Patent Application
20230175045
This application contains a Sequence Listing that has been submitted electronically as an XML file named 47706-0322001_SL_ST26.xml. The XML file, created on Dec. 1, 2022, is 3,772 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
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).
Chromatin structure can be different between cells in a biological sample or between biological samples from the same tissue. Assaying differences in accessible chromatin can be indicative of transcriptionally active sequences, e.g., genes, in a particular cell. Exemplary methods for assaying differences in accessible chromatin include, but are not limited to, nucleosome occupancy and methylome sequencing (NOMe-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), DNase I hypersensitive sites sequencing (DNAse-seq), Hi-C sequencing, RNA-seq, bisulfate sequencing (BS-seq), DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), chemical-labeling-enabled C-to-T conversion sequencing (CLEVER-seq), chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq), cleavage under targets and release using nuclease (CUT & Run), cleavage under targets and tagmentation (CUT & Tag), scCUT & Tag, high-spatial-resolution chromatin modification state profiling by sequencing (hsrChST-seq), and single cell sequencing varieties thereof. Further understanding the transcriptionally active regions within chromatin will enable identification of which genes contribute to a cell's function and/or phenotype.
The present disclosure generally describes methods for spatially analyzing genomic DNA present in a biological sample.
Methods have been developed to study epigenomes, e.g., CUT & Run or CUT & Tag sequencing. These assays help identify regulators (e.g., cis regulators and/or trans regulators) that contribute to dynamic cellular phenotypes. While CUT & Run and CUT & Tag sequencing have been valuable in defining epigenetic variability within a cell population, conventional applications of these methods are limited in their ability to spatially resolve the two- and three-dimensional structures and associated genes that promote cellular variation. Although spatial methods are also known, additional and/or alternative methods are still needed. In particular, methods that can simultaneously assess epigenomes and gene expression in a tissue sample would be useful.
Thus, the present disclosure relates generally to the spatial tagging and analysis of nucleic acids. In some instances, provided herein are methods that utilize a transposome to fragment genomic DNA and to capture the fragmented DNA on a spatial array, thus revealing epigenomic insights regarding the structural features contributing to cellular regulation within the spatial context of a biological sample.
Provided herein are methods for determining location of accessible genomic DNA in a biological sample, the method comprising: (a) providing the biological sample on an 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) adding an antibody specific to a chromatin protein in the biological sample and binding the antibody to the chromatin protein; (c) binding a transposome-binding moiety complex to the antibody, wherein the transposome-antibody-binding moiety complex comprises: (i) a transposase, (ii) an antibody-binding moiety, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (d) generating fragmented genomic DNA; (e) adding a plurality of splint oligonucleotides to the biological sample, wherein a portion of a splint oligonucleotide hybridizes to a portion of the capture domain; (f) hybridizing the splint sequence of the fragmented genomic DNA to the splint oligonucleotide hybridized to the capture domain; and (g) determining (i) the spatial barcode or a complement thereof, of the capture probe and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, to determine the location of the accessible genomic DNA in the biological sample.
In some embodiments, steps (b) and (c) are performed at the same time, and wherein the antibody and the transposome-antibody-binding moiety are combined to form a multi-complex.
In some embodiments, the splint oligonucleotide comprises about 12 to about 40 nucleotides.
In some embodiments, step (b), through step (f) are performed at substantially the same time.
In some embodiments, the methods described herein further comprise ligating the splint sequence of the fragmented genomic DNA to the capture domain of the capture probe.
In some embodiments, the methods described herein further comprise gap filling and ligation between the 3′ end of the transposon and the 5′ end of the fragmented genomic DNA.
In some embodiments, the methods described herein further comprise extending the 3′ end of the captured fragmented genomic DNA using the capture probe as a template, wherein the gap filling, ligation, and extension occur at the substantially the same time.
In some embodiments, the functional sequence of the second transposon end sequence comprises a primer sequence.
In some embodiments, the antibody-binding moiety is protein A, protein G, or functional derivatives thereof.
In some embodiments, the transposase is a Tn5 transposase enzyme, a Mu transposase enzyme, a Tn7 transposase enzyme, a Vibhar species transposase, or functional derivatives thereof.
In some embodiments, the methods described herein further comprise extending a 3′ end of the capture probe using the fragmented genomic DNA as a template, wherein the extending step is performed using a DNA polymerase having strand displacement activity.
In some embodiments, the methods described herein further comprise staining the biological sample, optionally wherein the staining comprises hematoxylin and eosin (H&E) staining or immunofluorescence staining.
In some embodiments, the biological sample is permeabilized prior to step (b), wherein permeabilization is chemical or enzymatic, and wherein the chemical permeabilization condition comprises a detergent, optionally wherein the detergent is one or more of NP-40, polysorbate-20, and digitonin.
In some embodiments, the enzymatic permeabilization condition comprises a protease of the group consisting of a pepsin, a collagenase, a proteinase K, or combinations thereof.
In some embodiments, the biological sample is a fresh tissue sample or section, a frozen tissue sample or section, or a fixed tissue sample or section.
In some embodiments, the fixed tissue sample or section is a formalin-fixed, paraffin embedded (FFPE) tissue sample or section.
In some embodiments, the capture probe further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or combinations thereof.
In some embodiments, the methods described herein further comprise determining the location of an mRNA in the biological sample, the method comprising: hybridizing the mRNA or a portion thereof to the capture domain; and determining (i) the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the mRNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the mRNA in the biological sample.
In some embodiments, hybridizing the mRNA or a portion thereof to the capture domain is performed concurrent with or after step (b).
In some embodiments, determining (i) the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the mRNA, or a complement thereof occurs concurrent with step (g).
In some embodiments, determining (i) spatial barcode or a complement thereof, and (ii) all or part of a sequence of the mRNA, or a complement thereof comprises sequencing.
Also provided herein are kits for determining the location of accessible genomic DNA in a biological sample comprising: (a) an 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 complex comprising: (i) an antibody-binding protein, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (c) instructions for performing any one of the methods described herein.
Also provided herein are kits for determining abundance and location of accessible genomic DNA in a biological sample comprising: (a) an 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 multi-complex comprising: (i) an antibody-binding protein, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, (iv) a second transposon end sequence comprising a functional sequence, and (v) an antibody that binds to a chromatin protein in the biological sample; and (c) instructions for performing any one of the methods described herein.
Also provided herein are compositions for determining abundance and/or location of accessible genomic DNA in a biological sample comprising: (a) an 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 complex comprising: (i) an antibody-binding protein, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence; and (c) an antibody bound to a chromatin protein in the biological sample, wherein the antibody is additionally bound to the complex from step (b).
Also provided herein are methods for determining the location of accessible genomic DNA in a biological sample, the method comprising: (a) providing the biological sample on a first substrate; (b) binding an antibody specific to a chromatin protein in the biological sample to the chromatin protein; (c) binding a transposome-binding moiety complex to the antibody, wherein the transposome-antibody-binding moiety complex comprises: (i) a transposase, (ii) an antibody-binding moiety, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (d) generating fragmented genomic DNA; (e) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (f) adding a plurality of splint oligonucleotides to the biological sample, wherein a portion of a splint oligonucleotide hybridizes to a portion of the capture domain; (g) hybridizing the splint sequence of the fragmented genomic DNA to the splint oligonucleotide hybridized to the capture domain; and (h) determining (i) the spatial barcode or a complement thereof, of the capture probe and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, to determine the location of the accessible genomic DNA in the biological sample.
In some embodiments, the aligning 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; applying a reagent medium to the first substrate and/or the second substrate; and operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array, and such that the portion of the biological sample and the portion of the array contact the reagent medium.
In some embodiments, the reagent medium comprises a permeabilization agent selected from trypsin, pepsin, elastase, or proteinase K.
In some embodiments, at least one of the first substrate and the second substrate further comprise a spacer disposed on the first substrate or the second substrate, wherein when at least the portion of the biological sample is aligned with at least a portion of the array such that the portion of the biological sample and the portion of the array contact the reagent medium, the spacer is disposed between the first substrate and the second substrate and is configured to maintain the reagent medium within a chamber formed by the first substrate, the second substrate, and the spacer, and to maintain a separation distance between the first substrate and the second substrate, wherein the spacer is positioned to surround an area on the first substrate on which the biological sample is disposed and/or the array disposed on the second substrate, wherein the area of the first substrate, the spacer, and the second substrate at least partially encloses a volume comprising the biological sample.
In some embodiments, steps (b) and (c) are performed at the same time, and the antibody and the transposome-antibody-binding moiety are combined to form a multi-complex.
In some embodiments, the transposase is a Tn5 transposase enzyme, a Mu transposase enzyme, a Tn7 transposase enzyme, a Vibhar species transposase, or functional derivatives thereof.
Also provided herein are methods for determining abundance and/or location of accessible genomic DNA in a biological sample, the method comprising (a) providing the biological sample on an 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) adding an antibody that binds to a chromatin protein; (c) binding a transposome-antibody-binding moiety complex to the antibody, thereby generating fragmented genomic DNA, wherein the transposome—antibody-binding moiety complex comprises: (i) a transposase, (ii) an antibody-binding moiety (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (d) adding a plurality of splint oligonucleotides to the biological sample, wherein a portion of a splint oligonucleotide of the plurality of splint oligonucleotides hybridizes to a portion of the capture domain; (e) hybridizing the splint sequence of the fragmented genomic DNA to the splint oligonucleotide and hybridizing the splint oligonucleotide to the capture probe; (f) ligating the splint sequence of the fragmented genomic DNA to the capture domain; and (g) determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the abundance and/or location of the accessible genomic DNA in the biological sample.
Also provided herein are methods for determining abundance and/or location of accessible genomic DNA in a biological sample, the method comprising: (a) providing the biological sample on an 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) complexing an antibody that binds to a chromatin protein in the biological sample to a transposome-antibody-binding moiety complex, generating a multi-complex, wherein the multi-complex comprises: (i) a transposase, (ii) an antibody-binding moiety, (iii) the first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, (iv) the second transposon end sequence comprising a functional sequence, and (v) the antibody; and (c) adding the multi-complex to the biological sample, thereby generating fragmented genomic DNA; (d) adding a plurality of splint oligonucleotides to the biological sample, wherein a portion of a splint oligonucleotide of the plurality of splint oligonucleotides hybridizes to a portion of the capture domain; (e) hybridizing the splint sequence of the fragmented genomic DNA to the splint oligonucleotide and hybridizing the splint oligonucleotide to the capture probe, (f) ligating the transposon splint sequence of the fragmented genomic DNA to the capture domain; and (g) determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the abundance and/or location of the accessible genomic DNA in the biological sample.
In some instances, the splint oligonucleotide comprises about 12 to about 40 nucleotides.
In some instances, step (b), step (c), step (d), and step (e) are performed at substantially the same time.
In some instances, the ligating utilizes a DNA ligase.
In some instances, any of the methods described herein further comprise performing gap filling and ligation between the 3′ end of a transposon and a 5′ end of a fragmented genomic DNA. In some instances, any of the methods described herein further comprise extending the 3′ end of the captured fragmented genomic DNA using the capture probe as a template.
In some instances, the gap filling, ligation, and extension occur at the same time.
In some instances, the functional sequence of the second transposon end sequence is a primer sequence.
In some instances, the antibody-binding moiety is protein A or functional derivatives thereof. In some instances, the antibody-binding moiety is protein G or functional derivatives thereof. In some instances, the transposase is a Tn5 transposase enzyme, a Mu transposase enzyme, a Tn7 transposase enzyme, a Vibhar species transposase, or functional derivatives thereof.
In some instances, any of the methods described herein further comprise extending a 3′ end of the capture probe using the fragmented genomic DNA as a template. In some instances, the extending step is performed using a DNA polymerase having strand displacement activity. In some instances, the determining step (g) comprises sequencing (i) all or part of the sequence of the spatial barcode or a complement thereof, and (ii) all or part of the sequence of the fragmented genomic DNA or a complement thereof.
In some instance, any of the methods herein further comprise staining the biological sample, optionally with hematoxylin and eosin (H&E) staining or immunofluorescence staining.
In some instances, adding the antibody or the multi-complex to the biological sample is performed under a chemical permeabilization condition, under an enzymatic permeabilization condition, or both. In some instances, the chemical permeabilization condition comprises a detergent. In some instances, the detergent is one or more of NP-40, polysorbate-20, and digitonin. In some instances, adding the antibody or the multi-complex to the biological sample is performed after an enzymatic pre-permeabilization condition. In some instances, the enzymatic pre-permeabilization condition comprises a protease. In some instances, the protease is a pepsin, a collagenase, a proteinase K, and combinations thereof.
In some instances, the biological sample is a fresh tissue sample, a frozen tissue sample, or a fixed tissue sample. In some instances, the biological sample is a formalin-fixed, paraffin embedded (FFPE) tissue sample. In some instances, the biological sample is a tissue section.
In some instances, the array comprises one or more features. In some instances, the capture probe further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or combinations thereof.
In some instances, any of the methods herein further comprise determining abundance and location of an analyte in the biological sample, the method comprising: hybridizing the analyte or a portion thereof to the capture domain; and determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the analyte, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the abundance and the location of the analyte in the biological sample.
In some instances, the analyte is RNA. In some instances, the RNA is mRNA.
In some instances, hybridizing the analyte or a portion thereof to the capture domain is performed at the same time as step (e).
In some instances, determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the analyte, or a complement thereof occurs at the same time as step (g). In some instances, determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the analyte, or a complement thereof comprises sequencing (i) all or part of the sequence of the spatial barcode or a complement thereof, and (ii) all or part of the sequence of the fragmented genomic DNA or a complement thereof.
Also provided herein are kits for determining a location of accessible genomic DNA in a biological sample comprising: (a) an 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 complex comprising: (i) an antibody-binding moiety, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (c) instructions for performing any one of the methods described herein.
Also provided herein are kits for determining abundance and/or location of accessible genomic DNA in a biological sample comprising: (a) an 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 multi-complex comprising: (i) an antibody-binding moiety, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, (iv) a second transposon end sequence comprising a functional sequence, and (v) an antibody that binds to a chromatin protein; and (c) instructions for performing any one of the methods described herein.
Also provided herein are compositions for determining abundance and/or location of accessible genomic DNA in a biological sample comprising: (a) an 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; and (b) a complex comprising: (i) an antibody-binding moiety, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence.
Also provided herein are compositions for determining abundance and/or location of accessible genomic DNA in a biological sample comprising: (a) an 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; and (b) a multi-complex comprising: (i) an antibody-binding moiety, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, (iv) a second transposon end sequence, and (v) an antibody that binds to a chromatin protein.
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 “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 instances of the features of this disclosure are described herein. However, it should be understood that such instances 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 instances described herein are also within the scope of this disclosure.
The following drawings illustrate certain instances of the features and advantages of this disclosure. These instances are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.
Epigenomic methods such as CUT & Run or CUT & Tag methodologies help identify regulators (e.g., cis regulators and/or trans regulators) that contribute to dynamic cellular phenotypes. While CUT & Run and CUT & Tag methodologies have been valuable in defining epigenetic variability within a cell population, conventional applications of these methods are limited in their ability to spatially resolve the two- and three-dimensional structures and associated genes that promote cellular variation. Methods that can simultaneously assess epigenomes and localize gene expression in a tissue sample would be useful.
Thus, the present disclosure relates generally to the spatial tagging and analysis of nucleic acids. In some instances, provided herein are methods that utilize a transposome to fragment genomic DNA and to capture the fragmented DNA on a spatial array, thus revealing epigenomic insights regarding the structural features contributing to cellular regulation within the spatial context of a biological sample.
Spatial analysis methodologies described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.
Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Pat. Nos. 11,447,807, 11,352,667, 11,168,350, 11,104,936, 11,008,608, 10,995,361, 10,913,975, 10,774,374, 10,724,078, 10,640,816, 10,494,662, 10,480,022, 10,364,457, 10,317,321, 10,059,990, 10,041,949, 10,030,261, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, and 7,709,198; U.S. Patent Application Publication Nos. 2020/0239946, 2020/0080136, 2020/0277663, 2019/0330617, 2020/0256867, 2020/0224244, 2019/0085383, and 2013/0171621; PCT Publication Nos. WO2018/091676, WO2020/176788, WO2017/144338, and WO2016/057552; Non-patent literature references Rodrigues 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 F, dated January 2022); and/or the Visium Spatial Gene Expression Reagent Kits—Tissue Optimization User Guide (e.g., Rev E, dated February 2022), both of which are available at the 10× 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 PCT Publication No. WO2020/176788 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-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. 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 instances, the biological sample is an organism (e.g., a small organism such as a parasite). In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue or sample). Additional methods of fixation and preparation are provided in this application.
In some instances, the biological sample is fixed using PAXgene. PAXgene is a formalin-free, non-cross-linking fixative that preserves morphology and biomolecules. It is a mixture of different alcohols, acid, and a soluble organic compound. Ergin B. et al., J Proteome Res. 2010 Oct. 1; 9(10):5188-96 appears to have first developed and described PAXgene. Kap M. et al., PLoS One.; 6(11):e27704 (2011) and Mathieson W. et al., Am J Clin Pathol.; 146(1):25-40 (2016) both describe and evaluate PAXgene for tissue fixation. 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 PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
The following embodiments can be used with any of the methods described herein. In some embodiments, the biological sample is imaged. In some embodiments, the biological sample is visualized or imaged using bright field microscopy. In some embodiments, the biological sample is visualized or imaged using fluorescence microscopy. Additional methods of visualization and imaging are known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. In some embodiments, the sample is stained and imaged prior to adding the primer to the biological sample.
In some embodiments, the method includes staining the biological sample. In some embodiments, the staining includes the use of hematoxylin and eosin. In some embodiments, a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation.
In some embodiments, the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Briefly, in any of the methods described herein, the method includes a step of permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of the extension products to the capture probes on the array. In some embodiments, the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, and methanol), a detergent (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), an enzyme (an endopeptidase, an exopeptidase, a protease), or combinations thereof In some embodiments, the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X-100™, Tween-20™, or combinations thereof. In some embodiments, the endopeptidase is pepsin. In some embodiments, the endopeptidase is Proteinase K. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference.
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 instances, the capture probe includes a homopolymer sequence, such as a poly(T) sequence. In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
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 A1, WO 2022/061152 A2, and WO 2022/140028 A1.
During the exemplary sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the capture probes (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., array slide 104) is in a superior position to the first substrate (e.g., slide 103). In some embodiments, the first substrate (e.g., slide 103) may be positioned superior to the second substrate (e.g., slide 104). A reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates. The reagent medium may be a permeabilization solution which permeabilizes and/or digests the biological sample 102. In some embodiments wherein the biological sample 102 has been pre-permeabilized, the reagent medium is not a permeabilization solution. In some embodiments, analytes (e.g., mRNA transcripts) and/or analyte derivatives (e.g., intermediate agents; e.g., ligation products) of the biological sample 102 may release from the biological sample, actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106. Alternatively, in certain embodiments, migration of the analyte or analyte derivative (e.g., intermediate agent; e.g., ligation product) from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration). Exemplary methods of electrophoretic migration are described in WO 2020/176788, and US. Patent Application Pub. No. 2021/0189475, each of which is hereby incorporated by reference.
As further shown, one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including spatially barcoded capture probes 106). The one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.
In some embodiments, the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and 500 microns, between about 2 microns and 400 microns, between about 2 microns and 300 microns, between about 2 microns and 200 microns, between about 2 microns and 100 microns, between about 2 microns and 25 microns, or between about 2 microns and 10 microns), measured in a direction orthogonal to the surface of first substrate that supports the biological sample. In some instances, the separation distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In some embodiments, the separation distance is less than 50 microns. In some embodiments, the separation distance is less than 25 microns. In some embodiments, the separation distance is less than 20 microns. The separation distance may include a distance of at least 2 μm.
The sandwiching process methods described above can be implemented using a variety of hardware components. For example, the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sample handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in, e.g., US. Patent Application Pub. No. 2021/0189475, and PCT Publ. No. WO 2022/061152 A2, each of which are incorporated by reference in their entirety.
In some embodiments of a sample holder, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a biological sample. The first retaining mechanism can be configured to retain the first substrate disposed in a first plane. The sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane. The sample holder can further include an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism can be configured to align the first and second members along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane. The adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and/or move the first member along an axis orthogonal to the first plane.
In some embodiments, the adjustment mechanism includes a linear actuator. In some embodiments, the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and/or the second member. In some embodiments, the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and/or the second member. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0.1 mm/sec. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member with an amount of force of at least 0.1 lbs.
In some aspects, when the sample handling apparatus 200 is in an open position (e.g., in
In some aspects, after the first member 204 closes over the second member 210, an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and/or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration). The adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration.
In some embodiments, the biological sample (e.g., sample 102 from
In some embodiments, during the permeabilization step, the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and/or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas.
Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate.
In some embodiments, the first substrate and/or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate.
While
It may be desirable that the reagent medium be free from air bubbles between the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present between the substrates may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and/or elimination of air bubbles between the two substrates (e.g., slide 303 and slide 304) during a permeabilization step (e.g., step 104). In some aspects, it may be possible to reduce or eliminate bubble formation between the substrates using a variety of filling methods and/or closing methods. In some instances, the first substrate and the second substrate are arranged in an angled sandwich assembly as described herein. For example, during the sandwiching of the two substrates (e.g., the slide 303 and the slide 304), an angled closure workflow may be used to suppress or eliminate bubble formation.
At step 410, the dropped side of the angled substrate 406 contacts the reagent medium 401 first. The contact of the substrate 406 with the reagent medium 401 may form a linear or low curvature flow front that fills uniformly with the slides closed.
At step 415, the substrate 406 is further lowered toward the substrate 402 (or the substrate 402 is raised up toward the substrate 406) and the dropped side of the substrate 406 may contact and may urge the liquid reagent toward the side opposite the dropped side and creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the substrates. As further shown, the spring may begin to compress as the substrate 406 is lowered.
At step 420, the reagent medium 401 fills the gap between the substrate 406 and the substrate 402. The linear flow front of the liquid reagent may form by squeezing the 401 volume along the contact side of the substrate 402 and/or the substrate 406. Additionally, capillary flow may also contribute to filling the gap area. As further shown in step 420, the spring may be fully compressed such that the substrate 406, the substrate 402, and the base are substantially parallel to each other.
In some embodiments, the reagent medium (e.g., 105 in FIG IA) comprises a permeabilization agent. In some embodiments, following initial contact between the biological sample and a permeabilization agent, the permeabilization agent can be removed from contact with the biological sample (e.g., by opening sample holder). Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution).
In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. In some embodiments, the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, pepsin, elastase, and proteinase K. In some embodiments, the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises an RNase. In some embodiments, the Rnase is selected from Rnase A, Rnase C, Rnase H, and Rnase I. In some embodiments, the reagent medium comprises one or more of sodium dodecyl sulfate (SDS), proteinase K, pepsin, N-lauroylsarcosine, RNAse, and a sodium salt thereof.
In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG is from about PEG 2K to about PEG 16K. In some embodiments, the PEG is PEG 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 11K, 12K, 13K, 14K, 15K, or 16K. In some embodiments, the PEG is present at a concentration from about 2% to 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v/v).
In certain embodiments a dried permeabilization reagent is applied or formed as a layer on the first substrate or the second substrate or both prior to contacting the biological sample and the array. For example, a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.
In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1 minute, about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45 minutes, or about an hour. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1-60 minutes.
In some instances, the device is configured to control a temperature of the first and second substrates. In some embodiments, the temperature of the first and second members is lowered to a first temperature that is below room temperature.
There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.
In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). 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 the 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 by a reverse transcriptase. In some embodiments, the capture probe is extended using one or more DNA polymerases. In some embodiments, the extended capture probes include the sequence of the capture domain 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., sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction).
Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. 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 PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Spatial information can provide information of medical importance. For example, the methods described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder. Exemplary methods for identifying spatial information of biological and/or medical importance can be found in U.S. Patent Application Publication Nos. 2021/0140982, 2021/0198741, and 2021/0199660.
Spatial information can provide information of biological importance. For example, the methods described herein can allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor or proximity based analysis); determination of up- and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in healthy and diseased tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).
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 PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of PCT Publication No. WO2020/176788 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 PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
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 non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.
In some embodiments, the spatial barcode 505 and functional sequences 504 is common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 506 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.
In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
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 PCT Publication No. WO2020/176788 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):e128. 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 binding capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap 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). In some instances, the ligation product is removed using heat. In some instances, the ligation product is removed using KOH. The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.
A non-limiting example of templated ligation methods disclosed herein is depicted in
In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of PCT Publication No. WO2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.
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 described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.
When sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.
Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed . . . ” of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and/or the Visium Spatial Gene Expression Reagent Kits—Tissue Optimization User Guide (e.g., Rev E, dated February 2022).
In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of PCT Publication No. WO2020/123320.
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 (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.
In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Publication No. WO2021/102003 and/or U.S. Patent Application Publication No. 2021/0150707, 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 PCT Publication No. WO2020/053655 and spatial analysis methods are generally described in PCT Publication No. WO2021/102039 and/or U.S. Patent Application Publication No. 2021/0155982, 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 PCT Publication Nos. WO2020/123320, WO 2021/102005, and/or U.S. Patent Application Publication No. 2021/0158522, 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.
The human body includes a large collection of diverse cell types, each providing a specialized and context-specific function. Understanding a cell's chromatin structure can reveal information about a cell's function. Open chromatin, or accessible chromatin, or accessible genomic DNA, is often indicative of transcriptionally active sequences, e.g., genes, in a particular cell. Further understanding the transcriptionally active regions within chromatin will enable identification of which genes contribute to a cell's function and/or phenotype.
Methods have been developed to study epigenomes, e.g., chromatin accessibility assays (ATAC-seq, CUT & RUN, Cleavage Under Targets and Tagmentation (CUT & Tag)) or identifying proteins associated with chromatin e.g., (ChIP-seq). These assays help identify, for example, regulators (e.g., cis regulators and/or trans regulators) that contribute to dynamic cellular phenotypes. See, e.g., CUT & RUN and CUT & Tag described in Skene and Henikoff, eLife 2017; 6:e21856; Meers et al., eLIFE (2019) 8:e46314; and Kaya-Okur et al., Nature Communications (2019):10, 1930, each of which is incorporated by reference in its entirety. See also, e.g., single-cell CUT & Tag described in Bartosovic et al., Nature Biotechnology (2021):39, 825-835; Deng et al. bioRxiv (2021) preprint doi: https://doi.org/10.1101/2021.03.11.434985, each of which is incorporated by reference in its entirety. While CUT & RUN and CUT & Tag have been invaluable in defining epigenetic variability within a cell population, conventional applications of these methods are limited in their ability to spatially resolve the three dimensional structures and associated genes that promote cellular variation.
Thus, the present disclosure relates generally to the spatial tagging and analysis of nucleic acids. In some instances, provided herein are methods that utilize a transposase enzyme fused to an antibody-binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, fusion of protein A and protein G, or functional equivalent thereof), to engage and fragment, for example, the accessible (e.g., open chromatin) genomic DNA and enable the simultaneous capture of DNA and RNA from a biological sample, thus revealing epigenomic insights regarding the structural features contributing to cellular regulation.
In some instances of any of the spatial analysis methods described herein, CUT & Tag based methods or methods based on assays for transposase-accessible chromatin using sequencing (ATAC-seq) is used to generate genome-wide chromatin accessibility maps. These genome-wide accessibility maps can be integrated with additional genome-wide profiling data (e.g., RNA-seq, ChIP-seq, Methyl-Seq) to produce gene regulatory interaction maps that facilitate understanding of transcriptional regulation. For example, interrogation of genome-wide accessibility maps can reveal the underlying transcription factors and the transcription factor motifs responsible for chromatin accessibility at a given genomic location. Correlating changes in chromatin accessibility with changes in gene expression (RNA-seq), changes in transcription factor (TF) binding (e.g., ChIP-seq) and/or changes in DNA methylation levels (e.g., Methyl-seq) can identify the transcription regulation driving these changes. In disease states, there is often an imbalance in this transcriptional regulation. Thus, analyzing both chromatin accessibility and, for example, gene expression using spatial analysis methods enables identification of causes underlying the imbalances in transcriptional regulation.
Accordingly, provided herein are methods for determining a location of accessible genomic DNA in a biological sample. In some instances, the method includes (a) providing the biological sample on an 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) contacting a plurality of splint oligonucleotides to the biological sample, wherein a portion of a splint oligonucleotide of the plurality of splint oligonucleotides hybridizes to a portion of the capture domain; (c) contacting one or more antibodies, for example, antibodies that recognize a portion of a histone to the biological sample, (d) contacting a transposome complex to the biological sample, thereby generating fragmented and tagged genomic DNA (e.g., tagmented genomic DNA), wherein the transposome complex includes (i) an antibody-binding moiety (e.g. an antibody-binding protein), (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of the splint oligonucleotide, and (iv) the second transposon end sequence comprising a functional sequence (collectively, (ii), (iii) and (iv) form a transposome); (d) hybridizing a transposon splint sequence of the fragmented genomic DNA to the splint oligonucleotide and ligating the transposon splint sequence of the fragmented genomic DNA to the capture domain, thereby generating a capture probe that is ligated to the fragmented genomic DNA; and (e) determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine and correlate the location of the accessible genomic DNA in the biological sample.
Also provided herein are methods for determining and correlating a location of accessible genomic DNA in a biological sample. In some instances, the method includes (a) providing the biological sample on an 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) adding an antibody that binds to a chromatin protein to the biological sample; (c) binding a transposome - antibody-binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, or a fusion protein thereof) complex (“the complex”) to the antibody, thereby generating fragmented genomic DNA, wherein the complex comprises: (i) a transposase, (ii) an antibody-binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, or a fusion protein thereof), (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of the a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (d) adding a plurality of splint oligonucleotides to the biological sample, wherein a portion of a splint oligonucleotide of the plurality of splint oligonucleotides hybridizes to a portion of the capture domain; (e) hybridizing the splint sequence of the fragmented genomic DNA to the splint oligonucleotide and hybridizing the splint oligonucleotide to the capture probe; (f) ligating the splint sequence of the fragmented genomic DNA to the capture domain; and (g) determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the accessible genomic DNA in the biological sample.
In any of the methods described herein, binding a transposome - antibody-binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, or a fusion protein thereof) complex (“the complex”) to the antibody, thereby generating fragmented genomic DNA, is performed at room temperature. For example, room temperature can include any temperature between 22° C. to 30° C. In some instance, room temperature is 26° C., 27° C., or 28° C.
In any of the methods described herein, binding a transposome—antibody-binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, or a fusion protein thereof) complex (“the complex”) to the antibody, thereby generating fragmented genomic DNA further comprises incubating the multi-complex added to the biological sample for one or more hours (e.g. 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 hours), for example, at room temperature.
In another embodiment of any of the methods described herein, binding a transposome—antibody-binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, or a fusion protein thereof) complex (“the complex”) to the antibody, thereby generating fragmented genomic DNA further comprises incubating the multi-complex added to the biological sample for one or more days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, or 14 days), for example, at room temperature.
In some instances, step (d) and the step of determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine and correlate the location of the accessible genomic DNA in the biological sample are performed sequentially. In some instances, step (d) and the step of determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine and correlate the location of the accessible genomic DNA in the biological sample are performed simultaneously. For example, some tagmented DNA fragments can be captured with non-ligated transposon end sequences still hybridized. In such examples, the non-ligated transposon end sequences are released after capture of the tagmented DNA. In some instances, the non-ligated transposon end sequences are released prior to capture by the capture domain.
Also provided herein are methods for determining genomic DNA accessibility including (a) a biological sample on an 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) contacting the biological sample with one or more antibodies, for example that will recognize and bind a portion of a histone, (d) contacting a transposome and/or transposome complex to the biological sample to insert transposon end sequences into accessible genomic DNA, thereby generating fragmented genomic DNA; (c) binding the transposon end sequences of the fragmented genomic DNA to the capture domain of the capture probe; (d) releasing one or more transposon end sequences not bound to the capture domain; (e) determining (i) all or a portion of a sequence of the spatial barcode or a complement thereof, and (ii) all or a portion of a sequence of the fragmented genomic DNA, or a complement thereof, and using the determined sequences of (i) and (ii) to determine a location of the accessible genomic DNA in the biological sample.
In some instances, steps (c) and (d) are performed sequentially. In some instances, steps (c) and (d) are performed simultaneously. For example, some tagmented DNA fragments can be captured with one or more transposon end sequences still hybridized. In such examples, the one or more transposon end sequences are released after capture of the tagmented DNA. In some instances, the one or more transposon end sequences are released prior to capture by the capture domain.
In some instances, provided herein are methods for spatial analysis of nucleic acids (e.g., genomic DNA, mRNA) in a biological sample. In some instances, an array is provided, wherein the array comprises a plurality of capture probes. In some instances, the capture probes may be attached directly to the substrate (e.g., an array comprising a substrate comprising a plurality of capture probes). In some instances, the capture probes may be attached indirectly to the substrate. For example, the capture probes can be attached to features on the substrate. In some instances, a feature is a bead. In some instances, the capture probes comprise a spatial barcode and a capture domain. In some instances, the capture probe can be partially double stranded. In some instances, the capture probe can bind a complementary oligonucleotide. In some instances, the complementary oligonucleotide (e.g., splint oligonucleotide) can have a single stranded portion. In some instances, the single stranded portion can bind fragmented (e.g., tagmented) DNA. In some instances, a biological sample is treated under conditions sufficient to make nucleic acids in cells of the biological sample (e.g., genomic DNA) accessible to transposon insertion (e.g., tagging the DNA fragments with transposon ends). In some instances, a transposon end sequence and a transposase enzyme (collectively, a transposome) are provided to the biological sample such that the transposon end sequence can be inserted into the genomic DNA of cells present in the biological sample. In some instances, the transposase enzyme of the transposome complex fragments the genomic DNA and transposon ends are attached to the ends of the genomic DNA fragments (e.g., “tagmenting”).
In some instances, the biological sample comprising nucleic acids (e.g., genomic DNA, mRNA) is contacted to the substrate such that a capture probe can interact with the fragmented and tagged (e.g., tagmented) genomic DNA. In some instances, the biological sample comprising nucleic acids (e.g., genomic DNA, mRNA) is contacted with the substrate such that the capture probe can interact with both the tagmented genomic DNA and the mRNA present in the biological sample (e.g., a first capture probe can bind genomic DNA, a second capture probe can bind mRNA).
In some instances, the location of the capture probe on the substrate can be correlated to a location in the biological sample, thereby spatially determining the location of the tagmented genomic DNA. In some instances, the location of the capture probe on the substrate can be correlated to a location in the biological sample, thereby spatially determining the location of the tagmented genomic DNA and mRNA in the biological sample.
In some instances, where spatial determining the location of analytes includes a concurrent analysis of different types of analytes from a single cell or a subpopulation of cells within a biological sample (e.g., a tissue section), an additional layer of spatial information can be integrated into the genome regulatory interaction maps. In some instances, the spatial determining of analytes can be done on whole genomes. In some instances, the spatial profiling can be done on an immobilized biological sample.
In some instances, the genome-wide chromatic accessibility maps can be generated by spatial CUT & Tag methods. Without being bound by theory, CUT & Tag uses a transposome that includes a hyperactive Tn5 transposase, or another transposase, fused to Protein A (pA-Tn5) or Protein G (pG-Tn5), thereby creating a protein A or protein G, or a combination thereof-transposome complex. The pA-Tn5 or pG-Tn5 complex further is loaded with sequencing adapters to bind to a target protein that is bound to genomic DNA. Non-limiting examples include those of SEQ ID NOs: 1-2, and other modified varieties known in the art. (See, for example, U.S. Pat. Nos. 9,790,476; 10,035,992; and 10,544,403, which are incorporated in their entirety herein). After binding, the complex cuts (i.e., digests) the DNA under the target protein. Released are DNA fragments that are ready for fragment capture on an array described herein, PCR enrichment, and DNA sequencing. For a detailed description of CUT & Tag, see, for e.g., Kaya-Okur et al. Nature Communications (2019):1930, which is incorporated by reference in its entirety.
In some instances, the genome-wide chromatin accessibility maps generated by spatial CUT & Tag can be used for cell type identification. For example, traditional cell type classification relies on mRNA expression levels but chromatin accessibility can be more adept at capturing cell identity. Furthermore, in some instances, correlations between transcriptionally active regions (e.g., open chromatin, accessible) with expression profiles (e.g., expression profiles of mRNA) can be determined in a spatial manner.
In some instances, the genome-wide chromatic accessibility maps can be generated by spatial ATAC-seq. In some instances, the genome-wide chromatin accessibility maps generated by spatial ATAC-seq can be used for cell type identification. Without being bound by theory, ATAC-seq maps a chromatin protein by binding of a specific antibody, and then tethering a Protein A/Micrococcal Nuclease (pA-MNase) fusion protein in permeabilized cells without cross-linking. MNase is activated by addition of calcium, and fragments are released into the supernatant for extraction of DNA, library preparation and paired-end sequencing. For a detailed description of ATAC-seq, see, for e.g., Skene and Henikoff (2017). Elife 6, e21856.
In some instances, the genome-wide chromatin accessibility maps generated by spatial ATAC-seq can be used for cell type identification. As indicated above for use with spatial CUT&Tag, traditional cell type classification can rely on mRNA expression levels but chromatin accessibility can be more adept at capturing cell identity. Furthermore, in some instances, correlations between transcriptionally active regions (e.g., open chromatin, accessible) with expression profiles (e.g., expression profiles of mRNA) can be determined in a spatial manner.
The present disclosure generally describes methods of tagmenting genomic DNA to generate DNA fragments in a biological sample. In some examples, a chemical or enzymatic “pre-permeabilization” of biological samples immobilized on a substrate can be employed to make DNA in the biological sample accessible to a transposase enzyme (e.g. a transposome or a transposome antibody complex). In some instances, permeabilizing the biological sample can be a two-step process (e.g., pre-permeabilization treatment, followed by a permeabilization treatment). In some instances, permeabilizing the biological sample can be a one-step process (e.g., a single permeabilization treatment sufficient to permeabilize the cellular and nuclear membranes in the biological sample).
In some instances, pre-permeabilization can include an enzymatic or chemical condition. In some instances, pre-permeabilization can be performed with an enzyme (e.g., a protease). In some instances, in a non-limiting way, the protease can include trypsin, pepsin, dispase, papain, accuses, or collagenase. In some instances, pre-permeabilization can include an enzymatic treatment with pepsin. In some instances, pre-permeabilization can include pepsin in 0.5M acetic acid. In some instances, pre-permeabilization can include pepsin in Exonuclease-1 buffer. In some instances, the pH of the buffer can be acidic. In some instances, pre-permeabilization can include enzymatic treatment with collagenase. In some instances, pre-permeabilization can include collagenase in HBSS buffer. In some instances, the HBSS buffer can include bovine serum albumin (BSA). In some instances, pre-permeabilization can last for about 1 to minute to about 20 minutes. In some instances, pre-permeabilization can last for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19 minutes. In some instances, pre-permeabilization can last for about 10 minutes to about one hour. For example, in some instances, pre-permeabilization can last for about 20, about 30, about 40, or about 50 minutes.
In some instances, permeabilizing the biological sample comprises an enzymatic treatment. In some instances, the enzymatic treatment can be a pepsin enzyme, or a pepsin-like enzyme treatment. In some instances, the enzymatic treatment can be protease treatment. In some instances, enzymatic treatment can be performed in the presence of reagents. In some instances, the enzymatic treatment (e.g., pre-permeabilization) can include contacting the biological specimen with an acidic solution including a protease enzyme. In some instances, the reagent can be HCl. In some instances, the reagent can be acetic acid. In some instances, the concentration of HCl can be about 100mM. In some instances, the about 100mM HCl can have a pH of around, or about 1.0. In some instances, the additional reagent can be 0.5M acetic acid, having a pH of around, or about 2.5. It is noted that enzymatic treatment of the biological sample can have different effects on tagmentation. For example, enzymatic treatment with pepsin and 100 mM HCl can result in tagmentation of chromatin regardless of chromatin accessibility. In some instances, enzymatic treatment with pepsin and 0.5M acetic acid can result in tagmentation of chromatin that can retain a nucleosomal pattern indicative of tagmentation.
In some instances, the enzymatic treatment can comprise contacting the biological sample with a reaction mixture (e.g., solution) comprising an aspartyl protease (e.g., pepsin) in an acidic buffer, e.g., a buffer with a pH of about 4.0 or less, such as about 3.0 or less, e.g., about 0.5 to about 3.0, or about 1.0 to about 2.5. In some instances, the aspartyl protease is a pepsin enzyme, pepsin-like enzyme, or a functional equivalent thereof. Thus, any enzyme or combination of enzymes in the enzyme commission number 3.4.23.1.
In some instances, the enzymatic treatment (e.g., pre-permeabilization) can be performed using collagenase. In some instances, enzymatic treatment with collagenase can provide access to the genomic DNA for the transposase, transposome, or transposome antibody complex while preserving nuclear integrity. In some instances, pre-permeabilization (e.g., enzymatic treatment) with collagenase yields nucleosomal patterns generally associated with tagmentation. Collagenases can be isolated from Clostridium histolyticum. In some instances, enzymatic treatment with a zinc endopeptidase (e.g., collagenase) with reagents and under conditions suitable for proteolytic activity comprises a buffered solution with a pH of about 7.0 to about 8.0 (e.g., about 7.4). Collagenases are zinc endopeptidases and can be inhibited by either EDTA or EGTA, or both. Therefore, in some instances, the biological sample can be contacted with a zinc endopeptidase (e.g., collagenase) in the absence of a chelator of divalent cations, (e.g., EDTA, EGTA). In some instances, it can be useful to stop the zinc endopeptidase (e.g., collagenase) and the permeabilization step can be stopped (e.g., inhibited) by contacting the biological sample with a chelator of divalent cations (e.g., EDTA, EGTA).
In some instances, the zinc endopeptidase is a collagenase enzyme, collagenase-like enzyme, or a functional equivalent thereof. In such instances, any enzyme or combination of enzymes in the enzyme commission number 3.4.23.3 can be used in accordance with materials and methods described herein. In some instances, the collagenase is one or more collagenases from the following group, (UniProtKB/Swiss-Prot accession numbers): P43153/COLA_CLOPE; P43154/COLA_VIBAL; Q9KRJO/COLA_VIBCH; Q56696/COLA_VIBPA; Q8D4Y9/COLA_VIBVU; Q9X721/COLG_HATHI; Q46085/COLH_HATHI; Q899Y1/COLT_CLOTE URSTH and functional variants and derivatives thereof (described herein), or a combination thereof.
Methods of permeabilizing biological samples are well known in the art. It will be known to a person skilled in the art that different sources of biological samples can be treated with different reagents (e.g., proteases, RNAses, detergents, buffers) and under different conditions (e.g., pressure, temperature, concentration, pH, time). In some instances, permeabilizing the biological sample can comprise reagents and conditions to sufficiently disrupt the cell membrane of the biological sample to capture nucleic acid (e.g., mRNA). In some instances, permeabilizing the biological sample can comprise reagents and conditions to sufficiently disrupt the nuclear membrane of the biological sample to capture nucleic acid (e.g., genomic DNA). In some instances, commercially available proteases isolated from their native (e.g., animal, microbial) source can be used. In some instances, proteases produced recombinantly (e.g., bacterial expression system) can be used. In some instances, pre-permeabilizing and permeabilizing a biological sample can be a one-step process (e.g., enzymatic treatment). In some instances, pre-permeabilizing and permeabilizing a biological sample can be a two-step process (e.g., enzymatic treatment, followed by chemical or detergent treatment).
In some instances, the chemical permeabilization conditions comprise contacting the biological specimen with an alkaline solution, e.g. a buffered solution with a pH of about 8.0 to about 11.0, such as about 8.5 to about 10.5 or about 9.0 to about 10.0, e.g. about 9.5. In some instances, the buffer is a glycine-KOH buffer. Other buffers are known in the art.
In some instances, a biological sample can be treated with a detergent following an enzymatic treatment (e.g., permeabilization following a pre-permeabilization step). Detergents are known in the art. Any suitable detergent can be used, including, in a non-limiting way NP-40 or equivalent, Digitonin, Tween-20, IGEPAL-40 or equivalent, Saponin, SDS, Pitsop2, or combinations thereof. In some instances, a biological sample can be treated with other chemicals known to permeabilize cellular membranes. As further exemplified in the examples below, detergents described herein can be used at a concentration of between about 0.01% to about 0.1%. In some instances, detergents described herein can be used at a concentration of about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, or about 0.9%. In some instances, detergents described herein can be used at a concentration of about 1.1% to about 10% or more. In some instances, detergents described herein can be used at a concentration of about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9%, or about 10%.
Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for biological sample permeabilization can generally be used in connection with the biological samples described herein.
Different sources of biological samples can be treated with different reagents (e.g., proteases, RNAses, detergents, buffers) and under different suitable conditions (e.g., pressure, temperature, concentration, pH, time) to achieve sufficient pre-permeabilization and permeabilization to capture nucleic acids (e.g., genomic DNA, mRNA).
In some instances, the reaction mixture (e.g., solution) including the proteases described herein can contain other reagents, (e.g., buffer, salt, etc.) sufficient to ensure that the proteases are functional. For instance, the reaction mixture can further include an albumin protein, (e.g., BSA). In some instances, the reaction mixture (e.g., solution) including the collagenase enzyme (or functional variant or derivative thereof) includes an albumin protein, (e.g., BSA).
In some instances, there is one or more wash steps between pre-permeabilization and permeabilization of a biological sample. For example, it may be preferential to wash as much of the pre-permeabilization solution off of the biological sample prior to adding a permeabilization solution. Therefore, in some instances the biological sample is washed, for example with a SSC wash solution, after pre-permeabilization to remove the pre-permeabilization reagents and before applying permeabilization reagents to the biological sample. In some instances, the permeabilization solution is removed from the biological sample prior to the addition of the transposome or transposome complex reagents for tagmentation of the released genomic DNA. One or more washes may also be performed post permeabilization and pre-tagmentation, for example using a SSC solution. In some instances, there are no wash steps between permeabilization of the biological sample and tagmentation of the genomic DNA.
Transposase enzymes and transposons can be utilized in methods of spatial genomic analysis. Generally, transposition is the process by which a specific genetic sequence (e.g., a transposon sequence) is relocated from one place in a genome to another. Many transposition methods and transposable elements are known in the art (e.g., DNA transposons, retrotransposons, autonomous transposons, non-autonomous transposons). One non-limiting example of a transposition event is conservative transposition. Conservative transposition is a non-replicative mode of transposition in which the transposon is completely removed from the genome and reintegrated into a new locus, such that the transposon sequence is conserved, (e.g., a conservative transposition event can be thought of as a “cut and paste” event) (See, e.g., Griffiths A. J., et. al., Mechanism of transposition in prokaryotes. An Introduction to Genetic Analysis (7th Ed.). New York: W. H. Freeman (2000)).
In one example, transposition can occur when a transposase enzyme binds a sequence flanking the ends of the transposome (e.g., a recognition sequence, e.g., a mosaic end sequence). A transposome (e.g., a transposition complex) forms and the endogenous DNA can be manipulated into a pre-excision complex such that, for example, two transposase enzymes can interact to form a dimer when the transposase is a Tn5 transposase or a tetramer when the transposase is a Mu transposase. In some instances, when the transposases interact with the DNA, double stranded breaks are introduced into the DNA resulting in the insertion of the transposon sequence. The transposase enzymes can locate and bind a target site in the DNA, create a double stranded break, and insert the transposon end sequence (See, e.g., Skipper, K.A., et. al., DNA transposon-based gene vehicles-scenes from an evolutionary drive, J Biomed Sci., 20: 92 (2013) doi:10.1186/1423-0127-20-92). Canonical transposases include Tn5. Alternative cut and paste transposases include Tn552 (College, et al, J. BacterioL, 183: 2384-8, 2001; Kirby C et al, Mol. Microbiol, 43: 173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res., 22: 3765-72, 1994 and International Publication WO 95/23875), Transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol, 204:27-48, 1996), Tn/O and IS10 (Kleckner N, et al, Curr Top Microbiol Immunol, 204:49-82, 1996), Mariner transposase (Lampe D J, et al, EMBO J., 15: 5470-9, 1996), Tel (Plasterk R H, Curr. Topics Microbiol. Immunol, 204: 125-43, 1996), P Element (Gloor, G B, Methods Mol. Biol, 260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265: 18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown, et al, Proc Natl Acad Sci USA, 86:2525-9, 1989), and retrotransposon of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989). More examples include ISS, TnlO, Tn903, IS911, and engineered versions of transposase family enzymes (Zhang et al, (2009) PLoS Genet. 5:e1000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J. Microbiol. Methods 71:332-5).
Transposome-mediated fragmentation and tagging (“tagmentation”) is a process of transposase-mediated fragmentation and tagging of DNA. A transposome is a complex of a transposase enzyme and DNA which comprises a transposon end sequence (also known as “transposase recognition sequence” or “mosaic end” (MEs)). In some methods of spatial genomic analysis, DNA is fragmented in such a manner that a functional sequence such as a sequence complementary to a capture domain of a capture probe (e.g., capture domain of a splint oligonucleotide) is inserted into the fragmented DNA (e.g., the fragmented DNA is “tagged”), such that the sequence (e.g. an adapter) can hybridize to the capture probe. In some instances, the capture probe is present on a substrate. In some instances, the capture probe (e.g., a capture probe and a splint oligonucleotide) is present on a feature. A transposome dimer, in the case of the Tn5 transposome, is able to simultaneously fragment DNA based on its transposon recognition sequences and ligate the transposon sequences from the transposome to the fragmented DNA (e.g., tagmented DNA). This system has been adapted using hyperactive transposase enzymes and modified DNA molecules (adaptors) comprising MEs to fragment DNA and tag both strands of DNA duplex fragments with functional DNA molecules (e.g., primer binding sites). For instance, the Tn5 transposase may be produced as purified protein monomers. Tn5 transposase is also commercially available (e.g., manufacturer Illumina, Illumina.com, Catalog No. 15027865, TD Tagment DNA Buffer Catalog No. 15027866). These can be subsequently loaded with the oligonucleotides of interest, e.g., ssDNA oligonucleotides containing MEs (e.g., transposon sequences) for Tn5 recognition and additional functional sequences (e.g., Nextera adapters, e.g., primer binding sites) are annealed to form a dsDNA mosaic end oligonucleotide (MEDS) that is recognized by Tn5 during dimer assembly (e.g., transposome dimerization). In some instances, a hyperactive Tn5 transposase can be loaded with adapters (e.g., oligonucleotides of interest) which can simultaneously fragment and tag a genome with the sequences. As used herein, the term “tagmentation” refers to a step in the Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) or a step in the CUT & Tag assay. (See, e.g., Buenrostro, J. D., Giresi, P. G., Zaba, L. C, Chang, H. Y., Greenleaf, W. J., Transposition of native chromatin for fast and sensitive epi genomic profiling of open chromatin, DNA-binding proteins and nucleosome position, Nature Methods, 10 (12): 1213-1218 (2013)). ATAC-seq identifies regions of open chromatin using a hyperactive prokaryotic Tn5-transposase, which preferentially inserts into accessible chromatin and tags the sites with adaptors (Buenrostro, J. D., et. al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods, 10: 1213-1218 (2013)).
As used herein “accessible chromatin” or “open chromatin” or “accessible genomic DNA” refers to portions of a genome that are nucleosome-depleted regions that can be bound by proteins and play various roles in nuclear organization, gene transcription, and are generally considered transcriptionally active regions of DNA (Zhang, Q., et al., Genome-wide open chromatin regions and their effects on the regulation of silk protein genes in Bombyx mori, Scientific Reports, 7: 12919 (2017).
In some instances, the step of fragmenting the genomic DNA in cells of the biological sample comprises contacting the biological sample containing the genomic DNA with the transposase enzyme (e.g., a transposome or transposome antibody complex, e.g., a reaction mixture (e.g., solution)) including a transposase, transposome, or transposome antibody complex), under any suitable conditions. In some instances, such suitable conditions result in the tagmentation of the genomic DNA of cells present in the biological sample. Typical conditions will depend on the transposase enzyme and/or antibody complexed to the transposome used and can be determined using routine methods known in the art. Therefore, suitable conditions can be conditions (e.g., buffer, salt, concentration, pH, temperature, time conditions) under which the transposase enzyme is functional, e.g., in which the transposase enzyme displays transposase activity, particularly tagmentation activity, in the biological sample.
The term “functional”, as used herein in reference to transposase enzymes, is meant to include instances in which the transposase enzyme can show some reduced activity relative to the activity of the transposase enzyme in conditions that are optimum for the enzyme, e.g., in the buffer, salt and temperature conditions recommended by the manufacturer. Thus, the transposase can be considered to be “functional” if it has at least about 50%, e.g., at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, activity relative to the activity of the transposase in conditions that are optimum for the transposase enzyme.
In one non-limiting example, the reaction mixture comprises a transposome and/or a transposome antibody complex in a buffered solution (e.g., Tris-acetate) having a pH of about 6.5 to about 8.5, e.g., about 7.0 to about 8.0 such as about 7.5. Additionally or alternatively, the reaction mixture can be used at any suitable temperature, such as about 10° to about 55° C., e.g., about 10° to about 54°, about 11° to about 53° , about 12° to about 52°, about 13° to about 51°, about 14° to about 50°, about 15° to about 49° , about 16° to about 48°, about 17° to about 47° C., e.g., about 10°, about 12°, about 15°, about 18°, about 20° , about 22°, about 25°, about 28°, about 30°, about 33°, about 35°, about or 37° C., preferably about 30° to about 40° C., e.g., about 37° C. In some instances, the transposome can be contacted with the biological sample for about 10 minutes to about one hour. In some instances, the transposome can be contacted with the biological sample for about 20, about 30, about 40, or about 50 minutes. In some instances, the transposome can be contacted with the biological sample for about 1 hour to about 4 hours.
In some instances, the transposase enzyme of the transposome complex is a Tn5 transposase, or a functional derivate or variant thereof (See, e.g., Reznikoff et al, WO 2001/009363, U.S. Pat. Nos. 5,925,545, 5,965,443, 7,083,980, and 7,608,434, and Goryshin and Reznikoff, J. Biol. Chem. 273:7367, (1998), which are herein incorporated by reference). In some instances, the Tn5 transposase is a hyper Tn5 transposase, or a functional derivate or variate thereof (U.S. Pat. No. 9,790,476, incorporated herein by reference). For example, the Tn5 transposase can be a fusion protein (e.g., a Tn5 fusion protein). Tn5 is a member of the RNase superfamily of proteins which includes retroviral integrases. The Tn5 transposon is a composite transposon in which two near-identical insertion sequences (IS50L and IS50R) flank three antibiotic resistance genes. Each IS50 contains two inverted 19-bp end sequences (ESs), an outside end (OE) and an inside end (IE). Wild-type Tn5 transposase enzyme is generally inactive (e.g., low transposition event activity). However, amino acid substitutions can result in hyperactive variants or derivatives. In one non-limiting example, amino acid substitution, L372P, substitutes a leucine amino acid for a proline amino acid which results in an alpha helix break, thus inducing a conformational change to the C-terminal domain. The alpha helix break separates the C-terminal domain and N-terminal domain sufficiently to promote higher transposition event activity (See, Reznikoff, W. S., Tn5 as a model for understanding DNA transposition, Mol Microbiol, 47(5): 1199-1206 (2003)). Other amino acid substitutions resulting in hyperactive Tn5 are known in the art. For example, the improved avidity of the modified transposase enzyme (e.g., modified Tn5 transposase enzyme) for the repeat sequences for OE termini (class (1) mutation) can be achieved by providing a lysine residue at amino acid 54, which is glutamic acid in wild-type Tn5 transposase enzyme (See U.S. Pat. No. 5.925,545). The mutation strongly alters the preference of the modified transposase enzyme (e.g., modified Tn5 transposase enzyme) for OE termini, as opposed to IE termini. The higher binding of this mutation, known as EK54, to OE termini results in a transposition rate that is about 10-fold higher than is seen with wild-type transposase enzyme (e.g., wild type Tn5 transposase enzyme). A similar change at position 54 to valine (e.g., EV54) also results in somewhat increased binding/transposition for OE termini, as does a threonine to proline change at position 47 (e.g., TP47; about 10-fold higher) (See U.S. Pat. No. 5.925,545).
Other examples of modified transposase enzymes (e.g., modified Tn5 transposase enzymes) are known. For example, a modified Tn5 transposase enzyme that differs from wild-type Tn5 transposase enzyme in that it binds to the repeat sequences of the donor DNA with greater avidity than wild-type Tn5 transposase enzyme and also is less likely than the wild-type transposase enzyme to assume an inactive multimeric form (U.S. Pat. No. 5,925,545, which is incorporated by reference in its entirety). Furthermore, techniques generally describing introducing any transposable element (e.g., Tn5) from a donor DNA (e.g., adapter sequence, e.g., Nextera adapters (e.g., top and bottom adapter) into a target are known in the art. (See, e.g., U.S. Pat. No. 5,925,545). Further study has identified classes of mutations resulting in a modified transposase enzyme (e.g., modified Tn5 transposase enzyme) (See, U.S. Pat. No. 5,965,443, which is incorporated by reference in its entirety). For example, a modified transposase enzyme (e.g., modified Tn5 transposase enzyme) with a “class 1 mutation” binds to repeat sequences of donor DNA with greater avidity than wild-type Tn5 transposase enzyme. Additionally, a modified transposase enzyme (e.g., modified Tn5 transposase enzyme) with a “class 2 mutation” is less likely than the wild-type Tn5 transposase enzyme to assume an inactive multimeric form. It has been shown that a modified transposase enzyme that contains both a class 1 and a class 2 mutation can induce at least about 100-fold (+10%) more transposition than the wild-type transposase enzyme, when tested in combination with an in vivo conjugation assay as described by Weinreich, M. D., “Evidence that the cis Preference of the Tn5 Transposase is Caused by Nonproductive Multimerization,” Genes and Development 8:2363-2374 (1994), incorporated herein by reference (See e.g., U.S. Pat. No. 5,965,443). Further, under sufficient conditions, transposition using the modified transposase enzyme (e.g., modified Tn5 transposase enzyme) may be higher. A modified transposase enzyme containing only a class 1 mutation can bind to the repeat sequences with sufficiently greater avidity than the wild-type Tn5 transposase enzyme such that a Tn5 transposase enzyme induces about 5- to about 50-fold more transposition than the wild-type transposase enzyme, when measured in vivo. A modified transposase enzyme containing only a class 2 mutation (e.g., a mutation that reduces the Tn5 transposase enzyme from assuming an inactive form) is sufficiently less likely than the wild-type Tn5 transposase enzyme to assume the multimeric form that such a Tn5 transposase enzyme also induces about 5- to about 50-fold more transposition than the wild- type transposase enzyme, when measured in vivo (See U.S. Pat. No. 5,965,443)
Other methods of using a modified transposase enzyme (e.g., modified Tn5 transposase enzyme are further generally described in U.S. Pat. Nos. 5,965,443 and 9,790,476. For example, a modified transposase enzyme could provide selective markers to target DNA, to provide portable regions of homology to a target DNA, to facilitate insertion of specialized DNA sequences into target DNA, to provide primer binding sites or tags for DNA sequencing, or to facilitate production of genetic fusions for gene expression. Studies and protein domain mapping, as well as, to bring together other desired combinations of DNA sequences (combinatorial genetics) (U.S. Pat. No. 5,965,443).Still other methods of inserting a transposable element (e.g., transposon) at random or semi-random locations in chromosomal or extra-chromosomal nucleic acid are known. For example, methods including a step of combining in a biological sample nucleic acid (e.g., genomic DNA) with a synaptic complex that comprises a Tn5 transposase enzyme complexed with a sequence comprising a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase enzyme and a transposable element (e.g., transposon) under conditions that mediate transposition events into the genomic DNA. In this method, a synaptic complex can be formed in vitro under conditions that disfavor or prevent synaptic complexes from undergoing a transposition event. The frequency of transposition (e.g., transposition events) can be increased by using either a hyperactive transposase enzyme (e.g., a mutant transposase enzyme) or a transposable element (e.g., transposon) that contains sequences well adapted for efficient transposition events in the presence of a hyperactive transposase enzyme (e.g., hyperactive Tn5 transposase enzyme), or both (U.S. Pat. No. 6,159,736, which is incorporated herein by reference).
In some instances, the transposome can be fused to (e.g., joined with, linked to, associated with, complexed to) anantibody-binding moiety to form a transposome-antibody binding moiety (e.g. an antibody-binding protein that contains an antibody-binding moiety, such as protein A, protein G, or a fusion protein thereof) complex, or a “transposome complex”. In some instances, the transposome is fused to protein A. In some instances, the transposome is fused to protein G. In some instances, the transposome is fused to a fusion protein of protein A and protein G (protein A/G). A variety of linkers (e.g. amino acid linkers) that are flexible or rigid can be used. Linkers can include cyclopeptide linkers, disulfide linkers, and protease-sensitive linkers that are cleavable. In some instances, the transposome is fused to the antibody with a DDDKEF(GGGGS)4 linker. Other fusion protein linkers are described in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369, which is incorporated by reference in its entirety. The transposome can also be fused to the antibody or protein binding moiety post translational linkers, such as generation of covalent or non-covalent bonds.
An antibody can bind to a target protein that may be associated with chromatin (e.g. bound to), for example a transcription factor, histones, histone PTMs, nucleosomes, ChAPs, and DNA methylation, or any protein binding moiety. A protein that binds in a general manner to antibodies, such as protein A, protein G, a fusion protein, a fusion between protein A and protein G, protein L, protein Y, or functional derivatives thereof, can be used to bind to the antibody that is bound to the target protein. The protein biding moiety can include biotin, glutathione-S-transferase (GST), etc. In some instances, the antibody binding protein can be protein A, or a functional derivative thereof. In some instances, the antibody binding protein is protein G, or a functional derivative thereof.
Methods, compositions, and kits for treating nucleic acid, and in particular, methods and compositions for fragmenting and tagging DNA using transposon compositions are described in detail in U.S. Patent Application Publication No. US 2010/0120098, U.S. Patent Application Publication No. US2011/0287435, and Satpathy, A. T., et. al., Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T-cell exhaustion, Nat Biotechnol., 37, 925-936 (2019), the contents of which are herein incorporated by reference in their entireties.
Any transposase enzyme with tagmentation activity, e.g., any transposase enzyme capable of fragmenting DNA and inserting oligonucleotides (e.g., adapters, e.g. Nextera index adapters) to the ends of the fragmented (e.g., tagmented) DNA, can be used. In some instances, the transposase is any transpose capable of conservative transposition. In some instances, the transposase is a cut and paste transposase. Other kinds of transposase are known in the art and are within the scope of this disclosure. For example, suitable transposase enzymes include, without limitation, Mos-1, HyperMu™, Ts-Tn5, Ts-Tn5059, Hermes, Tn7, a Vibhar species transposase (See e.g., U.S. Patent Application No. 20120301925A1 and WO 2015/069374, the contents of which are herein incorporated by reference in their entireties), or any functional variant or derivative of the previously listed transposase enzymes.
In some instances, a hyperactive variant of the Tn5 transposase enzyme is capable of mediating the fragmentation of double-stranded DNA and ligation of synthetic oligonucleotides (e.g., Nextera adapters) at both 5′ ends of the DNA in a reaction that takes a short period of time (e.g., about 5 minutes). However, as wild-type end sequences have a relatively low activity, they are sometimes replaced in vitro by hyperactive mosaic end (ME) sequences. A complex of the Tn5 transposase with 19-bp ME facilitates transposition, provided that the intervening DNA is long enough to bring two of these sequences close together to form an active Tn5 transposase enzyme homodimer.
In some instances, the Tn5 transposase enzyme, or functional variant or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ. ID NO. 1. In some instances, the Tn5 transposase enzyme, or functional variant or derivative thereof, comprises an amino acid sequence having a sequence identity of at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NO. 1.
In some instances, the transposase enzyme is a Mu transposase enzyme, or a functional variant or derivative thereof.
The adaptors (e.g., Nextera adaptors) in the complex with the transposase enzyme (e.g., that form part of the transposome, e.g., MEDS described herein) can include partially double stranded oligonucleotides. In some instances, there is a first adapter and a second adapter. In some instances, the first adapter can be complexed with a first monomer. In some instances, the second adapter can be complexed with a second monomer. In some instances, the first monomer complexed with the first adapter and the second monomer complexed with the second monomer can be assembled to form a dimer. In some instances, the double stranded portion of the adaptors contains transposon end sequences (e.g., Mosaic End (ME)) sequences. In some instances, the single stranded portion of the adaptors (e.g., Nextera index adapters) (5′ overhang) contains the functional domain or sequence to be incorporated in the tagmented DNA. In some instances, the adapters can be Nextera adapters (e.g., index adapter) (for example, reagents including, Nextera DNA Library Prep Kit for ATAC-seq (no longer available), TDE-1 Tagment DNA Enzyme (Catalog No. 15027865), TD Tagment DNA Buffer (Catalog No. 15027866), available from Illumina, Illumina.com). In some instances, the sequence incorporated into the tagmented DNA is a sequence complementary to a capture domain of a capture probe. In some instances, the sequence complementary to the capture domain of the capture probe is a transposon end sequence. In such instances, the functional domain is on the strand of the adaptor that will be ligated to the capture probe. In other words, the functional domain can be located upstream (e.g., 5′ to) the ME sequence, e.g., in the 5′ overhang of the adapter.
The adaptors (e.g., Nextera index adapters, e.g., first and second adapters) ligated to the tagmented DNA can be any suitable sequence. For example, the sequence can be a viral sequence. In some instances, the sequence can be a CRISPR sequence. In some instances, the adaptor (e.g., oligonucleotides) ligated to the tagmented DNA can be a CRISPR guide sequence. In some instances, the CRISPR guide sequence can target a sequence of interest (e.g., genomic locus of interest e.g., gene specific).
In some instances, the ME sequence is a Tn5 transposase recognition sequence. In some instances, the mosaic end (e.g., ME) sequence is a Mu transposase recognition sequence.
In some instances, a composition comprising a transposase enzyme (e.g., any transposase enzyme described herein) complexed with adapters (e.g., first and second adapters complexed with first and second monomers, respectively) comprising transposon end sequences (e.g., mosaic end sequences) is used in a method for spatially tagging nucleic acids in a biological sample. In some instances, a composition comprising a transposase enzyme further comprises a domain that binds to a capture probe as described herein (e.g., Nextera adapter, e.g., first adapter) and a second adapter is used in a method for spatially tagging nucleic acids of a biological sample, such as any of the methods described herein.
In some instances, the transposase enzyme can be in the form of a transposome comprising adaptors (MEDS) in which the 5′ overhang can be phosphorylated. In some instances, the adaptors (e.g., Nextera adaptors, e.g., first and second adapters) may be phosphorylated prior to their assembly with the transposase enzyme to form the transposome. In some instances, phosphorylation of adaptors can occur when complexed with a transposase enzyme (e.g., phosphorylation in situ in the transposome).
In some instances, the 5′ overhang of the adaptor is not phosphorylated prior to its assembly in the transposome. In such instances, the 5′ overhang can have accessible 5′ hydroxyl groups outside of the mosaic-end transposase sequence. In some instances, phosphorylation of the 5′ overhang of the assembled transposome complexes can be achieved by exposing these 5′ ends of transposome complexes to a polynucleotide kinase (e.g., T4-polynucleotide kinase (T4-PNK)) in the presence of ATP.
In some instances, tagmenting genomic DNA of the biological sample with a transposome (e.g., any of the transposomes described herein) can comprise a further step of phosphorylating the 5′ ends of the adaptors (e.g., the 5′ overhangs of the Nextera adaptors, e.g., MEDS) in the transposome complex.
In some instances, methods provided herein comprise a step of providing a transposome that has been treated to phosphorylate the 5′ ends of the adaptors (e.g., the 5′ overhangs of the Nextera adaptors (e.g., first and second adapters), e.g., MEDS) in the transposome complex, thus fragmenting the biological sample with a transposome that has been treated to phosphorylate the 5′ ends of the adaptors in the transposome complex.
In some instances, the transposome (e.g., the transposase dimer complexed with adapters) can be linked to an antibody or peptide with a protein binding domain. In some instances, the antibody is protein A, protein G, a fusion protein of all or parts of protein A and protein G, or derivatives thereof.
Any suitable enzyme and/or conditions can be used to phosphorylate the 5′ ends of the adaptors (e.g., the 5′ overhangs of the adaptors, e.g., MEDS) in the transposome complex, e.g., T4-PNK or T7-PNK. In some instances, the phosphorylation reaction can be carried out by contacting the transposome with a polynucleotide kinase (e.g., T4-PNK or T7-PNK) in a buffered solution (e.g., Tris-HC1, pH about 7.0 to about 8.0, e.g., about 7.6) at about 20 to about 40° C., e.g., about 25 to about 37° C., for about 1 to about 60 minutes, e.g., about 5 to about 50, about 10 to about 40, about 20 to about 30 minutes.
In some instances, gap filling and ligating breaks can be performed on the fragmented (e.g., tagmented) DNA. For example, the Tn5 transposition event results in a 9 base pair gap between an inserted transposon end sequence and the genomic DNA. In some instances, the gap filling is performed between the inserted transposon end sequence and fragmented genomic DNA.
In some instances, the transposon end sequences adjacent to the 9 base pair gap followed by the fragmented genomic DNA are released. In some examples, the transposon end sequences adjacent to the gap are released (e.g., removed) from the fragmented genomic DNA (e.g., released from the complementary transposon end sequence). In some instances, the released transposon end sequences are not ligated to the splint oligonucleotide (e.g., non-ligated transposon end sequences). In some instances, the non-ligated transposon end sequences are released with a heat gradient. In some instances, the ligated transposon end sequences are ligated to the capture probe. In some instances, the splint oligonucleotide is hybridized to the capture domain of the capture probe, or a portion thereof, and the remaining transposon end sequence (e.g., ligated transposon end sequence). In some instances, a gap filling reaction is performed. In some instances, gap filling occurs between the splint oligonucleotide and the fragmented genomic DNA. For example, a gap filling polymerase can fill the gap between the splint oligonucleotide and the fragmented genomic DNA (e.g., a portion of which included the released transposon end sequence).
In some instances, the non-ligated transposon end sequences (e.g., transposon end sequences adjacent to 9 base pair gap) are released with a heat gradient from about 20° C. to about 90° C., from about 25° C. to about 85° C., from about 30° C. to about 80° C., from about 35° C. to about 75° C., from about 40° C. to about 75° C., from about 45° C. to about 75° C., 50° C. to about 75° C., or about 50° C. to about 70° C. In some instances, releasing the non-ligated transposon end sequences occurs at 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 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., or about 90° C.
In some instances, releasing the non-ligated transposon end sequences (e.g., transposon end sequences adjacent to the 9 base pair gap) are released with a heat gradient for about 10 minutes to about 150 minutes, from about 20 minutes to about 140 minutes, from about 30 minutes to about 130 minutes, from about 40 minutes to about 120 minutes, from about 40 minutes to about 110 minutes, from about 50 minutes to about 110 minutes, from about 60 minutes to about 100 minutes, from about 70 minutes to about 90 minutes, from about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about 170 minutes, about 175 minutes, about 180 minutes, about 185 minutes, about 190 minutes, about 195 minutes, about 200 minutes, about 205 minutes, about 210 minutes, about 215 minutes, about 220 minutes, about 225 minutes, about 230 minutes, about 235 minutes, about 240 minutes, about 245 minutes, or about 250 minutes.
In some instances, spatially tagging the genomic DNA can be performed by insertion of the transposon sequence into the genomic DNA with adapters described herein. An amplification step can be performed with primers to the adapters (e.g., inserted adapters into the genomic DNA). The amplified products can contain accessible genomic DNA which can be spatially tagged by methods described herein.
In some instances, spatially tagging the genomic DNA can be performed by transposome complexes immobilized on the surface of the substrate. In some instances, spatially tagging the genomic DNA can be performed by transposome complexes immobilized on a feature (e.g., a bead). In some instances, the transposome complexes are assembled prior to adding the biological sample to the substrate or features. In some instances, the transposome complexes are assembled after adding the biological sample to the substrate or features on a substrate. For example, a spatially barcoded substrate (e.g., array) can include a plurality of capture probes that include a Mosaic End sequence (e.g., a transposase recognition sequence). The Mosaic End sequence can be at the 3′ end of the capture probe (e.g., the capture probe is immobilized by its 5′ end and the Mosaic End sequence is at the 3′ most end of the capture probe). The Mosaic End sequence can be a Mosaic End sequence for any of the transposase enzymes described herein. The Mosaic End sequence (e.g., a transposase recognition sequence) can be hybridized to a reverse complement sequence (e.g., oligonucleotide). For example, the reverse complement sequence (e.g., reverse complement to the Mosaic End sequence) can hybridize to the Mosaic End sequence thereby generating a portion of double stranded DNA on the capture probe. The reverse complement to the Mosaic End sequence (e.g., oligonucleotide) can be provided to the spatially barcoded array prior to the biological sample being provided to the substrate. In some instances, the reverse complement to the Mosaic End sequence can be provided after the biological sample has been provided to the substrate. Transposase enzymes can be provided to the substrate and assemble at the double stranded portion of the capture probe (e.g., reverse complement oligonucleotide and the Mosaic End sequence hybridized to each other) thereby generating a transposome complex. For example, a transposome homodimer can be formed at the double stranded portion of the capture probe. Additionally, a transposase and antibody, or a transposase complexed to an antibody or protein binding moiety) can be provided to the substrate and assembled at the double stranded portion of the capture probe (e.g., reverse complement oligonucleotide and the Mosaic End sequence hybridized to each other) thereby generating a transposome complex. A biological sample can be provided to the substrate such that the position of the capture probe on the substrate can be correlated with a position (e.g., location) in the biological sample. The transposome complexes or transposome complex can fragment (e.g., tagment) and spatially tag the genomic DNA.
In some instances, spatially tagging genomic DNA can be performed by hybridizing a single stranded capture probe to the tagmented DNA. In some instances the single stranded capture probe can be a degenerate sequence. In some instances, the single stranded capture can be a random sequence. The single stranded capture probe can have a functional domain, a spatial barcode, a unique molecular identifier, a cleavage domain, or combinations thereof. The single stranded capture probe (e.g., random sequence, degenerate sequence) can non-specifically hybridize tagmented genomic DNA, thereby spatially capturing the tagmented DNA. Methods for extension reactions are known in the art and any suitable extension reaction method described herein can be performed.
In other instances, tagmented DNA can be captured by the capture domain of the capture probe by binding (e.g., hybridizing) a poly(A) tailed tagmented DNA (e.g., by including a sequence complementary to a capture probe on one or more adaptors or recognition sequences (e.g., X1 as shown in
In some instances, after fragmenting the genomic DNA, gap filing (e.g., no strand displacement) polymerases and ligases can repair gaps and ligate breaks in the tagmented DNA. In some instances, a sequence complementary to the capture domain can be introduced to the fragmented DNA. For example, a poly(A) tail can be added to the tagmented DNA, such that the capture domain (e.g., poly(T) sequence) of the capture probe can bind (e.g., hybridize) to the poly(A) tailed tagmented DNA (See, e.g., WO 2012/140224, which is incorporated herein by reference). In some instances, a poly(A) tail is added to the tagmented by a terminal transferase enzyme. In some instances, the terminal transferase enzyme is a terminal deoxynucleotidyl transferase (TdT), or a mutant variant thereof. TdT is an independent polymerase (e.g., it does not require a template molecule) that can catalyze the addition of deoxynucleotides to the 3′ hydroxyl terminus of DNA molecules. Other template independent polymerases are known in the art. For example, Polymerase θ, or a mutant variant thereof, can be used as a terminal transferase enzyme (See, e.g., Kent, T., Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining, eLIFE, 5: e13740 doi: 10.7554/eLife.13740, (2016)). Other methods of introducing a poly(A) tail are known in the art. In some instances, a poly(A) tail can be introduced to the tagmented DNA by a non-proof reading polymerase. In some instances, a poly(A) tail can be introduced to the fragmented DNA by a polynucleotide kinase.
In some instances, the TDT enzyme will generate tagments with a 3′ poly(A) tail, thereby mimicking the poly(A) tail of an mRNA. In some instances, the capture domain (e.g., poly(T) sequence) of the capture probe would interact with the poly(A) tail of the mRNA and the generated (e.g., synthesized) poly(A) tail added to the fragmented (e.g., tagmented) DNA, thereby simultaneously capturing the fragmented DNA (e.g., tagmented DNA) and the mRNA transcript. The generated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g., tagmented DNA) could be between about 10 nucleotides to about 30 nucleotides long. The generated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g., tagmented DNA) could be about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, or about 29 nucleotides long.
As used herein, the term “splint oligonucleotide” refers to an oligonucleotide that, when hybridized to other polynucleotides, acts as a “splint” (e.g., splint helper probe) to position the polynucleotides next to one another so that they can be ligated together. In some instances, the splint oligonucleotide is DNA or RNA. The splint oligonucleotide can include a nucleotide sequence that is partially complementary to nucleotide sequences from two or more different oligonucleotides. In some instances, the splint oligonucleotide assists in ligating a “donor” oligonucleotide and an “acceptor” oligonucleotide. In some instances, an RNA ligase, a DNA ligase, or other ligase can be used to ligate two nucleotide sequences together.
In some instances, the splint oligonucleotide can be between about 10 and about 50 nucleotides in length, e.g., between about 10 and about 45, about 10 and about 40, about 10 and about 35, about 10 and about 30, about 10 and about 25, or about 10 and about 20 nucleotides in length. In some instances, the splint oligonucleotide can be between about 15 and about 50, about 15 and about 45, about 15 and about 40, about 15 and about 35, about 15 and about 30, about 15 and about 30, or about 15 and about 25 nucleotides in length.
In some instances, the fragmented DNA can include a sequence that is added (e.g., ligated) during fragmentation of the DNA. For example, during a transposition event (e.g., a Tn5 transposition event) an additional sequence (e.g., transposon end sequences) can be attached (e.g., covalently attached, e.g., via a ligation event) to the fragmented DNA (e.g., fragmented genomic DNA, e.g., tagmented genomic DNA). In some instances, the splint oligonucleotide can have a sequence that is complementary (e.g., a capture domain) to the fragmented DNA (e.g., fragmented genomic DNA, e.g., fragmented genomic DNA that includes a sequence that is added during fragmentation of the DNA, e.g. a first adapter attached during fragmentation of the DNA, e.g., a transposon end sequence) and a sequence that is complementary to the capture domain of the capture probe. In some instances, the splint oligonucleotide can be viewed as part of the capture probe. For example, the capture probe can be partially double stranded where a portion of the capture probe can function as a splint oligonucleotide that binds a portion of the capture probe (e.g., dsDNA portion) and can have a single strand portion that can bind (e.g., capture domain) the fragmented DNA (e.g., fragmented genomic DNA e.g., tagmented, e.g., an adapter attached during fragmentation of the DNA, e.g., a Nextera adapter). The first adapter sequence (e.g., the sequence attached to the fragmented DNA complementary to the capture domain, e.g., Nextera adapter) can be any suitable sequence. In some instances, the adapter sequence can be between about 15 and 25 nucleotides long. In some instances, the adapter sequence can be about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 nucleotides long.
In some instances, a splint oligonucleotide can include a sequence that is complementary (e.g., capture domain) to the first adapter attached to the fragmented DNA (e.g., tagmented DNA). In some instances, the splint oligonucleotide includes a sequence that is not perfectly complementary to the first adapter (e.g., Nextera adapter) attached to the fragmented DNA (e.g., tagmented DNA), but is still capable of hybridizing the first adapter sequence (e.g., sequence complementary to the capture domain) ligated on to the fragmented DNA (e.g., Nextera adapter).
Any of a variety of capture probes having capture domains that hybridize to a splint oligonucleotide can be used in accordance with materials and methods described herein. As described herein, a capture domain is a domain on a capture probe capable of hybridizing the splint oligonucleotide to form a partially double stranded capture probe. For example, a single stranded capture probe can have a sequence complementary (e.g., capture domain) to a portion of the splint oligonucleotide, such that a partially double stranded capture probe is formed with a single stranded portion capable of binding the inserted transposon end sequences. In some instances, a splint oligonucleotide includes a sequence that is complementary (e.g., at least partially complementary) to the capture domain of the capture probe.
In some instances, the splint oligonucleotide includes a sequence that is not perfectly complementary to the capture domain of the capture probe, but is still capable of hybridizing the capture domain of the capture probe. In some instances, the splint oligonucleotide can hybridize to both the transposon end sequence (e.g., additional sequence attached to the tagmented DNA) and the capture domain of the capture probe via its sequence complementary to the capture domain. In such instances, where the splint oligonucleotide can hybridize to both the transposon end sequence (e.g., Nextera adapter, additional sequence attached to the fragmented DNA e.g., tagmented DNA), and the capture domain of the capture probe, the splint oligonucleotide can be viewed as part of the capture probe.
In some instances, the splint oligonucleotide can have a capture domain that is homopolymeric. For example, the capture domain can be a poly(T) capture domain.
In some instances, a splint oligonucleotide can facilitate ligation of the tagmented DNA and the capture probe. Any variety of suitable ligases known in the art or described herein can be used. In some instances, the ligase is T4 DNA ligase. In some instances, the ligation reaction can last for about 1 to about 5 hours. In some instances, the ligation reaction can last for about 2, about 3, or about 4 hours. In some instances, after ligation, strand displacement polymerization can be performed. In some instances, a DNA polymerase can be used to perform the strand displacement polymerization. In some instances, the DNA polymerase is DNA polymerase I.
The present disclosure describes methods for permeabilizing biological samples under conditions sufficient to allow tagmentation of genomic DNA. The genomic DNA, or accessible chromatin, also known as open chromatin, can be tagmented with a transposome complex (e.g. a transposase and an antibody-binding moiety, bound to an antibody bound to, for example, a histone; or a multi-complex of a transposase, antibody-binding moiety, and an antibody, the complex bound to, for example, a histone). The tagmented genomic DNA can be captured via a capture probe (e.g., a capture probe and a splint oligonucleotide), however, at times it can be useful to simultaneously capture tagmented genomic DNA and other nucleic acids (e.g., mRNA). For example, expression profiles of transcripts can be correlated, anti-correlated, or not correlated with open (e.g. accessible) chromatin. Put another way, the presence of transcripts can correlate with open chromatin (e.g., accessible chromatin) corresponding to the genes (e.g., genomic DNA) from which the RNA transcripts were transcribed.
The present disclosure also describes methods regarding the simultaneous capture of tagmented genomic DNA and mRNA or protein on spatially barcoded arrays from biological samples (e.g., fresh or frozen tissue samples). Methods of detecting RNA molecules having poly(A) sequences, proteins, and derivatives of RNA molecules (e.g., RTL products) have been described previously in WO 2020/176788 and in U.S. Patent Application Publication Nos. 2020/0277663, 2021/0285046 and 2021/0285036, each of which is incorporated by reference in its entirety.
For example, multiplex capture (e.g. capturing genomic DNA, DNA, RNA and/or mRNA) can be performed on a spatially barcoded array having a plurality of capture probes immobilized on a substrate surface. The plurality of capture probes can have substantially the same capture sequence (e.g. a poly(T) capture sequence) and can capture both tagmented gDNA or mRNA.
Alternatively, multiplex capture can be performed on a spatially barcoded array having multiple pluralities of capture probes immobilized on a substrate surface (e.g. different types of capture probes). In some instance, tagmented genomic DNA (e.g. tagmented with a transposome complex or a multi-complex) can be captured on the array using a splint oligo, as described above. In some instances, the splint oligo is capable of hybridizing to both a capture probe on an array and to the tagmented DNA. In some instances, the capture probes have capture sequences that are specific to either tagmented DNA or RNA molecules or derivatives of RNA molecules (e.g. RTL products). In some cases, the capture probes for the RNA molecules or derivatives of RNA molecules (e.g. RTL products) are poly(T) sequences.
In any of the methods described herein, the feature with a plurality of capture probes can be on a substrate. The capture probes can have spatial barcodes corresponding to a position (e.g., location) on the substrate. In some instances, the capture probes can further have a unique molecular identifier, one or more functional domains, and a cleavage domain, or combinations thereof. In some instances, the capture probe includes a capture domain. In some instances, the capture probe can be a homopolymeric sequence. For example, in a non-limiting way, the homopolymeric sequence can be a poly(T) sequence. In some instances, nucleic acid (e.g., mRNA) can be captured by the capture domain by binding (e.g., hybridizing) of poly(A) tails of mRNA transcripts. Tagmented genomic DNA can also be captured by the capture domain of the capture probe by binding (e.g., hybridizing) a poly(A) tailed tagmented genomic DNA (e.g., by including a sequence complementary to a capture probe on one or more adaptors or recognition sequences (e.g., X1 as shown in
In some instances, after fragmenting the genomic DNA and optionally capturing RNA molecules having poly(A) sequences or derivatives of RNA molecules (e.g. via RTL products), gap filing (e.g., no strand displacement) polymerases and ligases can repair gaps and ligate breaks in the tagmented DNA. In some instances, a sequence complementary to the capture domain can be introduced to the fragmented DNA. For example, a poly(A) tail can be added to the tagmented DNA, such that the capture domain (e.g., poly(T) sequence) of the capture probe can bind (e.g., hybridize) to the poly(A) tailed tagmented DNA (See, e.g., WO 2012/140224, which is incorporated herein by reference). In some instances, a poly(A) tail is added to the tagmented DNA by a terminal transferase enzyme. In some instances, the terminal transferase enzyme is a terminal deoxynucleotidyl transferase (TdT), or a mutant variant thereof. TdT is an independent polymerase (e.g., it does not require a template molecule) that can catalyze the addition of deoxynucleotides to the 3′ hydroxyl terminus of DNA molecules. Other template independent polymerases are known in the art. For example, Polymerase θ, or a mutant variant thereof, can be used as a terminal transferase enzyme (See, e.g., Kent, T., Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining, eLIFE, 5: e13740 doi: 10.7554/eLife.13740, (2016)). Other methods of introducing a poly(A) tail are known in the art. In some instances, a poly(A) tail can be introduced to the tagmented DNA by a non-proof reading polymerase. In some instances, a poly(A) tail can be introduced to the fragmented DNA by a polynucleotide kinase. The captured RNA molecules (e.g. via RTL products) lack gaps and are substantially not affected by the gap-filling process used on the tagmented DNA.
In some instances, the TDT enzyme will generate tagments with a 3′ poly(A) tail, thereby mimicking the poly(A) tail of an mRNA. In some instances, the capture domain (e.g., poly(T) sequence) of the capture probe would interact with the poly(A) tail of the mRNA and the generated (e.g., synthesized) poly(A) tail added to the fragmented (e.g., tagmented) DNA, thereby simultaneously capturing the fragmented DNA (e.g., tagmented DNA) and the mRNA transcript. The generated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g., tagmented DNA) could be between about 10 nucleotides to about 30 nucleotides long. The generated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g., tagmented DNA) could be about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, or about 29 nucleotides long.
In some instances, provided herein are methods of multiplexing that capture both gDNA and RNA. In some instances, in addition to methods of associating gDNA (either using a splint oligonucleotide or via hybridization directly), the methods of detecting RNA include hybridizing the analyte (e.g., RNA) or a portion thereof to the capture domain; and determining (i) all or part of a sequence of the spatial barcode or a complement thereof, and (ii) all or part of a sequence of the analyte, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the abundance and the location of the analyte in the biological sample. Methods of RNA capture have been described previously in WO 2020/176788 and in U.S. Patent Application Publication No. 2020/0277663, each of which is incorporated by reference. These methods generally include tissue staining and imaging, cDNA synthesis, second strand synthesis and denaturation, cDNA amplification, library construction, and sequencing.
In some instances, the methods can include determining abundance and location of a second analyte in the biological sample by detecting an analyte derivative such as an RTL product. In some instances, at the same time as capture of gDNA, the multiplex methods can include contacting a first templated ligation (RTL) probe and a second RTL probe with the biological sample, wherein the first RTL probe and the second RTL probe each comprise sequences that are substantially complementary to adjacent sequences of the second analyte, and wherein the second probe comprises a capture probe capture domain that is complementary to all or part of the capture domain; (b) hybridizing the first probe and the second probe to the second analyte; (c) generating an RTL ligation product by ligating the first probe and the second probe; (d) releasing the RTL ligation product from the second analyte; (e) hybridizing the RTL ligation product to the capture domain; and (f) determining (i) all or part of the sequence of the RTL ligation product bound to the capture domain, or a complement thereof, and (ii) all or part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify determine the location of the second analyte in the biological sample.
Detection of gDNA using methods described herein can be achieved concurrently with RNA templated ligation. In some cases, sample processing for determining the location of tagmented gDNA can occur before, after, or concurrently with the sample processing for determining the location of RNA or RNA-derived products (e.g. via RTL products). In some instances, both RTL probes and transposome complexes or RTL probes and multi-complexes can be added to the biological sample on a spatially barcoded array at the same time. In some instances, RTL probes are added to the biological sample on a spatially barcoded array before the transposome complexes or multi-complexes are added. In some instances, RTL probes are added to the biological sample on a spatially barcoded array after the transposome complexes or multi-complexes are added.
In some instances, one of the RTL probes includes a poly(A) sequence, or a complement thereof. In some instances, the poly(A) sequence, or a complement thereof, is on the 5′ end of one of the RTL probes. In some instances, the poly(A) sequence, or a complement thereof, is on the 3′ end of one of the RTL probes. In some instances, one RTL probe includes a degenerate or UMI sequence. In some instances, the UMI sequence is specific to a particular target or set of targets. In some instances, the UMI sequence, or a complement thereof, is on the 5′ end of one of the RTL probe. In some instances, the UMI sequence, or a complement thereof, is on the 3′ end of one of the RTL probe.
After addition of the RTL probes, RTL probes hybridize to target mRNA and are ligated together. After ligation of the RTL probes, an endonuclease such as RNAse H is added to the sample. RNAse H digests both the RNA analyte and the undesirable RNA. In some instances, at least one of the RTL probes includes a probe capture sequence such as a poly-A sequence, an oligo-d(T) sequence, or a particular capture sequence (in the setting of targeted RNA analysis).
In some instances, the first RTL probe hybridizes to an analyte and a second RTL probe hybridizes to an analyte in proximity to the first RTL probe. Hybridization can occur at a target having a sequence that is 100% complementary to the probe RTL probe(s). In some instances, hybridization can occur at a target having a sequence that is at least (e.g., at least about) 80%, at least (e.g., at least about) 85%, at least (e.g., at least about) 90%, at least (e.g., at least about) 95%, at least (e.g., at least about) 96%, at least (e.g., at least about) 97%, at least (e.g., at least about) 98%, or at least (e.g., at least about) 99% complementary to the RTL probe (s). After hybridization, in some instances, the first RTL probe is extended. After hybridization, in some instances, the second RTL probe is extended. For example, in some instances a first RTL probe hybridizes to a target sequence upstream of a second RTL probe, whereas in other instances a first RTL probe hybridizes to a target sequence downstream of a second RTL probe.
In some instances, methods disclosed herein include a wash step after hybridizing the first and the second RTL probes. The wash step removes any unbound probes and can be performed using any technique known in the art. In some instances, a pre-Hybridization buffer is used to wash the sample. In some instances, a phosphate buffer is used. In some instances, multiple wash steps are performed to remove unbound probes. For example, it is advantageous to decrease the amount of unhybridized probes present in a biological sample as they may interfere with downstream applications and methods.
In some instances, after hybridization of the RTL probes (e.g., the first and the second RTL probes) to the target analyte, the RTL probes are ligated together, creating a single ligated probe that is complementary to the target analyte. Ligation can be performed enzymatically or chemically, as described herein. For example, the first and second RTL probes are hybridized to the first and second target regions of the analyte, and the RTL probes are subjected to a ligation reaction to ligate them together. For example, the probes may be subjected to an enzymatic ligation reaction using a ligase (e.g., T4 RNA ligase (Rn12), a SplintR ligase, or a T4 DNA ligase). See, e.g., Zhang L., et al.; Archaeal RNA ligase from Thermoccocus kodakarensis for template dependent ligation RNA Biol. 2017; 14(1): 36-44 for a description of KOD ligase.
In some instances, adenosine triphosphate (ATP) is added during the ligation reaction. DNA ligase-catalyzed sealing of nicked DNA substrates is first activated through ATP hydrolysis, resulting in covalent addition of an AMP group to the enzyme. After binding to a nicked site in a DNA duplex, the ligase transfers this AMP to the phosphorylated 5′-end at the nick, forming a 5′-5′ pyrophosphate bond. Finally, the ligase catalyzes an attack on this pyrophosphate bond by the OH group at the 3′-end of the nick, thereby sealing it, whereafter ligase and AMP are released. If the ligase detaches from the substrate before the 3′ attack, e.g., because of premature AMP reloading of the enzyme, then the 5′ AMP is left at the 5′-end, blocking further ligation attempts. In some instances, ATP is added at a concentration of about 1 μM, about 10 μM, about 100 μM, about 1000 μM, or about 10000 μM during the ligation reaction.
In some instances, cofactors that aid in ligation of the RTL probes are added during the ligation process. In some instances, the cofactors include magnesium ions (Mg2+). In some instances, the cofactors include manganese ions (Mn2+). In some instances, Mg2+ is added in the form of MgCl2. In some instances, Mn2+ is added in the form of MnCl2. In some instances, the concentration of MgCl2 is at about 1 mM, at about 10 mM, at about 100 mM, or at about 1000 mM. In some instances, the concentration of MnCl2 is at about 1 mM, at about 10 mM, at about 100 mM, or at about 1000 mM.
In some instances, after ligation of the first and second RTL probes to generate a ligation product, the ligation product is released from the analyte. At this stage of the method, (1) the ligation product is hybridized to the analyte, and (2) the gDNA has been tagmented. To release the ligation product from the analyte, an endoribonuclease is used. An endoribonuclease such as RNAse H specifically cleaves RNA in RNA:DNA hybrids. Thus, not only does RNAse H cleave the hybridization of the ligation product to the analyte (releasing the ligation product), RNAse H also cleaves the undesirable RNA. In some instances, the ligation product is released enzymatically. In some instances, an endoribonuclease is used to release the ligation product from the analyte. In some instances, the endoribonuclease is one or more of RNase H. In some instances, the RNase H is RNase H1 or RNase H2.
In some instances, after generation of a ligation product from the RTL probes (e.g., a first RTL probe and second RTL probe), the biological sample is permeabilized. In some instances, permeabilization occurs using a protease. In some instances, the protease is an endopeptidase. Endopeptidases that can be used include but are not limited to trypsin, chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamyl endopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN), endopeptidase LysC and endopeptidase LysN. In some instances, the endopeptidase is pepsin. In some instances, permeabilization is performed using proteinase K.
In some instances, the ligation product includes a capture probe binding domain, which can hybridize to a capture probe (e.g., a capture probe immobilized, directly or indirectly, on a substrate). In some instances, methods provided herein include contacting a biological sample with a substrate, wherein the capture probe is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). In some instances, the capture probe includes a spatial barcode and the capture domain. In some instances, the capture probe binding domain of the ligation product binds (e.g., hybridizes) to the capture domain. After hybridization of the ligation product to the capture probe, the ligation product is extended at the 3′ end to make a copy of the additional components (e.g., the spatial barcode) of the capture probe. In some instances, methods of ligation product capture as provided herein include permeabilization of the biological sample such that the capture probe can more easily hybridize to the ligation product (i.e., compared to no permeabilization). In some instances, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents provide for the extension of the capture probe using the ligation product as a template, as well as the extension of the ligation product using the capture probe as a template thereby producing nucleic acid molecules that comprise target analyte sequences, or complements thereof, and sequences of the capture probe, or complements thereof such as spatial barcodes, functional sequences, UMIs, etc.
The resulting nucleic acid molecule 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 nucleic acid molecule can be amplified via PCR prior to library construction. The nucleic acid molecule can then be enzymatically fragmented and size-selected in order to optimize the amplicon size. P5, P7, i7, and i5 can be used as sample indexes, and 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 as sequencing primer sites.
Post-hybridization steps are also provided in Stahl P. L., et al., Visualization and analysis of gene expression in tissue sections by spatial transcriptomics Science, vol. 353, 6294, pp. 78-82 (2016), which in incorporated herein by reference).
In any of the methods described herein, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a fresh tissue sample, or a frozen tissue sample. In some instances, the tissue sample is the FFPE tissue sample. In some instances, the biological sample is deparaffinized. Deparaffinization can be achieved using any method known in the art. For example, in some instances, the biological samples is treated with a series of washes that include xylene and various concentrations of ethanol. In some instances, methods of deparaffinization include treatment of xylene (e.g., three washes at 5 minutes each). In some instances, the methods further include treatment with ethanol (e.g., 100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 10 minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, two washes 10 minutes each). In some instances, after ethanol washes, the biological sample can be washed with deionized water (e.g., two washes for 5 minutes each). It is appreciated that one skilled in the art can adjust these methods to optimize deparaffinization.
In some instances, the biological sample is decrosslinked. In some instances, the biological sample is decrosslinked in a solution containing TE buffer (comprising Tris and EDTA). In some instances, the TE buffer is basic (e.g., at a pH of about 9). In some instances, decrosslinking occurs at about 50° C. to about 80° C. In some instances, decrosslinking occurs at about 70° C. In some instances, decrosslinking occurs for about 1 hour at 70° C. Just prior to decrosslinking, the biological sample can be treated with an acid (e.g., 0.1M HCl for about 1 minute). After the decrosslinking step, the biological sample can be washed (e.g., with 1× PBST).
Protein A is a 40-60 kDA surface protein found in Staphylococcus aureus cell walls. Its ability to bind immunoglobulins and IgG proteins, the heavy chain within the Fc region of most immunoglobulins, from many mammalian species makes it a useful protein for complexing with antibodies. It has the ability to interact and associate with a number of antibodies to create multiple options for protein-transposome-antibody complexing. Protein G is an immunoglobulin binding protein found in Streptococcal bacteria, but differs from Protein A in its binding properties. Protein G is a cell surface binding protein of 40-65 kDa, that binds to the Gav and Fc region of multiple antibodies, and indeed it finds utility in purifying antibodies because of its wide ability for antibody binding. Associating either protein A, protein G, or a protein A/G fusion protein with the transposase to create a protein A/G-Tn5 transposome can include generating a contiguous nucleic acid sequence via, e.g., standard cloning techniques. Adapters to be inserted into the transposome are added by complexing the protein A/G transposase complex with the associated transposon sequences as known in the art.
For example, Kaya-Okur et al (2019, Nature Communications 10:1930 CUT&Tag for efficient epigenomic profiling of small samples and single cells (incorporated herein by reference), provides a method for generating a protein A-Tn5 transposome complex by first generating a C-terminal fusion protein with protein A separated from the transposase by a 26 amino acid long flexible peptide linker, followed by transposome generation with the generated protein A-transposase fusion protein complexed with the transposon mosaic ends. Another method for generating a protein A-Tn5 transposome complex can be found in Bartosovic et al (2021, Nat Biotech, doi.org/10.1038/s41587-021-00869-9, incorporated herein by reference). Commercially available protein A-Tn5 transposase complexes for generating a protein A fused transposomes are available, for example from Vazyme (Hyperactive pA-Tn5 Transposase for CUT&Tag).
The present disclosure is not limited by the methods in which a protein A or protein G-transposome complexes are generated, further the present disclosure is not limited by the methods of generating an antibody-transposome complex, where the antibody-transposome complex comprises an antibody bound to protein A or protein G of a protein A-transposome complex or a protein G-transposome complex.
Also provided herein are compositions including capture probes, transposome complexes, tagmented DNA, splint oligonucleotides, and one or more polymerases. In some instances, a splint oligonucleotide is hybridized to the capture domain of a capture probe. In some instances, a splint oligonucleotide is hybridized to a transposon end sequence of fragmented (e.g., tagmented) genomic DNA. In some instances, a splint oligonucleotide is hybridized to the captured domain of a capture probe and a transposon end sequence of fragmented genomic DNA. In some instances, the composition comprises one or more transposon end sequences. In some instances, one or more transposon end sequences are ligated to the capture probe. In some instances, one or more transposon sequences are released from the fragmented DNA either before or after ligation of the fragmented genomic DNA to the capture probe. In some instances, the composition includes a ligase (e.g., T4 DNA ligase). In some instances, the composition includes a gap filling polymerase. In some instances, the composition includes a DNA polymerase.
In some instances, the capture domain of the capture probe binds the transposon end (e.g., without the facilitation by a splint oligonucleotide). In some instances, the composition includes a strand-displacing polymerase. In some instances, the composition includes a gap filling polymerase.
In some instances, the composition includes an array comprising a plurality of capture probes. In some instances, a capture probe of the plurality of capture probes include a spatial barcode and a capture domain.
In some instances, the composition includes a transposome. In some instances, the composition includes a protein binding protein-transposome complex. In some instance, the transposome complex includes a protein binding moiety, a transposase, a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and a second transposon end sequence.
Also provided herein are kits that include one or more reagents to detect one or more analytes described herein. In some instances, the kit includes an array comprising a plurality of capture probes comprising a spatial barcode and a capture domain. In some instances, the kit includes a transposome complex comprising an antibody, a transposase, a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and a second transposon end sequence comprising a functional sequence. In some instances, the kit includes a plurality of probes (e.g., RD probes, a first RTL probe, a second RTL probe, one or more spanning probes, and/or a third oligonucleotide).
A non-limiting example of a kit used to perform any of the methods described herein includes: (a) an 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 transposome complex comprising: (i) an antibody, (ii) a transposase, (iii) a first transposon end sequence comprising a splint sequence that is substantially complementary to a portion of a splint oligonucleotide, and (iv) a second transposon end sequence comprising a functional sequence; and (c) instructions for performing any one of the methods described herein.
In some instances of any of the kits described herein, the kit includes a second RTL probe that includes a preadenylated phosphate group at its 5′ end and a first RTL probe comprising at least two ribonucleic acid bases at the 3′ end.
qPCR and Analysis
Also provided herein are methods and materials for quantifying capture efficiency. In some instances, quantification of capture efficiency includes quantification of captured fragments (e.g., genomic DNA fragments, e.g., tagmented DNA fragments) from any of the spatial analysis methods described herein. In some instances, quantification includes PCR, qPCR, electrophoresis, capillary electrophoresis, fluorescence spectroscopy and/or UV spectrophotometry. In some instances, qPCR includes intercalating fluorescent dyes (e.g., SYBR green) and/or fluorescent labeled-probes (e.g., without limitation, Taqman probes or PrimeTime probes). In some instances, a NGS library quantification kit is used for quantification. For example, quantification can be performed using a KAPA library quantification kit (KAPA Biosystems), qPCR NGS Library Quantification Kit (Agilent), GeneRead Library Quant System (Qiagen), and/or PerfeCTa NGS Quantification Kit (Quantabio). In some instances that use qPCR for quantification, qPCR can include, without limitation, digital PCR, droplet digital (ddPCR), and ddPCR-Tail. In some instances that use electrophoresis for quantification, electrophoresis can include, without limitation, automated electrophoresis (e.g., TapeStation System, Agilent, and/or Bioanalzyer, Agilent) and capillary electrophoresis (e.g., Fragment Analyzer, Applied Biosystems). In some instances that use spectroscopy for quantification, the spectroscopy can include, without limitation, fluorescence spectroscopy (e.g., Qubit, Thermo Fisher). In some instances, NGS can be used to quantify capture efficiency.
In some instances, quantitative PCR (qPCR) is performed on the captured tagments. In some instances, the fragmented (e.g., tagmented) DNA is amplified, by any method described herein, before capture. For example, after capture of the fragmented DNA (e.g., tagmented DNA), ligation and strand displacement hybridization qPCR can be performed.
Methods of staining a biological sample (e.g., immunofluorescence, immunohistochemistry, H&E) are known in the art and are provided herein. In some instances, the biological sample can be imaged.
An antibody binding moiety-transposome complex (
An antibody that binds, for example, a histone is applied to the biological sample and the antibody is allowed to bind to its target antigen, The transposome complex is applied to a biological sample which is located on a spatial array, and the protein A or protein G moiety binds to the antibody that is bound to the target protein (such as a histone). The genomic DNA, or open chromatin, of the cell of the tissue can undergo tagmentation, mediated by the transposome complex. The open chromatin or open genomic DNA that may be present between histones is available for a potential transpositional event as the transposase cuts the genomic DNA and inserts the transposon ends into the cut genomic DNA (
The resulting tagmented gDNA in the biological sample can interact with capture probes on the spatial array, each capture probe having a capture domain and a spatial barcode, wherein the tagmented gDNA is attached to the capture probe using a splint oligonucleotide (
After the tagmented gDNA of Example 1 interacts with capture probes on the spatial array, with each capture probe having a capture domain and a spatial barcode, the tagmented gDNA is attached to the capture probe using a splint oligonucleotide (
The gaps can be filled, for example, by a “gap-filling” step which includes incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic acid molecules of interest, followed by ligation of the two now adjacent ends. As such, a non-interrupted nucleic acid is produced that contains at least the UMI, capture sequence, transposon sequences with mosaic ends, genomic DNA, another transposon sequence, and sequencing primer binding sites (
A multi-complex of a transposome, an antibody-binding moiety, and an antibody complex (
The multi-complex with the antibody that binds, for example, a histone, is applied to the biological sample located on a spatial array. The antibody of the multi-complex binds to its target antigen, such as a target protein (e.g., histone). The genomic DNA, or open chromatin, of the cell of the biological sample, such as a tissue, can undergo tagmentation, mediated by the transposome of the multi-complex. The open chromatin or open genomic DNA that may be present between histones is available for a potential transpositional event as the transposase cuts the genomic DNA and inserts the transposon ends into the cut genomic DNA (
The resulting tagmented gDNA in the biological sample can interact with capture probes on the spatial array, each capture probe having a capture domain and a spatial barcode, wherein the tagmented gDNA is attached to the capture probe using a splint oligonucleotide, as described in Example 1 (
This application claims priority to U.S. Provisional Patent Application No. 63/285,677, filed Dec. 3, 2021. The entire content of the foregoing application is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63285677 | Dec 2021 | US |
