WO2024015578A1

WO2024015578A1 - Methods for determining a location of a target nucleic acid in a biological sample

Info

Publication number: WO2024015578A1
Application number: PCT/US2023/027779
Authority: WO
Inventors: Mohammadreza MIRZAZADEH; Zaneta ANDRUSIVOVA; Ludvig Ale Larsson; Leire Alonso GALICIA; Xesus Manoel ABALO PINEIRO; Joakim Lundeberg
Original assignee: 10X Genomics, Inc.
Priority date: 2022-07-15
Filing date: 2023-07-14
Publication date: 2024-01-18

Abstract

The present disclosure relates to methods, compositions, and kits for determining the location of target RNA in a biological sample and/or rescuing target RNA, from which spatial gene expression analysis is difficult to determine, from fresh-frozen biological samples.

Description

METHODS FOR DETERMINING A LOCATION OF A TARGET NUCLEIC ACID IN A BIOLOGICAL SAMPLE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/389,566, filed on July 15, 2022 and U.S. Provisional Patent Application No. 63/403,538, filed on September 2, 2022. The contents of each of these applications are incorporated herein by reference in their entireties.

BACKGROUND

Cells within a tissue 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, signaling, and cross-talk with other cells in the tissue.

Spatial heterogeneity has been previously studied using techniques that typically provide data for a handful of analytes in the context of intact tissue or a portion of a tissue (e.g., tissue section), or provide significant analyte data from individual, single cells, but fails to provide information regarding the position of the single cells from the originating biological sample (e.g., tissue).

Spatial transcriptomics has enabled precise genome-wide mRNA expression profiling in various biological samples. Successful performance of spatial transcriptomic methods generally target poly (A) tails of mRNA and rely on the availability of biological samples with high quality RNA. However, some biological samples have been found to be suboptimal for spatial analysis via such methods, including fresh-frozen biological samples, thus additional methods are still needed.

SUMMARY

Spatial transcriptomics has enabled precise genome-wide mRNA expression profiling in various biological samples. At present methods, compositions, and kits for spatially capturing analytes from a biological sample (e.g., a fresh-frozen) biological sample have been previously described. However, there remains a need to spatially capture and/or rescue analytes from biological samples from which it has proved challenging to obtain useful spatial information. Moreover, spatial transcriptomic methods require a careful sample screening process to ensure high quality data to conserve time and costs, so improved methods that have an increased likelihood of providing valuable results without first understanding the quality of a biological sample are still needed. The present disclosure features methods, compositions, and kits for determining the location of nucleic acids from fixed, fresh-frozen biological samples via capture of proxies of target nucleic acids (e.g., templated-ligation probes) by a plurality of capture probes, where a capture probe of the plurality of capture probes includes a capture domain and a spatial barcode. The present disclosure also features methods, compositions, and kits for rescuing nucleic acids from fixed, fresh-frozen biological samples. The methods described herein show improved capture of target mRNA from fresh-frozen biological samples with moderate- to-low RNA quality that have been fixed, by demonstrating the disclosed methods on challenging biological samples including human lung, colon, small intestine, childhood brain tumor samples, and mouse bone and cartilage tissue samples, which have proven to be challenging biological samples from which to obtain spatial transcriptomic data.

Provided herein are methods for rescuing degraded target RNA for determining spatial gene expression in a fresh-frozen biological sample, the method including: (a) providing a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) fixing the fresh-frozen biological sample; (c) heating the fresh-frozen biological sample; (d) contacting the fresh-frozen biological sample with: (i) a first probe, where the first probe includes a first functional domain and a first sequence that is substantially complementary to a portion of the target RNA, and (ii) a second probe, where the second probe includes a second sequence that is substantially complementary to a portion of the target RNA and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array; (e) hybridizing the first probe and the second probe to the target RNA and ligating the first probe to the second probe to generate a proxy of the target RNA; and (f) hybridizing the proxy of the target RNA to the capture domain of the capture probe on the spatial array, thereby rescuing degraded target RNA for determining spatial gene expression in the fresh-frozen biological sample.

In some embodiments, the fresh-frozen biological sample is disposed on the spatial array including a plurality of capture probes. In some embodiments, the fresh-frozen biological sample is disposed on a substrate. In some embodiments, the method includes aligning the substrate with the spatial array, such that at least a portion of the fresh-frozen biological sample is aligned with at least a portion of the spatial array.

In some embodiments, the target RNA is mRNA.

In some embodiments, the spatial array includes one or more features selected from the group consisting of: a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead.

In some embodiments, the first functional domain is a first sequencing handle. In some embodiments, the capture sequence is substantially complementary to the capture domain or a portion thereof.

In some embodiments, the plurality of capture probes includes a cleavage domain, one or more functional domains, a unique molecular identifier, or combinations thereof.

In some embodiments, the method includes imaging the fresh-frozen biological sample. In some embodiments, the method includes staining the fresh-frozen biological sample. In some embodiments, the staining includes eosin and hematoxylin. In some embodiments, the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a biolurmnescent compound, or a combination thereof.

In some embodiments, the fixing includes the use of methanol-free formalin. In some embodiments, the methanol-free formalin is at about 2% (v/v) to about 6% (v/v). In some embodiments, the methanol-free formalin is at about 4% (v/v). In some embodiments, the fixing is performed from about 5 minutes to about 20 minutes. In some embodiments, the fixing is performed for about 10 minutes. In some embodiments, the fixing is performed at about 15°C to about 25°C. In some embodiments, the fixing is performed at about 20°C.

In some embodiments, the heating in step (c) is performed after the fixing in step (b). In some embodiments, the heating is performed at about 25°C to about 45°C. In some embodiments, the heating is performed at about 37°C. In some embodiments, the heating is performed from about 5 minutes to about 45 minutes. In some embodiments, the heating is performed from about 15 minutes to about 30 minutes. In some embodiments, the heating is performed for about 20 minutes.

In some embodiments, the ligating of the first probe to the second probe is performed using a ligase, where the ligase is selected from the group consisting of: Tth DNA ligase, Taq DNA ligase, Thermococcus sp. DNA ligase, AMPLIGASE, PBCV-1 DNA Ligase, and Chlorella virus DNA Ligase. In some embodiments, the method includes migrating the proxy of the target RNA to the spatial array, where the migrating includes electrophoresis.

In some embodiments, the method includes permeabilizing the fixed, fresh-frozen biological sample, where the permeabilizing includes the use of a protease.

In some embodiments, the fresh-frozen biological sample is a fresh-frozen tissue section.

In some embodiments, the method includes, after step (c), a step of treating the fresh- frozen biological sample with an RNase.

In some embodiments, the method includes in step (e) extending the capture probe thereby generating an extended capture probe. In some embodiments, the method includes in step (e) generating a second strand hybridized to the capture probe, where the second strand includes a sequence complementary to the spatial barcode and a nucleic acid sequence corresponding to the proxy of the target RNA.

In some embodiments, the method includes step (g) determining (i) the sequence of the spatial barcode, or a complement thereof, and (ii) the sequence of all or a portion of the proxy of the target RNA, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target RNA in the fresh-frozen biological sample. In some embodiments, the determining in step (g) includes sequencing. In some embodiments, the sequencing is high-throughput sequencing.

Also provided herein are methods for determining the location of a target RNA in a fresh-frozen biological sample, the method including: (a) providing a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) fixing the fresh-frozen biological sample; (c) heating the fresh-frozen biological sample; (d) contacting the fresh-frozen biological sample with: (i) a first probe, where the first probe includes a first functional domain and a first sequence that is substantially complementary to a portion of the target RNA, and (ii) a second probe, where the second probe includes a second sequence that is substantially complementary' to a portion of the target RNA and a capture sequence that is substantially complementary' to the capture domain of the capture probe on the spatial array; (e) hybridizing the first probe and the second probe to the target RNA and ligating the first probe to the second probe to generate a proxy of the target RNA; (f) hybridizing the proxy of the target RNA to the capture domain of the capture probe on the spatial array, extending the capture probe thereby generating an extended probe, and generating a second strand hybridized to the capture probe, where the second strand includes a sequence complementary to the spatial barcode and a nucleic acid sequence corresponding to the proxy of the target RNA; and (g) determining (i) the sequence of the spatial barcode, or a complement thereof, and (li) all or a portion of the proxy of the target RNA, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target RNA in the fresh- frozen biological sample.

In some embodiments, the target RNA is mRNA.

In some embodiments, the method includes imaging the fresh-frozen biological sample In some embodiments, the method includes staining the fresh-frozen biological sample. In some embodiments, the staining includes eosin and hematoxylin. In some embodiments, the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

In some embodiments, the heating in step (c) is perfomied after the fixing in step (b). In some embodiments, the heating is performed at about 25°C to about 45°C. In some embodiments, the heating is performed at about 37°C. In some embodiments, the heating is performed from about 5 minutes to about 45 minutes. In some embodiments, the heating is performed from about 15 minutes to about 30 minutes. In some embodiments, the heating is performed for about 20 minutes.

In some embodiments, the determining in step (g) includes sequencing. In some embodiments, the sequencing is high-throughput sequencing.

In some embodiments, the ligating of the first probe to the second probe is performed using a ligase selected from the group consisting of: Tth DNA ligase, Taq DNA ligase, Thermococcus sp. DNA ligase, AMPLIGASE, PBCV-1 DNA Ligase, and Chlorella virus DNA Ligase.

In some embodiments, the method includes migrating the proxy of the target RNA to the spatial array, where the migrating includes electrophoresis.

Also provided herein are compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain: (b) a fixed, fresh-frozen biological sample disposed on the spatial array where the biological sample has been heated while disposed on the spatial array; and (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence that is hy bridized to a portion of a target RNA, and a second probe of the plurality of probes, where the second probe includes a second sequence that is hybridized to a portion of the target RNA and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array, where the first probe and the second probe of the plurality of probes are ligated, thereby generating a plurality of proxies of the target RNA, and an RNase.

In some embodiments, the composition includes one or more proxies of the target RNA that are not hybridized to the target RNA.

In some embodiments, the capture sequence of one or more proxies of the target RNA is hybridized to the capture domain of the capture probe on the spatial array.

In some embodiments, the composition includes extended capture probes, where an extended capture probe is generated using the proxy of the target RNA as a template. In some embodiments, the composition includes extended proxies of the target RNA, where an extended proxy of the target RNA is generated using the capture probe as a template.

In some embodiments, the target RNA is mRNA.

In some embodiments, the fixed, fresh-frozen biological sample is fixed in about 4% (v/v) methanol-free formalin.

Also provided herein are kits including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a fixative; (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence that is substantially complementary to a portion of a target nucleic acid, a second probe of the plurality of probes, where the second probe includes a second sequence that is substantially complementary' to a portion of the target nucleic acid and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array; and (d) instructions for performing any of the methods described herein.

In some embodiments, the kit includes one or more permeabilization reagents. In some embodiments, the one or more permeabilization reagents includes one or more proteases.

In some embodiments, the kit includes one or more polymerases.

In some embodiments, the fixative is methanol-free formalin. In some embodiments, the methanol-free formalin is about 4% (v/v).

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 ordinary 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, 1 , 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 every 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. 1 is a schematic diagram showing an example of a barcoded capture probe, as described herein.

FIG. 2 is an exemplary workflow for practicing RNA rescue (RR) in a tissue sample for downstream spatial analy te analysis.

FIGs. 3A-B shows exemplary genes per spot violin plot expression data comparing RR tissue preparation protocol against standard Visium fresh-frozen (FF) tissue preparation protocol for A) prostate cancer tissue sections, and B) mouse brain tissue sections. The X axis shows the duplicate assays (Reps) and the Y axis is number of unique genes/spot.

FIGs. 4A-B shows exemplary human lung tissue spatial transcriptomics data using the A) RR tissue preparation protocol compared to the B) standard Visium FF tissue preparation protocol for human lung with an RNA Integrity Number (RIN)=7. 1. Top left is an image of the H&E stained tissue section, top right are the data for unique genes/spot and UMI counts/spot violin plots (Y axis is number counted), bottom left is a heat map of the unique genes in the tissue section, bottom right are the viable data (keep) and the data that was not considered useful (discard).

FIG. 5 shows exemplary human colon tissue data for the RR protocol (RRST) compared to standard Visium protocol (Standard) performed on tissue sections with RIN=5.1. The pictures (left) represent heat maps of unique genes for the standard Visium data (n=l) and n=2 replicate RR protocol data, and violin plots (right) for numbers of unique genes for each condition and replicate (n=2 RR protocol).

FIG. 6 shows exemplary data from the RR protocol (RRST) compared to standard Visium protocol (Standard) in evaluating the spatial gene expression in pediatric brain tumor (medulloblastoma) tissue sections. Two replicates for each protocol per sample were evaluated and violin plots of number of unique genes per spot are shown. For each sample (sample 1 and sample 2), the left two replicate data are from tissue sections processed using the standard Visium protocol, and the right two replicate data are from tissue sections processed using the RR protocol. X axis is replicate number and Y axis is number of unique genes on a log 10 scale.

FIG. 7 shows exemplary data for mouse bone/cartilage tissue sections when practicing the RR protocol. Two tissue samples, day 4 and day 11 postnatal, n=2 were assayed for gene expression, the violin plots show the numbers of unique genes per spot for each replicate.

FIG. 8 is a schematic diagram depicting an exemplary analyte transfer process between a first substrate comprising a biological sample and a second substrate comprising a spatially barcoded 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.

FIGs. 10A-B are schematic diagrams depicting exemplary analyte transfer embodiments. FIG. 10A shows an exemplary analyte transfer process, or sandwiching process, where a first substrate including a biological sample and a second substrate are brought into proximity with one another and a liquid reagent drop is introduced on the second substrate in proximity to the capture probes and in between the biological sample. FIG. 10B shows a fully formed sandwich configuration creating a chamber formed from one or more spacers, the first substrate, and the second substrate including spatially barcoded capture probes.

FIGs. 11A-C show exemplary human small intestine spatial transcriptomic data.

FIG. 11A shows an H&E stained small intestine tissue section (top) and spots colored by five major tissue regions: mucosa, Tertiary Lymphoid Tissue (TLS), submucosa, muscularis, and serosa (bottom). FIG. 11B shows data for unique genes/spot in the five major regions of the small intestine tissue prepared from the same fresh-frozen OCT block at about 1 month, about 6 months, and about 2 years after preparation. The standard Visium FF tissue preparation protocol was used at 1 month and 6 months and the RRST protocol was performed at about 2 years after preparation. FIG. 11C shows spatial visualization of five enterocyte markers (ANPEP; RBP2; DGAT1; FABP2; and APOB) with small intestine tissue sections at about 1 month, about 6 months, and about 2 years after initial preparation of the fresh-frozen OCT block compared with H&E stained tissue section for each time period. The standard Visium FF tissue preparation protocol was used at 1 month and 6 months and the RRST protocol was performed at about 2 years after preparation.

DETAILED DESCRIPTION

Spatial transcriptomics generally describes the field of charting genome-wide mRNA expressions within the spatial context of a tissue and has grown immensely in recent years. This technology allows researchers to look at the spatial architecture of tissues and interactions of cells within the spatial context of a tissue. Additional technological advances in next-generation sequencing (NGS) and imaging-based approaches have enhanced spatial transcriptomics ability to measure expression levels and have been adopted to generate insights in neuroscience, developmental biology, plant biology, and to investigate a range of disease contexts, including various forms cancer.

Methods, compositions, and kits for spatially capturing analytes from a biological sample (e.g., a fresh-frozen) biological sample have been previously described. However, there remains a need to spatially capture analytes from biological samples, such as fresh frozen or fixed biological samples, from which it has proved challenging to obtain useful spatial information. The present disclosure features methods, compositions, and kits for the spatial capture of proxies of target nucleic acids (e.g., templated-ligation probes) by a plurality of capture probes, where a capture probe of the plurality of capture probes includes a capture domain and a spatial barcode. The methods described herein show improved capture and/or rescue of target nucleic acids (e.g., mRNA) from biological samples with moderate-to- low RNA quality, as well as, several biological samples including human lung, colon, small intestine, childhood brain tumor samples (e.g., medulloblastoma), and mouse bone and cartilage tissue samples which have proven to be challenging biological samples from which to obtain spatial transcriptomic data. The methods described herein include fixing and heating fresh-frozen biological samples either directly on a spatial array or on a substrate that is then aligned with a spatial array.

Spatial analysis methodologies and compositions 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 and compositions 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. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663 , 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, 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; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the lOx Genomics Support Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in their entireties. 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. 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-lmked), 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. 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, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). 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 (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabihzed 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)(13) or the Exemplary Embodiments Section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or more analytes 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 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

FIG. 1 is a schematic diagram showing an exemplary capture probe, as described herein. As show n, the capture probe 102 is optionally coupled to a feature 101 by a cleavage domain 103, such as a disulfide linker. The capture probe can include a functional sequence 104 that is useful for subsequent processing. The functional sequence 104 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 105. The capture probe can also include a unique molecular identifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode 105 as being located upstream (5’) of UMI sequence 106, it is to be understood that capture probes wherein UMI sequence 106 is located upstream (5’) of the spatial barcode 105 is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain 107 to facilitate capture of a target analyte. In some embodiments, the capture probe comprises one or more additional functional sequences that can be located, for example between the spatial barcode 105 and the UMI sequence 106, between the UMI sequence 106 and the capture domain 107, or following the capture domain 107. 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 capture handle sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. Such splint oligonucleotide, in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence of a nucleic acid analyte, and/or a capture handle sequence described herein. 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 105 and functional sequences 104 is common to all of the probes attached to a given feature (e.g., a bead, a well, a spot on an array). In some embodiments, the UMI sequence 106 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.

In some instances, a capture probe and a nucleic acid analyte (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 in the other nucleic acid sequence. The complementary residues within a particular complementary nucleic acid sequence need not always be contiguous with each other, and 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 in 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 complementary to residues in the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the residues of one nucleic acid sequence are complementary' to residues in the other nucleic acid sequence. 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. During this process, one or more analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) are 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, WO 2022/061152, and WO 2022/140028.

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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). 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 ligations products that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 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 reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode 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., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions 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 No. 2021/0140982A1, U.S. Patent Application No. 2021/0198741A1, and/or U.S. Patent Application No. 2021/0199660.

Spatial information can provide information of biological importance. For example, the methods and compositions 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 analysis); determination of up- and/or dow n-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 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).

Typically, for spatial array-based methods, a substrate functions 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

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, 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

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 plurality 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

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. 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 two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR 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 between 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). 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.

A non-limiting example of templated ligation methods disclosed herein is depicted in FIG. 9A. In one scenario, 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 targethybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a target-hybridization sequence 905 and a capture domain (e.g., a poly-A sequence) 906, the first probe 901 and a second probe 904 hybridize 910 to an analyte 907. A ligase 921 ligates 920 the first probe to the second probe thereby generating a ligation product 922. The ligation product is released 930 from the analyte 931 by digesting the analyte using an endoribonuclease 932, for example. The biological sample is permeabilized 940 and the ligation product 941 is able to hybridize to a capture probe on the substrate. Methods and composition for spatial detection using templated ligation have been described in PCT Publ. 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. The same process of probe hybridization and ligation can occur in a biological sample that is not on an arrayed substrate, but instead is on a plain slide, for example. Once the ligation product is formed, the ligation product can migrate to a second substrate that has arrayed capture probes thereon. For example, the biological sample on a slide that includes ligation products can be placed, or sandwiched, face to face with an arrayed area of a slide wherein the ligation products are subsequently migrated to the arrayed capture area on the second substrate.

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 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 bind to the captured ligated probe (i.e., compared to no permeabilization). In some embodiments, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can extend the capture probes 9011 to produce spatially-barcoded full-length cDNA

9012 and 9013 from the capture probes and the captured ligation products. Extension reagents (e.g., dNTPs, enzymes) can be added to the biological sample on the slide to initiate extension of the captured ligation products and the capture probes.

In some embodiments, methods provided herein include permeabilization of the biological sample such that the capture probes can more easily capture the ligation products (i.e., compared to no permeabilization). In some embodiments, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can extend the capture probes 9011 to produce spatially-barcoded full-length cDNA 9012 and

9013 from the captured ligation products.

In some embodiments, the extended ligation products can be denatured 9014 from the capture probes 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, and can be used as flowcell sequencing capture probes (P5 and P7) sample sequencing indexes (i5 and i7). The amplicons can then be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 sequences that are incorporated into the extension products as sequencing primer sites.

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 analytes they capture 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 spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods descnbed 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, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some nonlimiting examples of the workflows described herein, the sample can be immersed .. . ” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020). 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 WO 2020/176788 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 WO 2020/123320.

Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or 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 (in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perfomi 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 WO 2021/102003 and/or U.S. Patent Application Serial No. 16/951,854, each of which is incorporated herein by reference in their entireties.

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- and/or three-dimensional map of the analyte presence and/or level are described in WO 2021/067514 and spatial analysis methods are generally described in WO 2021/102039 and/or U.S. Patent Application Serial No. 16/951,864, each of which is incorporated herein by reference in their entireties.

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 WO 2020/123320, WO 2021/102005, and/or U.S. Patent Application Serial No. 16/951,843, each of which is incorporated herein by reference in their entireties. 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.

Spatial Analysis in Fixed, Fresh-Frozen Biological Samples

The present disclosure features methods, compositions, and kits for the spatial capture of proxies of target nucleic acids (e.g., templated-ligation probes) by a plurality of capture probes, where a capture probe of the plurality of capture probes includes a capture domain and a spatial barcode. The methods described herein show improved capture of target nucleic acids (e.g., mRNA) from fresh-frozen biological samples with moderate-to-low RNA quality' that have been fixed, as well as, several biological samples including human lung, colon, small intestine, childhood brain tumor samples (e.g., medulloblastoma), and mouse bone and cartilage tissue samples which have proven to be challenging biological samples from which to obtain spatial transcriptomic data. The methods described herein include fixing fresh- frozen biological samples and heating the fixed, fresh-frozen biological sample either directly on a spatial array or on a substrate that is then aligned with a spatial array. In some examples, fixing is performed with methanol-free formalin instead of a methanol-based fixation.

Provided herein are methods for determining the location of analytes present in fixed, fresh-frozen biological samples. Also provided herein are methods of rescuing target nucleic acids from fixed, fresh-frozen biological samples. While accessing analytes in fresh-frozen biological samples can be performed, efficiently capturing and/or amplifying analytes from certain types of biological samples for spatial analysis can present challenges. For example, RNA (e.g., mRNA) in some fresh-frozen biological samples is generally of moderate quality based on its RNA Integrity Number (RIN) score. As used herein, RNA of moderate quality generally has a RIN score between about 5 to about 7. RNA of moderate quality may have missing or truncated poly(A) tails that reduce the efficiency of capture with a poly(T) capture domain of a capture probe on the spatial array. In another example, some biological samples have proven challenging to obtain spatial information with capture probes designed to capture the poly(A) tail of mRNA including human lung, colon, small intestine, childhood brain tumor samples (e.g., medulloblastoma), and mouse bone and cartilage tissue samples. As such, capture and/or amplification efficiency of such target nucleic acids (e.g., mRNA) with spatial arrays described herein can be reduced for any of the above reasons. The “rescue” or “rescuing” of target nucleic acids relates to the capture of the nucleic acid, such as RNA, that may lack a poly(A) tail, or where the poly(A) tail may be truncated, or where the RIN score for RNA integrity suggests that that integrity of the RNA is moderate to high, but in reality, its capture in a spatial setting does not prove to be the case. As such, the terms “rescue” or “rescuing” of target nucleic acids means methods and compositions for capture and spatial determinations of target nucleic acids from a biological sample that might otherwise have failed using established methods. Also, provided herein are methods for improving the efficiency of capture and/or amplification of target nucleic acids (e.g., nucleic acid, any of the nucleic acids described herein including rescued nucleic acids) in fixed, fresh-frozen biological samples.

Also, provided herein are methods for determining the location of a target RNA in a fresh-frozen biological sample, the method including: (a) providing a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) fixing the fresh-frozen biological sample; (c) heating the fresh-frozen biological sample; (d) contacting the fresh-frozen biological sample with: (i) a first probe, where the first probe includes a first functional domain and a first sequence that is substantially complementary to a portion of the target RNA, and (ii) a second probe, where the second probe includes a second sequence that is substantially complementary to a portion of the target RNA and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array; (e) hybridizing the first probe and the second probe to the target RNA and ligating the first probe to the second probe to generate a proxy of the target RNA; (f) hybridizing the proxy of the target RNA to the capture domain of the capture probe on the spatial array, extending the capture probe thereby generating an extended probe, and generating a second strand hybridized to the capture probe, where the second strand includes a sequence complementary to the spatial barcode and a nucleic acid sequence corresponding to the proxy of the target RNA; and (g) determining (i) the sequence of the spatial barcode, or a complement thereof, and (ii) all or a portion of the proxy of the target RNA, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target RNA in the fresh- frozen biological sample.

The fresh-frozen biological sample can be any of the biological samples described herein. For example, in some embodiments, the fresh-frozen biological sample is a tissue sample (e.g., a tissue section). In other embodiments, the fresh-frozen biological sample is a clinical sample (e.g., whole blood, blood-derived products, blood cells, cultured tissue, cultured cells, or a cell suspension). In some embodiments, the fresh-frozen biological sample is an organoid, embryonic stem cells, pluripotent stem cells, or any combination thereof. Non-limiting examples of an organoid include 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, a retinal organoid, or any combination thereof. In other example embodiments, the fresh-frozen biological sample can include diseased cells, fetal cells, immune cells, cellular macromolecules, organelles, extracellular polynucleotides, or any combination thereof.

Non-limiting examples of target nucleic acids include nucleic acids such as DNA or RNA. Non-limiting examples of target DNA analytes include genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and viral DNA.

Non-limiting examples of target RNA analytes include various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNA can be a transcript (e.g., present in a tissue section). The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or singlestranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA). The RNA can be from an RNA virus, for example RNA viruses from Group III, IV or V of the Baltimore classification system. The RNA can be from a retrovirus, such as a virus from Group VI of the Baltimore classification system.

In some embodiments, the array includes one or more features. In some embodiments, features are directly or indirectly attached or fixed to a substrate. In some embodiments, the features are not directly or indirectly attached or fixed to a substrate, but instead, for example, are disposed within an enclosed or partially enclosed three dimensional space (e.g., wells or divots). For example, the plurality of capture probes can be located on features on a substrate. In some embodiments, features include, but are not limited to, a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead (e.g., a hydrogel bead). In some embodiments, the fresh-frozen biological sample can be fixed. In some embodiments, the biological sample is fixed directly on the spatial array. In some embodiments, the biological sample is fixed on a substrate (e.g., a slide) and aligned with the spatial array (e g., sandwiched). In some embodiments, fixing the fresh-frozen biological sample includes the use of methanol-free formalin. In some embodiments, the methanol-free formalin is at least about 0.1% (v/v), at least about 0.5% (v/v), at least about 1% (v/v), at least about 1.5% (v/v) at least about 2% (v/v), at least about 2.5% (v/v), at least about a 3% (v/v), at least about 3.5% (v/v), at least about 4% (v/v), at least about 4.5% (v/v), at least about 5% (v/v), at least about 5.5% (v/v), at least about 6% (v/v), at least about 7% (v/v), at least about 8% (v/v), at least about 9% (v/v), at least about 10% (v/v), at least about 11% (v/v), at least about 12% (v/v), at least about 13% (v/v), at least about 14% (v/v), at least about 15% (v/v), at least about 16% (v/v), at least about 17% (v/v), at least about 18% (v/v), at least about 19% (v/v), at least about 20% (v/v), at least about 21% (v/v), at least about 22% (v/v), at least about 23% (v/v), at least about 24% (v/v), at least about 25% (v/v), or more. In some embodiments, the methanol-free formalin is between about 0.1% (v/v) and about 10% (v/v), between about 0.5% (v/v) and about 9.5%, between about 1% (v/v) and about 9% (v/v), between about 1.5% (v/v) and about 8.5% (v/v), between about 2% (v/v) and about 8.0% (v/v), between about 2.5% (v/v) and 7.5% (v/v), between about 3% (v/v) and about 7.5% (v/v), between about 3.5% (v/v) and about 7.0% (v/v), or between about 4% (v/v) and about 6.5% (v/v).

In some embodiments, the fresh-frozen biological sample is fixed for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes or more. In some embodiments, the fresh-frozen biological sample is fixed at about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51 °C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C or more.

In some embodiments, the fresh-frozen biological sample is fixed for about 10 minutes at about 20°C in about 4% methanol-free formalin.

In some embodiments, the fresh-frozen biological sample is heated. In some embodiments, the fresh-frozen biological sample is heated directly on the spatial array. In some embodiments, the fresh-frozen biological sample is heated on a substrate (e.g., a slide) and aligned with the spatial array (e.g., sandwiched). In some embodiments, the fresh-frozen biological sample is heated after the fresh-frozen biological sample is fixed. In some embodiments, the fresh-frozen biological sample is heated to about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51 °C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81 °C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C or more.

In some embodiments, the fresh-frozen biological sample is heated for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes or more.

In some embodiments, the fresh-frozen biological sample is heated for about 20 minutes at about 37°C.

In some embodiments, the fresh-frozen biological sample can be stained. In some embodiments, the fresh-frozen biological sample is stained after fixation. In some embodiments, the fresh-frozen biological sample is stained before fixation. In some embodiments, the staining includes optical labels as described herein, including, but not limited to, fluorescent (e.g., fluorophore), radioactive (e.g., radioisotope), chemiluminescent (e.g., a chemiluminescent compound), a bioluminescent compound, calorimetric, or colorimetric detectable labels. In some embodiments, the staining includes a fluorescent antibody directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample. In some embodiments, the staining includes an immunohistochemistry stain directed to a target analyte (e.g., cell surface or intracellular proteins) in the fresh-frozen biological sample. In some embodiments, the staining includes a chemical stain, such as hematoxylin and eosin (H&E) or periodic acid-schiff (PAS). In some embodiments, staining the fresh-frozen biological sample includes the use of a biological stain including, but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, safranin, or any combination thereof. In some embodiments, significant time (e.g., days, months, or years) can elapse between staining and/or imaging the biological sample.

In some embodiments, the fresh-frozen biological sample is imaged. In some embodiments, the fresh-frozen biological sample is imaged after fixation. In some embodiments, the fresh-frozen biological sample is imaged before fixation. In some embodiments, imaging includes one or more of expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. In some embodiments, the fixed, fresh-frozen biological sample is permeabilized. Permeabilization of a fixed, fresh-frozen (e.g., methanol-free formalin) biological sample can occur on a substrate where the substrate is aligned with the array such that at least a portion of the fixed, fresh-frozen biological sample is aligned with at least a portion of the array or directly on an array including a plurality of capture probes. In some embodiments, the fresh- frozen biological sample is permeabilized with a protease. In some embodiments, the protease is one or more of pepsin. Proteinase K, and collagenase.

The fresh-frozen biological sample can be applied to any of the variety of arrays described herein. In some embodiments, the plurality of capture probes include in a 5’ to a 3’ direction, a spatial barcode and a capture domain. In some embodiments, the capture domain hybridizes to a capture sequence. In some embodiments, the capture domain is a poly(T) capture domain. In some embodiments, the capture domain is not a poly(T) sequence. For example, the capture domain and the capture sequence of the second probe can be any sequence as long as the sequences are substantially complementary to one another to facilitate hybridization.

In some embodiments, a capture probe can include one or more functional domains, and/or a cleavage domain. A functional domain typically includes a functional nucleotide sequence for a downstream analytical step in the overall analysis procedure. In some embodiments, the functional domain can include a sequencing handle. In some embodiments, the functional domain can include an amplification (e g., PCR) handle. In some embodiments, a capture probe can include a unique molecular identifier as described herein. In some embodiments, the unique molecular identifier is located 5’ to the capture domain in the capture probe.

As described above, the location of target nucleic acids in a fixed, fresh-frozen biological sample can be identified using templated ligation probes. For example, the method can include contacting the fixed, fresh-frozen biological sample with a first probe and a second probe. In some embodiments, the first probe can have in a 5’ to a 3’ direction: a first functional domain and a first sequence that is substantially complementary to a portion of the target nucleic acid. In some embodiments, the first probe can have a 3’ end that includes a 3’ diribo sequence. In some embodiments, the first probe can have a 3’ end that includes a 3’ deoxynucleotide sequence. For example, the first probe can be a DNA/RNA hybrid probe. In some embodiments, the first probe can be a DNA probe. In some embodiments, the second probe can have in a 5’ to a 3’ direction: a second sequence that is substantially complementary' to a portion of the target nucleic acid and a capture sequence. In some embodiments, the capture sequence can be a sequence complementary to a capture domain (e.g., any of the capture domains described herein) of a capture probe. In some embodiments, the second probe can have a 5‘ end phosphate. In some embodiments, the first sequence and the second sequence can bind specifically to the target nucleic acid (e.g., mRNA).

In some embodiments, the 3’ end of the first probe can be ligated to the 5’ end of the second probe thereby generating a proxy of the target nucleic acid (e.g., a ligation product). In some embodiments, the first probe and the second probe can be ligated with a ligase. In some embodiments, the ligase can be any ligase capable of ligating RNA and DNA together. In some embodiments, the ligase can be any DNA ligase. In some embodiments, the ligase can be T4 RNA ligase 2. Other enzymes appropriate for the ligation step are know n in the art and include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNTM DNA ligase, New England Biolabs), AmpligaseTM (available from Lucigen, Middleton, WI), SplintR (available from New England Biolabs, Ipswich, MA) (also known as PBCV-1 DNA Ligase), or Chlorella virus DNA Ligase.

In some embodiments, prior to contacting the fresh-frozen biological sample with a spatial array and after ligation of the first probe and the second probe, the fresh-frozen biological sample can be treated with an RNase. In some embodiments, the fresh-frozen biological sample is contacted with an RNase while disposed on the spatial array. In some embodiments, the RNase is one or more of RNase A, RNase I, RNase H, and RNase P. In some embodiments, the RNase is RNase H.

In some embodiments, the unligated probes (e g., unligated first probes and unligated second probes) can be removed (e.g., washed) from the array. In some embodiments, the unligated probes (e.g., unligated first probes and unligated second probes) can be blocked from binding the capture probes of the array. For example, the unligated second probe including the capture sequence (e.g., the sequence complementary to the capture domain) can bind (e.g., hybridize) to the capture domain (e.g., any of the capture domains described herein). In some embodiments, when the unligated second probe binds to the capture domain it can block ligated probes from binding (e.g., hybridizing) with the capture domain. Thus, the capture sequence of the second probe can be blocked until the portion complementary to the target nucleic acid is hybridized to the target nucleic acid, and optionally, ligated to the first probe, before the blocking probe is removed.

In some embodiments, capture domains of the capture probes on the array can be blocked to prevent unligated second probes from binding. In some embodiments, after blocking the capture domain, the second probe, and or the proxy of the target nucleic acid an exonuclease can be added to the biological sample. In some embodiments, the exonuclease can be RecJf. In some embodiments, the exonuclease can be a 5’ to 3’ riboexonuclease. In some embodiments, the riboexonuclease can be Terminator™ nuclease. In some embodiments, the exonuclease can be a combination of RecJf and Terminator™ nuclease. In some embodiments, an endonuclease can be added to the fixed, fresh-frozen biological sample. In some embodiments, the endonuclease is RNase HI or RNase HII. In some embodiments, the endonuclease can nick unligated first probes non-specifically bound (e.g., hybridized) to the capture probe, or portion thereof.

In some embodiments, the 3’ end of the capture probe and/or the proxy of the target nucleic acid can be extended to add a sequence that corresponds (e.g., complementary) to a portion of the proxy of the target nucleic acid, the spatial barcode, the unique molecular identifier, one or more functional domains, and the complement of the capture sequence. In some embodiments, a second strand can be generated (e.g., a second strand sequence complementary to the capture probe). In some embodiments, the second strand can include a sequence complementary to the spatial barcode and the proxy of the target nucleic acids. In some embodiments, the extension products including the proxies of the target nucleic acids (e.g., the ligated first probe and second probe) and the complementary sequences of the barcode, UMI etc. are removed (e.g., melted away with increased temperature) and the extended target nucleic acid proxy can be used to generate a sequencing library . In some embodiments, after the ligated product is removed, second strand synthesis can be performed on the extended capture probe (e g., by any of the methods described herein). The generated second strand can be removed (e.g., melted away with increased temperature) and used to generate a sequencing library. In some embodiments, the generated second strand can be removed with KOH.

In some embodiments, the sequence of the spatial barcode or a complement thereof and the sequence of all or a portion of the target nucleic acid, or a complement thereof, can be determined. In some embodiments, the determined sequences can be used to identify the location of the target nucleic acid in the fixed, fresh-frozen biological sample. In some embodiments, determining the sequences can be performed by any of the sequencing methods described herein, including high-throughput sequencing.

In some embodiments, the first functional domain can be a first sequencing handle. In some embodiments, the capture sequence is a sequence substantially complementary to the capture domain, or a portion thereof. In some embodiments, a primer can be hybridized to at least a portion of the first functional domain of the capture probe.

The resulting extension products from the proxies of the target nucleic acids (e.g., ligation products) can be denatured from the capture probe template and transferred (e.g., to a clean tube) for amplification, and/or library construction as described herein. The spatially - barcoded, full-length extension products can be amplified via PCR prior to library construction. The extension products can then be enzymatically fragmented and size-selected in order to optimize the amplicon size. P5, P7, i7, and i5 can be incorporated into the library as for downstream sequencing, and additional library sequencing regions, such as TruSeq Read 2, can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. The fragments can then be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 sequences as sequencing primer sites. In some instances, the library is sequenced using any method described herein, such that different sequencing domains specific to other sequencing methods and techniques can be incorporated into a capture probe or introduced during library preparation. In some instances, the sequence of the analyte or proxies thereof is determined via sequencing. In some instances, the sequencing is high-throughput sequencing. In some instances, the spatial barcode is sequenced, providing the location of the analyte.

Nucleic acids present in a biological sample can often be degraded or fragmented in a moderate quality (e.g., RIN score of about 5 to 7) fresh-frozen biological samples. For example, DNA and/or RNA can be degraded or fragmented for a variety of reasons including: stringent permeabilization conditions, cellular processes and autolysis, other physical perturbations, and/or staining processes when biological samples are stained. The degradation or fragmentation of DNA and/or RNA can greatly affect the efficiency of capture of DNA and/or RNA sequences for spatial array assays.

As used herein, the term “RNA Integrity Number” or “RIN” refers to an indication of RNA quality based on an integrity score, the degree for which RNA from a sample is degraded or not (for example, as found in Schroeder, A., et al., The RIN: an RNA integrity number for assigning integrity values to RNA measurements, BMC Molecular Biology, 7:3 (2006) and Ahlfen, S.V., et al., Determinants of RNA Quality from FFPE Samples, PLoS ONE, 2(12): el261 (2007), Mueller, O , et al, RNA Integrity Number (RIN) - Standardization of RNA Quality Control, Agilent Technologies (2004), all of which are incorporated herein by reference). For example, a biological sample with a RIN score of about 1 includes RNA that is fully degraded, whereas a biological sample with a RIN score of about 10 includes RNA that is not degraded. In some embodiments, a RIN score can be calculated for a biological sample, one or more regions of a biological sample, or a single cell. In some embodiments, a RIN score can be calculated prior to performing any of the methods described herein on a similar biological sample. For example, a RIN score can be determined for a first biological sample and the second (e.g., next or adjacent) biological sample (e.g., tissue section) can be used to perform any of the methods described herein. In some examples, a RIN score of a first biological sample can be used to approximate the RIN value of the second (e.g., similar or adjacent) biological sample.

As used herein, the term “moderate quality biological sample” refers to a biological sample with a RIN score between about 5 to about 7.5. In some embodiments, a moderate quality biological sample can have a RIN score of less than 7.5, less than 7.0, 6.5, less than 6.0, less than 5.5, or less than 5.0. In some embodiments, the RIN value can be determined for a biological sample (e.g., a tissue section) before or after the biological sample is used for to perform any of the methods described herein. For example, a RIN value can be previously determined on a different (e.g., similar or adjacent) biological sample than the biological sample that is used to perform any of the methods described herein. In some embodiments, spatial analysis as described herein can be performed on samples with a RIN score between about 4 to about 10. In some embodiments, a moderate quality biological sample can have a RIN score of about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, a biological sample with a moderate RIN score may not comprise capturable RNA for an established spatial transcriptomics assays, whereas following the methods described herein would rescue that RNA, capturing it for spatial transcriptomics assays thereby providing useful information from that biological sample that might otherwise be lost.

Sandwich Configurations

FIG. 8 is a schematic diagram depicting an exemplary' analyte transfer, or sandwiching, process 804 between a first substrate comprising a fresh-frozen biological sample (e.g., a tissue section 802 on a slide 803) and a second substrate comprising a spatially barcoded array, e g., a slide 804 that is populated with spatially-barcoded capture probes 806. During the exemplary' sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the fresh-frozen biological sample is aligned with at least a portion of the array (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., slide 804) is in a superior position to the first substrate (e.g., slide 803). In some embodiments, the first substrate (e.g., slide 803) may be positioned superior to the second substrate (e.g., slide 804). A reagent medium 805 (e.g., permeabilization solution) within a gap 807 between the first substrate (e.g., slide 803) and the second substrate (e.g., slide 804) creates a permeabilization buffer which permeabilizes or digests the biological sample 802 and the proxies of the target nucleic acids 808 generated in the biological sample 802 may release, actively or passively migrate (e g., diffuse) across the gap 807 toward the capture probes 806, and hybridize to the capture probes 806. In some embodiments, the fresh-frozen biological sample is fixed (e.g., by any of the fixation methods described herein) prior to permeabilization. In some embodiments, the fresh-frozen biological sample is heated on the substrate prior to permeabilization.

After the proxies of the target nucleic acids 808 hybridize to the capture probes 806, an extension reaction may occur, thereby generating a spatially barcoded library. A polymerase can be used to generate an extension product library associated with a particular spatial barcode. Non-limiting examples of DNA polymerases include T7 DNA polymerase; Bsu DNA polymerase; and E.coli DNA Polymerase pol I. Barcoded extension product libraries can be mapped back to a specific spot on a capture area of the capture probes 806. This data can be subsequently layered over a high-resolution microscope image of the biological sample (e.g., a fresh-frozen biological sample), making it possible to visualize the data within the morphology of the tissue in a spatially-resolved manner. In some embodiments, the extension reaction can be performed separately from the sample handling apparatus described herein that is configured to perform the exemplary analyte transfer sandwiching process 804. The sandwich configuration of the sample 802, the first substrate (e.g., slide 803) and the second substrate (e.g., slide 804) can provide advantages over other methods of spatial analysis and/or analyte capture or proxies thereof (e.g., ligation product(s)). For example, the sandwich configuration can reduce a burden of users to develop in house tissue sectioning and/or tissue mounting expertise. Further, the sandwich configuration can decouple sample preparation/tissue imaging from the barcoded array (e.g., spatially -barcoded capture probes 806) and enable selection of a particular region of interest of analysis (e.g., for a tissue section larger than the barcoded array). The sandwich configuration also beneficially enables spatial analysis without having to place a biological sample (e.g., tissue section) 802 directly on the second substrate (e.g., slide 804). In some embodiments, the analyte transfer sandwiching process comprises: mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate; mounting the second substrate on a second member of the support device, the second member configured to retain the second substrate, applying a reagent medium to the first substrate and/or the second substrate, the reagent medium comprising a permeabilization agent, operating an alignment mechanism (also referred to herein as an adjustment mechanism) of the support device to move the first member and/or the second member such that a portion of the biological sample is aligned (e g., vertically aligned) with a portion of the array of capture probes and within a threshold distance of the array of capture probes, and such that the portion of the biological sample and the capture probe contact the reagent medium, wherein the permeabilization agent releases the extension product(s) from the biological sample.

The analyte transfer 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). 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 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 a to the plane or 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. 10A shows an exemplary sandwiching process 1000 where a first substrate (e.g., slide 1003), including a fresh-frozen biological sample 1002 (e.g., a tissue section), and a second substrate (e.g., slide 1004 including spatially barcoded capture probes 1006) are brought into proximity with one another. As shown in FIG. 10A a liquid reagent drop (e.g., permeabilization solution 1005) is introduced on the second substrate in proximity to the capture probes 1006 and in between the biological sample 1002 and the second substrate (e.g., slide 1004 including spatially barcoded capture probes 1006). The permeabilization solution 1005 can release proxies of target nucleic acids that can be captured (e.g., hybridized) by the capture probes 1006 of the array. As further shown, one or more spacers 1010 can be positioned between the first substrate (e.g., slide 1003) and the second substrate (e.g., slide 1004 including spatially barcoded capture probes 1006). The one or more spacers 1010 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 1010 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.

FIG. 10B shows a fully formed sandwich configuration creating a chamber 1050 formed from the one or more spacers 1010, the first substrate (e.g., the slide 1003), and the second substrate (e.g., the slide 1004 including spatially barcoded capture probes 1006) in accordance with some example implementations. In FIG. 10B, the liquid reagent (e g., the permeabilization solution 1005) fills the volume of the chamber 1050 and can create a permeabilization buffer that allows proxies of target nucleic acids to diffuse from the biological sample 1002 toward the capture probes 1006 of the second substrate (e.g., slide 1004). In some aspects, flow of the permeabilization buffer may deflect proxies of the target nucleic acids from the biological sample 1002 and can affect diffusive transfer of the proxies of the target nucleic acids for spatial analysis. A partially or fully sealed chamber 1050 resulting from the one or more spacers 1010, the first substrate, and the second substrate can reduce or prevent flow from undesirable convective movement of transcripts and/or molecules over the diffusive transfer from the biological sample 1002 to the capture probes 1006

Kits Also provided herein are kits including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a fixative; (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence that is substantially complementary to a portion of a target nucleic acid, a second probe of the plurality of probes, where the second probe includes a second sequence that is substantially complementary to a portion of the target nucleic acid and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array; and (d) instructions for performing any of the methods described herein.

In some embodiments, the kit includes one or more penneabilization reagents. In some embodiments, the one or more penneabilization reagents comprises one or more proteases. Many proteases are known in the art, including but not limited to, pepsin, Proteinase K, and collagenase.

In some embodiments, the kit includes one or more polymerases. For example, nonlimiting examples of DNA polymerases include T7 DNA polymerase; Bsu DNA polymerase; and E.coh DNA Polymerase pol I.

In some embodiments, the kit includes one or more blocking probes. In some embodiments, the kit includes an RNase. In some embodiments, the RNase is one or more of RNase A, RNase I, RNase H, and RNase P. In some embodiments, the RNase is RNase H.

Compositions

The present disclosure also features compositions of the methods described herein. Also provided herein are compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a fixed, fresh-frozen biological sample disposed on the spatial array where the biological sample has been heated while disposed on the spatial array; and (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence that is hybridized to a portion of a target RNA, and a second probe of the plurality of probes, where the second probe includes a second sequence that is hybridized to a portion of the target RNA and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array, where the first probe and the second probe of the plurality of probes are ligated, thereby generating a plurality of proxies of the target RNA, and an RNase.

Also provided herein are compositions including: (a) a spatial array including a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a fixed, fresh-frozen biological sample disposed a substrate, such that the substrate is aligned with the spatial array, such that at least a portion of the fixed, fresh-frozen biological sample is aligned with at least a portion of the spatial array; and (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence hybridized to a portion of a target nucleic acid, and a second probe of the plurality of probes, where the second probe includes a second sequence that is hybridized to a portion of the target nucleic acid and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array.

Also provided herein are compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a fixed, fresh-frozen biological sample disposed on the spatial array; and (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence hybridized to a portion of a target nucleic acid, and a second probe of the plurality of probes, where the second probe includes a second sequence that is hybridized to a portion of the target nucleic acid and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array, where the first probe and the second probe are ligated, thereby generating a proxy of the target nucleic acid.

Also provided herein are compositions including: (a) a spatial array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a fixed, fresh-frozen biological sample disposed a substrate, such that the substrate is aligned with the spatial array, such that at least a portion of the fixed, fresh-frozen biological sample is aligned with at least a portion of the spatial array; and (c) a plurality of probes, where a first probe of the plurality of probes includes a first functional domain and a first sequence hybridized to a portion of a target nucleic acid, and a second probe of the plurality of probes, where the second probe includes a second sequence that is hybridized to a portion of the target nucleic acid and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array, where the first probe and the second probe are ligated, thereby generating a proxy of the target nucleic acid.

In some embodiments, where a proxy of the target nucleic acid is generated, the target nucleic acid is released. In some embodiments, the target nucleic acid is released using heat. In some embodiments, the target nucleic acid (e.g., RNA) is released via denaturation. In some embodiments, the target nucleic acid is released using an RNase. In some embodiments, the RNase is one or more of RNase A, RNase I, RNase H, and RNase P. In some embodiments, the RNase is RNase H.

In some embodiments, the capture sequence of the proxy of the target nucleic acid is hybridized to the capture domain of the capture probe on the spatial array. In some embodiments, the capture probe is extended using the proxy of the target nucleic acid as a template, thereby generating an extended probe. In some embodiments, the proxy of the target nucleic acid is extended using the capture probe as a template.

In some embodiments, the target nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the target nucleic acid is DNA. In some embodiments, the DNA is genomic DNA.

In some embodiments, the first functional domain is a first sequencing handle.

In some embodiments, the capture sequence is substantially complementary to the capture domain or a portion thereof.

In some embodiments, the plurality of capture probes includes a cleavage domain, one or more functional domains, a unique molecular identifier, and combinations thereof

Methods and compositions provided herein are further described in the following examples, which do not limit the scope of the methods and compositions described in the claims.

EXAMPLES

Example 1: Tissue sample preparation and spatial analysis

Experiments were run comparing tissue samples prepared using the RNA rescue (RR) protocol against established spatial transcriptomics tissue preparation protocols for fresh frozen and FFPE samples. All replicates were serial tissue sections from the same tissue sample.

Initially, tissue samples were evaluated for RNA quality. RNA was extracted from a tissue section of a tissue sample using RNeasy Mini Kit (Qiagen) and an RNA Integrity Number, or RIN score, was determined using an Agilent Bioanalyzer.

A control protocol was performed on tissue sections from a sample being tested. A standard Visium fresh frozen (FF) spatial gene expression protocol (CG000239 Visium Spatial Gene Expression User Guide, Rev F), where mRNA is captured directly on a spatial array, served as the experimental control to which the RR protocol was compared.

For the RR protocol being evaluated, FF tissue samples were cryo-sectioned to 10 pm thickness, placed onto slides and stored at -80°C prior to processing. After removal from the freezer, the slides were incubated for 1 min. at 37°C followed by tissue fixation in a 4% methanol -free formaldehyde solution for 10 min. at room temperature. After fixation, the tissue sections were washed with PBS and incubated at 37°C for 20 min. Following incubation, the slides with fixed tissue sections were allowed to come to room temperature prior to H&E staining and subsequent imaging.

After imaging, the tissue slides were washed with Milli-Q water, air dried, and placed into a Visium gene expression slide cassette. Briefly, the tissues were treated with 0.01M HC1 for 1 min. at room temperature, washed in PBS, and gene expression library preparation steps followed per manufacturer’s user guide (CG000407 Visium Spatial Gene Expression for FFPE User Guide); with the exception of the decrosslinking steps which were not done. The target template ligation probes were added to the tissue sections and pre-hybridization for 15 min. at room temperature was performed followed by probe hybridization overnight per the established protocol in the User Guide. The Visium Gene Expression for FFPE protocol uses pairs of RNA targeted templated ligation probes that hybridize to RNA targets, the two probes in a pair are ligated and capture of the ligation product on a spatial array serves as a proxy of the targeted RNA, unlike the standard Visium protocol where mRNA is directly captured via its poly(A) tail.

Sequencing libraries generated from the standard Visium protocol and the RR protocol were sequenced on aNextSeq 2000 sequencing instrument (Illumina). Data from sequencing were processed using the SpaceRanger software and the sequencing reads were aligned to the human or mouse reference genomics (GRCh38 or mmlO, version 32, ensemble 98). Experiments were performed to test the sensitivity of the RR protocol to recover transcriptomic profiles from an FF mouse brain sample (RIN=9) and a clinical FF prostate tumor sample (RIN > 9), samples designated as not being particularly problematic or challenging in generating sequencing libraries using standard Visium protocols. Results showed that the RR protocol produced robust sequencing libraries with an approximately 2- fold increase in the number of genes detected per spot compared to the standard Visium protocol. Additionally, there was high concordance (R=0.9) between the two protocols in both the mouse brain and the human prostate cancer data, thereby demonstrating the high similarity of data between the standard Visium and the RR protocol regardless of tissue type. The data suggests that the RR protocol is comparable to, and may exhibit a higher capture efficiency, compared to the standard Visium protocol.

Experiments were subsequently performed using tissue samples that were found to be problematic and challenging in generating good or high quality spatial transcriptomics data. It is contemplated that a number of factors could contribute to low sample quality, such as nature of the tissue itself, RNA degradation processes perhaps due to surgical procedures, sensitivity to freeze/thaw events during tissue sections, etc. As such, the RR protocol was applied to several challenging tissue types that had performed poorly using the standard Visium protocol as previously described.

Adult human lung tissue samples

Two human lung samples (RIN=6.8 and RIN=7.1) were obtained where the standard Visium protocol yielded poor quality data even though the RIN score would suggest the RNA was of good quality. Data generated from the two samples showed a 2-fold and 10-fold, respectively, increase in the number of detected genes per spot (FIGs. 4A-B, RR compared to standard Visium protocol, respectively, data shown for the tissue sample RIN=7.1). Additionally, even when a permissive cutoff threshold of 300 unique genes was set to include as many spots as possible in the analysis from both conditions, approximately 80% (21% for tissue sample RIN=6.8) of the spot data was discarded when using the standard Visium protocol compared to approximately 2.3% (1.3% for tissue sample RIN=6.8) of the spot data being discarded when the RR protocol was followed. Cluster analysis detected 11 clusters in the RR derived data and nine clusters in the standard Visium derived data. However, differential expression analysis highlighted distinct marker genes for each of the 11 RR derived clusters, whereas three of the nine clusters derived from the standard Visium data were difficult to distinguish one from the other. Further, when evaluating the top markers detected from four different tissue areas in the RR derived data (airway epithelium, megakaryocyte enriched, smooth muscle and glands), the top markers were consistently higher in the RR derived data compared to the standard Visium derived data, consistent with the higher quality and complexity of the data from the RR derived tissue sections.

Adult human colon tissue samples

Experiments were run comparing the RR protocol with standard Visium protocol using human colon tissue samples which had previously been shown to fail gene expression analysis using the standard Visium protocol.

Adult human colon tissue samples were collected, where the samples demonstrated degraded mRNA in the gut tissue sections. Gut tissue samples are highly delicate, filled with digestive enzymes and a microbiome of varying quality and quantity which can lead to rapid RNA degradation. Two colon tissue samples from two different subjects (RIN=4.5 and RIN=5.1) were processed using the RR protocol. Data showed that the RR protocol recovered good quality and reproducible results (FIG. 5) as compared to the standard Visium protocol.

Adult human small intestine tissue samples

Experiments were run comparing the RR protocol with standard Visium protocol using adult human small intestine tissue samples. It was noted that when practicing the standard Visium protocol on human small intestine tissue samples, there was RNA degradation over time even when the samples were stored and handled properly. As such, these tissue samples were evaluated using the RR protocol.

An adult human intestinal ileum tissue sample was obtained (RIN <7) and the standard Visium protocol was performed on tissue sections (n=4) within a few weeks of biopsy. At this initial timepoint, high quality gene expression data was obtained from the mucosa, Tertiary Lymphoid Tissue (TLS), submucosa, muscularis, and serosa layers of the tissue sample. FIG. 11A shows an exemplary H&E stained human small intestine tissue section (top) and spots colored by five major tissue listed above (bottom). However, surprisingly upon repeat of the same standard Visium protocol using tissue sections from the same biopsy sample six months later, there was almost a complete loss of spatial gene expression data in the mucosal and submucosal layers (FIG. 11B). Previous studies confirm the experimental findings, where the muscularis layer RNA spatial gene expression was found to be less variable over time compared to the mucosal and submucosal layers of the intestine.

The human intestinal ileum tissue sample that failed at the six month time point was re-evaluated at two years using the RR protocol, to determine if the spatial gene expression that was lost for the mucosal and submucosal layers could be rescued to some degree even two years after storage of the tissue. Surprisingly, even after two years of storage, the human intestinal ileum tissue sample using the RR protocol generated spatial gene expression data in all tissue layers, albeit with lower quality than the initial experiment using the fresh biopsy sample (FIG. 11B). More specifically, the second attempt with the standard Visium protocol at about 6 months after sample preparation resulted in an average of 159 unique genes per spot in the mucosa, whereas the RR protocol performed about 2 years after sample preparation resulted in an average of 814 unique genes per spot in the mucosa. Also, a large percentage of the expression data generated by the standard Visium protocol came from mitochondrial transcripts, ribosomal transcripts, and IncRNA (data not shown). Most genes (1272) were only detected in the initial dataset (about 1 month after sample preparation) and 466 genes were detected in both the initial dataset and the dataset generated at 2 years after sample preparation. In contrast, only 31 differentially expressed genes were detected in the standard Visium dataset generated after about 6 months after preparation. FIG. 11C shows spatial expression data of five enterocy te markers identified from the Gut Cell Atlas: alanyl aminopeptidase (ANPEP) (Ensembl: ENSG00000166825); retinol binding protein 2 (RBP2) (Ensembl: ENSG000001 14113); di acylglycerol O-Acyltransferase (DGAT1 ) (Ensembl: ENSG00000185000); fatty acid binding protein 2 (FABP2) (Ensembl: ENSG00000145384); and apolipoprotein B (APOB) (Ensembl: ENSG00000145384). These enterocyte markers were clearly visible in the mucosa in the 1 month (standard Visium) and 2 year dataset (RR protocol), but not in the 6 month (standard Visium) dataset.

It is contemplated that rapidly proliferating epithelial cells in the gut contain more RNases than the muscular layer, which may lead to the RNA degradation seen in the mucosal and submucosal layers over time and over multiple freeze/thaw cycles of the tissue sample, thereby negatively impacting the RNA quality.

The adult human intestinal ileum tissue sample data provides several insights; even within an intestinal tissue sample RNA degradation can be variable in different tissue types in the same tissue section and, the lack or absence of spatial gene expression data in these samples over time can be rescued to some extent by practicing the RR protocol. Pediatric human medulloblastoma brain tumor samples

Experiments were run comparing the RR protocol with the standard Visium protocol using pediatric medulloblastoma brain tumor tissue samples. Samples such as pediatric brain tumor tissue samples (e.g., medulloblastoma) are very precious and there is generally little sample for experimental analysis. However, evaluating these samples is critical in developing diagnostic and therapeutic strategies for patient treatment. As the sample sizes can be extremely limiting, the use of a portion of the sample for evaluating whether an assay should or should not be performed would be of better use in performing an assay where the outcome is expected to be reproducible regardless of quality of starting material.

Initially , standard Visium protocols were run on tissue sections from two different medulloblastoma brain tumor tissue samples, RIN=7.0 and RIN=7.1, to gain insights into the gene expression profiles of the tissue samples. However, even though the RIN scores of both samples were moderate and suggested RNA of sufficient quality to perform the standard Visium protocol, the data contained low gene and UMI counts, suggesting that the RNA was of lower quality and potentially degraded more than expected given the RIN scores, rendering the samples unusable.

The two samples were subsequently processed using the RR protocol. Data yielded an unexpected 12 to 100-fold increase for the number of genes detected per spot over the standard Visium data (FIG. 6). Surprisingly, utilizing the RR protocol for these samples rescued the RNA such that useful data could be derived from the spatial assay and used for biological interpretation. Gene clustering analysis was performed on the sample data from the RR protocol compared to the standard Visium protocol. Gene clustering analysis for the RR derived data identified five larger clusters and the differential expression analysis supported the existence of all five clusters. However, gene cluster analysis of the standard Visium derived data identified only three larger clusters, and the three larger clusters were weakly supported by differential expression analysis. Further, tissue sample data from sample 1 using the standard Visium protocol yielded too few spots and genes to analyze, whereas the RR protocol was able to rescue the sample such that unique genes per spot were substantially increased akin to those seen in sample 2 (FIG. 6).

Mouse bone/cartilage tissue samples

Experiments were run comparing the RR protocol with the standard Visium protocol using mouse bone/cartilage tissue samples. Bone/cartilage tissue sections can be challenging to evaluate in a spatial transcriptomics assay given the permeability and tissue adherence issues these types of samples present. The mouse bone/ cartilage tissue samples were obtained postnatal at 4 and 11 days, before and after formation of secondary ossification centers at the end of the tibia and femur in the knee joint.

Standard Visium protocol data was extremely poor (data not shown), while the RR protocol generated good quality data allowing for spatial gene expression profiling (FIG. 7). The number of unique genes per spot for each replicate regardless of day 4 or day 11 postnatal was consistently high. Additionally, gene expression data revealed that the median number of genes per spot and the mean reads per spot varied greatly between the different protocols as seen in Table 1.

Table 1 -Spatial transcriptomics data for mouse bone/ cartilage tissue sections

As such, experiments performed in evaluating the RR protocol using myriad diverse tissue samples demonstrate that the RR protocol can be effective in generating spatial gene expression data from challenging fresh frozen tissue samples even when such samples have performed poorly or have failed spatial transcriptomic profiling using known spatial transcriptomic assays.

Claims

WHAT IS CLAIMED IS:

1. A method for rescuing degraded target RNA for determining spatial gene expression in a fresh-frozen biological sample, the method comprising:

(a) providing a spatial array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (h) a capture domain;

(b) fixing the fresh-frozen biological sample;

(c) heating the fresh-frozen biological sample;

(d) contacting the fresh-frozen biological sample with:

(i) a first probe, wherein the first probe comprises a first functional domain and a first sequence that is substantially complementary to a portion of the target RNA, and

(ii) a second probe, wherein the second probe comprises a second sequence that is substantially complementary to a portion of the target RNA and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array;

(e) hybridizing the first probe and the second probe to the target RNA and ligating the first probe to the second probe to generate a proxy of the target RNA; and

(f) hybridizing the proxy of the target RNA to the capture domain of the capture probe on the spatial array, thereby rescuing degraded target RNA for determining spatial gene expression in the fresh-frozen biological sample.

2. The method of claim 1, wherein the fresh-frozen biological sample is disposed on the spatial array comprising a plurality of capture probes.

3. The method of claim 1, wherein the fresh-frozen biological sample is disposed on a substrate.

4. The method of claim 3, wherein the method further comprises aligning the substrate with the spatial array, such that at least a portion of the fresh-frozen biological sample is aligned with at least a portion of the spatial array.

5. The method of claim 1, wherein the target RNA is mRNA.

6. The method of any one of claims 1-5, wherein the spatial array comprises one or more features selected from the group consisting of: a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead.

7. The method of any one of claims 1-6, wherein the first functional domain is a first sequencing handle.

8. The method of any one of claims 1-7, wherein the capture sequence is substantially complementary to the capture domain or a portion thereof.

9. The method of any one of claims 1-8, wherein the plurality of capture probes further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or combinations thereof.

10. The method of any one of claims 1-9, wherein the method further comprises imaging the fresh-frozen biological sample.

1 1 . The method of any one of claims 1 -10, wherein the method further comprises staining the fresh-frozen biological sample.

12. The method of claim 11, wherein the staining comprises eosin and hematoxylin.

13. The method of claim 11, wherein the staining comprises the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

14. The method of any one of claims 1-13, wherein the fixing comprises the use of methanol-free formalin.

15. The method of claim 14, wherein the methanol-free formalin is at about 2% (v/v) to about 6% (v/v).

16. The method of claim 15, wherein the methanol-free formalin is at about 4% (v/v).

17. The method of any one of claims 1-16. wherein the fixing is performed from about 5 minutes to about 20 minutes.

18. The method of claim 17, wherein the fixing is performed for about 10 minutes.

19. The method of any one of claims 1-18, wherein the fixing is performed at about 15°C to about 25°C.

20. The method of claim 19, wherein the fixing is performed at about 20°C.

21. The method of any one of claims 1-20, wherein the heating in step (c) is performed after the fixing in step (b).

22. The method of claim 21, wherein the heating is performed at about 25°C to about 45 °C.

23. The method of claim 22, wherein the heating is performed at about 37°C.

24. The method of any one of claims 1-23, wherein the heating is performed from about 5 minutes to about 45 minutes.

25. The method of claim 24, wherein the heating is performed from about 15 minutes to about 30 minutes.

26. The method of claim 25, wherein the heating is performed for about 20 minutes.

27. The method of any one of claims 1-26, wherein the ligating of the first probe to the second probe is performed using a ligase, wherein the ligase is selected from the group consisting of: Tth DNA ligase, Taq DNA ligase, Thermococcus sp. DNA ligase, AMPLIGASE, PBCV-1 DNA Ligase, and Chlorella virus DNA Ligase.

28. The method of any one of claims 1-27. wherein the method further comprises migrating the proxy of the target RNA to the spatial array, wherein the migrating comprises electrophoresis.

29. The method of any one of claims 1-28, wherein the method further comprises permeabilizing the fixed, fresh-frozen biological sample, wherein the permeabilizing comprises the use of a protease.

30. The method of any one of claims 1-29, wherein the fresh-frozen biological sample is a fresh-frozen tissue section.

31. The method of any one of claims 1-30, wherein the method further comprises, after step (c), a step of treating the fresh-frozen biological sample with an RNase.

32. The method of any one of claims 1-31, wherein the method further comprises in step (e) extending the capture probe thereby generating an extended capture probe.

33. The method of any one of claims 1-32, wherein the method further comprises in step (e) generating a second strand hybridized to the capture probe, wherein the second strand comprises a sequence complementary to the spatial barcode and a nucleic acid sequence corresponding to the proxy of the target RNA.

34. The method of any one of claims 1-33, wherein the method further comprises step (g) determining (i) the sequence of the spatial barcode, or a complement thereof, and (ii) the sequence of all or a portion of the proxy of the target RNA, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target RNA in the fresh-frozen biological sample.

35. The method of claim 34, wherein the determining in step (g) comprises sequencing.

36. The method of claim 35, wherein the sequencing is high-throughput sequencing.

37. A method for determining the location of a target RNA in a fresh-frozen biological sample, the method comprising:

(a) providing a spatial array comprising 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) fixing the fresh-frozen biological sample;

(c) heating the fresh-frozen biological sample;

(d) contacting the fresh-frozen biological sample with:

(e) hybridizing the first probe and the second probe to the target RNA and ligating the first probe to the second probe to generate a proxy of the target RNA;

(f) hybridizing the proxy of the target RNA to the capture domain of the capture probe on the spatial array, extending the capture probe thereby generating an extended probe, and generating a second strand hybridized to the capture probe, wherein the second strand comprises a sequence complementary to the spatial barcode and a nucleic acid sequence corresponding to the proxy of the target RNA; and

(g) determining (i) the sequence of the spatial barcode, or a complement thereof, and (ii) all or a portion of the proxy of the target RNA, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target RNA in the fresh- frozen biological sample.

38. The method of claim 37, wherein the fresh-frozen biological sample is disposed on the spatial array comprising a plurality of capture probes.

39. The method of claim 37, wherein the fresh-frozen biological sample is disposed on a substrate.

40. The method of claim 39, wherein the method further comprises aligning the substrate with the spatial array, such that at least a portion of the fresh-frozen biological sample is aligned with at least a portion of the spatial array.

41. The method of any one of claims 37-40, wherein the target RNA is mRNA.

42. The method of any one of claims 37-41, wherein the spatial array comprises one or more features selected from the group consisting of: a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead.

43. The method of any one of claims 37-42, wherein the first functional domain is a first sequencing handle.

44. The method of any one of claims 37-43, wherein the capture sequence is substantially complementary to the capture domain or a portion thereof.

45. The method of any one of claims 37-44, wherein the plurality of capture probes comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or combinations thereof.

46. The method of any one of claims 37-45, wherein the method further comprises imaging the fresh-frozen biological sample.

47. The method of any one of claims 37-46, wherein the method further comprises staining the fresh-frozen biological sample.

48. The method of claim 47, wherein the staining comprises eosin and hematoxylin.

49. The method of claim 47, wherein the staining comprises the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

50. The method of any one of claims 37-49, wherein the fixing comprises the use of methanol-free formalin.

51. The method of claim 50, wherein the methanol-free formalin is at about 2% (v/v) to about 6% (v/v).

52. The method of claim 51, wherein the methanol-free formalin is at about 4% (v/v).

53. The method of any one of claims 37-52, wherein the fixing is performed from about 5 minutes to about 20 minutes.

54. The method of claim 53, wherein the fixing is performed for about 10 minutes.

55. The method of any one of claims 37-54, wherein the fixing is performed at about 15°C to about 25°C.

56. The method of claim 55, wherein the fixing is performed at about 20°C.

57. The method of any one of claims 37-56, wherein the heating in step (c) is performed after the fixing in step (b).

58. The method of claim 57, wherein the heating is performed at about 25°C to about 45 °C.

59. The method of claim 58, wherein the heating is performed at about 37°C.

60. The method of any one of claims 37-59, wherein the heating is performed from about 5 minutes to about 45 minutes.

61. The method of claim 60, wherein the heating is performed from about 15 minutes to about 30 minutes.

62. The method of claim 61, wherein the heating is performed for about 20 minutes.

63. The method of any one of claims 37-62, wherein the determining in step (g) comprises sequencing.

64. The method of claim 63, wherein the sequencing is high-throughput sequencing.

65. The method of any one of claims 37-64, wherein the ligating of the first probe to the second probe is performed using a ligase selected from the group consisting of: Tth DNA ligase, Taq DNA ligase, Thermococcus sp. DNA ligase, AMPLIGASE, PBCV-1 DNA Ligase, and Chlorella virus DNA Ligase.

66. The method of any one of claims 37-65, wherein the method further comprises migrating the proxy of the target RNA to the spatial array, where the migrating comprises electrophoresis.

67. The method of any one of claims 37-66, wherein the method further comprises permeabilizing the fixed, fresh-frozen biological sample, wherein the permeabilizing comprises the use of a protease.

68. The method of any one of claims 37-67, wherein the fresh-frozen biological sample is a fresh-frozen tissue section.

69. The method of any one of claims 37-68, wherein the method further comprises, after step (c), a step of treating the fresh-frozen biological sample with an RNase.

70. A composition comprising:

(a) a spatial array comprising 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 fixed, fresh-frozen biological sample disposed on the spatial array wherein the biological sample has been heated while disposed on the spatial array; and

(c) a plurality of probes, wherein a first probe of the plurality of probes comprises a first functional domain and a first sequence that is hybridized to a portion of a target RNA, and a second probe of the plurality of probes, wherein the second probe comprises a second sequence that is hybridized to a portion of the target RNA and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array, wherein the first probe and the second probe of the plurality of probes are ligated, thereby generating a plurality of proxies of the target RNA; and

(d) an RNase.

71. The composition of claim 70, further comprising one or more proxies of the target RNA that are not hybridized to the target RNA.

72. The composition of any one of claims 70-71, wherein the capture sequence of one or more proxies of the target RNA is hybridized to the capture domain of the capture probe on the spatial array.

73. The composition of claim 72, further comprising extended capture probes, wherein an extended capture probe is generated using the proxy of the target RNA as a template.

74. The composition of claim 73, further comprising extended proxies of the target RNA, wherein an extended proxy of the target RNA is generated using the capture probe as a template.

75. The composition of any one of claims 70-74, wherein the target RNA is mRNA.

76. The composition of any one of claims 70-75, wherein the spatial array comprises one or more features selected from the group consisting of: a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead.

77. The composition of any one of claims 70-76, wherein the first functional domain is a first sequencing handle.

78. The composition of any one of claims 70-77, wherein the capture sequence is substantially complementary to the capture domain or a portion thereof.

79. The composition of any one of claims 70-78, wherein the plurality of capture probes comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or combinations thereof.

80. The composition of any one of claims 70-79, wherein the fixed, fresh-frozen biological sample is fixed in about 4% (v/v) methanol-free formalin.

81. A kit comprising:

(b) a fixative;

(c) a plurality of probes, wherein a first probe of the plurality of probes comprises a first functional domain and a first sequence that is substantially complementary to a portion of a target nucleic acid, a second probe of the plurality of probes, wherein the second probe comprises a second sequence that is substantially complementary to a portion of the target nucleic acid and a capture sequence that is substantially complementary to the capture domain of the capture probe on the spatial array; and

(d) instructions for performing any of the methods of claims 1-69.

82. The kit of claim 81, wherein the kit further comprises one or more permeabilization reagents.

83. The kit of claim 82, wherein the one or more permeabilization reagents comprises one or more proteases.

84. The kit of any one of claims 81-83, wherein the kit further comprises one or more polymerases.

85. The kit of any one of claims 81-84, wherein the fixative is methanol-free formalin.

86. The kit of claim 85, wherein the methanol-free formalin is about 4% (v/v).