instances, the first probe and second probe are selected from PRDM4, GTF2A1, SLC30A5. VPS33B, IZUMO1R, DDO. OR7D2, UQCRH, ZNF841, IL18RAP, MYB, NBPF3. SLC25A51, SKA1, OR6C2. SMIM29, IGKC, FAM72B, NSL1, CAMKMT. RIDA, OLFML1, and ZFYVE16, where the probe name indicates the target nucleic acid analyte to which the probe is designed to hybridize.

In some embodiments, the compositions, systems, and/or kits disclosed herein include tools and/or reagents for harvesting, staining, crosslinking, decrosslinking, and/or imaging biological samples.

An additional non-limiting example of a composition, system, and/or kit used to perform any of the methods described herein includes: (a) a substrate comprising a plurality of capture probes comprising a spatial barcode and a capture domain; (b) a plurality of first probes and second probes, wherein a first probe and a second probe each comprises sequences that are substantially complementary to a chimeric nucleic acid, and wherein the second probe comprises a capture binding domain; and (c) instructions for performing any of the methods disclosed herein.

EXAMPLES

Example 1. Identification of Chimeric Ligation Products in Samples with Blood Clots

In preparation for templated ligation probe hybridization, human spleen, tonsil, and kidney FFPE tissue sections were obtained and placed on a first substrate. Each tissue section was deparaffinized, H&E stained, and imaged. Next, the tissue sections were hematoxylin- destained with three HC1 solution washes. The sections were then decrosslinked to remove formalin induced adducts, and washed.

A whole transcnptome panel of RNA templated ligation (RTL) probes (e.g., containing a first probe and a second probe of multiple probe pairs) were contacted with the tissue sections and allowed to hybridize to adjacent sequences of an analyte (e.g., an RNA molecule) in the tissue sections. The RTL probes were then ligated together using a ligase, thereby creating a connected probe (e.g., a ligation product), as illustrated in FIG. 9A. The chimeric connected probe (e.g., a chimeric ligation product) included a capture probe binding domain. The probe pairs were designed to hybridize to a single gene of the human transcriptome.

In another group (as shown in FIGs. 12D and 12E). in addition to incubation of the tissue sections with RTL probes, the tissue sections were incubated with a plurality of oligonucleotide-tagged antibodies that target proteins, followed by washing. The antibodies were tagged with oligonucleotides that have (i) a sequence complementary to a capture probe capture domain of a capture probe, and (ii) a barcode sequence that uniquely identifies the antibody. In this cohort, the connected probes and antibody oligonucleotide tags were released from the tissue section together.

After ligation of the RTL probes (and/or incubating of the tissue sections with the oligonucleotide-tagged antibodies), the first substrate then was aligned with the second substrate, such that at least a portion of the tissue section was aligned with at least a portion of the capture probes (e g., aligned in a sandwich configuration). The connected probes (and/or oligonucleotides from the oligonucleotide-tagged antibodies) were released from the tissue sections by contacting the tissue sections with a reagent medium having a protease (e.g.. proteinase K) and an RNAse H. Then, the connected probes (and/or oligonucleotides from the oligonucleotide-tagged antibodies) were allowed to migrate (e.g., diffuse) toward and hybridize to the capture probes on an array on a second substrate.

Following hybridization, the capture probes, connected probes, and/or oligonucleotides from the oligonucleotide-tagged antibodies were extended, sequencing libraries were prepared and sequenced, and the results were analyzed computationally.

As shown in FIG. 12A, a blood clot (indicated by the arrow) in the human spleen sample was observed by a pathologist using a histological stain. A pair of replicates (serial tissue sections) was run for each test condition. In one group (FIGs. 12B and 12C), no oligonucleotide-tagged antibodies were used (i.e.. only RTL probes were added to the tissue section). In a second group (FIGs. 12D and 12E), oligonucleotide-tagged antibodies were used in addition to RTL probes. When reviewing the ligation products via sequencing for each of the four replicates show n in FIGs. 12B-12E, it was observed that there w as an increase in first probe/second probe mismatches (chimeric ligation products) in the vicinity of the blood clot. Mismatches were identified when the first probe targeting one analyte and the second probe targeting a different analyte were detected in a single ligation product. As shown in FIGs. 12B-12E, there was an increase in various mismatched ligation products around the area of the blood clot in FIG 12A. The increase in mismatched ligation products included the following mismatched probe pairs: PRDM4/GTF2A1; SLC30A5/VPS33B; IZUM01R/DD0; OR7D2/UQCRH; ZNF841/IL18RAP; and SLC25A51/SKA1, as shown below in Table 2. In addition, as shown in FIG. 12F, two of three areas (labeled Al and A3) of the human spleen tissue section that were identified as having blood clots were associated with a high number of chimeric ligation products.

Table 2. Mismatched Probe Pairs Identified in Blood Clots of Human Spleen Samples.

Given that similar mismatched ligation products were detected whether oligonucleotide-tagged antibodies were used or not, this indicates that the chimeric/mismatched ligation products are not due to the presence (or absence) of oligonucleotide-tagged antibodies. On the other hand, the increase in chimeric ligation products is the result of mismatching of RTL first and second probes in an area with a blood clot or pool of blood/red blood cell. Thus, these data demonstrate that there is an increase in chimeric ligation products in areas of blood clots or pools of blood/red blood cells in a biological sample.

This phenomena was further extended to additional human samples. First, using the same methods described above in this example, an archived human tonsil sample was examined. Indeed, as shown in FIGs. 13A-13B, there was a significant increase in the number of chimeric ligation products around an area (outlined and shown using an arrow" in FIG. 13A) having blood clots in the tonsil sample. In particular, it was observed that there was an increase in mismatched ligation probes with the following mismatched probe pairs: MYB/NBPF3; SLC25A51/SKA1; SLC30A5/VPS33B; OR6C2/SMIM29; IGKC/IGKC (the later being mismatched probes from different probe pairs targeting the same gene).

Compared to the human spleen replicates shown in FIGs. 12A-12F, there was an overlap in two mismatched probe pairs (SLC25A51/SKA1; and SLC30A5/VPS33B), demonstrating that at least these two chimeric ligation products could be detected in samples from different organs.

Second, a human kidney tissue section was examined. As shown in FIG. 14A, the kidney tissue section had multiple areas where blood was pooled (shown with arrows in FIG. 14A). In six replicates (FIGs. 14B-14G) of the kidney tissue sample, chimeric ligation products were detected in each of the replicates, demonstrating that another tissue can be used to detect mismatched probes as a proxy for blood clots in a biological sample. Table 3 below shows the mismatched probe pairs identified in blood clots of human kidney sample.

Table 3. Mismatched Probe Pairs Identified in Blood Clots of Human Kidney Sample.

Claims

WHAT IS CLAIMED IS.

1. A method of determining presence of a blood clot or pool of blood in a biological sample, the method comprising:

(a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe in the plurality of probes each comprise sequences that are substantially complementary to different nucleic acid analytes in the biological sample or wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100, 150. 200, 250. 300, 350, 400. 450, 500 nucleotides, or more apart;

(b) generating a chimeric ligation product by ligating the first probe and the second probe; and

(c) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

2. The method of claim 1, wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 100 nucleotides or more apart.

3. The method of claim 1 or 2, wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 200 nucleotides or more apart.

4. The method of any one of claims 1-3, wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 300 nucleotides or more apart.

5. The method of anyone of claims 1-4, wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 400 nucleotides or more apart.

6. The method of anyone of claims 1-5, wherein the first probe and the second probe in the plurality of probes each comprise sequences that are substantially complementary to a first sequence and a second sequence in the same target nucleic acid that are 500 nucleotides or more apart.

7. A method for determining a location of a chimeric nucleic acid in a biological sample, the method comprising:

(a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to the chimeric nucleic acid present in the biological sample, and wherein the second probe comprises a capture probe capture domain sequence;

(b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample;

(c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid;

(d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality of capture probes; and

(e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the chimeric nucleic acid in the biological sample.

8. A method of determining a location of a blood clot or pool of blood in a biological sample, the method comprising:

(a) contacting a plurality of probes with the biological sample, wherein a first probe and a second probe of the plurality of probes comprise sequences that are substantially complementary to a chimeric nucleic acid in the biological sample, and wherein the second chimeric probe comprises a capture probe capture domain sequence;

(b) hybridizing the first probe and the second probe to the chimeric nucleic acid in the biological sample; (c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid;

(d) hybridizing the chimeric ligation product to a capture domain of a capture probe, wherein the capture probe further comprises a spatial barcode and is comprised in an array comprising a plurality⁷ of capture probes; and

(e) determining (i) all or part of the sequence of the chimeric ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the blood clot or pool of blood in the biological sample.

9. A method of determining presence of a blood clot or pool of blood in a biological sample, the method comprising:

(a) contacting a plurality⁷ of probes with the biological sample, wherein a first probe and a second probe of the plurality⁷ of probes comprise sequences that are substantially complementary⁷ to a chimeric nucleic acid in the biological sample;

(b) hybridizing the first probe and the second probe to the chimeric nucleic acid;

(c) generating a chimeric ligation product by ligating the first probe and the second probe hybridized to the chimeric nucleic acid; and

(d) determining the presence of the chimeric ligation product, or a complement thereof, in the biological sample, wherein a location where the chimeric ligation product is detected corresponds to or indicates a location of the blood clot or pool of blood in the biological sample.

10. The method of anyone of claims 1-9, wherein the location where the chimeric ligation product is detected corresponds to or indicates a location of one or more red blood cells in the biological sample.

11. The method of any one of claims 1-10, further comprising mounting the biological sample on a first substrate.

12. The method of any one of claims 1-11, further comprising amplifying the chimeric ligation product.

13. The method of any one of claims 1-12, wherein the determining step comprises: hybridizing a detectable probe to the chimeric ligation product; and detecting the presence of the detectable probe.

14. The method of claim 13, wherein the detectable probe comprises a fluorescent label or a chromogenic label.

15. The method of any one of claims 1-14, wherein a first region of the biological sample has a number of chimeric ligations products that is greater than a number of chimeric ligation products (A) in a second region of the biological sample or (B) in a test sample compared to a reference sample.

16. The method of any one of claims 1-15, wherein the chimeric ligation product comprises a probe pair selected from the group consisting of:

PRDM4-GTF2A1;

SLC30A5-VPS33B;

IZUM01R-DD0;

OR7D2-UQCRH;

ZNF841-IL18RAP;

MYB-NBPF3;

SLC25A51-SKA1;

OR6C2-SM1M29;

IGKC-IGKC;

FAM72B-NSL1;

CAMKMT-RIDA;

OLFML1-ZFYVE16; and any combination thereof.

17. The method of any one of claims 1-16, wherein the contacting the plurality of probes with the biological sample comprises contacting the biological sample with 5000 or more probe pairs, wherein a probe pair of the 5000 or more probe pairs comprises the first probe and the second probe.

18. The method of any one of claims 1-17, wherein the contacting the plurality of probes with the biological sample comprises contacting the biological sample with 100 or more probe pairs, wherein a probe pair of the 100 or more probe pairs comprises the first probe and the second probe.

19. The method of any one of claims 1-18, wherein the contacting the plurality of probes with the biological sample comprises contacting the biological sample with a single pair of probes that is capable of forming chimeric ligation products in the presence of blood clots or pools of blood in the biological sample.

20. The method of any one of claims 1-19, wherein the contacting the plurality of probes with the biological sample comprises contacting the biological sample with two or more pairs, (e.g.. two pairs, three pairs, four pairs, five pairs, six pairs, seven pairs, eight pairs, nine pairs, ten or more pairs), of probes that are capable of forming chimeric ligation products in the presence of blood clots or pools of blood in the biological sample.

21. The method of claims 18 or 19, wherein the single pair or two or more pairs of probes are selected from the group consisting of:

PRDM4-GTF2A1;

SLC30A5-VPS33B;

IZUMO1R-DDO;

OR7D2-UQCRH;

ZNF841 -IL18RAP;

MYB-NBPF3;

SLC25A51-SKA1;

OR6C2-SMIM29;

IGKC-IGKC;

FAM72B-NSL1;

CAMKMT-RIDA;

OLFML1-ZFYVE16; and any combination thereof.

22. The method of any one of claims 7, 8, and 10-21, wherein the capture probe capture domain sequence is substantially complementary to the capture domain of the capture probe.

23. The method of any one of claims 1-22, wherein the first probe further comprises a primer sequence.

24. The method of any one of claims 7, 8, and 10-21, wherein the capture probe capture domain sequence comprises a poly-adenylated sequence or a complement thereof.

25. The method of any one of claims 1-24, wherein the first probe and/or the second probe is a DNA probe.

26. The method of any one of claims 1-25, wherein the first probe and the second probe hybridize to adjacent sequences on the chimeric nucleic acid.

27. The method of claim 26, wherein the adjacent sequences abut one another.

28. The method of claim 27, wherein the adjacent sequences are at least 1, 2, 3. 4, 5, 6. 7, 8, 9, 10, or more nucleotides away from one another.

29. The method of claim 28, further comprising generating an extended first probe, wherein the extended first probe comprises a sequence substantially complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second chimeric probe.

30. The method of claim 28, further comprising generating an extended second probe using a polymerase, wherein the extended second probe comprises a sequence substantially complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe.

31. The method of any one of claims 7, 8, or 10-30, wherein the array comprising the plurality of capture probes is on the first substrate.

32. The method of any one of claims 7, 8, or 10-30, wherein the array comprising the plurality of capture probes is on a second substrate.

33. The method of claim 32, wherein 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 array of capture probes.

34. The method of claim 33, further comprising, when the biological sample is aligned with at least a portion of the array of capture probes, releasing the chimeric ligation product from the biological sample.

35. The method of any one of claims 1-34, further comprising: contacting the biological sample with a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents comprises an analyte binding moiety and a capture agent barcode domain, wherein the analyte binding moiety specifically binds to a protein from the biological sample, and wherein the capture agent barcode domain comprises an analyte binding moiety barcode and a capture handle sequence; and hybridizing the capture handle sequence to a second capture domain of a second capture probe on the array of capture probes, wherein the second capture probe further comprises a second spatial barcode.

36. The method of claim 35, further comprising determining the sequence of (i) the capture agent barcode domain: and (ii) the second spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the location of the protein in the biological sample.

37. The method of any one of claims 1-36, wherein the generating the chimeric ligation product comprises ligating the first probe to the second probe using enzymatic ligation or chemical ligation, wherein the enzymatic ligation is via a ligase.

38. The method of any one of claims 29-36, wherein the generating the chimeric ligation product comprises ligating the extended first probe to the second probe using enzymatic ligation, wherein the enzymatic ligation is via a ligase.

39. The method of claim 37 or 38, wherein the ligase is one or more of a T4 RNA ligase (Rnl2). a Chlorella virus ligase, a single-stranded DNA ligase, or a T4 DNA ligase.

40. The method of claim 38, wherein the generating the chimeric ligation product comprises ligating the extended first probe to the second probe using chemical ligation.

41. The method of any one of claims 1-40, further comprising releasing the chimeric ligation product.

42. The method of claim 41. wherein the releasing the chimeric ligation product comprises contacting the biological sample with a reagent medium comprising an agent for releasing the chimeric ligation product.

43. The method of claim 42, wherein the agent for releasing the chimeric ligation product comprises a nuclease.

44. The method of claim 43, wherein the nuclease comprises an RNase, optionally wherein the RNase is selected from RNase A, RNase C, RNase H, or RNase I.

45. The method of any one of claims 42-44, wherein the reagent medium further comprises a permeabilization agent, optionally wherein the permeabilization agent comprises a protease.

46. The method of claim 45, wherein the protease is selected from trypsin, pepsin, elastase, or proteinase K.

47. The method of any one of claims 42-46, wherein the reagent medium further comprises a detergent.

48. The method of any one of claims 42-47, wherein the reagent medium further comprises polyethylene glycol (PEG).

49. The method of any one of claims 1-48, wherein the capture domain comprises a poly(T) sequence.

50. The method of any one of claims 1-49, wherein the capture domain comprises a sequence substantially complementary to a sequence in the first probe or the second probe.

51. The method of any one of claims 1-50, wherein the capture probe further comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, and combinations thereof.

52. The method of any one of claims 7-51, wherein the chimeric nucleic acid is DNA.

53. The method of claim 52, wherein the DNA is genomic DNA.

54. The method of any one of claims 7-51, wherein the chimeric nucleic acid is RNA.

55. The method of claim 54, wherein the RNA is mRNA.

56. The method of any one of claims 1-55, wherein the biological sample is a tissue sample.

57. The method of claim 56, wherein the tissue sample is a solid tissue sample.

58. The method of claim 57. wherein the solid tissue sample is a tissue section.

59. The method of any one of claims 1-58, wherein the biological sample is a fixed tissue sample.

60. The method of claim 59, wherein the fixed tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample, preferably an FFPE tissue section.

61. The method of claim 60, wherein the FFPE tissue sample or tissue section is deparaffinized and decrosshnked prior to step (a).

62. The methods of any one of claims 1-58, wherein the biological sample is a fresh- frozen tissue sample, preferably a fresh-frozen tissue section.

63. The methods of any one of claims 1-62, wherein the biological sample is fixed and stained prior to step (a).

64. The method of any one of claims 1-63, wherein the biological sample is stained using immunofluorescence, immunohistochemistry, hematoxylin, and/or eosin.

65. The method of any one of claims 1-64, further comprising imaging the biological sample.

66. The method of any one of claims 1-65, wherein the biological sample is obtained from a subject having deep vein thrombosis, a pulmonary⁷ embolism, or an arterial thrombosis.

67. The method of any one of claims 1-65, wherein the biological sample is obtained from a subject having antiphospholipid antibody syndrome, Factor V Leiden, a prothrombin gene mutation, a Protein C deficiency, a Protein S deficiency, hemophilia, or an ATIII deficiency.

68. The method of any one of claims 1-65, wherein the biological sample is derived from skin, liver, brain, lung, heart, kidney, spleen, pancreas, and/or tonsils of a subject.

69. The method of any one of claims 66-68. wherein the subject is selected from a mouse, a rat, a rabbit, a guinea pig, an ungulate, a horse, a sheep, a pig, a goat, a cow, a cat, or a dog.

70. The method of claim 69, wherein the subject is a human.

71. The method of any one of claims 1-70, further comprising extending the chimeric ligation product using the capture probe as a template, thereby generating an extended chimeric ligation product.

72. The method of any one of claims 1-70, further comprising extending the capture probe using the chimeric ligation product as a template, thereby generating an extended capture probe.

73. The method of claim 71 or 72, wherein determining the sequence of (i) and (ii) comprises determining the sequence of the extended chimeric ligation product, or a complement thereof, or the extended capture probe, or the complement thereof.

74. The method of any one of claims 71-73, wherein the determining comprises sequencing the extended chimeric ligation product, or a complement thereof, or the extended capture probe, or the complement thereof.

75. A method of diagnosing a subject with a blood clot, the method comprising performing the method of any one of claims 1-74, and diagnosing the subject as having a blood clot.

76. The method of claim 75, wherein the subject has had a mini-stroke.

77. A method of diagnosing a subject with a one or more of antiphospholipid antibody syndrome, Factor V Leiden, a prothrombin gene mutation, a Protein C deficiency, a Protein S deficiency, hemophilia, or an ATIII deficiency, the method comprising performing the method of any one of claims 1-74, and diagnosing the subject as having one or more of anti phospholipid antibody syndrome, Factor V Leiden, a prothrombin gene mutation, a Protein C deficiency, a Protein S deficiency, hemophilia, or an ATIII deficiency.

78. A method of identifying a subject as having an increased likelihood of having a blood clot comprising performing any one of the methods of any one of claims 1-74, and identifying a subject as having an increased likelihood of having the blood clot.

79. The method of any one of claims 75-78, further comprising administering to the subject one or more anticoagulant medications.

80. The method of claim 79. wherein the one or more anticoagulant medication is selected from aspirin, warfarin, heparin, low-molecular weight heparin, fondaparinux, rivaroxaban, apixaban, dabigatran, or any combination thereof.

81. The method of any one of claims 75-80, wherein the blood clot is removed using minimally invasive interventional radiology.

82. A system comprising:

(a) a support device configured to retain a first substrate and a second substrate, wherein a biological sample is placed on the first substrate, and wherein the second substrate comprises a plurality of capture probes, wherein a capture probe of the plurality’ of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;

(b) a delivery’ means to deliver a first probe and a second probe to the biological sample, wherein the first probe and the second probe each comprise a sequence that is substantially complementary' to adjacent sequences of an analyte, wherein the second probe comprises a capture probe binding domain, and wherein the first probe and the second probe are capable of being ligated together to form a chimeric ligation product; and

(c) a location within the first substrate or the second substrate comprising a reagent medium comprising a permeabilization agent and optionally an agent for releasing the chimeric ligation product.

83. The system of claim 82, wherein the permeabilization agent is pepsin or proteinase K.

84. The system of claim 82 or 83, wherein the agent for releasing the chimeric ligation product is an RNAse, optionally wherein the RNAse is selected from RNase A, RNase C, RNase H, or RNase I.

85. The system of any one of claims 82-84, further comprising an alignment mechanism on the support device to align the first substrate and the second substrate.

86. The system of claim 85, wherein the alignment mechanism comprises a linear actuator, wherein the first substrate comprises a first member and the second substrate comprises a second member, and optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to a plane or the first member and/or the second member, and/or 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, and/or 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, and/or 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.

87. The system of any one of claims 82-86, wherein the chimeric ligation product comprises a probe pair selected from the group consisting of:

PRDM4-GTF2A1;

SLC30A5-VPS33B;

IZUM01R-DD0;

OR7D2-UQCRH;

ZNF841-IL18RAP;

MYB-NBPF3;

SLC25A51-SKA1;

OR6C2-SMIM29;

IGKC-IGKC;

FAM72B-NSL1;

CAMKMT-RIDA;

OLFML1-ZFYVE16; and any combination thereof.

88. A kit comprising:

(a) a support device configured to retain a first substrate and a second substrate, wherein a biological sample is placed on the first substrate, and wherein the second substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;

(b) a delivery means to deliver a first probe and a second probe to the biological sample, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to adjacent sequences of an analyte, wherein the second probe comprises a capture probe binding domain, and wherein the first probe and the second probe are capable of being ligated together to form a chimeric ligation product; (c) a location within the first substrate or the second substrate comprising a reagent medium comprising a permeabilization agent and optionally an agent for releasing the chimeric ligation product; and

(d) instructions for performing the method of any one of claims 1- 74.

89. The kit of claim 88, wherein the permeabilization agent is pepsin or proteinase K.

90. The kit of claim 88 or 89, wherein the agent for releasing the chimeric ligation product is an RNAse, optionally wherein the RNAse is selected from RNase A, RNase C, RNase H, or RNase I.

91. The kit of any one of claims 88-90, further comprising an alignment mechanism on the support device to align the first substrate and the second substrate.

92. The kit of claim 91, wherein the alignment mechanism comprises a linear actuator, wherein the first substrate comprises a first member and the second substrate comprises a second member, and optionally wherein: the linear actuator is configured to move the second member along an axis orthogonal to a plane or the first member and/or the second member, and/or 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, and/or 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, and/or 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.